1 Department of Biochemistry and Biophysics and Cardiovascular Research
Institute, Programs in Developmental Biology, Genetics, and Human Genetics,
University of California San Francisco, 1550 Fourth street, San Francisco, CA
94143, USA
2 Department of Molecular, Cellular, and Developmental Biology, University of
California Los Angeles, Los Angeles, CA 90095, USA
Author for correspondence (e-mail:
didier_stainier{at}biochem.ucsf.edu)
Accepted 14 September 2005
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SUMMARY |
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Key words: Endothelial cell, Migration, Endoderm, VEGF, Angioblast, Zebrafish
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Introduction |
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Several signaling molecules and transcription factor genes have been
implicated in the development of the vertebrate vasculature: vegf, vegfr1,
vegfr2, vegfr3, tie1, tie2, angiopoietin1, angiopoietin2, ephrinB2, ephB4,
scl, fli1, ets1, runx, semaphorins and plexins have been analyzed in
zebrafish, amphibians, birds and mammals, and found to display similar
temporal and spatial expression patterns
(Fouquet et al., 1997;
Liao et al., 1997
;
Gering et al., 1998
;
Liang et al., 1998
;
Liao et al., 1998
;
Lyons et al., 1998
;
Kataoka et al., 2000
;
Lawson et al., 2001
;
Pham et al., 2001
;
Habeck et al., 2002
;
Torres-Vazquez et al., 2004
).
For example, flk1/vegfr2 is strongly expressed in developing
angioblasts/endothelial precursors in zebrafish
(Liao et al., 1997
), and its
level of expression significantly decreases as vessels mature, as observed in
Xenopus, chick and mouse (Quinn
et al., 1993
; Cleaver et al.,
1997
; Eichmann et al.,
1998
).
In addition to exhibiting similar expression patterns, the function of
these genes appears to be conserved in all vertebrates. For example, Vegf and
Hedgehog (Hh) signaling have been shown to be crucial for vascular development
in several vertebrate model systems. Vegf regulates the migration and survival
of endothelial cells. Mice lacking a functional Vegf signal show various
developmental defects, including a reduced number of endothelial cells and a
failure to form a functional vasculature
(Carmeliet et al., 1996;
Ferrara et al., 1996
)
(reviewed by Carmeliet and Storkebaum,
2002
). Similar phenotypes have been reported in Xenopus
and chick embryos with compromised Vegf signaling
(Cleaver and Krieg, 1998
). In
zebrafish, Vegf appears to be crucial for angioblast formation as well as for
the subsequent differentiation into arterial endothelial cells
(Nasevicius and Ekker, 2000
;
Lawson et al., 2002
). Another
example of functional conservation in vascular development is Hh. In zebrafish
embryos, Shh in the notochord appears to regulate vegf expression in
the somites, which in turn regulates vascular development
(Lawson et al., 2002
). In
chick and mice, Hh signaling also appears to be important in vascular
development (Vokes et al.,
2004
).
Anatomically, vascular development in zebrafish proceeds in a fashion
analogous to what is observed in other vertebrates. Studies using
microangiography (Isogai et al.,
2001) or transgenic lines that specifically express green
fluorescent protein (GFP) in endothelial cells
(Motoike et al., 2000
;
Lawson et al., 2002
) have
delineated vascular development in zebrafish. The major axial vessels in
zebrafish, the dorsal aorta (DA) and posterior cardinal vein (PCV), form as
the result of angioblast migration from the lateral plate mesoderm (LPM) and
subsequent coalescence in the midline
(Torres-Vazquez et al., 2003
).
Two waves of angioblast migration have been observed and it has been
hypothesized that the endothelial precursors that migrate during the initial
wave contribute to the DA, while those that migrate during the second wave
contribute to the PCV (Torres-Vazquez et
al., 2003
). This pattern of angioblast migration to the midline
resembles the previously reported process of vasculogenesis in quail embryos
documented with the QH1 antibody
(Pardanaud et al., 1987
).
