1 Centre Oncologia Molecular, IDIBELL-Institut de Recerca Oncologica,
Hospitalet, Barcelona 08907, Spain
2 Department of Immunology and Oncology, Centro Nacional de
Biotecnología, CSIC. Darwin, 3. Campus de Cantoblanco, Madrid 28049,
Spain
* Author for correspondence (e-mail: abigas{at}iro.es)
Accepted 22 December 2004
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SUMMARY |
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Key words: Notch, Mouse, Rbpsuh
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Introduction |
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The developmental origin and the genetic program of embryonic HSC emergence
in the YS and the P-Sp/AGM in some aspects are divergent. Yolk sac blood cells
originate simultaneously with the surrounding endothelial cells, consistent
with the idea of developing from a common progenitor or hemangioblast
(Palis and Yoder, 2001). By
contrast, P-Sp/AGM hematopoietic cells emerge in close association to the
presumably differentiated aortic endothelium. The lineage relationships and
molecular events leading to their differentiation are not completely
understood. Immunohistochemical analyses of the AGM region reveal overlapping
expression of hematopoietic and endothelial markers in the clusters of cells
that emerge from the ventral wall of the aorta. However, Aml1/Cbfa2 (Runx1 -
Mouse Genome Informatics) transcription factor has been shown specifically to
be involved in the development of intra-embryonic hematopoiesis without
affecting the main vasculature (North et
al., 1999
). The analysis of recently developed transgenic mice,
which enable specific labeling of emerging HSCs, provides supportive evidence
that true HSCs originate among the cells residing in the endothelial layer
(Ma et al., 2002
). Besides
Aml1 (North et al.,
2002
), Gata2 (Tsai et
al., 1994
; Tsai and Orkin,
1997
) and Scl (Tal1 - Mouse Genome Informatics)
(Porcher et al., 1996
;
Robb et al., 1996
) are also
expressed in hematopoietic clusters and endothelial-like cells lining the
ventral wall of the dorsal aorta at E10-11 and there is now strong evidence
that all these transcription factors are important for the onset of definitive
hematopoiesis in the embryo.
Signaling through the Notch receptors is a widely used mechanism for cell
fate specification and pattern formation in embryonic development and
adulthood (Artavanis-Tsakonas et al.,
1999; Lai, 2004
;
Lewis, 1998
). The interaction
between Notch receptors and ligands results in the cleavage of the
intracellular domain of Notch that translocates to the nucleus and together
with RBPj
(Rbpsuh - Mouse Genome Informatics) activates gene
transcription. The best-characterized Notch-target genes are the orthologs of
the Hairy and enhancer of split (Hes) and Hes-related (Hrt) proteins (for a
review, see Iso et al., 2003
).
Notch family members have been identified in several hematopoietic cell types
from diverse origin and there is now strong evidence that they participate in
the control of hematopoietic differentiation in many different lineages
(Han et al., 2002
;
Radtke et al., 1999
;
Stier et al., 2002
).
The first evidence showing the involvement of Notch in the onset of
embryonic hematopoiesis has recently been published, confirming that
development of hematopoietic cells from the hemogenic endothelium is a
Notch1-regulated event and it is impaired in Notch1-deficient embryos
(Hadland et al., 2004;
Kumano et al., 2003
). We show
here that this is an RBPj
-dependent event, since
RBPj
mutant embryos also lack intra-embryonic hematopoiesis.
Endothelial cells are not affected, as previously seen in the Notch1
mutant embryos. We identify several Notch family members showing distinct
expression patterns in presumptive E9.5 and 10.5 hemogenic endothelium,
suggesting that different Notch signals may operate in this system. We also
present evidence that Notch1 directly regulates the expression of
Gata2, thus suggesting that one of the first events in embryonic
hematopoietic determination consists in the activation of Gata2
expression by Notch1/RBPj
.
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Materials and methods |
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Cell lines
32Dcl3 wild-type (32D-wt) and activated Notch1-expressing 32D cells
(32D-N1IC) have been extensively characterized
(Bigas et al., 1998;
Milner et al., 1996
). Cells
were maintained in Iscove's 10% fetal bovine serum (FBS) and 10%
IL-3-conditioned media.
