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DEVELOPMENTAL DYNAMICS 214:323–336 (1999)
Early Hematopoiesis and Developing Lymphoid Organs
in the Zebrafish
CATHERINE E. WILLETT,1* ALFONSO CORTES,2 ADELINA ZUASTI,3 AND AGUSTIN G. ZAPATA2
Department, Massachusetts Institute of Technology, Cambridge, Massachusetts
2Department of Cell Biology, Faculty of Biology, Complutense University, Madrid, Spain
3Department of Cell Biology, School of Medicine, University of Murcia, Murcia, Spain
1Biology
ABSTRACT
In zebrafish, the transparent
and rapidly developing embryo and the potential
for genetic screening offer a unique opportunity
to investigate the early development of the vertebrate immune system. Here we describe the initial appearance of various blood lineages and the
nature of accumulating hematopoietic tissue in
the thymus and kidney, the main lymphoid organs of adult teleosts. The ultrastructure of the
first site of hematopoiesis, the intermediate cell
mass (ICM), is described from the 5-somite stage,
about 11.5 hours post-fertilization (hpf) until 24
hpf. The ICM gives rise to the primitive erythroid
lineage, which accounts for all circulating erythrocytes for the first 4 days pf. From 24 to 72 hpf, a
few developing granulocytes are seen close to the
yolk sac walls and in the caudal axial vein. The
heart, previously proposed as an early bloodforming organ in zebrafish, did not contain hematopoietic cells. The thymic primordium, consisting of two layers of epithelial cells, appears at 60
hpf. By 65 hpf, it is colonized by immature lymphoblasts. The thymus gradually accumulates lymphocytes, and the lymphocytes and epithelial
cells progressively differentiate for 3 weeks pf. At
96 hr, the pronephros contains hematopoietic
cells, mainly developing erythrocytes and granulocytes. The amount of renal hematopoietic tissue increases rapidly; however, lymphocytes were
not detected until 3 weeks pf. Dev Dyn
1999;214:323–336.
r 1999 Wiley-Liss, Inc.
Key words: zebrafish development; hematopoiesis; thymus; kidney
INTRODUCTION
In adult vertebrates, erythroid, myeloid, and lymphoid cells share a common progenitor. It is thought
that these common stem cells gives rise to two subsequent progenitors, one capable of generating erythroid
and myeloid lineages, the other generating lymphoid
lineages. The myeloid lineage includes monocytes (macrophages) and granulocytes. Hematopoietic tissue can
therefore be erythropoietic, myelopoietic, lymphopoietic, or a combination, depending on the nature of the
progenitor cells comprising it.
r 1999 WILEY-LISS, INC.
In all vertebrates, the site of hematopoiesis changes
during development. In mammals, chicken, and Xenopus laevis, the first site of hematopoiesis is in the tissue
derived from the ventral mesoderm surrounding the
yolk sac—the primitive blood islands in mammals and
chicken, and the ventral blood islands of Xenopus
(reviewed in Orkin, 1995; Robb, 1997). This stage of
hematopoiesis is called ‘‘primitive,’’ and this tissue
gives rise only to embryonic red blood cells (RBCs) and
some myeloid lineages. The next site of hematopoiesis
differs among species; however, the origin of the tissue
in all cases is dorsal mesoderm: the para-aortic splanchopleura and the related aorta-gonad-mesonephros
region (AGM) in mammals (Godin et al., 1995; Medvinsky and Dzierzak, 1996) and chicken (Dieterlen-Lievre
and Le Douarin, 1993); the dorso-lateral plate (DLP) in
Xenopus (Chen and Turpen, 1995). This dorsallyderived hematopoietic tissue is capable of giving rise to
additional blood lineages, including lymphoid (Cumano
et al., 1996; Medvinsky and Dzierzak, 1996), and is
called ‘‘definitive’’ hematopoiesis.
Sites of hematopoiesis during the development of
several teleost species have been described (Al-Adhami
and Kunz, 1976; Al-Adhami and Kunz, 1977; Ellis,
1977; Iuchi and Yamamoto, 1983; Secombes et al., 1983;
Zapata et al., 1995). There are three classes of teleost
embryos, based on the initial site of hematopoiesis. In
some species, such as angelfish (Al-Adhami and Kunz,
1976; Colle-Vandevelde, 1962) and Blennius gattorugine (Colle-Vandevelde, 1963), initial hematopoiesis
occurs exclusively in the yolk sac blood islands. In other
species such as zebrafish, the first and only hematopoietic site during embryogenesis is the intraembryonic
ICM (Al-Adhami and Kunz, 1977; Glomski et al., 1992;
this communication). In yet other species, such as
killifish (Stockard, 1915a; Stockard, 1915b), rainbow
trout (Iuchi and Yamamoto, 1983), and Lebistes reticulatus (Colle-Vandevelde, 1961a; Colle-Vandevelde, 1961b)
early hematopoiesis occurs at both locations. Analogous
Grant sponsor: National Institutes of Health; Grant number: 2 RO1
AI08054; Grant sponsor: Spanish Ministry of Education and Culture;
Grant number: PB94–0332.
*Correspondence to: Catherine E. Willett, Massachusetts Institute
of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139.