Secondary vessel formation by angiogenesis follows shortly after the
coalescence of angioblasts at the midline. As previous studies have primarily
focused on vascular development after the onset of circulation (24 hpf), the
mechanisms of angioblast migration and coalescence, as well as subsequent
vascular tube formation, remain largely unexplored.
During early development, angioblasts migrate to the midline in response to
an unidentified attractive signal. In Xenopus embryos, the endoderm
appears to be required for proper vascular tube formation
(Vokes and Krieg, 2002), as
surgical removal of this tissue severely affected vascular tube formation. In
quail and mouse embryos, migrating angioblasts are in close contact with the
endoderm, indicating that the endoderm is required for vascular development
(Vokes and Krieg, 2002
;
Vokes et al., 2004
). However,
it is not clear from these studies how the endoderm affects vascular tube or
lumen formation and whether it has a direct or indirect role in these
processes.
In this report, we identify distinct steps of early vascular development at
single cell resolution. We examine how angioblasts migrate from the LPM to the
midline, how these cells coalesce to form a vascular cord, and how this
transient structure is stabilized by cell-cell junctions and later lumenized.
In addition, we analyze the role of Vegf signaling in these processes. We find
that downregulation of Vegf signaling, while affecting the number of
angioblasts does not appear to affect their migratory behavior. In order to
further understand the function of the endoderm in vascular development, we
analyzed the migratory path of angioblasts relative to the endoderm in
wild-type embryos. Furthermore, we delineated vascular development in
casanova (cas) and bonnie and clyde (bon)
mutant embryos, which show either a complete absence (cas) or strong
reduction (bon) of the endoderm
(Alexander et al., 1999;
Kikuchi et al., 2000
). To our
surprise, these mutant embryos formed wild-type like vascular tubes,
suggesting that the endoderm plays a more limited role in vascular tube and
lumen formation than previously thought.
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Materials and methods |
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Approximately 6.5 kb of upstream sequence of the zebrafish flk1
gene (Liao et al., 1997;
Thompson et al., 1998
) was
amplified from wild-type genomic DNA based on the sequence information
previously deposited (from 6410 to 3 of the transcriptional
start site, GenBank Accession Number AY045466)
(Chan et al., 2002
), and
subcloned into the pCRII TOPO vector (Invitrogen). This fragment has been
shown to drive endothelial-specific expression of the green-red coral
fluorescent protein (G-RCFP) (Cross et
al., 2003
). The EcoRI cleaved fragment from this
construct was placed 5' upstream of the enhanced green fluorescent
protein (EGFP) vector (Clontech) to drive endothelial specific expression of
EGFP (Fig. 1A). Approximately
300 pg of this linearized construct was microinjected into one-cell stage
embryos to generate transgenic lines as previously described
(Motoike et al., 2000
).
Embryos were screened for transient expression of EGFP under a Leica
epifluorescence microscope. Embryos showing specific expression of EGFP in the
vasculature were raised to adulthood and crossed to identify founder fish with
germline integration, from which stable transgenic lines were established.
Heterozygous carriers of the cas
(Alexander and Stainier, 1999)
or bon mutations (Stainier et
al., 1996
) were crossed to Tg(flk1:EGFP)s843
zebrafish to generate Tg(flk1:EGFP)s843/+; cas/+ double
heterozygotes and Tg(flk1:EGFP)s843/+; bon/+ double
heterozygotes, respectively. Homozygous embryos were obtained by incrossing
these fish. Heterozygous carriers of
Tg(her5:GFP)ne2067 zebrafish
(Tallafuss and Bally-Cuif,
2003
) were crossed to Tg(flk1:EGFP)s843
zebrafish to generate Tg(flk1:EGFP)s843;
Tg(her5:GFP)ne2067 embryos.
Microinjections and chemical treatment
Microinjections of Morpholinos (MO) were performed as previously described
(Nasevicius and Ekker, 2000;
Horne-Badovinac et al., 2001
).
The sequence of the vegf MO used is
5'-CTCGTCTTATTTCCGTGACTGTTTT3'
(Ober et al., 2004
;
Parker et al., 2004
), and the
sequence of the plcg1 MO is
5'-ATTAGCATAGGGAACTTACTTTCG-3'
(Lawson et al., 2003
).