RT-PCR
Total RNA from dissected wild-type and RBPj mutant
embryonic P-Sp was isolated using TRIzol Reagent (Invitrogen). Poly-AT Tract
System IV (Promega) and RT-First Strand cDNA Synthesis kit (Amersham Pharmacia
Biotech) was used to obtain mRNA and cDNA respectively. PCR product was
analyzed at 35 and 40 cycles to avoid saturation. Quantity One software
(Biorad) was used for densitometry. Oligonucleotide sequences will be given
under request.
Hematopoietic colony assay
The P-Sp from E9.5 wild-type and RBPj mutant embryos was
digested in 0.1% collagenase (Sigma) in PBS, 10% FBS and 10% IL3- and stem
cell factor (SCF)-conditioned medium for 1 hour at 37°C. One hundred
thousand cells were plated in 1% methylcellulose (Stem Cell Technologies) plus
Iscove's with 10% FBS, 10% IL3- and SCF-conditioned medium, 2.5% L-glutamine,
0.1% monothioglycerol (Sigma), 1% Pen/Strep (Biological Industries), 2 IU/ml
erythropoietin (Laboratorios Pensa), 20 ng/ml GM-CSF (PeproTech) and 100 ng/ml
of G-CSF (Aventis Pharma). After 7 days, the presence of hematopoietic
colonies was scored under a microscope. For liquid cultures, the P-Sp region
was dissected from embryos and dissociated by gentle pipetting. One hundred
thousand cells were plated in Iscove's with 10% FBS, 10% IL3- and
SCF-conditioned medium, 0.1% monothioglycerol, 2.5% L-glutamine and 1%
Pen/Strep. Non-adherent cells were recovered and analyzed after 6 days.
Flow cytometry analysis
For flow cytometry (FACS) assay, 75,000 non-adherent cells were stained
with anti-CD45-FITC or IgG-FITC (Pharmingen). Cells were analyzed by
FACScalibur (Becton&Dickinson) and WinMDI2.8 software. Dead cells were
excluded by 7-aminoactinomicin-D staining.
Immunostaining
Wild-type and RBPj null embryos (E9.5) were frozen in
tissue-tek OCT (Sakura) and sectioned (10 µm). Slides were fixed with
-20°C methanol for 15 minutes and blocked-permeabilized in 10% FBS, 0.3%
Surfact-AmpsX100 (Pierce) and 5% non-fat milk in PBS for 90 minutes at
4°C. Samples were stained with rat anti-PECAM (Pharmingen) at 1:50 in 10%
FBS, 5% non-fat milk in PBS overnight and HRP-conjugated rabbit anti-rat
antibody (Dako) at 1:100 for 90 minutes and developed with Cy3-coupled
tyramide (PerkinElmer). Sections were mounted in Vectashield medium with
4'6-diamidino-2-phenylindole (DAPI) (Vector).
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) analysis was performed as described
previously (Aguilera, 2004). In
brief, crosslinked chromatin from 32D cells or whole E9.5 embryos was sheared
by sonication with a UP50H Ultrasonic Processor (2 minutes, four times),
incubated overnight with anti-N1 antibody (sc-6014) or
-N1
(Huppert et al., 2000
) and
precipitated with protein G/A-Sepharose. Cross-linkage of the co-precipitated
DNA-protein complexes was reversed, and DNA was used as a template for
semiquantitative PCR to detect the mouse Gata2IG (from
-435 to -326), Hes1 (from -175 to +13), ß-globin (from +125 to
+309) promoters. PCR primers will be given under request.
Whole-mount in-situ hybridization
Whole-mount in-situ hybridization (WISH) was performed according to
standard protocols (de la Pompa et al.,
1997). For histological analysis, embryos were fixed overnight at
4°C in 4% paraformaldehyde, dehydrated and embedded in Paraplast (Sigma).
Embryos were sectioned in a Leica-RM2135 at 7 µm.
Double in-situ hybridization
Wild-type embryos (E10.5) were frozen in OCT and sectioned (10 µm).