E-mail: kptnkate@mit.edu
Received 5 November 1998; Accepted 23 December 1998
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WILLETT ET AL.
to yolk sac hematopoiesis of other vertebrates, both the
yolk sac blood islands and ICM of teleosts, which are
derived from ventral lateral mesoderm, are thought to
be sites of primitive hematopoiesis. Recent reports in
zebrafish, describing expression patterns of transcription factors known to be involved in murine hematopoiesis, support the idea that the ICM is the initial site of
blood formation and gives rise primarily to primitive
erythrocytes (Detrich et al., 1995; Liao et al., 1998;
Thompson et al., 1998).
In zebrafish, cells comprising the ICM enter the
developing circulatory system between 24 and 30 hr
post-fertilization (hpf). Recent expression studies of
transcription factors involved in hematopoiesis suggest
that a few pluripotent hematopoietic cells subsequently
exist at other locations, most notably the ventral wall of
the dorsal aorta and a region in the tail posterior to the
ICM and ventral to the axial vein, the ‘‘ventral vein
region’’ (Liao et al., 1998; Thompson et al., 1998; H.
Kawasaki and C. Amemiya, personal communication;
our unpublished results). These putative hematopoietic
foci may constitute the initial site of definitive hematopoiesis in zebrafish. Later, hematopoietic tissue is seen
in the kidney (Al-Adhami and Kunz, 1977; Willett et al.,
1997b). The teleost kidney is the equivalent of bone
marrow in other vertebrates and is the major site of
hematopoiesis through adulthood (reviewed in Zapata
et al., 1995).
The zebrafish has recently shown promise as a model
organism for the study of vertebrate embryogenesis—
the transparent, free-living, and rapidly developing
embryos are ideal for observation of early developmental processes (Kimmel, 1989). An additional advantage
of zebrafish as a model system is the ability to perform
large-scale screens for mutations affecting embryogenesis (Driever et al., 1996; Hafter et al., 1996).
To initiate analysis of the zebrafish immune system,
we cloned the zebrafish rag1 and rag2 genes, which
encode the recombinase responsible for recombination
of the Ig and TCR genes. Expression of rag1 identified
the thymus as the first lymphoid organ to emerge
during zebrafish ontogeny (Willett et al., 1997a; Willett
et al., 1997b).
A previous histological description performed by light
microscopy had identified primitive hematopoietic cells
in the ICM at 10 somites; hematopoietic cells were first
described in the pronephros on day 4 pf, with ‘‘all
lineages’’ visible by day 8 pf (Al-Adhami and Kunz,
1977). It was not clear, however, whether they included
lymphocytes in the description, and the thymus was not
mentioned. In our initial studies by light microscopy,
we described isolated cells of possible hematopoietic
nature in the pronephros at 90 hpf, although significant
hematopoietic tissue did not accumulate until 2–3
weeks pf (Willett et al., 1997b). In contrast, accumulation of lymphoid cells in the thymus occurred by 72 hpf,
supporting the observation that, in most teleosts, the
thymus is the first organ to become lymphoid (Zapata et
al., 1997).
A detailed description of the development of the
zebrafish immune system is required for phenotypic
characterization of any mutants that might be isolated
in future screens. The following specific points are
addressed here: (1) the timing of lymphocyte infiltration and maturation within the thymus, (2) the timing
of differentiation of the epithelial components of the
thymus, (3) the possible presence of hematopoietic
tissue in the heart, and (4) the discrepancy between our
observations and the report of Al-Adhami and Kunz
regarding the timing of appearance of hematopoietic
lineages in the pronephros. We have thus extended our
previous results and present a detailed histological
description by light and electron microscopy of the
zebrafish immune system from 5 somites through 3
weeks of development.
RESULTS
Initial Site of Hematopoiesis
As early as 11 hr post-fertilization (the 5-somite
stage), cells can be identified in an intraembryonic
region between the somites and the yolk sac, which
presumably corresponds to the intermediate cell mass
(ICM) described by other authors (Al-Adhami and
Kunz, 1977; Detrich et al., 1995; Fig. 1A,B). This area
contains primitive hematopoietic precursors (Fig. 1B)
in addition to cells that will give rise to the pronephros
and trunk blood vessels. By 20 hpf, these cells have
migrated medially and have aggregated into two bilaterally symmetric bands between the notochord and
endoderm (Fig. 1C), as previously noted (Detrich et al.,
1995). The 20 hpf ICM contains proerythroblasts, which
exhibit an electron-dense nucleus with clumps of condensed chromatin and a cytoplasm filled by ribosomes
and large, electron-lucent mitochondria (Fig. 1D). These
cells also contain ropheocytic vesicles, which are involved in iron transport, and are a hallmark of erythroid cells. Myeloblasts are not evident. At 24 hpf,
numerous erythroblasts appear, closely associated with
primitive endothelial cells proximal to the developing
pronephric ducts (Fig. 2A). Erythroblasts, round or
polygonal in shape, possess clumps of condensed chromatin arranged in an electron-dense nuclear matrix.
The cytoplasm contains ropheocytic vesicles and is
filled with polyribosomes (Fig. 2B). Between 24 and 30
hpf, the ICM disappears as cells are taken up by the
nascent circulatory system. At 42 hpf, a few cells that
morphologically resemble erythroblasts and promonocytes were identified in the ventral axial vein region of
the tail, posterior to the ICM (Fig. 2C,D). By electron
microscopy (EM), there are circulating cells in the axial
vein (Fig. 2C); however, there were also some primitive
cells in the connective tissue surrounding the blood
vessel (Fig. 2D).