One-cell or two-cell stage embryos were injected with 4 ng of either MO and
raised at 28°C until harvested.
Embryos were treated with 1.5 µM of the Vegfr antagonist SU5416
(Calbiochem) as previously described (Fong
et al., 1999; Serbedzija et
al., 1999
). As a control, embryos from the same batch were treated
with 1% DMSO. Embryos were treated from 6 hpf until they were harvested for
fixation.
Immunohistochemistry and in situ hybridization
Immunohistochemistry was performed as previously described
(Trinh and Stainier, 2004).
Briefly, embryos were fixed overnight with 2% paraformaldehyde and embedded in
4% NuSieve GTG low melting agarose. Embedded embryos were cut with a VT1000S
vibratome (Leica) into 250 µm sections. Sections were processed in PBDT [1%
BSA, 1% DMSO, and 0.1% Triton X-100 in PBS (pH 7.3)]. The following antibodies
were used at the following dilutions: rabbit polyclonal anti-Fibronectin
(Sigma) at 1:200 (Trinh and Stainier,
2004
), mouse IgG anti-ß-catenin (Sigma) at 1:100
(Horne-Badovinac et al.,
2003
), mouse IgG anti-Zona Occludin-1 (BD Transduction Laboratory)
(Horne-Badovinac et al., 2003
)
at 1:200, goat anti-EphrinB2 (R&D System) at 1:100, and mouse IgG
anti-Claudin5 (Zymed) at 1:100. Filamentous actin was visualized with
rhodamine phalloidin (Molecular Probes) at 1:100
(Trinh and Stainier, 2004
).
Nuclei were visualized with TOPRO (Molecular Probes) at 1:10000
(Oomman et al., 2004
).
Processed samples were mounted in Vectashield (Vector Laboratories) and the
images were acquired using a Zeiss LSM5 Pascal confocal microscope.
Whole-mount in situ hybridization was performed as previously described
(Alexander and Stainier, 1999).
Riboprobes for flk1 (Liao et al.,
1997
), ephrinB2a (Chan
et al., 2001
; Lawson et al.,
2001
), VE-cadherin
(Larson et al., 2004
), and
flt4 (Thompson et al.,
1998
) were prepared with Ambion mMessage Machine. Embryos were
mounted in benzylbenzoate:benzyl alcohol and documented with a Zeiss
Axiocam.
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Results |
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The GFP expression pattern in Tg(flk1:EGFP)s843 appears
to be specific to the vasculature and to faithfully recapitulate the
endogenous expression of flk1
(Liao et al., 1997).
Furthermore, analyzing GFP expression in these embryos allows higher
resolution analyses than previously used methods, such as detecting alkaline
phosphatase activity (Childs et al.,
2002
; Parker et al.,
2004
) (Fig. 1D-K).
The expression of GFP in the endothelial precursors can be detected as early
as 12 hpf (data not shown). At this stage, GFP-positive cells are scattered
within the LPM. At 14 hpf, GFP expression, like flk1 expression, is
detected in bilateral stripes along the anteroposterior axis of the embryos,
and these stripes merge at the midline at approximately 17 hpf (see Fig. S1 in
the supplementary material). In addition, similar to the endogenous expression
of flk1 (Lawson et al.,
2001
), the intensity of GFP expression in the arterial endothelial
cells appears stronger than that in the venous endothelial cells. The
expression of GFP persists in the vasculature, even in adult fish, similar to
what is observed in Tg(fli1:EGFP)y1
(Lawson and Weinstein, 2002
)
transgenic and Tg(Tie2:GFP)s849
(Motoike et al., 2000
) lines.
