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Blood First Edition Paper, prepublished online September 17, 2007; DOI 10.1182/blood-2007-06-095398
Origins and unconventional behavior of neutrophils in developing zebrafish
Running head:
Patrolling neutrophils in developing zebrafish
Dorothée Le Guyader,1 Michael J. Redd,2 Emma Colucci-Guyon,1 Emi Murayama,1 Karima
Kissa,1 Valérie Briolat,1 Elodie Mordelet,1 Agustin Zapata,3 Hiroto Shinomiya4 and Philippe
Herbomel1,*
1
Unité Macrophages et Développement de l'Immunité, CNRS-URA 2578, Institut Pasteur, 25
rue du Dr Roux, 75724 Paris cedex 15, France
2
Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake City, Utah
84112
3
Department of Cell Biology, Faculty of Biology, Complutense University, 28040 Madrid,
Spain
4
Department of Immunology and Host Defenses, Ehime University School of Medicine,
Shigenobu, Ehime 791-0295, Japan
*Correspondence to: Philippe Herbomel
Unité Macrophages et Développement de l'Immunité,
Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France
[email protected]
phone: 33 1 44 38 95 29 ; fax : 33 1 45 68 89 21
1
Copyright © 2007 American Society of Hematology
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Abstract
The first leukocytes that arise in the development of vertebrate embryos are the primitive
macrophages, which differentiate in the yolk sac, and then quickly invade embryonic tissues. These
macrophages have been considered to constitute a separate lineage giving rise to no other cell type.
Using an in vivo photoactivatable cell tracer in the transparent zebrafish embryo, we demonstrate that
this lineage also gives rise to an equal or higher number of neutrophilic granulocytes. Surprisingly, the
differentiation of these primitive neutrophils occurs only after primitive myeloid progenitors have
dispersed in the tissues. By 2 days post-fertilization, these neutrophils have become the major
leukocyte type found wandering in the epidermis and mesenchyme. Like the primitive macrophages,
all primitive and larval neutrophils express PU.1 and L-plastin, they are highly attracted to local
infections, yet only a small fraction of them phagocytose microbes, and to a much lesser extent per
cell than the macrophages. They are also attracted to variously stressed or malformed tissues,
suggesting a wider role than anti-microbial defence.
2
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Introduction
1
In all vertebrate embryos examined so far, the first leukocytes to appear are macrophages . They
belong to a specific, so-called 'primitive macrophage' lineage that arises from mesoderm and
differentiates in the yolk sac in parallel with the primitive wave of erythrocytes. In zebrafish as well as
in xenopus embryos, these primitive macrophages were found to originate from the rostral-most lateral
2,3
mesoderm, adjacent to the cardiac field . They differentiate in the neighboring yolk sac between the
20- and 30-somite stages (24 hpf), just before the onset of blood circulation, and then quickly spread
4
throughout the mesenchyme of the embryo .
The next type of leukocyte that appear in zebrafish development are neutrophilic granulocytes
(neutrophils). These cells have been documented by electron microscopy in the trunk and tail by 48
5,6
hpf . Searching for a molecular marker of neutrophils, two groups cloned the same zebrafish gene
5,7
through similarity with the mammalian myeloperoxidase (mpo) gene . Sequence comparisons
revealed that this gene was equally close to four related mammalian peroxidases – myelo-, eosinophil lacto- and salivary peroxidase (three of which lie on the same human chromosome within 100 kb) –
strongly suggesting that the diversification of these peroxidases through gene duplications occurred in
5
tetrapods after their divergence from fishes. Therefore, Lieschke et al. appropriately named the
zebrafish gene 'myeloid peroxidase' (mpx), rather than myeloperoxidase. In adult zebrafish, mpx was
found to be expressed in neutrophils of the kidney
5,7
(the definitive hematopoietic organ in fish) and
5
spleen . In embryos, mpx was found expressed in dispersed leukocytes, which were assumed to be
5,7
neutrophils . However, in mammals, eventhough myeloperoxidase is expressed most strongly in
neutrophils, it is also expressed during monocytic differentiation, and has been used as a marker of
immature cells of the monocyte/macrophage as well as primitive macrophage lineages, that becomes
8
down-regulated upon terminal differentiation . Since in zebrafish embryos, many cells of the primitive
macrophage lineage are likely to be not yet terminally differentiated, they may express the mpx gene,
yet not necessarily become granulocytes. Consistent with this, in Xenopus embryos, all cells of the
3
primitive macrophage lineage were found to express xpox2, the likely frog homolog of mpx .
In the present study, we have investigated the embryological origins of neutrophilic granulocytes, their
subsequent deployment in tissues throughout zebrafish development, and their behavior upon
experimental infections. We find that the primitive macrophage lineage also gives rise to neutrophils,
most of which acquire their granules only once in the tissues. These primitive neutrophils, as well as
larval neutrophils later born in the tail, are distributed mostly in sub-epidermal mesenchyme, rather
than concentrated in the blood as in mammals. They steadily circulate within the tissues, and are
quickly attracted to any local infection, as well as to stressed tissues. Yet they are barely phagocytic
relative to the other myeloid tissue leukocytes, the primitive macrophages, suggesting their main
functions may be other than phagocytosis.
3
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Materials and methods
Zebrafish stocks and embryo treatments
Wild-type AB and spadetailb104 and mindbombta52b mutant zebrafish were raised and maintained as
described
9,10
11
. The anti-runx1 morpholino
dose (1 ng) injected in 1- to 4-cell stage embryos
suppressed larval hematopoiesis (Kissa et al., submitted) but preserved blood circulation.