Early Myelopoiesis
Although not seen in the ICM, cells of the myeloid
lineage can be found surrounding the yolk sac as early
as 24 hpf (data not shown). Fig. 3 shows myeloblasts
HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT
325
Fig. 1. First appearance of hematopoietic tissue in the intermediate
cell mass (ICM). A, B: Transverse section of 11 hpf embryo (5 somites). C,
D: 20 hpf embryo. A: The forming ICM at 5 somites (circled) contains
precursors that will give rise to the first hematopoietic cells, the pronephros, and blood vessels. NT, neural tube; Nc, notochord; S, somite; YS,
yolk sac. Light micrograph, magnification: 120 ⫻. B: Primitive hematopoietic cell in forming ICM. arrows, polyribosomes; n, nucleus; m, mitochondria. Electron micrograph, magnification: 7,800 ⫻. C: ICM (circled) is
dorsal and medial to the pronephric ducts (PD). Note the abundance of
erythroblasts (arrows). S, somite; YS, yolk sac. Light micrograph, magnification: 250 ⫻. D: Erythroblasts (Eb) can be seen in the ICM adjacent to
the pronephric duct (PD). Note the high electron density of the nucleus (n)
and the abundance of ribosomes and large mitochondria (m) in the
cytoplasm of erythroblasts. Primitive erythroblasts contain ropheocytic
vesicles (arrow). Electron micrograph, magnification: 5,900 ⫻. Plane of
sections diagrammed at right.
and neutrophilic myelocytes at 34 and 48 hpf. Whereas
numerous circulating erythroblasts occur inside blood
vessels, maturing granulocytes are seen mostly outside
the circulatory system, in loose connective tissue surrounding the yolk sac at 34 hpf (Fig. 3A), and adjacent
to the first renal tubules at 48 hpf (Fig. 3B), although
some images support the migration of myelocytes or
young neutrophils through the blood vessel walls (Fig.
3C). Mature neutrophils can be identified in different
tissues at 72 hpf (data not shown).
resembling lymphocytes or primitive hematopoietic cells
similar to those described by Al-Adhami and Kunz
(1977) seemed to occur in the endocardial walls (Fig.
4E). However, at higher magnifications by EM, these
cells were found to be irregular endocardial cells joined
together by desmosomes, specialized epithelial cell
junctions (Fig. 4F).
Circulating Erythroblasts in the Heart
A previous description of hematopoiesis in zebrafish
by light microscopy suggested that the zebrafish heart
functions as a transitory blood-forming organ, between
the time of disappearance of the ICM and the onset of
hematopoiesis in the kidney (Al-Adhami and Kunz,
1977). We therefore examined the histology of the heart
from 24 hpf onward. In all stages examined, circulating
erythroblasts could easily be identified (Fig. 4A,B,C),
sometimes closely associated with the endocardium
(Fig. 4A,C,D). At lower magnifications, small cells
The Thymus
We previously reported the appearance of the thymic
primordium by light microscopy at 54 hpf (Willett et al.,
1997b). Figs. 5A,B show the primordium at 60 hpf as a
small outgrowth of the pharyngeal epithelium, devoid
of lymphocytes. By EM, the region containing the
primordium can be seen to consist of two layers of
epithelial cells surrounded by an incipient but continuous basement membrane (Fig. 5B). Flat, electron-dense
epithelial cells line the pharyngeal cavity, joined together by well-developed desmosomes. Adjacent to the
pharyngeal epithelium, electron-lucent epithelial cells
constitute the thymic primordium. These epithelial
cells are irregular or polygonal, contain numerous
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WILLETT ET AL.
Fig. 2. ICM at 24 hpf (A, B) and ventral vein region of tail at 42 hpf (C,
D); transverse sections. A: Erythroblasts (Eb) are closely associated with
primitive endothelial cells (En) adjacent to the pronephric duct (PD).
Electron micrograph, magnification: 2,600 ⫻. The box in (A) is magnified
in (B). Both the nucleus (n) and cytoplasm of erythroblasts are electrondense. The cytoplasm has abundant ribosomes, electron-lucent, round
mitochondria (m), and occasional ropheocytic vesicles (arrows). Electron
micrograph, magnification: 8,500 ⫻. C: Erythroblasts (Eb) and presump-
tive promonocyte (arrow) in lumen of axial vein in tail. Electron micrograph, magnification: 3,300 ⫻. D: Primitive hematopoietic cell, presumably a promonocyte or early promyelocyte (Pm), in connective tissue
adjacent to axial vein, surrounded by fibroblasts (Fb). Two lobes of the
nucleus (n) can be seen, along with numerous mitochondria (m) and
rough endoplasmic reticulum (r). Electron micrograph, magnification:
5,500 ⫻.
ribosomes, large electron-lucent mitochondria, and
rough endoplasmic reticulum. Nascent desmosomes
join these primitive epithelial cells together (Fig. 5C).
At 65 hpf, the thymic primordium is slightly larger,
and by EM, the first lymphoid cells can be seen (Fig.
5D,E). The thymic epithelial cells arranged along the
pharyngeal epithelium are relatively primitive, and the
basement membrane is still poorly formed. Lymphoblasts can be distinguished, between epithelial cells, by
their round shape, high electron density, and a small
amount of cytoplasm filled with ribosomes, yet missing
rough endoplasmic reticulum.
The thymus grows slowly for the next few hours, and
the number of lymphoblasts increases gradually. At 72
hpf, the thymic primordium still contains few lymphoid
cells. However, the connections between epithelial cells
have matured (data not shown). Thymic epithelial cells
now contain an irregular nucleus with few peripheral
areas of condensed chromatin (data not shown).
