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Blood First Edition Paper, prepublished online July 17, 2008; DOI 10.1182/blood-2008-03-145011
Carboxypeptidase A5 identifies a novel mast cell lineage in the zebrafish providing new
insight into mast cell fate determination
J. Tristan Dobson,1,2 Jake Seibert,1 Evelyn M. Teh,1,2 Sahar Daas,1 Robert B. Fraser,1,4 Barry H.
Paw,5 Tong-Jun Lin,1,2, 3 and Jason N. Berman1,2, 3
Institutional affiliations:
1
IWK Health Centre, Depts. of 2Microbiology and Immunology, 3Pediatrics and 4Pathology,
Dalhousie University, Halifax, Nova Scotia, Canada and 5Brigham & Women’s Hospital,
Division of Hematology and Children’s Hospital Boston, Division of Hematology-Oncology,
Harvard Medical School, Boston, Massachusetts, USA
Correspondence: Jason N Berman MD FRCPC FAAP
Address:
Division of Pediatric Hematology/Oncology
Departments of Pediatrics and Microbiology/Immunology
Dalhousie University
IWK Health Centre
PO Box 9700, 5850/5980 University Avenue
Halifax, Nova Scotia B3K 6R8 Canada
Phone: 902-470-8048 (office)
902-470-8840/8841 (lab)
Fax: 902-470-7216
Email: [email protected]
Short title: Cpa5 identifies zebrafish mast cells
Jason N. Berman is supported by a Dalhousie University Clinical Scholar Award and a Canadian
Institutes of Health Research – Nova Scotia Health Research Foundation Regional Partnership
Award.
Copyright © 2008 American Society of Hematology
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Abstract:
Mast cells (MCs) play critical roles in allergy and inflammation, yet their development remains
controversial due to limitations posed by traditional animal models. The zebrafish provides a
highly efficient system for studying vertebrate hematopoiesis. We have identified zebrafish MCs
in the gill and intestine, which resemble their mammalian counterparts both structurally and
functionally. Carboxypeptidase A5 (cpa5), a MC specific enzyme, is expressed in zebrafish
blood cells beginning at 24 hours post fertilization (hpf). At 28 hpf, co-localization is observed
with pu.1, mpo, l-plastin and lysozyme C, but not fms or cepbα, identifying these early MCs as a
distinct myeloid population arising from a common granulocyte/monocyte progenitor.
Morpholino “knockdown” studies demonstrate transcription factors gata-2 and pu.1, but not
gata-1 or fog-1 as necessary for early MC development. These studies validate the zebrafish as
an in vivo tool for studying MC ontogeny and function, with future capacity for modeling human
MC diseases.
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Introduction:
Mast cells (MCs) play central roles in allergic and inflammatory reactions.1,2 Stimulation
of cell surface receptors, such as C-KIT and the high affinity IgE receptor,1,2 results in the release
of mediators from cytoplasmic granules, including tryptase and histamine.2 MC number and
function are regulated by their development, proliferation, migration and survival.1 Barriers to
understanding these processes include accessibility and imaging limitations posed by traditional
animal models. The zebrafish has proven itself to be a robust and highly conserved model for
studying vertebrate hematopoiesis.3 Here, we provide the first evidence that the zebrafish
possesses MC equivalents that share structural and functional characteristics with their
mammalian counterparts. Furthermore, we demonstrate the utility of the zebrafish as an in vivo
tool in dissecting the contribution of transcription factors to MC development.
Materials and methods
Zebrafish were maintained, bred and developmentally staged according to Westerfield.4
Use of zebrafish in this study was approved by the Dalhousie University Animal Care
Committee. Zebrafish gills and intestine were fixed in 10% neutral buffered formalin and
standard staining procedures were applied (Figure 1A-F). Immunohistochemisty was facilitated
by antigen retrieval (Figure 1I,J). For electron microscopy, tissues were fixed overnight in 2%
glutaraldehyde in 0.1M caccodylate and post-fixed in 1% osmium tetroxide. Thin sections
(90nm) were stained in 25% uranyl acetate in methanol and lead citrate.