E. coli bacteria expressing DsRed
12
or GFP
13
were microinjected either in the caudal vein at 52 hpf or
in the otic cavity at 3-3.5 dpf. Overnight stationary-phase cultures (109 bacteria/ml) of E.coli
expressing GFP
P
13
or DsRed
12
were concentrated 4x for intravenous injection or 2x for otic vesicle
injection. Zebrafish larvae were injected using a Picospritzer III microinjector (Parker Hannifin,
Fairfield, NJ) and a mechanical micromanipulator (M-152; Narishige, Tokyo, Japan); bacteria were
loaded in a pulled borosilicate glass capillary (GC100F-15; Harvard Apparatus, Holliston, MA). For
intravenous injection, 3-5 nl were injected under a pressure of 40 psi and injection time of 40 msec.
For the ear injections shown in Figure 7, 0.5-1nl were injected under a pressure of 20 psi and injection
time of 20 msec. For the ear injections shown in Figure S5, an injection pressure of 40 psi and
injection time of 40 msec were used.
Sudan Black staining
Embryos were fixed with 4% methanol-free formaldehyde (Polysciences, Warrington, PA) in
phosphate-buffered saline (PBS) for 2 hrs at room temperature, rinsed in PBS, incubated in Sudan
Black (380B; Sigma-Aldrich, Saint-Quentin Fallavier, France) for 20 min., washed extensively in 70%
ethanol in water, then progressively rehydrated to PBS, 0.1% Tween (PBT).
Tyramide-based detection of endogenous peroxidase activity
Fluorescein isothiocyanate (FITC)- and Cyanine 3 (Cy3)- conjugated tyramides were synthesized as
described
14,15
. Embryos fixed as above were washed in PBS, incubated in 1/100 tyramide in (PBS,
0,1M imidazole, 0,001% H2O2) in the dark for 10-30 min., then washed 3x10 min. in PBT. The
reaction was stopped by incubation in 2% H202 in PBT for 30 min.
Immunohistochemistry
The generation of rabbit anti-zebrafish and anti-mouse L-plastin antibodies have been described
16,17
.
Both antibodies gave the same results and were used indifferently. Anti zebrafish PU.1 antibody was
generated by injecting rabbits with a fusion protein comprised of the amino-terminal 190 amino acids
of zebrafish PU.1 fused to glutathione S-transferase. The resulting anti-sera were purified over an
affinity column bearing the same 190 amino acids of zebrafish PU.1.
4
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18
Whole-mount immunohistochemistry was performed as described , omitting the acetone treatment
which destroys the SB staining. Briefly, following fixation as indicated above, larvae are treated with
18
collagenase , washed in PBDT (PBT, 1%DMSO), incubated for 3 hrs in PBDT containing 10% sheep
serum (PBDTS), then with the primary antibody in PBDTS overnight at 4°C, washed in PBDT 2x15
min.; then, when relevant, endogenous peroxidase is inactivated by a 50 min. incubation in PBDT
containing 2% H202. Embryos are rinsed for 3 hrs in PBDT, incubated in PBDTS for 2-3 hrs, then
incubated with the secondary antibody overnight at 4°C, then washed in PBDT then PBT for 2 hrs.
The secondary rabbit antibody used was coupled either to Alexa 488 (1:200; Invitrogen, CergyPontoise, France), or to horseraddish peroxidase (HRP) (1:800; Amersham, Les-Ulis, France), in
18
which case revelation was performed using aminoethylcarbazol (AEC) as a substrate . Rabbit
antibodies against mouse or zebrafish L-plastin, zebrafish PU.1, and GFP (#598; MBL, Watertown,
MA) were used at 1:500, 1:800, and 1:300 dilution, respectively.
Cell tracing
Embryos were injected at the 1- to 4-cell stage with 1 nl of a 1:3 mix of fixable and non-fixable caged
fluorescein-dextran 10.000 (Invitrogen) together with 1/10 v/v rhodamine-dextran 10.000 (all from 50
mg/ml stock solutions in 120 mM KCl), and then let to develop in the dark, except for their
manipulation, during which illumination was kept minimal. Only those embryos that displayed
homogeneous rhodamine fluorescence were used in uncaging experiments. Fluorescein uncaging was
performed through the epifluorescence port of a Nikon 90i microscope, using a Micropoint pulsed
laser generating 365 nm light (Photonic Instruments, Saint-Charles, IL), and a 40x water immersion
objective.
The targeted primitive myeloid progenitors in the 20 hpf yolk sac are visualized by DIC as large non
adherent round cells just beneath the yolk sac epidermis, next to the lateral and anterior borders of the
2
pericardium . These characteristics together with the very simple histology of the 20 hpf yolk sac (one
2
giant yolk cell covered by an epidermal monolayer, with the myeloid progenitors inbetween ) make
the labelling of myeloid progenitors easily achievable at single-cell resolution.
To detect simultaneously granulocytes and uncaged fluorescein at 72 hpf, we used either of two
methods: i) Sudan Black staining followed by anti-fluorescein immunohistochemistry revealed in
19
AEC , as indicated above except that an HRP-conjugated anti-fluorescein antibody (1:300; Roche,
Neuilly-sur-Seine, France) was used; ii) Cy3-tyramide based detection of endogenous peroxidase,
followed by incubation with HRP-conjugated anti-fluorescein antibody as above, then detection of
HRP activity with FITC-tyramide as above.
Microscopy
5
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All fixed embryos, larvae, and juveniles were transferred gradually from PBT to 100% glycerol for
conservation and microscopic observation. Live specimens were anesthesized with tricaine in embryo
9
medium , and observed in depression slides. VE-DIC / fluorescence microscopy was performed either
on a Reichert Polyvar2 (under a 40x/1.00 NA or 100x/1.32 NA oil objective) or a Nikon 90i (under a
60x/1.00 NA water immersion objective) microscope (Nikon-France, Champigny-sur-Marne, France);
VE-DIC images were generated using a 3-CCD video camera (HVD-25, Hitachi, Leeds, UK) tuned as
described
2,21
, and captured with the BTVPro software; fluorescence images were captured with a
DS5c digital camera driven by the NIS software (Nikon-France). Electron microscopy was performed
6
as described previously . Confocal fluorescence microscopy was performed on a Leica SPE (LeicaFrance, Rueil-Malmaison, France), with a 40x/1.15 NA oil immersion objective.