The thymus contains many more lymphoid cells at 96
hpf (Fig. 6A). Lymphoblasts are seen as large cells with
a nucleus containing few areas of condensed chromatin,
a prominent nucleolus, numerous polyribosomes, a
poorly developed Golgi complex, and some cisternae of
rough endoplasmic reticulum (Fig 6B). The cytoplasm
of the thymic epithelial cells contain bundles of microfilaments, and frequent desmosomes occur between
these epithelial cells, two characteristic features of
maturing epithelial cells (Fig. 6C).
On day 7 pf, increased numbers of small (mature)
lymphocytes, as well as additional types of epithelial
cells, are found in the thymus (Fig. 6D), as is seen in the
HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT
327
Fig. 3. First appearance of myeloid lineages. A: 34 hpf embryo.
Myeloid cells are found outside of the blood vessel (BV), both close to the
yolk sac (YS), and next to connective tissue (CT) of the body wall. arrows,
myeloblasts; arrowheads, neutrophilic promyelocytes. Electron micrograph, magnification: 2,300 ⫻. B: 48 hpf embryo. Neutrophilic myelocyte
(see arrows in box) in the connective tissue surrounding the ciliated
pronephric duct (PD), between the yolk sac (YS), and yolk sac blood
vessel (BV; Duct of Cuvier). Electron micrograph, magnification: 2,900 ⫻.
C: Higher magnification of the neutrophilic myelocyte boxed in (B). Note
the immature cytoplasmic granules and the Golgi complex (g). Cell
processes (arrows) crossing two endothelial cells (En) suggest that the
cell is migrating into the blood circulation. Electron micrograph, magnification: 10,200 ⫻. Plane of section and locations of micrographs diagrammed at upper right. G, glomerulus; I, intestine; n, nucleus; Nc,
notochord; Nt, neural tube; PD, pronephric duct; S, somite; YS, yolk sac.
trout thymus (Castillo et al., 1991). Numerous medium
and small lymphocytes are seen as round, electrondense cells with abundant clumps of condensed chromatin and a small cytoplasm filled with ribosomes. Electron-dense epithelial cells are seen between the
lymphoid cells. Secretory epithelial cells containing a
heterogeneous population of electron-dense, cytoplas-
mic granules and a well developed Golgi complex are
now found (Fig. 6E).
By 3 weeks pf, both electron lucent epithelial cells
(Fig. 7A) and epithelial cysts (Fig. 7B) are seen, as has
been reported in the thymus of other fish (Zapata et al.,
1996), marking the morphological maturation of the
thymic stroma which began 1 week pf. The pale epithe-
328
WILLETT ET AL.
Fig. 4. Circulating erythroblasts in heart. A: Transverse
section through 24 hpf heart showing erythroblasts (arrows)
closely associated with endocardial cells (Ec). Light micrograph, magnification 1,000 ⫻. B: Transverse section through
60 hpf heart showing immature erythrocytes (RBC) within the
heart chambers. Ec, endocardial cells. Light micrograph,
magnification: 800 ⫻. C: Transverse section through 72 hpf
heart showing pooled erythrocytes and erythroblasts in
atrium and ventricle. GA, gill arches; H, heart; Ph, pharynx.
Inset: magnification of boxed region. Circulating erythroblasts (arrows) are evident. Light micrograph, magnification:
170 ⫻; inset, 600 ⫻. D: Circulating erythroblasts (Eb) in
close association with the endocardium (Ec) at 72 hpf. Mc,
myocardium. Electron micrograph, magnification: 3,400 ⫻.
E: Lymphoid-like cells in endocardium (arrows) at 72 hpf.
Electron micrograph, magnification: 3,400 ⫻. Boxed area in
(E) is magnified in (F). F: Lymphoid-like cells are actually
irregular endocardial cells connected together by desmosomes, specialized cell junctions (arrows). Electron micrograph, magnification: 13,200 ⫻.
lial cells have the same general features as the epithelial cells described above at 72 hpf and 96 hpf, but they
have lost electron-dense cytoplasm and appear devoid
of most membranous organelles. They contain bundles
of cytoplasmic filaments (Fig. 7C) and are connected by
desmosomes to the neighboring electron-dense epithelial cells (Fig. 7D).
The Kidney
Well-developed renal tubules and glomeruli are observed at 72 hpf; however, the first hematopoietic cells
do not appear in the zebrafish pronephros until 96 hpf
(Fig. 8A,B). Hematopoietic tissue is organized into cell
cords surrounding blood vessels, between the renal
tubules and glomeruli. By day 7 pf, fibroblastic reticular cells line the sinusoidal blood vessels; this constitutes the supporting apparatus for the developing
hematopoietic cells (Fig. 8C,D), as occurs in the kidney
of other teleosts (Meseguer et al., 1991; Zapata, 1979).