Bromophenol blue and 10 ug Compound 48/80 or saline were injected intraperitoneally
(IP) and blood extracted by cardiac puncture after 2.5 minutes. Tryptase activity was measured
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in plasma spectrophotometrically at 415 nm by the release of p-nitroanilide from N-benzoyl-DLarginine-p-nitroanilide (BAPNA), a tryptase substrate.
Digoxogenin- or fluorescein isothiocyanate (FITC)-labeled antisense mRNA probes for
zebrafish carboxypeptidase A5 (cpa5), α-globin, cebpα, pu.1, myeloperoxidase (mpo), l-plastin,
lysozyme C, fms, gata-2 and gata-1, were synthesized according to the published literature.5-8
Whole-mount single or double mRNA in situ hybridization (ISH) assays were adapted from
standard protocol.5 Images were taken on a Leica MZ16F with a Leica DFC 490 camera (5X
objective). Cpa5-FITC-labeled Fast Red-stained 28 hour and 7 day embryos were dissociated
using Blendzyme 3 and a strained cell suspension was centrifuged for 10 minutes at 4000 rpm
and re-suspended in 400 µL of 0.9 X PBS/5% FBS for fluorescent activated cell sorting (FACS)
and cytospin.
Five micron sections of intestinal tissue were de-paraffinized with xylene and rehydrated
with graded alcohols. ISH (whole mount protocol) was performed adding 400ug/mL levamisole
following NBT-BCIP staining (methyl green. counter-stain).
Gata-1, gata-2, and friend of gata-1 (fog-1) morpholinos and controls were purchased
from Genetools LLC (Philomath, OR): Pu.1: morpholino and control9 were kindly provided by
Dr. Jennifer Rhodes (DFCI, Boston, MA). Morpholinos were diluted to a working concentration
(gata-1 1.0 mM, gata-2 1.0 mM, fog-1 0.8 mM, pu.1 0.5 mM) with 1% phenol red and 1 nL was
injected into zebrafish embryos at the 1-4 cell stage.
Results and Discussion:
MCs were identified in gill and intestinal sections of adult wild type zebrafish - anatomic
equivalents to well-established sites where MCs reside as part of the innate immune system in
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mammals.2 These cells contain an ovoid eccentric nucleus and prominent eosinophilic granules
on H&E staining, which stained positively with PAS, a feature shared with the
eosinophil/basophil population previously identified amongst the hematopoietic elements of the
zebrafish kidney.5 Mammalian MCs and basophils similarly share some common structural
features.10 Granules appeared metachromatic following toluidine blue staining, a pathognomonic
characteristic of mammalian MCs11 (Figure 1A-F). Electron microscopy demonstrated an
eccentrically placed nucleus and dense homogenous granules closely approximating the
appearance of murine MCs (Figure 1G,H). Immunohistochemistry demonstrated a positive
reaction to polyclonal anti-human C-KIT and monoclonal anti-human mast cell tryptase (Figure
1I,J). Intraperitoneal injection of Compound 48/80, an agent shown to induce MC degranulation
in both mammals and other teleost fish,12,13 resulted in increased numbers of activated
degranulating intestinal MCs and a significant elevation in plasma tryptase levels compared with
saline-injected controls (Figure M-O). Tryptase release is a reliable reflection of MC burden or
reaction severity,14 suggesting that the zebrafish can serve as a robust in vivo system for
evaluating vertebrate MC responses.
We identified zebrafish cpa5, the protein product of which shares 64% identity with
human CPA1 expressed in exocrine pancreas and 38% identity with CPA3 found in human MCs.