It appears that all endothelial cells express GFP as the expression pattern of
Tg(flk1:EGFP)s843 is indistinguishable from that of
VE-cadherin, a known pan-endothelial cell marker in zebrafish (see
Fig. S2 in the supplementary material)
(Larson et al., 2004
).
|
Formation of the vascular cord by angioblast migration to the midline
Using the Tg(flk1::EGFP)s843 line, we analyzed the
distinct phases of early vascular development in zebrafish, focusing on two
crucial events: the coalescence of angioblasts to the midline and the
lumenization of the vascular tube. Angioblasts first appear within the LPM
around 12 hpf (data not shown). These cells then migrate as individual cells
to the midline directly on top of the endodermal layer
(Fig. 2). It appears that there
are at least two distinct waves of angioblast migration during vascular
development. The initial wave of migration begins at 14 hpf, while the
second wave begins at
16 hpf (Fig.
3A,B). Upon completion of the initial wave of migration,
angioblasts aggregate at the midline to form a cord-like structure, which is
situated directly dorsal to the endoderm and ventral to the hypochord
(Fig. 3C). We name this
cellular aggregate the vascular cord. Although cells in the vascular cord are
located in close proximity to one another, they do not appear to form any
detectable cell-cell junctions, suggesting that the vascular cord is initially
a simple cluster of angioblasts, rather than an organized structure. Within
this aggregate, fibronectin is deposited between the angioblasts
(Fig. 3C), further supporting
the idea that these cells have not yet formed a cohesive structure at this
stage. However, within the next 2 hours, cell-cell junctions appear (see
below) and, subsequently, a lumen is formed.
|
|
Vascular tube formation and angioblast differentiation
Cell-cell junctions between angioblasts first form at 17 hpf.
Immunostaining with anti-zona occluding 1 (ZO1) antibody clearly shows
junctions formed between adjacent angioblasts
(Fig. 4A,B). At the same time,
adherens junctions are present, as indicated by the localized deposition of
ß-catenin, which is heightened at the focal point of cell-cell contacts
(see Fig. S4 in the supplementary material). At 30 hpf, when vascular
development is complete and active circulation is established, ZO1 deposition
can be detected in both the DA and PCV
(Fig. 4C). Although the
expression of ZO1 is pan-endothelial, that of another junctional protein,
claudin 5, appears to be more restricted. The expression of claudin 5 can be
detected
1 hour later than that of ZO1
(Fig. 4D). Whereas ZO1 appears
to mark cell-cell junctions in both arterial and venous endothelial cells,
claudin 5 seems to mark cell-cell junctions only in arterial endothelial cells
(Fig. 4D-F).
|
The number of angioblasts is not critical for their migration to the midline
In order to test the role of Vegf signaling in the migration of angioblasts
to the midline, we investigated this process in embryos with compromised Vegf
signaling. We first blocked the activity of Vegf by injecting morpholino
antisense oligonucleotides (MO) targeting vegf (Nasevicius et al.,
2000) or its downstream effector plcg1 (phospholipase C )
(Lawson et al., 2003
).
|
Despite this reduction in their number, the angioblasts appeared to migrate to the midline at approximately the same time as their counterparts in uninjected embryos, and formed a wild-type like vascular cord. Analyses of both transverse sections (Fig. 6A-F) and whole-mount in situ hybridization with the pan-endothelial marker cdh5 (VE-cadherin) (Fig. 6G-I) indicated that angioblasts in vegf MO- or plcg1 MO-injected embryos behave normally.
To verify the results from the MO experiments, we employed the
pharmacological reagent SU5416, a widely used antagonist of VEGF signaling
(Fong et al., 1999). When
embryos were treated from 6 hpf onwards, SU5416 caused a dramatic reduction in
the number of angioblasts, as was observed in vegf MO- or
plcg1 MO-injected embryos (data not shown). However, despite this
reduction, the angioblasts in SU5416-treated embryos migrated to the midline,
suggesting that the Vegf signal is dispensable for this process (data not
shown).
The endoderm is required for proper temporal regulation of angioblast migration, but not for angioblast differentiation
Several studies have suggested a pivotal role for the endoderm in vascular
development. Surgical removal of the endoderm in Xenopus and avian
embryos resulted in the failure to form functional vascular tubes
(Vokes and Krieg, 2002). In
order to further investigate how the endoderm regulates vascular tube
formation, we analyzed angioblast migration in a previously described
zebrafish mutant, cas, which is completely devoid of endoderm
(Alexander and Stainier, 1999
;
Alexander et al., 1999
).