Fibroblastic reticular cells and cells lining the sinusoidal blood vessels are both electron-lucent, the former
being very irregular and extremely flat, the latter
HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT
329
Fig. 5. Thymic primordium 60 hpf (A, B, C) and 65 hpf (D, E). A:
Thymic primordium (boxed) lies in a dorsal pocket of the pharynx, ventral
to the otic vesicle (Ov). Ph, pharyngeal epithelium. Light micrograph,
magnification: 225 ⫻. B: Boxed area in (A), magnified. Thymic primordium
consists of two epithelial cell layers, the pharyngeal epithelium (Ph), and
thymic epithelium (TE). Pharyngeal epithelial cells are joined by desmosomes (arrow). Limit of thymic primordium indicated by arrowheads. m,
large mitochondria; r, rough endoplasmic reticulum. Electron micrograph,
magnification: 9,400 ⫻. C: Incipient desmosomes (arrows) between
thymic epithelial cells. Electron micrograph, magnification: 54,500 ⫻. D:
By 65 hpf, lymphoblasts (Lb) have infiltrated between epithelial cells (TE).
Ph, pharyngeal epithelium. Electron micrograph, magnification: 4,100 ⫻.
E: Lymphoblasts are distinguished by patches of condensed chromatin.
Thymic epithelial cells have prominent mitochondria (m) and rough
endoplasmic reticulum (r). TE, epithelial cells. Arrows mark basement
membrane. Electron micrograph, magnification: 12,200 ⫻.
exhibiting an elongated nucleus with peripheral areas
of condensed chromatin, and sometimes, a small, compact nucleolus (Fig. 8D). It was difficult to observe a
basement membrane separating these cell types. At
these early stages, the renal hematopoietic tissue consists of erythroblasts, myeloblasts, and heterophilic
myelocytes, which form clusters or cords of cells (Fig.
8C).
During the next several days, the amount of hematopoietic tissue greatly increases (Fig. 9A). However, even
at 2 weeks, the types of cells found in the pronephros
remain remarkably unchanged; erythroblasts and myeloblasts are still the most frequent blood cell lineages
found (Fig. 9B,C). Erythroblasts, at different stages of
development, are easily identifiable by the high electron density of both their nuclei and cytoplasm (Fig.
9B). Characteristic of erythroblasts is a nucleus with
many patches of condensed chromatin and a small
nucleolus. In addition, hemoglobin, which leaks from
the cytoplasm, accumulates in the nuclear matrix and
confers high electron density to the nucleus, which
distinguishes these cells from myeloblasts or lymphoblasts. The hemoglobin-filled cytoplasm contains large,
electron-lucent vacuoles, as a consequence of the degradation of membranous organelles (i.e., mitochondria,
lysosomes). Macrophages can be seen with degenerated
erythroid cells in their cytoplasm (Fig. 9B). Myeloblasts
can be seen as polygonal cells, larger than erythroblasts
or lymphoblasts, with few patches of condensed chromatin, an active, big nucleolus, and a cytoplasm filled with
abundant ribosomes and a few electron-lucent mitochondria (Fig. 9C). All developmental stages of the neutrophilic cell lineage could be identified at 2 weeks pf.
At 3 weeks pf, the pronephros has enlarged and
contains more patches of hematopoietic tissue, interspersed among increased numbers of renal tubules
(Fig. 9D). The first lymphoid cells are seen in the
pronephros at this time (Fig. 9E,9F). Lymphoblasts are
identified as being smaller than myeloblasts, with a
small cytoplasm filled by ribosomes and a few clumps of
330
WILLETT ET AL.
Fig. 6. Thymic primordium 96 hpf (A,B,C) and day 7 pf (D,E). A:
Thymus (circled) contains many lymphoid cells. Ph, pharyngeal epithelium. Light micrograph, magnification: 630 ⫻. B: Lymphoblasts contain a
nucleus (n) with clumps of condensed chromatin, a nucleolus (nu), and
cytoplasm filled with abundant ribosomes. m, mitochondria; g, Golgi
complex; r, cisternae of rough endoplasmic reticulum. Electron micrograph, magnification: 11,100 ⫻. C: Filaments (arrows) in, and desmosomes (arrowheads) between, two thymic epithelial cell processes.
Electron micrograph, magnification: 21,600 ⫻. D: Increasing numbers of
medium and small lymphocytes (arrowheads) occur among thymic
epithelial cells (stars). A connective tissue capsule (demarcated by
arrows) surrounds the thymus. Ph, pharyngeal epithelium. Electron
micrograph, magnification: 4,100 ⫻. E: Thymic epithelial cells have begun
to differentiate into different cell types, including secretory epithelial cell
shown here. stars, basement membrane; arrowheads, desmosomes;
arrows, secretory granules. Electron micrograph, magnification: 13,500 ⫻.
condensed chromatin (Fig. 9E). Small lymphocytes
have very electron-dense cytoplasm, filled by free ribosomes and devoid of membranous organelles, which
forms a narrow rim around a large nucleus that has
abundant patches of condensed chromatin (Fig. 9F).
Hematopoietic cells in ICM are evident at the 5-somite stage (10–11 hpf; Fig. 1A,B). The morphological
features of these early progenitor cells are not sufficiently defined to distinguish hematopoietic tissue from
vascular and pronephric progenitors, which also originate in this region (Gering et al., 1998; Liao et al., 1998;
Liao et al., 1997). By 20 hpf, proerythroblasts are clearly
distinguished (Fig. 1C,D). Accumulation of hematopoietic
tissue in the ICM reaches its maximum development at 16
to 24 hpf. Erythroblasts within the ICM are taken up into
the nascent circulatory system around 24 hpf and begin to
circulate as immature erythrocytes (Fig. 2; Al-Adhami and
Kunz, 1977; Detrich et al., 1995; Weinstein et al., 1996);
final maturation of these erythrocytes occurs in circulation.