Zebrafish cpa5 pancreatic expression has been previously identified15 and embryonic blood cell
expression demonstrated in a large-scale ISH screen.16 In adults, cpa5 expression was restricted
to morphologically identified MCs and pancreatic tissue (Figure 1K,L). In embryos, cpa5
expression was restricted to hematopoietic cells present in the anterior lateral paraxial mesoderm
and in smaller numbers around the intermediate cell mass (sites of embryonic hematopoiesis3,6),
beginning at 24 hpf. Cpa5 expressing cells reached a peak by 28 hpf where they congregated at
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both sites of embryonic hematopoiesis and in circulation over the yolk sac, persisting through 7
dpf. By 72 hpf, cpa5 expression could also be observed in the pancreas (Figure S1). Other
zebrafish homologues of mammalian carboxypeptidases, including cpa1 and cpb1, were not
expressed in zebrafish blood cells (data not shown). FACS analysis and cytospin of cpa5-FITClabeled Fast Red-stained cells revealed a predominance of cells with a morphological appearance
in keeping with mammalian MCs17(Figure 1P-S, S2). Cpa5 expression co-localized in a
proportion of embryonic myeloid cells expressing the early myeloid transcription factor, pu.1, as
well as mpo,5,18 l-plastin6 and lysozyme-C.19 These latter three genes were previously
characterized as granulocyte (mpo) or monocyte (l-plastin, lysozyme C) specific, but more recent
zebrafish data has implicated a more pan-myeloid expression profile.8,20,21 Interestingly, no colocalization was observed between cpa5 and fms, a gene exclusively expressed on monocytes,8
or for cpa5 and cepb-α, a transcription factor required for neutrophil and basophil cell
fate22(Figure 2A, S3). These data establish cpa-5 expressing cells as a unique myeloid
subpopulation arising from a cell with both granulocyte and monocyte potential, in keeping with
the model of mastopoiesis posed by Arinobu et al.22 This model contends that MCs and
basophils arise from a common granulocyte/monocyte progenitor, with cepb-α functioning as the
transcriptional switch determining cell fate.
We used a morpholino-based strategy to interrogate the roles of several transcription
factors in MC development. We demonstrated that gata-2 and pu.1 are both required for early
MC development in zebrafish, as gata-2 and pu.1 morpholino-injected morphants demonstrate
severely decreased to absent cpa5 expression. Gata-1 morphants paradoxically demonstrated
abundant cpa5 positive cells, likely due to unopposed pu.1 expression.9 This supposition was
supported by the absence of cpa5 expression in compound gata-1/pu.1 morphants.
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Alternatively, cpa5- positive MC progenitors may accumulate at the expense of mature MCs that
require gata-1 expression, as observed in Gata-1 low mice.23 Fog-1 has recently been suggested
to antagonize MC development.24 25 Zebrafish fog-1 morphants maintained cpa5 expression,
confirming fog-1 is dispensable for early MC development. Interestingly, a large expansion of
cpa5-positive cells was seen when gata-1 was simultaneously “knocked down”, suggesting the
permissive effect of fog-1 inhibition on MC progenitor development may be enhanced in the
absence of gata-1 (Figure 2B,C).
With the discovery of zebrafish MC counterparts, we have contributed to the
establishment of a complete myeloid cell repertoire in this species and demonstrated that the
developmental and technical opportunities afforded by the zebrafish for studying other lineages
can be applied to MC biology. Continuation of these efforts has the potential for significantly
expanding our understanding of vertebrate mastopoiesis and MC function. Moreover, these
studies set the stage for harnessing the transgenic capabilities of the zebrafish to model
inflammatory and malignant human MC diseases with the future capacity for high-throughput
inhibitor screening.
Acknowledgements
The authors would like to thank Patricia Colp for assistance with immunohistochemistry,
Marlene Henry for assistance with electron microscopy, and Sarah Bugden for assistance with
FACS analysis. We would like to thank Alan Cantor, John Kanki and Jean Marshall for their
critical review of the manuscript, Jennifer Rhodes and Leonard Zon for helpful discussion and
Jocelyn Jaques for administrative assistance. J.N.B is supported by a Dalhousie University
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Clinical Scholar Award and a Canadian Institutes of Health Research – Nova Scotia Health
Research Foundation Regional Partnership Award.
Author contributions
J.T.D performed research and analyzed data. J.S. performed research. E.M.T. performed
research and analyzed data. S.D. performed research. R.B.F. contributed pathology expertise
and provided reagents. B.H.P contributed reagents and analyzed data. T.J.L. designed research
and analyzed data. J.N.B. designed research, analyzed data and wrote the paper. Conflict of
Interest Disclosure: Authors have no conflicts of interest to disclose.