Results
Deployment and dynamics of granulocytes from embryo to juvenile fish
Sudan black B is a classical lipid stain that stains the granules of granulocytes much more avidly than
any other cell structures. Thus, staining followed by extensive 70% ethanol washes results in a highly
20
specific staining of the granules of granulocytes . We found this staining method to be strikingly
efficient when applied to whole fixed zebrafish embryos, larvae, and even juvenile (over 1-month old)
fish (Figure 1). At medium magnification, the staining nicely delineates the often amoeboid
granulocytes throughout the embryo, leaving their nucleus unstained (Figure 1A,b-c; B,d, C,b-c). At
high magnification, the individually stained granules can be discerned wherever the cell is flat enough.
(Figure 1A,d and Figure S1J) Combining the Sudan black (SB) staining with video-enhanced
Differential Interference Contrast (VE-DIC) microscopy reveals the precise histological location of
these cells in whole-mount embryos (Figure 1A,b-d; B,c-f; C,b-c).
The very first detectable SB stained granules appear by 33-35 hpf, already in diverse locations (Figure
S1A-J): in amoeboid cells in the yolk sac, between pericardium and epidermis, in the cephalic
mesenchyme, and the ventral tail. The small number of granules per cell and their frequent clustering
close to the nucleus (Figure S1H,I) suggests most of these cells are still immature neutrophils, i.e.
myelocytes
6,21
.
By 2-4 dpf, heavily stained amoeboid cells are present in all embryos (Figure 1A,1B), mostly in the
ventral moiety (Figure 1B,a-b): in the cephalic mesenchyme, notably in contact with muscles (Figure
1B,c), in the epidermis and around the base of olfactory pits and neuromasts of the lateral line (Figure
1A,c; B,e-f); medially in the ventral tail (Figure 1Ae), and sub-epidermally in the trunk and tail. In the
next days of larval development (Figure 1C,D), their number increases, especially in the ventral head
(jaw and gills), along the gut and pronephric ducts (Figure 1C,b-c), and prominently in the ventral tail,
i.e. the Caudal Hematopoietic Tissue (CHT) that we recently characterized
6
19
(Figure 1C,d). By 7 dpf,
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most larvae display a local concentration of SB+ granulocytes in the pronephros around the glomerulus
(Figure 1C,a), i.e. where definitive hematopoiesis has just been initiated, and all of them do so by 10
dpf. The number of stained cells in the pronephros increases steadily over the next days and weeks
(Figure 1D,b-c; E,a,c,f).
Unlike primitive macrophages, these SB stained granulocytes were never seen in the brain and retina
(Figure Db).
In juvenile (1-month old) fish, whole-mount SB staining reveals the entire population of granulocytes
throughout the fish (Figure 1E). Access to deep tissues is demonstrated by the heavy staining of the
entire head kidney, resolved into individual leukocytes at higher magnification (Figure 1E,a,c,f, and
data not shown). We noted numerous, mostly amoeboid granulocytes in the gills (Figure 1Ed), below
or within the epidermis, in the fins (Figure 1Eb), at the midline between the somitic muscles (not
shown), around the nasal pits (Figure 1Ee), and dorsally in the head, between the skull and epidermis
(Figure 1Ec).
In embryos and larvae, electron microscopy revealed bona fide neutrophilic granulocytes in the same
5,6
locations as SB staining, i.e. not only in the ventral tail as previously documented , but also in
peripheral tissues, including the epidermis (Figure S2). Figure S2B shows a sub-epidermal neutrophil
in extended contact with a macrophage.
We found that in live larvae, the granules of these neutrophils are readily noticeable through VE-DIC
microscopy, for they are refractile and constantly moving in Brownian motion in the cytoplasm
(Movies 1-3). Just like with SB staining, the first granules were discerned in vivo by 35 hpf, clustered
in the indentation of a kidney-shaped nucleus (Figure S1K); such cells could be followed undergoing
21
mitosis (Figure S1L-P, arrow), indicative of myelocytes . From 48 hpf, leukocytes with numerous
mobile granules were found at all the same locations as the SB stained cells (Figure 2A-H). When the
cell is moving, its granules are moved collectively by the cytoplasmic flow that accompanies the cell's
protrusive activity and anticipates its motility (Movie 1). In the mesenchyme these cells can migrate
quite rapidly (15-20 µm/min.). Even within the epidermis, at these stages still a monolayer, we found
them wandering by slipping between epidermal cells at a velocity of 10 µm/min (Figure 2A and
Movie 1). In fact, by 48 hpf, all leukocytes moving within the epidermis display these granules. In
4
contrast, all leukocytes resting and dendritically spread between epidermal cells , lack granules, and
are likely to be primitive macrophages, that by 48 hpf have stopped wandering to settle down between
epidermal cells.
Some neutrophils were found in the circulation; Figure 2D and Movie 2 show one neutrophil
interacting with one, then two macrophages in the duct of Cuvier. VE-DIC microscopy also readily
reveals that unlike the primitive macrophages, these neutrophils do not phagocytose cell debris. Nor
are they particularly endocytic: in some embryo batches, the primitive macrophages take up large
7
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amounts of methylene blue (present in the standard growth medium of the embryos), whereas
neutrophils never do so (Figure 2E, Movie 3, and Figure 7).
Thus SB staining, electron microscopy, and DIC video-microscopy of live specimens altogether show
that neutrophils are abundant and rapidly moving in the mesenchyme and epidermis of healthy
embryos and larvae.