Sections were fixed in 4% paraformaldehyde for 10 minutes, digested with 1
µg/ml proteinase K (Roche) in 50 mmol/l TrisHCl pH 7.5, 5 mmol/l EDTA
buffer and permeabilized with 1% Surfact-Amps X100 (Pierce) in PBS. After
incubation with 3% H2O2 (Sigma) in PBS, slides were
prehybridized for 1 hour and hybridized overnight at 70°C with
fluorescein-tagged or digoxigenin-tagged probes. Anti-fluorescein and
anti-digoxigenin-POD antibodies (Roche) were used at 1:1000 in Blocking
reagent (Roche). Slides were developed using the tyramide amplification
system, TSA-Plus Cyanine3/Fluorescein System (PerkinElmer) and mounted in
glycerol:water.
Image acquisition
Images were acquired with an Olympus BX-60 for embryonic sections and with
a Leica MZ125 for whole embryos using a Spot camera and Spot3.2.4 software
(Diagnostic Instruments). Images for liquid cultures were acquired with an
Olympus IX-70 using a video camera and Image-Pro-Plus4.5.1 software. Adobe
Photoshop 6.0 software was used for photograph editing.
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Results |
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The Notch pathway is activated in the P-Sp/AGM aorta
To confirm that the Notch pathway is activated in the P-Sp/AGM aorta, we
next determined the expression of different Notch-target genes such as
Hes1 and Hes-related protein 1 and 2 (Hrt1 and
Hrt2). Consistent with previous reports, Hrt1 and
Hrt2 are expressed in endothelial cells of the aorta
(Nakagawa et al., 2000),
although their expression patterns are not completely homogenous, showing a
preferential ventral staining in the AGM region at E9.5 and 10.5
(Fig. 1D). We could not detect
Hes1 expression in E9.5 aorta, whereas a strong upregulation was
observed in few ventral cells and in hematopoietic clusters arising from the
endothelium at E10.5 (Fig. 1D).
Thus, different Notch-target genes display specific temporal and spatial
expression patterns in the aorta, suggesting that they could be playing
different roles in early hematopoietic/endothelial decisions.
RBPj mutant embryos display an aberrant expression of Notch receptors and ligands in the P-Sp/AGM region
There is strong evidence from a variety of systems that Notch signaling
participates in the transcriptional regulation of several Notch receptors and
ligands by positive (Barrantes et al.,
1999; Timmerman et al.,
2004
) or negative (Chitnis,
1995
; de la Pompa et al.,
1997
; Heitzler et al.,
1996
) feedback mechanisms. Since most of these regulatory networks
depend on the RBPj
transcription factor
(Heitzler et al., 1996
;
Timmerman et al., 2004
), we
investigated whether the expression of the different Notch family members is
affected in the aorta of RBPj
mutant embryos
(Oka et al., 1995
). We first
compared the expression by semi-quantitative RT-PCR of Notch receptors and
ligands in the dissected P-Sp/AGM region from wild-type and mutant embryos at
E9.5. We consistently observed a decrease in the expression of Notch1
in the RBPj
mutant embryos compared with the wild type, while
we did not detect important changes in the level of expression of
Notch4 or the different Notch ligands
(Fig. 2A).
|
Intra-embryonic hematopoiesis is impaired in the RBPj mutant embryos
To investigate whether Notch/RBPj signaling plays a role in
hematopoietic determination in the aorta, we next assayed the hematopoietic
activity contained in the P-Sp/AGM region of RBPj
mutant
embryos compared with wild type. Despite the presence of several developmental
abnormalities and disorganized vasculature, the majority of the RBPj
mutant embryos (more than 80%) display a regular fused aorta in the trunkal
region at E9.5 (Oka et al.,
1995
). As RBPj
mutants die at E10, we performed
direct hematopoietic colony assays with cells obtained from P-Sp/AGM at E9.5.
Hematopoietic colony forming cells (CFCs) of the different lineages were
generated in cell cultures from wild-type embryos whereas few rare colonies
were obtained from the cultures from RBPj
mutant littermates
in the same conditions (Fig.