These results suggest that the ICM is responsible for
‘‘primitive’’ hematopoiesis and is functionally analogous to
the yolk sac blood islands of mammals and birds and the
ventral blood islands of amphibians.
DISCUSSION
Recent results in zebrafish have demonstrated that
the ICM is the first embryonic area expressing molecules involved in early blood cell formation in mammals, including scl, GATA-1, GATA-2, c-myb, LMO2
and Ikaros (Detrich et al., 1995; Gering et al., 1998;
Liao et al., 1998; Thompson et al., 1998; H. Kawasaki
and C. Amemiya, personal communication; unpublished results of the authors). The observations presented here, by light and electron microscopy, confirm
those studies and show that the ICM contains hematopoeitic/erythropoietic progenitor cells.
HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT
331
Fig. 7. Different types of epithelial cells are seen in
3 week pf thymus. A: Electron-lucent (pale) epithelial
cell (star), surrounded by lymphoid cells. Electron
micrograph, magnification: 8,800 ⫻. B: An epithelial
cyst. Epithelial cell processes, which delimit the cavity
of the epithelial cyst, are marked by arrows. Electron
micrograph, magnification: 8,900 ⫻. C: Bundles of
microfilaments (arrows) in the cytoplasm of an electronlucent thymic epithelial cell. Electron micrograph, magnification: 48,800 ⫻. D: Desmosomes connect cell
processes of an electron-dense (small stars) and an
electron-lucent thymic epithelial cell (star). Electron
micrograph, magnification: 48,800 ⫻.
The location of hematopoietic tissue, from the time
the ICM or yolk sac blood islands disappear (24–30 hpf
in zebrafish) until the appearance of hematopoietic
tissue in the kidney (at 96 hpf in zebrafish; Fig 8), has
not been conclusively identified in teleosts. Thompson
et al. (1998) suggest that a lateral stripe of c-Mybexpressing cells may populate the ventral wall of the
dorsal aorta at 48 hpf. The dorsal aorta may therefore
represent the teleost equivalent of the aorta-gonadmesonephros region (AGM), which has been shown to
be the first site of definitive hematopoiesis in Xenopus
(Chen and Turpen, 1995), chicken (Dieterlen-Lievre
and Le Douarin, 1993), and mouse (Godin et al., 1995;
Medvinsky and Dzierzak, 1996), and has also been
described in elasmobranchs (Zapata et al., 1996). Definitive resolution of the identity of these cells awaits
lineage tracing experiments.
Liao et al. (1998) and Thompson et al. (1998) report
expression of hematopoietic markers that persists until
day 4 pf in the region of the tail posterior to the ICM
and ventral to the axial vein. At 42 hpf, a few erythro-
blasts and promonocytes are found at this location (Fig.
2C,2D); these cells are found in the axial vein as well as
in the mesenchyme surrounding the blood vessel. This
population of cells could represent a residual compartment of the ICM that might be capable of generating
hematopoietic precursors for a longer period during
embryogenesis than previously thought.
Between 24 and 48 hpf, a few myeloblasts and
heterophilic myelocytes appear between the yolk sac
and the body walls (data not shown; see Fig. 3).Mature
neutrophils are seen in different tissues, but not in the
blood circulation, at 72 hpf. Detrich et al (1995) reported the migration of erythroblasts from the ICM
onto the yolk sac between the ectoderm and the yolk
surface, the same location in which the myeloid cells
occur. Macrophages have also been reported between
the yolk sac and body walls, by light microscopy, in
living embryos from 24 hpf (P. Herbomel, personal
communication).
Al-Adhami and Kunz (1976, 1977) postulated the
existence of endocardial ‘‘stem-cell like’’ cells in both
332
WILLETT ET AL.
Fig. 8. Pronephros of 96 hpf (A, B) and day 7 pf (C, D) embryo. A:
Hematopoietic tissue between renal tubules (RT) and the forming glomerulus (G). BV, blood vessel; I, intestine; L, liver; M, muscle; Nc, notochord.
Dorsal is upper right corner. Transverse section, light micrograph, magnification: 180 ⫻. B: Magnification of boxed area in (A). Mature (arrows) and
developing (white arrowhead) erythrocytes can be seen. G, glomerulus.
Light micrograph, magnification: 540 ⫻. C: Hematopoietic cell cords are
arranged between blood vessels (BV) and sinusoidal blood vessels (SBV)
and consist of myeloid (MC) and erythroid cells (E). In the lumen of a
sinusoidal blood vessel, erythroblasts (Eb) are closely associated with
cells lining the lumen (stars). arrowheads, Fibroblastic reticular cells.
Electron micrograph, magnification: 3,700 ⫻. D: Box in (C), magnified.
Erythroblast (Eb) associated with fibroblastic reticular cells (arrowheads)
and cells lining the lumen (stars) of a sinusoidal blood vessel. Note the
similarities between the reticular cell and the lining cell, and the absence
of a basement membrane separating them. Electron micrograph, magnification: 8,300 ⫻.
zebrafish and angelfish that could become free hematopoietic stem cells. This suggests that the heart could be
the blood cell forming organ, between the time of
disappearance of hematopoiesis in the ICM and the
onset of blood cell formation in the pronephros. Our
analyses, by both light and electron microscopy, demonstrate the existence of primitive hematopoietic cells,
mainly erythroblasts, closely associated with, but not
attached to, the endocardial cells of zebrafish heart
from 24 hpf onward (Fig. 4). These cells are circulating
erythroblasts and immature erythrocytes, which, as
mentioned above, mature in circulation (Fig. 4A,B,C,D).