5
Are these cells the same as the peroxidase positive leukocytes detected previously ? To test this, we
combined SB staining with the detection of peroxidase activity, using fluorescent tyramide as a
substrate. This method revealed a 100% coincidence of SB staining with intense tyramide-based
labelling (Figure 3E-M) in all embryos examined (n=15). The only difference was that with the
fluorescent tyramides, we could easily detect peroxidase-positive leukocytes in the yolk sac, head
mesenchyme, and ventral tail already by 24 hpf (Figure 3A-D), i.e. ten hours before the first SB
stained granules appeared.
We then tested if SB staining coud be combined with whole-mount immunohistochemistry for
leukocyte proteins, using polyclonal antibodies that we developed against the zebrafish homologs of
the transcription factor PU.1 and the actin-bundling protein Leukocyte-specific plastin (L-plastin).
Immunostaining revealed the PU.1 protein throughout the differentiation sequence of the primitive
macrophage lineage from the rostral lateral mesoderm (Figure 4A,B), as previously found by in situ
22,23
hybridization
, and still at 2, 3, and 5 dpf in numerous tissue leukocytes (Figure 4C-L).
Intriguingly, the PU.1 protein localized to the nucleus until 18-somites, then to the whole cell, and
later on predominantly to the cytoplasm (Figure 4A-F). Immunostaining following SB staining was
successful and revealed that all granulocytes express PU.1 at 2 and 3 dpf (Figure 4G,H,J,K) (n=15
embryos/stage). The PU.1-positive, SB-negative leukocytes included macrophages of the retina and
brain (Figure 4I), and ramified leukocytes in the epidermis (Figure 4C,D) clearly corresponding to the
4
ramified sessile leukocytes observed in vivo by VE-DIC microscopy .
Immunostaining using antibodies against zebrafish
16
17
or mouse
L-plastin following SB staining
2,4
revealed that like macrophages , all granulocytes, as well as thymocytes, express L-plastin (Figure
24,25
S3; n=12 embryos/stage), as in mammals
.
Granulocytes arise from two origins in the zebrafish embryo
What is the embryological origin of all these tissue neutrophils ? We recently showed that the CHT is
a site of granulopoiesis between at least 3 and 14 dpf, seeded by the definitive hematopoietic
precursors arisen in the trunk beneath the dorsal aorta (the fish homolog of the Aorta-Gonad19
Mesonephros (AGM) area of amniote embryos) . This caudal granulopoiesis, which was documented
by electron microscopy, obviously accounts for many of the numerous SB+ cells seen in the CHT and
its surroundings from at least 3 dpf onwards and possibly earlier. Yet the first appearance of SB+
8
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leukocytes by 32-35 hpf simultaneously in the CHT, yolk sac and cephalic mesenchyme suggested
that the rostral "primitive macrophage lineage" might also be a source of granulocytes.
Therefore we undertook a cell tracing analysis of the clearly identifiable hematopoietic precursors of
the primitive macrophages present in the yolk sac before 24 hpf, which we previously named the "pre2
macrophages" . Embryos were injected at the 1-cell stage with the photoactivatable cell tracer caged
fluorescein-dextran. At the 22-somite stage (20 hpf), when mesodermal precursors had emigrated from
antero-cardiac mesoderm to the neighboring yolk sac, and evolved into the characteristic, round
2
(12µm large) blast-like "pre-macrophages" , we uncaged the fluorescein with a U.V. laser in single
pre-macrophages - 10 per embryo (Figure 5A). At 72 hpf the larvae were fixed, and we combined
immunodetection of the uncaged fluorescein (revealing the progeny of the initially photoactivated
cells) with a detection of granulocytes, either through their SB+ granules, or through their peroxidase
activity. Both approaches led to the same results. Figure 5B shows a typical outcome obtained with the
first detection scheme. From the 10 cells initially photoactivated in the yolk sac at 20 hpf, a progeny of
17 cells was detected at 72 hpf, dispersed around the eye, in the CHT, along the pronephric duct, and
dorsal to the somites. 11/17 of these cells were also stained by SB, hence were granulocytes. Figure
5C shows an example following the second detection scheme. From the 10 initially photoactivated
cells, a progeny of 34 cells was detected, mostly around the left eye, in the CHT, and dorsal to the
somites. 17/34 also displayed bright red fluorescence, indicative of peroxidase activity, hence of
granulocytic identity; the non granulocytic progeny included 5 microglial cells in the retina (e.g.
Figure 5C,f). Over 14 different embryos, the mean detected progeny was 20.1 cells, of which 57 ± 9 %
were granulocytes (Figure S4).
These cell tracing data demonstrate that the hematopoietic precursors of the primitive macrophages
also give rise to granulocytes, in even slightly higher number.
To distinguish the granulocytes from the two lineages, we will call 'primitive granulocytes' those
arisen from the same rostral lineage as the primitive macrophages, and "larval granulocytes" those
arisen from AGM-derived precursors that homed to the CHT, as this is essentially a site of larval
hematopoiesis.
We then turned to mutants and morphants that are defective in definitive, AGM-derived hematopoiesis
but not in the primitive, rostral myeloid lineage : the spadetail
23,26
27
and mindbomb
mutants, and
11,27
runx1 knock-down morphants
, obtained by injection of an anti-runx1 morpholino in the fertilized
eggs. In both spadetail and mindbomb mutants, and in runx1 morphants, SB staining revealed
numerous granulocytes throughout the head and in the yolk sac. They were present also in the trunk
and tail but in a pattern different from wild-type siblings (Figure 6, and data not shown): all confined
to superficial (epidermal and sub-epidermal) locations; none (in spadetail mutants) or very few (in
mindbomb mutants and runx1 morphants) were seen in the CHT (which lies deep at the midline,
ventrally). This pattern confirms the absence of granulopoiesis in the CHT and the functionality of the
9
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rostral primitive lineage in spadetail and mindbomb mutants and runx1 morphants. Since spadetail has
no blood circulation in the trunk and tail (but normal circulation in the head), its SB staining pattern
further shows that the neutrophils of rostral origin can easily disperse in the whole body up to the tail
tip in the absence of blood circulation (Figure 6B).