Angioblasts in Tg(flk1:EGFP)s843;cas mutant embryos
appear to differentiate normally (Fig.
7). Tg(flk1:EGFP)s843;cas mutant embryos and
their wild-type siblings are morphologically indistinguishable until 16 hpf.
However, angioblasts in these embryos show severe migration defects. At this
stage, the angioblasts in cas mutant embryos and their wild-type
siblings begin to migrate to the midline
(Fig. 7A,B, compare with
Fig. 3A,B). Angioblasts in
cas mutant embryos eventually reach the midline at 22 hpf, which is
significantly later than their wild-type counterparts
(Fig. 7C-D, compare with
Fig. 3C,D). Despite this
initial delay in migration, angioblasts in cas mutant embryos
eventually form two distinct axial vessels that are properly lumenized and
coated with fibronectin (Fig.
7E). Although the DA in these mutant embryos is similar to that in
wild-type siblings, the PCV appears to be dilated to various degrees as
observed in many mutants that lack circulation
(Sehnert et al., 2002).
|
|
|
Angioblasts in Tg(flk1:EGFP)s843;cas mutant embryos
undergo proper differentiation: the arterial specific marker, ephrin B2a, is
restricted to the dorsal aorta, as determined by in situ hybridization and
immunostaining (Fig. 8D-F),
while in situ hybridization with the venous specific marker flt4
detects this marker only in the cardinal vein (data not shown). Thus, it
appears that the differentiation of angioblasts into arterial or venous
endothelial fates does not require the endoderm. Similar phenotypes were
observed in cas MO-injected embryos, as well as in bon
mutant embryos, which lack most endodermal cells
(Alexander and Stainier, 1999;
Kikuchi et al., 2000
) (data
not shown). These results suggest that the endothelial phenotype in
cas mutant embryos is not allele specific or cas specific,
but rather reflects the role of the endoderm in vascular development, as
analyzed genetically.
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Discussion |
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In addition to the vasculature, the hindbrain and the pharyngeal region of
the transgenic embryos express GFP. The GFP expression in the pharyngeal
region might be related to the role of Vegfc in endoderm migration and
morphogenesis. It has been reported that embryos with compromised Vegfc
function show a split gut and duplicated liver primodia owing to defects in
endoderm migration (Ober et al.,
2004). As Vegfc can also interact with Vegfr2
(Joukov et al., 1996
;
Ober et al., 2004
), and the
major receptor for Vegfc, Vegfr3/Flt4, is not expressed in the pharyngeal
endoderm, it is plausible that the pharyngeal expression of Flk1 is crucial
for transducing Vegfc signaling during endoderm migration.
Angioblast migration and the factors regulating this process
Angioblasts migrate extensively during development. The absolute number of
angioblasts does not seem to be crucial for this process. Embryos injected
with vegf or plcg1 MOs do not display any obvious defects in
angioblast migration despite a significant reduction in their number. Similar
results were observed in embryos treated with SU5416, a chemical antagonist of
Vegf signaling. These data suggest that Vegf signaling is required for
angioblast formation, but is not necessary to direct angioblasts during their
migration to the midline. Other signals, such as Fgf or Pdgf, might be
responsible for the directionality of migration
(Yang and Moses, 1990;
Sa et al., 1995
;
Thommen et al., 1997
).
Alternatively, repulsive signals such as Semaphorins
(Shoji et al., 2003
;
Torres-Vazquez et al., 2004
)
might play a more significant role in angioblast migration than previously
thought.
Unlike the precardiac mesoderm, which migrates as a polarized
pseudo-epithelium, angioblasts appear to migrate as individual cells.
Throughout this migration process, fibronectin deposition is observed on the
ventral side of the angioblasts (data now shown), although it is not clear
whether the fibronectin provides a directional cue or is merely a substratum
for migration. Two fibronectin genes are present in the zebrafish genome
(Trinh and Stainier, 2004;
Sun et al., 2005
), and they
may have partially overlapping function in angioblast migration.