There are also immature cells in the endocardium itself
(Fig. 4E). However, upon inspection by EM, these cells
are found to be connected by desmosomes and are, in
fact, maturing endocardial cells (Fig. 4F). A similar
situation has recently been described in the developing
heart of the turbot (Padrós and Crespo, 1996). Liao et
al. (1998) observed scl-expressing cells in the heart at
day 4 pf; however, upon sectioning, these cells were
shown to be pooled circulating blood cells.
Maturation of lymphocytes in the thymus is dependent on interactions with the thymic epithelium; interestingly, the converse is also true (Hollander et al., 1995;
Shores et al., 1991; van Vleit et al., 1985). In teleosts,
several epithelial cell types have been described (reviewed
in Chilmonczyk, 1992). At the time of initial infiltration by
lymphoblasts, the thymic epithelial cells are relatively
undifferentiated. Differentiation of the various epithelial
cell types continues long after infiltration (Castillo et al.,
1991). While the function of different classes of thymic
epithelial cells has not been addressed in teleosts, certain
morphological similarities to classes of mammalian cell
types have been noted (Castillo et al., 1991).
In zebrafish, the thymic epithelium is first visible, by
light and electron microscopy, at 60 hpf, lining the
HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT
333
Fig. 9. Pronephros at 2 weeks (A, B, C) and 3 weeks (D, E, F) pf. A:
Hematopoietic tissue (circled) surrounding the renal tubules (RT) has
expanded. G, glomerulus; I, intestine; L, liver; M, muscle; Nc, notochord.
Light micrograph, magnification: 110 ⫻. B: Macrophage (Mp) containing
engulfed cell debris appears between erythroblasts. Note the high
electron density of both nuclei and cytoplasm of the erythroblasts and the
presence of big vacuoles (arrowheads), which represent degenerated
membranous organelles. Electron micrograph, magnification: 3,900 ⫻. C:
Renal hematopoietic tissue also contains myeloblasts (Mb), myelocytes
(Mc) and mature neutrophils (Ne). Myeloblasts are large cells with few
areas of condensed chromatin, an obvious nucleolus (nu), abundant
ribosomes and incipient rough endoplasmic reticulum (arrowheads).
Neutrophils contain abundant cytoplasmic granules (gr). arrow, Fibroblastic reticular cell; m, mitochondria. Electron micrograph, magnification:
5,400 ⫻. D: Pronephros has enlarged and contains increased hematopoietic tissue (circled) interspersed among renal tubules (RT) and blood
vessels (BV). Light micrograph, magnification: 100 ⫻. E: Several types of
hematopoietic cells are discernible, including small (SL), medium (ML),
and large (LL) lymphocytes, myeloblasts (Mb), mature neutrophils (Ne),
and mature acidophils (Ac). Electron micrograph, magnification: 3,200 ⫻.
F: Small lymphocytes (SL) exhibit high electron density, numerous clumps
of condensed chromatin, and a small rim of cytoplasm packed with
ribosomes. Electron micrograph, magnification: 12,300 ⫻.
dorsal pharynx (Fig. 5A,B,C); the thymic epithelium is
continuous with the pharyngeal epithelium. Lymphoblasts can first be distinguished in the thymic primordium by EM at 65 hpf (Fig. 5D,E). The epithelial cells
are still relatively immature. At 72 hpf, the number of
lymphoid cells has increased slightly, and the epithelial
cells show signs of maturation; some are developing
secretory capability, desmosomes are forming between
cells, and a basement membrane is forming. The hematopoietic transcription factor Ikaros can be detected in a
few of these thymic lymphocytes at 72 hpf (H. Kawasaki
and C. Amemiya, personal communication; unpublished results of the authors). By 92 hpf, the thymus
has enlarged considerably and contains many more
lymphoid cells (Fig. 6). At this approximate time, rag1
expression is first seen in the thymus (Willett et al.,
1997b). Lymphoblasts remain the most numerous lymphoid cell type, with increasing numbers of medium
and small lymphocytes during the first week pf. Epithe-
lial cells have mature desmosomes connecting them,
and the cytoplasm has developed bundles of microfilaments. From day 7 pf through 3 weeks pf, increasing
heterogeneity is seen in both the lymphocytic and
epithelial cell populations (Fig. 6C,D, and Fig. 7). The
sequential expression of Ikaros and rag1, as well as
increased heterogeneity, suggest progressive maturation of lymphocytes in the thymic primordium, which
correlates with the progressive maturation of thymic
epithelial cells.
The thymus is generally the first organ to become
lymphoid in teleosts (Castillo, 1990; Ellis, 1977; Secombes et al., 1983), although in some species, principally marine fish, the kidney contains hematopoietic
tissue before mature thymic lymphocytes are evident
(Padrós and Crespo, 1996; Tatner, 1996; Zapata et al.,
1996; Zapata et al., 1997). In trout, before hatching,
(which occurs at approximately 3 weeks pf ), the kidney
contains only developing erythrocytes and myeloid cells
334
WILLETT ET AL.
(Castillo, 1990). Preliminary in situ hybridization of
kidney tissue shows rag1-positive cells at hatching,
when the organ contains cells that are identifiable as
lymphoid by EM (Hansen and Zapata, 1998, and unpublished results). In trout, however, both IgM-expressing
cells (Castillo et al., 1993) and Ig VH expression are
detected much earlier (J. Hansen, personal communication). It is possible that, in trout, a few B cells appear
elsewhere in the early embryo before the appearance of
lymphocytes in the kidney.