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Figure Legends:
Figure 1: Zebrafish mast cells structurally and functionally resemble their mammalian
counterparts. (A: intestine, B: gill) Hematoxylin and eosin staining, (C: intestine, D: gill)
Periodic acid shift (PAS), (E: intestine, F: gill) Toluidine blue (100X objective). Black
arrowheads indicate the mast cells in each panel. Transmission electron microscopic images of
(G) a zebrafish intestinal mast cell (20,000X magnification) and (H) a mouse bone marrowderived mast cell (26,000X magnification) (Phillips 300 transmission electron microscope).
Zebrafish mast cells demonstrate a positive reaction to (I) a polyclonal antibody raised against
human CD117 (C-KIT) antigen (Dako Cytomation) and (J) a monoclonal anti-human mast cell
tryptase antibody (gills) (Dako Cytomation). Biotinylated Universal Linker (Dako Cytomation)
secondary antibody and 3,3’-diaminobenzidine (DAB) for chromogenic detection (hematoxylin
counterstain). RNA in situ hybridization using digoxigenin-labeled RNA anti-sense probe to
zebrafish cpa5 demonstrates positive staining in (K) intestinal mast cells and (L) pancreas (100X
objective). Adult zebrafish injected intraperitoneally with 10 ug of Compound 48/80
demonstrate (N) mast cell degranulation compared to (M) saline injected controls (PAS staining,
100X objective. Black arrowheads indicate the mast cells in each panel) and (O) increased
plasma tryptase levels compared to saline injected control fish. Presented as mean ± SEM of
three experiments with 4-6 fish per group, * p<0.05 (t-test). Cytospin of FACS sorted FITClabeled Fast Red-stained cpa5 positive cells isolated from zebrafish embryos at 7 dpf
demonstrate morphology consistent with mast cells (P) toluidine blue (Q) Wright-Giemsa. (R)
green channel (FITC), (S) red channel (Fast Red) (also Figure S2).
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Figure 2: Carboxypeptidase A5 (cpa5) identifies zebrafish mast cell progenitors (A) Double
whole mount in situ hybridization using a digoxigenin-labeled RNA anti-sense probe to zebrafish
cpa5 (blue) and FITC-labeled RNA anti-sense probe (red) to pu.1, (mpo, l-plastin, and lysozyme
C, see Figure S3) demonstrate co-expression of cpa5 in a proportion of cells (panel i tail, panel ii
head/yolk sac (5X objective)). Evidence of co-expression is shown by co-localization observed
in higher magnification images of selected cells (panel iii brightfield, panel iv fluorescence (10X
objective)). No co-localization is observed for fms and cebp-α (panel i brightfield, panel ii
fluorescence (8X objective)) (B) gata-2 and pu.1 are both required for the development of early
mast cells as evident by the absence of cpa5 expression in gata-2 and pu.1 morphants; whereas
gata-1 morphants paradoxically demonstrate increased numbers of cpa5 positive cells. Fog-1 is
dispensable for early mast cell development as evidenced by wild type cpa5 expression in fog-1
morphants. Compound gata-1/pu.1 morphants demonstrate an absence of cpa5 expression;
while compound fog-1/gata-1 morphants show a dramatic increase in numbers of cpa5 positive
cells. Lateral views, anterior left and dorsal at the top (28 hpf, 5X objective). Inset boxes
demonstrate a higher magnification view of the tail and the region around the intermediate cell
mass (C) Proposed model of transcription factor interactions required for early MC development
(solid lines represent established interactions, dotted lines represent potential interactions).
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Prepublished online July 17, 2008;
doi:10.1182/blood-2008-03-145011
Carboxypeptidase A5 identifies a novel mast cell lineage in the zebrafish
providing new insight into mast cell fate determination
J. Tristan Dobson, Jake Seibert, Evelyn M Teh, Sahar Daas, Robert B Fraser, Barry H Paw, Tong-Jun Lin
and Jason N Berman
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