Role of endoderm during early vascular development
Our study shows that the endoderm is required for the temporal regulation
of angioblast migration. Although angioblasts in endodermless embryos start
their migration to the midline at the same time as those in wild-type embryos,
they reach the midline much later than their wild-type counterparts.
Interestingly, the angioblasts in the endodermless mutant embryos display a
distinct behavior that is not observed in wild-type embryos. They appear to
aggregate during their migration to the midline and form ectopic cell-cell
junctions with each other. These angioblasts then migrate as groups and
maintain their cell-cell junctions during this process. It is possible that
the endoderm somehow prevents cell-cell contact between migrating angioblasts,
and thereby promotes their migration as individual cells. Once the angioblasts
in endodermless embryos reach the midline, they form a single aggregate as
observed in wild-type embryos. It is not clear how the cell-cell junctions
between endothelial precursors are redistributed once they reach the
midline.
The mechanism by which the endoderm regulates angioblast migration is also unclear. It is possible that the endoderm provides an unidentified attractant for angioblasts. However, it appears to be an undifferentiated homogenous cell layer at this stage. A more likely scenario may be that the endoderm functions as a substratum for the angioblasts to migrate upon, and prohibits them from aggregating.
Several studies have indicated that the endoderm is required for proper
vascular tubulogenesis (Palis et al.,
1995; Bielinska et al.,
1996
; Vokes and Krieg,
2002
). However, we found no evidence for a pivotal role of the
endoderm in vascular tube formation or angioblast differentiation in
zebrafish. Although the angioblasts in endodermless embryos show a distinct
phenotype and a delay during migration, they form comparatively normal
vascular tubes. Two distinct vascular tubes with proper junctional complexes
are formed. Furthermore, the expression patterns of the arterial
endothelial-specific marker ephrin B2a and the venous endothelial specific
marker flt4 (Lawson et al.,
2001
) appear to be unaltered in endodermless embryos, suggesting
that the endoderm is not required for angioblast differentiation into arterial
and venous endothelial cells.
The PCV in endodermless embryos appears to be dilated at later stages. We
observed a severe accumulation of blood cells in these dilated vessels that
might in fact be responsible for the dilation as many other zebrafish
mutations that affect circulation exhibit a similar phenotype
(Sehnert et al., 2002;
Lawson et al., 2003
). We
believe that the defects in cas and bon mutant embryos
reflect the function of the endoderm during vascular development, rather than
previously uncharacterized functions of these genes in the lateral plate
mesoderm or angioblasts. Previous studies have indeed shown that the
cas mutation affects the endoderm specific HMG transcription factor
Sox32 (Kikuchi et al.,
2001
).
Why is the endoderm dispensable for vascular tube formation in zebrafish,
unlike what appears to happen in other organisms? It is possible that the fast
development of zebrafish embryos allows us to monitor later stages of
development than previously observed in Xenopus or avian embryos. For
example, we analyzed vascular tube formation in endodermless embryos until 72
hpf. By contrast, previous studies (Vokes
and Krieg, 2002; Vokes et al.,
2004
) analyzed vascular tube formation until stage 34 in
Xenopus embryos, and the eight-somite stage in quail embryos, which
is approximately equivalent to 24 hpf for zebrafish embryos. A similar
temporal delay in endothelial precursor migration and vascular tube formation
as we observed in endodermless zebrafish embryos might occur in
Xenopus and avian embryos, but was misinterpreted because later stage
embryos were not examined. Alternatively, the temporal and spatial expression
patterns of molecular cues regulating angioblast migration might have evolved
differently.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/23/5199/DC1
* Present address: Foundation for Biomedical Research of the Academy of
Athens, Basic Research Center, Soranou Efesiou 4, 115 27 Athens, Greece
Present address: Eyetech Pharmaceutical, Eyetech Research Center, 35
Hartwell Avenue, Lexington, MA 02421, USA
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