Interestingly, in both mindbomb and spadetail, the granulocyte population still increased 2-fold
between 2 and 3 dpf (Figure 6C,D), indicating that once in the peripheral tissues, the cells of the
primitive, rostral lineage are still capable of proliferation and granulocytic differentiation.
Intriguingly, mindbomb embryos displayed a striking accumulation of SB+ granulocytes by 3 dpf on
the dorsal side of the head, sub-epidermally (Figure 6A), possibly related to the brain malformation in
this mutant, a malformation that however does not correlate with increased apoptosis, as revealed by
acridine orange vital staining (data not shown).
Primitive and larval granulocytes are barely phagocytic yet highly attracted to infected or
stressed tissues during zebrafish development
The mpx+ or peroxidase-positive leukocytes of zebrafish larvae -presumed neutrophils- have been
5
shown to be attracted to a caudal fin wound , but their behavior towards infections has not been
investigated. Surprisingly, when we injected E. coli bacteria in the blood by 52 hpf, the bacteria
immediately stuck to the macrophages, but not to the neutrophils present in the blood flow or in the
CHT. 30min-2hrs later, the macrophages were loaded with engulfed bacteria, but the neutrophils
contained none (Figure 7A,B). Only a few neutrophils in the CHT, close to the injection site, had
phagocytosed bacteria, though much smaller amounts than the macrophages (Figure 7C,D).
Since the primitive macrophages are able to sense and migrate to an infected body cavity to clear the
2
microbes , we devised a similar test for neutrophils, by injecting E. coli bacteria into another closed
space naturally devoid of leukocytes, the left ear, at 3 dpf. SB staining at various time points post
injection revealed that the neutrophils were massively attracted to the infected ear, where their number
peaked by 4-5 hpi (Figure 7E). Initially those of the head were first recruited, then the large caudal
population of the CHT also became visibly depleted by the recruitment (data not shown). Red
fluorescent tyramide-based staining of the peroxidase activity of the recruited neutrophils allowed us
to analyze in detail their relation with the green fluorescent bacteria, by confocal microscopy (Figure
7F). Again here, many bacteria were phagocytosed by peroxidase-negative macrophages, while only a
minority of the numerous recruited neutrophils phagocytosed bacteria - 2-10 per cell, instead of up to
100 per macrophage. Similar results were obtained upon injection of gram+ bacteria, or zymosan (data
not shown). As the production of extracellular thread-like "nets" by neutrophils from adult zebrafish
28
28
was recently described , we stained the infected embryos with DAPI , but failed to detect such
structures.
10
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The number of neutrophils in the ear gradually decreased past 5 hrs pi. In vivo VE-DIC microscopy,
4
which readily detects any type of cell death or damage , never revealed any trace of it among the
neutrophils (data not shown). They gradually left the site and most were gone by 24 hpi, whereas some
macrophages still remained in the cavity.
Interestingly, if the microinjection in the otic cavity was performed slightly less smoothly (see
Materials and Methods), injected sterile PBS attracted neutrophils to the same extent as injected
bacteria (Figure S5), further indicating that these neutrophils are exquisitely sensitive and attracted to
local perturbations of homeostasis. Figure S6 gives another example, which occured naturally in a 3weeks old wild-type larva that suffered some malformation, and displayed a striking accumulation of
neutrophils all over the outer and vitreal surface of both lenses, as well as around the intestinal bulb.
This massive mobilization had entirely depleted the kidney and CHT from their neutrophils.
Discussion
The results presented here clarify the identity, distribution, origins, and functional traits of neutrophils
through zebrafish development. SB staining of the granules that define these cells, along with their
visualization in vivo by VE-DIC microscopy, and by EM, in the tissues of the whole animal provides a
firm, unequivocal basis on which molecular and behavioral traits can be safely superimposed. Thus
doing, we find that from 48 hpf onwards, the granulocytes coincide completely with the peroxidase
7,23
positive leukocytes, as previously proposed. On the other hand, unlike previous claims
, all these
granulocytes express L-plastin, as well as PU.1, even once fully differentiated and wandering in
peripheral tissues.
The simultaneous appearance of granulocytes over the whole embryo by 48 hpf was intriguing. Our
cell tracing results demonstrate that they have two origins: not only the Caudal Hematopoietic Tissue
19
(CHT) seeded by the definitive HSCs born in the trunk , but also the rostral lineage that produced the
primitive macrophages a day earlier, from antero-cardiac mesoderm. The morphologically
2
homogeneous hematopoietic progenitors of the primitive macrophages, in the yolk sac , actually give
rise to both macrophages and granulocytes in similar numbers. So far, in vertebrates, especially in
mammals where they have been most studied, the primitive macrophages have always been considered
1
to constitute a separate lineage giving rise to no other cell type . Either the primitive granulocytes
found in the fish do not exist in mammals, or they have been overlooked because of the peculiar
features of their differentiation, which occurs almost a day after that of the primitive macrophages, and
mostly after emigration from the yolk sac and dispersal in embryonic tissues. In fact, to our
knowledge, the very notion of myeloid progenitors that emigrate to the peripheral tissues before
differentiating into neutrophilic granulocytes in situ is unprecedented. The peroxidase-positive
myeloid cells found in the yolk sac and embryonic tissues in the 24-32 hpf interval, i.e. before the
appearance of the first SB stained granules, are likely to represent such migrating progenitors, since
11
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we no longer detect perox+ SB- cells in the tissues (nor morphologically identifiable primitive
myeloid progenitors in the yolk sac) by 48 hpf. Still, it is not possible to know if these migrating mpx+
progenitors are already committed to a granulocytic fate, for mpx may be expressed by the progenitors
8
both of granulocytes and of macrophages, as myeloperoxidase is in mammals . Remarkably, despite
the absence of such progenitors in the tissues by 48 hpf, we still observed a doubling of the primitive
granulocyte population in the tissues between 48 and 72 hpf, in spadetail and mindbomb mutants. In
wild-type embryos, over half of the progeny at 72 hpf of primitive myeloid progenitors labelled in the
yolk sac at 20 hpf consisted in neutrophils, scattered all over the swimming larva. Altogether these
data suggest that such mature tissue neutrophils have a much longer lifespan than assumed so far for
neutrophils in mammals, and might even be capable of some proliferation.