Our results confirm the late appearance of lymphoid
cells in the zebrafish kidney (Figs. 8, 9; Willett et al.,
1997b). Although developing erythrocytes and granulocytes occur in the pronephros from 96 hours onward,
the first lymphocytes and lymphoblasts appear later
than 2 weeks pf. Consistent with these findings, expression of Ig heavy chain in zebrafish remains at low levels
until two weeks pf, when it increases slightly. It then
increases dramatically at 3 weeks pf (N. Danilova,
personal communication).
The nature of the relationship between the lymphoid
cell populations of thymus and kidney during teleost
ontogeny has been controversial. It has previously been
proposed that stem cells migrate from the pronephros
to the thymic rudiment to mature and differentiate
(Ellis, 1977; Nakanishi, 1991; Padrós and Crespo,
1996). However, these authors did not consider the
existence of other sites of hematopoiesis earlier than
the pronephros. Re-evaluation of results presented by
Padrós and Crespo (1996) suggest the existence of an
ICM in the turbot embryo. Our results suggest that
lymphoid progenitors found in the thymic primordium
of 72 hour-old zebrafish do not come from the pronephros, which initiates its hematopoietic activity between
72 and 96 hours. It is possible that lymphoid progenitors colonizing the thymic primordium derive from the
early hematopoietic centers, e.g., the ICM and/or putative AGM region (the ventral part of dorsal aorta).
On the basis of indirect and morphological evidence,
a thymic contribution to the lymphoid cell populations
that appear in the kidney during teleost ontogeny has
been suggested (Grace and Manning, 1980; Josefsson
and Tatner, 1993; Nakanishi, 1991). However, removal
of the thymus in rainbow trout at 4 to 24 days posthatching has no effect on the lymphocyte population in
the kidney (Manning et al., 1982). It is likely, therefore,
that colonization of the kidney by early hematopoietic
progenitors is similar to, but independent from, population of the thymus. Thompson et al. (1998) have proposed, but not shown, that progeny of the c-mybexpressing cells of the dorsal aorta may colonize the
kidney.
Hematopoiesis in zebrafish is typical of teleosts, and
more broadly, of vertebrates in general. The first site of
hematopoeisis, the ICM, is derived from lateral mesoderm and provides primitive progenitors, giving rise to
embryonic erythrocytes. The posterior ICM appears to
maintain hematopoietic potential for up to 4 days pf.
The dorsal aorta, a structure physically related to the
ICM, but developmentally later, may be the zebrafish
equivalent of the AGM, the first site of definitive
hematopoiesis in other vertebrates. Cells in this region
express transcription factors known to be important for
definitive hematopoiesis in mammals. Furthermore,
definitive hematopoiesis must begin at a location other
than the kidney, since lymphocytes appear in the
thymus before establishment of hematopoietic tissue in
the kidney. Lineage tracing experiments are required to
establish the source of lymphocytes that populate the
thymus, an experiment that will be facilitated by the
embryological advantages of zebrafish.
EXPERIMENTAL PROCEDURES
Animals
Wild-type zebrafish, Danio rerio, Tübingen strain,
were obtained from C. Nüsslein-Volhard (Tübingen,
Germany) and were reared at 28°C as described (Culp
et al., 1991; Lin et al., 1992).
Light and Electron Microscopy
Wild-type zebrafish, from 5-somite stage (about 11
hpf) to 3 weeks (11.5, 16, 20, 24, 34, 42, 72, 96 hr, and 1,
2, and 3 weeks post-fertilization), at least 3 fish from
each developmental stage, were fixed for 3–5 hours in
2.5% glutaraldehyde/0.1M sodium caccodylate at 4°C,
washed for equal time in 0.1M sodium caccodylate/5%
sucrose, and post-fixed for 2 hr in dark with 1% osmium
tetroxide/0.1M sodium caccodylate. After washing in
0.1M sodium caccodylate/5% sucrose, the fish were
dehydrated quickly in increasing concentrations of
ethanol to 100%. Fish were then washed twice with
propylene oxide, 5 min each. Propylene oxide was
replaced with a 2:1 mixture of propylene oxide:PB 812
(PB 812:24%(w/v) Polybed 812 resin, 50% (w/v) dodecenyl succinic anhydride, 1% (v/v) 2,4,6-tris(dimethylamino-methyl) phenol; all from PolySciences, Inc., Warrington, PA) overnight at room temperature, then a 1:1
mixture of the same components for 10 hr, and finally
pure PB 812 overnight. Blocks were polymerized at
70°C overnight in fresh PB 812. Sections were obtained
with a Reichert OM-U3 ultramicrotome. Semi-thin
sections stained with an alkaline solution of 1% toluidine blue were used for light microscopy. Ultra-thin
sections, obtained from selected areas on the semi-thin
sections, were double stained with uranyl acetate and
lead citrate, examined and photographed in a JEOL
10.10 electron microscope.
ACKNOWLEDGMENTS
We would like to thank Drs. Nadia Danilova, Valerie
Hohman, and Lisa Steiner for thoughtful comments.
A.C. and A.G. Z. were supported by grant PB94–0332
from the Spanish Ministry of Education and Culture.
C.E.W. was supported by NIH grant 2 ROI AI08054.
HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT
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