2C). We speculated that RBPj
mutant embryos
contained lower numbers of HSC that may be undetectable in the direct CFC
cultures. To test this possibility, we expanded the number of progenitors by
incubating cells from single wild-type P-Sp/AGM compared with pools of two or
three mutant P-Sp/AGM in liquid cultures with cytokines for 6 days. As shown
in Fig. 2D, liquid cultures
from both wild-type and mutant embryos formed equivalent stromal cell layers
after 6 days, although only wild-type cultures contained non-adherent,
round-shaped, hematopoietic-like cells
(Fig. 2D). By flow cytometry,
we demonstrated that liquid cell cultures from wild-type embryos contained
30-50% of CD45+ cells (Fig. 2E) that corresponded to the non-adherent population (data not shown). In
agreement with the absence of hematopoietic-like cells, this CD45+ population
was not detected in the mutant cultures
(Fig. 2E). Cells from wild-type
cultures generated CFCs with a predominant granulo-monocytic morphology,
although colonies from other lineages were also observed
(Fig. 2F). By contrast, we did
not observe any hematopoietic colonies from the RBPj
mutant
cultures (Fig. 2F). These
results indicate that Notch signaling through RBPj
is required
for the generation of the hematopoietic progenitors in the P-Sp/AGM.
Absence of hematopoietic cells and increase of endothelial cells in the P-Sp/AGM of RBPj mutant embryos
Difficulties in characterizing HSCs in the P-Sp/AGM endothelium reside in
the lack of specific HSC markers. In fact, endothelial markers were expressed
in all the cells in the P-Sp/AGM endothelium, including the cells that would
generate the HSCs. Thus, specific hematopoietic transcription factors such as
Aml1, Gata2 and Scl are widely used to identify these endothelial-like cells
that will generate the hematopoietic clusters
(Minegishi et al., 1999;
North et al., 1999
). These
hematopoietic markers are expressed in individual rare cells in the floor of
the dorsal aorta of the AGM region (North
et al., 2002
; Porcher et al.,
1996
; Tsai and Orkin,
1997
) (Fig. 3B). To
better understand the mechanisms by which definitive hematopoiesis is
abrogated in RBPJ
mutant embryos, we studied the expression of
these genes together with endothelial genes in the P-Sp/AGM region in
wild-type and mutant E9.5 embryos. RT-PCR showed reduced expression of the
hematopoietic transcription factors Aml1, Gata2 and Scl but
higher expression of the classical endothelial marker VE-cadherin
(VE-C) in dissected P-Sp/AGM regions of RBPj
mutants,
compared with wild-type embryos (Fig.
3A). Next, we investigated the expression of these transcription
factors specifically in the endothelium of the aorta using WISH. We observed
few cells expressing Aml1, Gata2 and Scl, mainly localized
in the ventral wall of the dorsal aorta in wild-type embryos as expected,
whereas no expression was detected in the aorta endothelium of
RBPj
mutant embryos (Fig.
3B). These results are consistent with the lack of hematopoietic
precursors in these mutants (Fig.
4). In addition, we detected expression of VE-C gene in a
multiple-layered endothelium in some regions of the aorta in the
RBPj
mutant embryos (Fig.
3C). The endothelial nature of these cells was confirmed by
PECAM/CD31 immunofluorescence staining. By contrast, in wild-type embryos
VE-C/PECAM-expressing cells were restricted to a one-cell layer in
the aorta (Fig. 3C). In
addition, we detected a moderate increased percentage of PECAM/CD31-positive
cells by flow cytometry in the mutant embryos (data not shown). These
observations may reflect that the impairment of hematopoietic determination in
the aorta results in an increase in the endothelial lineage.
|
|
Notch1+ Gata2+ cells in the P-Sp/AGM endothelium are Jag1+Jag2-
Different expression levels of Notch receptors and ligands dictate the
specification of different cell lineages (for a review, see
Lai, 2004). To investigate the
specific ligands that activate Notch1 in the presumptive hematopoietic cells
in the aorta, we performed double in-situ hybridizations. It is well
established that the ligands responsible for activating Notch1 are expressed
in cells adjacent to the Notch expressing one. We analyzed transverse sections
of E10.5 embryos simultaneously hybridized with specific probes for
Notch1 and the different ligands that are expressed in the aorta
endothelium at this developmental stage. We consistently observed that cells
expressing Notch1 (Notch1+) also expressed
Jag1 (Fig. 5A, upper
panels), whereas Jag2 was specifically detected in cells adjacent to
Notch1+ but not in the Notch1+
themselves (Fig. 5A, middle
panels). Dll4 showed a mixed pattern of co-expression with
Notch1, in which some cells simultaneously expressed both
Notch1 and Dll4 and other cells only expressed one of these
genes (Fig. 5A, lower panels).