19
Our cell tracing data about both the rostral/primitive (this work) and caudal/larval leukopoiesis
lead
to a coherent picture of how the cells from these two origins contribute to the total myeloid population.
When blood circulation starts, by 26 hpf, some of the primitive myeloid leukocytes born in the yolk
2
sac are taken by the blood that flows freely over the yolk surface . Many of them stop in the caudal
vein, and settle there and in the surrounding mesenchyme. As in the other tissues which these cells
19
colonize, some become macrophages -here the stromal macrophages of the CHT - while others start
differentiating into neutrophils by 32-35 hpf. From 33 hpf onwards, the definitive HSCs born in the
trunk between aorta and axial vein start seeding the CHT (Kissa et al., submitted), where they expand
19
and differentiate, leading to a steady larval granulopoiesis for at least several days .
We found that overall the embryonic granulocytes and macrophages share the same tissue
localizations, with the clearcut exception of the brain and retina, in which granulocytes are never seen.
In this respect, our previous finding that the colonization of brain and retina by macrophages requires
4
the M-CSF receptor takes a further meaning. It provides a straightforward explanation for the absence
of neutrophils in the CNS, since the M-CSF receptor is the very marker of the monocyte/macrophage
lineage among all leukocytes.
We found that these neutrophils are potently attracted to bacteria and able to cross epithelia to reach
them; yet surprisingly, only a minority of them phagocytose microbes, and many times less per cell
than the macrophages of the same embryological origin.
A macrophage-restricted expression of microbial sugar binding lectin receptors may be responsible for
the fast sticking of microbes to the macrophage surface but not to the neutrophil, observed in the
embryo's blood. Phagocytosis by the neutrophils could require an opsonization of the microbes by
antibodies or complement, that would be lacking in embryos/larvae. Alternatively, these cells may
29
display some intrinsic immaturity, as documented for perinatal neutrophils in mammals . However,
our preliminary data indicate that in juvenile zebrafish, neutrophils behave as in early larvae, with
strong attraction to but poor phagocytosis of injected bacteria (E.C. and P.H., work in progress). If
12
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they are barely phagocytic, what could be the function of these neutrophils patrolling in the tissues ?
Since they are attracted to microbes together with the macrophages, they might assist the latter in
killing the microbes, through some controlled degranulation, of a kind that would not compromise
their viability, for once the bacteria have been cleared by the macrophages, these neutrophils appear to
just leave and resume their wandering.
30
But their action may be broader than the release of microbicidal compounds upon infection , for we
found these cells to be also strongly attracted to an aseptic local tissue damage (in the otic cavity
following a larger PBS injection) or to some developmental abnormalities (the abnormal brain of
mindbomb mutants, or the abnormal lenses of a naturally occurred case). Overall, these long-lived
neutrophils steadily patrolling in the dermis and epidermis display a lifestyle that we so far attributed
4
to tissue resident macrophages . Along this line, a non-phagocytic, IL-1 producing resident population
of neutrophils was recently identified in the testis of the gilthead seabream, a seasonal breeding fish,
31
suggesting a role of these cells in seasonal testicular involution . Thus the possible roles in tissue
remodelling and homeostasis beyond microbe elimination that have been advocated for tissue
4
32,33
macrophages , notably on the basis of their extremely diverse secretory activity
, should be
investigated as well for fish tissue neutrophils. Their study might ultimately lead to uncovering
neutrophil subsets of similar functions in mammals.
Acknowledgments
D.L.G., M.J.R., E.C., E. Murayama, K.M., V.B., E. Mordelet, A.Z. and P.H. performed experiments,
analyzed data, and checked or improved the manuscript; H.S. contributed an important reagent (anti
mouse L-plastin antibody); P.H. drafted the manuscript.
We thank Isabelle Godin, Véronique Witko-Sarsat and Jean-Pierre Levraud for their critical reading of
the manuscript, and J.M. Ghigo and W. Bitter for the GFP and DsRed expressing E. coli bacteria,
respectively. This work was initially supported by an Avenir grant from Inserm to P.H. D.L.G. was
supported by a M.N.E.R.T. fellowship.
The authors declare no competing financial interests.
13
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Figure Legends
Figure 1 : Deployment of Sudan Black stained granulocytes from embryo to juvenile zebrafish.
(A) 48 hpf embryo: (a) Lateral view of the head; the granulocytes are mainly dispersed in the mesenchyme; (bd) Granulocytes in the yolk sac (b), head epidermis (c) and mesenchyme (d); (e) Lateral view of the tail, where
larval hematopoiesis is beginning. (B) 4 dpf larvae, head region: a) Ventro-lateral and b) ventral views, showing
the increasing number of granulocytes. (c-f) Examples of granulocytes (c) between two cephalic muscle fibers,
(d) in the mesenchyme, (e) in the epidermis (note the characteristic actin ridges of the overlying periderm in the
lower half of the image), (f) at the border of a neuromast. (C) 7 dpf larvae: (a) Granulocytes gathered around the
pronephric glomerulus (arrow), revealing the beginning of definitive granulopoiesis in the kidney. (b-c)
Granulocytes along the basal lamina of the gut (b) and the left pronephric duct (c); (d) lateral view of the tail
showing the growing granulocyte population in the CHT, the site of larval hematopoiesis. (D) 14 dpf larvae: (a)
Ventral and (b) dorsal views of the anterior region; note the paucity of granulocytes in the dorsal head, except
most anteriorly around the olfactory pits, and the increasing number of granulocytes in the kidney around the
glomerulus (arrow), also visible in lateral view in (c). (E) 30 dpf juvenile fish : (a) The head kidney (remnant of
the pronephros) is now full of granulocytes (arrow). (b-f) Granulocyte populations in the caudal fin (b), all over
the head (c), in the gills (d), in the left olfactory pit (e), and in the head kidney (f) (left lateral side). e, inner ear;
ca, caudal artery; g, pronephric glomerulus; k, kidney; m, muscle fiber; op, olfactory pit; pd, pronephric duct; r,
retina. Scale bars, 10µm in (Ab-d, Bc-f) ; 20µm in (Cb, c) ; 100µm in (Aa, Ba,b, Ca, Eb-f).
Figure 2 : In vivo imaging of neutrophilic granulocytes by video-enhanced DIC microscopy.
The nucleus of each neutrophil, whenever visible, is indicated by a black asterisk. (A) 48 hpf: a neutrophil
migrating within the epidermis (at this stage still a monolayer). Time is indicated in minutes and seconds. In this
24 min. sequence, the neutrophil moves successively along three long epidermal cells (e, e' and e''), at a mean
velocity of 10 µm/min; the worm-like elements visible in these cells are mitochondria. See also Movie 1. (B) 50
hpf: a neutrophil with visibly bilobed nucleus in addition to its granules, in contact with a Schwann cell (with
white asterisk on its nucleus) wrapped around the posterior lateral line nerve. (C) 48 hpf: two neutrophils on the
anterior aspect of the pericardium. (D) 50 hpf: still image from Movie 2. A neutrophil interacting with a
macrophage, itself interacting with three dying erythrocytes in the yolk sac circulation valley. (E) 56 hpf: a
macrophage that accumulated methylene blue from the outer medium, and three granulocytes, in the Caudal
Hematopoietic Tissue. See also Movie 3. (F) 5 dpf: two neutrophils along a blood vessel in the ventral head. (G)
56 hpf: a neutrophil in the mesenchyme along the retinal pigment cell layer. (H) 7 dpf: a neutrophil near a
neuromast of the ventral head (white asterisk). n, neutrophilic granulocyte; Mφ: macrophage; e-e'-e'', bulk
epidermal cells; m, epidermal mucous cell; er, degenerating primitive erythrocytes; pll, posterior lateral line
nerve; c, cartilage; mf, cephalic muscle fibers; s, somitic muscle; r, retina; rpc, retinal pigment cell layer. Scale
bars, 10 µm.
Figure 3. Sudan Black stained granulocytes coincide with peroxidase-positive leukocytes in zebrafish
embryos.
(A-D) At 24 hpf, 10 hrs before the first detection of SB stained granules, Cy3-tyramide based detection of
endogenous peroxidase activity already reveals numerous peroxidase-positive leukocytes in the yolk sac (A,C),
cephalic mesenchyme (B), and tail (A,D); note the unstained nuclei in (D). Unlike at later stages, the staining
appears concentrated in foci or granules (B,C). In (A), vertical lines indicate a picture element captured at a
slightly different focus. (E-M) 48 hpf embryos; detection of endogenous peroxidase activity with FITC-tyramide
followed by the detection of granules with SB demonstrates a perfect coincidence of the two stainings. (E-H)
High magnification of cells stained with both SB (E,G) and FITC-tyramide (F,H) near the ear. (I,J) Lateral right
views of the eye and ear regions. Arrowheads point at the three cells shown at higher magnification in (E-H).
(K-M) ventral tail (rostral to the left): DIC optics (K), fluorescence (M) and overlay (L). ca, caudal artery; e, ear;
r, retina. Scale bars, 15µm in (B and E), 40 µm in (K), 50 µm in (I, J).
Figure 4. All primitive and larval granulocytes express PU.1.
(A) Whole-mount immunodetection of PU.1 with a polyclonal antibody directed against zebrafish PU.1. Left
lateral view of a 14-somite embryo counterstained with DAPI, showing the immunostained cells in the rostralmost, 'myelopoietic' lateral mesoderm (arrow). At this early stage the staining is only nuclear. (B) Coimmunodetection of PU.1 (green) and L-plastin (red) in the yolk sac of a 24 hpf embryo; nuclei stained with
DAPI. (C-F) Immunostaining for PU.1 at 5 dpf; (C,D) Ramified non granulocytic myeloid leukocytes in the
trunk epidermis; an asterisk points at the nucleus, positionned at one end of the cell; (E) caudal fin; (F) Caudal
Hematopoietic Tissue (arrow, rostral end). (G-L) Immunostaining for PU.1 following Sudan Black staining at 48
hpf (G-I, L) and 72 hpf (J, K). All SB+ cells also express PU.1, e.g. in the pericardial region (G), posterior (H)
16
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and ventral to the ear (J), in the CHT (K); in (K), a vertical line separates two parts captured at a slightly
different focus. The PU.1+,SB-cells are likely macrophages, notably in the retina and brain (I). y, yolk sac; p,
pericardial area; e, inner ear; n, notochord; ca, caudal artery. Scale bars = 200 µm in (A), 15 µm in (B), 20 µm in
(C,G-L), 10 µm in (D), 5 µm in (E), 50 µm in (F).