Altogether, these results are consistent with a model in which Jag2 or Dll4
activate Notch1 in the ventral wall of the aorta. This event would initiate
the hematopoietic program in the Notch1+ cells by
activating the expression of Gata2
(Fig. 5B). However, and
considering that multiple ligands are simultaneously expressed in the
endothelium of the aorta, it is tempting to speculate that back and forward
signals between different members may occur.
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Discussion |
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RBPj-dependent Notch function in the generation of intra-embryonic hematopoiesis
The origin of definitive HSCs from an endothelial/hematopoietic common
progenitor known as hemangioblast is still controversial. While the yolk sac
is a primary site of hematopoietic development, several lines of evidence
support the idea that, under physiological conditions, HSCs are generated de
novo within the endothelium lining the ventral wall of the aorta of the
P-Sp/AGM region (Cai et al.,
2000; de Bruijn et al.,
2002
). Our work demonstrates that intra-embryonic hematopoiesis is
abolished in the RBPj
mutant embryos, presumably due to
impaired hematopoietic progenitor determination from endothelial-like
precursors in the aorta. This correlates with the absence of expression of
hematopoietic transcription factors in this region in the mutant embryos
compared with wild type. Furthermore, expression of classical endothelial
markers, such as VE-cadherin and PECAM, is increased in the embryonic aortas
of these mutants, suggesting that in the absence of Notch signaling, the
endothelial lineage is favored at the expense of the hematopoietic one. While
this work was in progress, it was reported that Notch1-deficient
embryos have impaired intra-embryonic hematopoiesis due to a defect in
hematopoietic determination from endothelial cells
(Kumano et al., 2003
), and
that Notch1-deficient embryonic stem cells cannot contribute to
definitive hematopoiesis in chimeric embryos
(Hadland et al., 2004
). Our
results are in agreement with a role of Notch1 in the onset of definitive
hematopoiesis through a transcriptional activation mechanism dependent on
RBPj
. Although the expression of other hematopoietic genes such as
Scl and Aml1 is severely affected in the
RBPj
mutants, we showed that only Gata2 is a direct
target of Notch1/RBPj
signaling. As Gata2 is required to maintain the
pool of undifferentiated hematopoietic progenitors
(Tsai and Orkin, 1997
), we
speculate and present evidence that the absence of Gata2 in the
RBPj
mutants could be responsible for the lack of
hematopoietic progenitors in these mutants and is likely in the
Notch1 mutants (Kumano et al.,
2003
). In agreement with this, the maintenance of undifferentiated
32D myeloid progenitors by Notch1 has been associated with Gata2
expression (Kumano et al.,
2001
). Our work demonstrates that most of the cells in the aorta
that express Notch1 simultaneously express Gata2. This
result, together with the demonstration by chromatin precipitation assays that
intracellular Notch1 associates with the Gata2 promoter, strongly
suggests that Notch1 may regulate the generation and maintenance of
hematopoietic progenitors by directly activating the expression of
Gata2.
Using in-situ hybridization, we detected high levels of expression of the
Hes1 gene in a few endothelial cells as well as in the hematopoietic
clusters of the aorta, thus suggesting that Notch activation is concomitant
with the formation of these clusters. The function of Hes1 in the maintenance
of HSC has not been studied in vivo; however, several pieces of evidence
confirm that Hes1 is regulating cell differentiation in different
hematopoietic cell types (Kawamata et al.,
2002; Kumano et al.,
2001
). These studies together with our results suggest that Hes1
could be involved in maintaining the immature phenotype of the hematopoietic
precursors budding from the aorta and/or in repressing the expression of
specific endothelial markers in these cells. The detection of other
Notch-target genes, such as Hrt1 and Hrt2 (E9.5), preceding
Hes1 expression confirms that Notch is active at this embryonic
stage. However, the role of these Hes-related proteins in the cellular
specification of the aorta remains to be determined.