Figure 5. In vivo cell labelling with a photo-activatable cell tracer demonstrates the double potential of the
primitive (rostral) myeloid progenitors.
(A) At 20 hpf, following injection of caged fluorescein-dextran in the embryo at the 1-cell stage, the fluorescein
was uncaged (hence its natural fluorescence was restored) with a pulsed UV (365 nm) laser beam in 10 primitive
myeloid progenitors, located along the left lateral border of the pericardium. 15 hours later (35 hpf), some of the
green-fluorescent labelled cells are observed in the yolk sac circulation valley (arrows). (B) 72 hpf;
immunodetection of uncaged fluorescein (AEC staining, red) following SB staining reveals the granulocyte vs.
macrophage nature of the progeny of the primitve myeloid progenitors photolabelled in (A). The drawing
recapitulates the locations of all red (uncaged fluorescein-positive) cells identified in this embryo; red dots
represent macrophages, stained only by AEC (6/17 cells); these are located along the retina, along the base of the
dorsal fin (b,c, red arrowheads), in the CHT. Black dots represent cells stained by both AEC and SB (11/17
cells), hence granulocytes; those are located around the eye, ventral to the trunk somites, in the CHT (a,d,e, black
arrowheads). Note that the dextran-coupled uncaged fluorescein revealed by AEC is in the whole cell, whereas
SB is confined to the cytoplasm, hence the red label is most apparent in the nucleus. Scale bars, 25 µm. (C) 72
hpf; fluorescent combined immunodetection of uncaged fluorescein (FITC-tyramide, green) with a detection of
the peroxidase activity of granulocytes (Cy3-tyramide, red), following uncaging of fluorescein in 10 primitive
myeloid progenitors in the yolk sac at 20 hpf. Green dots on the drawing represent macrophages (17/34 cells)
which are located in the head mesenchyme, around the eye, in the ganglion cell layer (rgc) of the retina (f),
dorsal to the somites (b,c), along the yolk tube (a), in the CHT (e,g, green arrowheads). Yellow dots and
arrowheads stand for granulocytes, stained in both green and red (17/34 cells). Those cells are located in the
head mesenchyme, dorsal to the somites (b,c), in the CHT (g). Scale bars, 50 µm.
Figure 6. Sudan Black stained granulocytes in mindbomb and spadetail mutants.
(A) Lateral and dorsal views of a 3 dpf mindbomb mutant. Note the accumulation of SB+ cells dorsally in the
head. (B) Lateral views of a 56 hpf and a 78 hpf spadetail mutants. Note the increase in SB+ cells all over the
body between the two stages, even though these mutants have no circulation in the trunk and tail (but normal
circulation in the head, data not shown). At both stages, all SB+ cells in the tail and trunk are superficial (mostly
sub-epidermal); none is in the CHT. (C,D) Histograms showing the number of SB+ granulocytes sorted by their
localisation throughout the embryo, in mindbomb at 48 and 72 hpf (C) and spadetail at 56 and 78 hpf (D)
mutants. In both mutants, the granulocyte population increases 2-fold between the two stages examined. Cell
numbers for the wild-type siblings are not shown since irrelevant to the point examined here.
Figure 7. Neutrophils are attracted to infection foci, yet much less phagocytic than macrophages, in
zebrafish larvae.
(A-D) In vivo observations in a swimming larva injected with red fluorescent E. coli in the caudal vein at 52 hpf.
Blue, green, and red arrowheads respectively point at macrophages, neutrophils, and erythrocytes. (A-D) are
video-enhanced DIC images, (A'-D') show the corresponding overlay of DIC and red fluorescence of the
bacteria; all images are at the same magnification. (A,A') 1h30 post injection, yolk sac circulation valley: two
macrophages, one of which loaded with methylene blue, have already phagocytosed many bacteria, while two
neutrophils nearby are free of them; the neutrophil marked by an asterisk at the border of its nucleus is also
displayed 45 seconds later in the lower left inset in A, to show its fast motility. The inset in A' shows 4 more
macrophages farther in the valley, two of which also loaded with methylene blue. (B,B'): 2h15 p.i., caudal
hematopoietic tissue (close to the site of bacteria injection): 4 macrophages, one loaded with methylene blue,
have phagocytosed many bacteria, whereas a neutrophil among them has none. (C,C') 2h30 p.i., CHT/ventral fin
junction: a neutrophil that phagocytosed bacteria; (D,D') images of the same neutrophil 8 min. later. In (A,A',B)
thin straight lines indicate picture elements captured at a slightly different focus. (E) SB staining of neutrophils
at 3 dpf, 5 hrs after microinjection of either PBS or E. coli bacteria in the left inner ear. (F) Confocal
fluorescence image of a portion of the left ear at 3 dpf, 5 hrs post injection of green fluorescent (GFP expressing)
E. coli in the ear cavity. After fixation, the peroxidase activity of neutrophils was revealed with Cy3-tyramide
(red), then GFP by anti-GFP immunohistochemistry with Alexa488 as fluorophore (green), and the nuclei were
stained with DAPI (blue). Two macrophages are highlighted by their large amount of phagocytosed bacteria,
while only some of the recruited neutrophils show a few small bacteria-containing phagosomes. The upper right
inset shows a neutrophil in an other focal plane, with four obvious such phagosomes. e, ear; oe, otic epithelium;
sc, semi-circular canal. Scale bars, 10µm.
17
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Prepublished online September 17, 2007;
doi:10.1182/blood-2007-06-095398
Origins and unconventional behavior of neutrophils in developing
zebrafish
Dorothee Le Guyader, Michael J Redd, Emma Colucci-Guyon, Emi Murayama, Karima Kissa, Valerie
Briolat, Elodie Mordelet, Agustin Zapata, Hiroto Shinomiya and Philippe Herbomel
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