Lateral inhibition or lateral induction in P-Sp/AGM hematopoietic determination
During the development of complex multicellular organisms, numerous
cell-cell signaling events are required for proper cell-fate determination.
Two different Notch signaling mechanisms have been proposed: lateral
inhibition and lateral induction (reviewed by
Lewis, 1998). Singling out an
individual cell or group of cells from initially equivalent cells is known as
lateral inhibition, whereas lateral induction implies the adoption of cellular
fates cooperatively. In lateral inhibition Notch activation leads to Delta
downregulation, while in lateral induction activation of Notch leads to Delta
upregulation. A typical example of lateral inhibition mediated by Notch is the
process of neurogenesis in Drosophila
(Artavanis-Tsakonas et al.,
1999
) and vertebrates (Chitnis,
1995
), while lateral induction occurs during wing margin
development in Drosophila (Panin
et al., 1997
), somite formation (reviewed by
Lewis, 1998
) and endocardial
development (Timmerman et al.,
2004
). To define whether the determination of hematopoietic cells
in the mid-gestation aorta is compatible with one of these mechanisms, it is
crucial to know the expression pattern of Notch receptors and ligands at this
stage, as well as the characterization of the aorta hematopoietic potential of
the different mutant embryos. Although Notch family members have been detected
in many adult and embryonic hematopoietic tissues, this is the first time that
E9.5-10.5 P-Sp/AGM aorta endothelium has been studied by single and double
in-situ hybridization and the expression of these genes has been analyzed on
transverse sections through the trunkal region. Our analysis reveals
co-expression of multiple Notch-family members in these cells at this
developmental stage, strongly suggesting that several Notch signals are likely
to be involved in hematopoietic determination. For example, Jag1 is
co-expressed with Notch1 in most of the endothelial cells, while the
Jag2 transcript is absent from these cells and specifically expressed
in the cells neighboring the Notch1+ ones. Moreover,
Jag1 is absent from the endothelium of RBPj
mutant
embryos, strongly suggesting that its expression depends on Notch1 activation
in this tissue.
An important question to be determined is how specific expression patterns
of Notch family members are acquired. For example, the endothelium covering
the aorta outside the AGM region has a very homogenous pattern of
Notch1 or Dll4 expression in the majority of cells (data not
shown), while the scattered expression pattern is restricted to the AGM aorta.
Considering this, it is tempting to speculate that the aorta endothelium
originates as a pool of equivalent Notch- and ligand-expressing cells and
lateral inhibition events will generate a `salt and pepper' expression pattern
that is reminiscent of that described for Drosophila neurogenesis
(Artavanis-Tsakonas and Simpson,
1991). Once Notch1 expression pattern in the P-Sp/AGM
aorta is established, hemogenic endothelial cells have to undergo
determination, proliferation and migration events that may require multiple
local interactions with neighboring cells. Our results are consistent with a
model in which expression of Notch1 in individual cells in the
ventral wall of the aorta leads to the activation of Gata2 that is
crucial for the generation of a pool of definitive HSCs. Loss of
Gata2 expression in the RBPj
-deficient embryos
results in the loss of the HSC pool and in the absence of definitive
hematopoiesis (Fig. 5B). This
model implies that, similarly to the situation in other developmental systems
(de Celis et al., 1991
),
Notch1 acts cell-autonomously in promoting an HSC fate in the P-Sp/AGM aorta
as previously proposed (Kumano et al.,
2003
). Our results support a role for Notch in the maintenance of
a population of stem cells (HSCs) that are critical for the definitive
hematopoiesis in the embryos and are consistent with the finding that
alterations in the Notch function are responsible for leukemias (reviewed by
Radtke and Raj, 2003
). Gaining
insight into the mechanism of Notch action will help to design therapeutical
approaches for the treatment of such complex diseases.
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ACKNOWLEDGMENTS |
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