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First published online 24 January 2007
doi: 10.1242/dev.003244
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Research Report |
1 Vertebrate Development Laboratory, Cancer Research UK London Research
Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
2 Department of Genetics, Box 8232, Washington University Medical School, 4566
Scott Avenue, St Louis, MO 63110, USA.
* Author for correspondence (e-mail: jonathan.leslie{at}cancer.org.uk)
Accepted 31 December 2007
SUMMARY
Notch signalling by the ligand Delta-like 4 (Dll4) is essential for normal vascular remodelling, yet the precise way in which the pathway influences the behaviour of endothelial cells remains a mystery. Using the embryonic zebrafish, we show that, when Dll4-Notch signalling is defective, endothelial cells continue to migrate and proliferate when they should normally stop these processes. Artificial overactivation of the Notch pathway has opposite consequences. When vascular endothelial growth factor (Vegf) signalling and Dll4-Notch signalling are both blocked, the endothelial cells remain quiescent. Thus, Dll4-Notch signalling acts as an angiogenic `off' switch by making endothelial cells unresponsive to Vegf.
Key words: Notch, Delta-like 4, Angiogenesis, Endothelial, Motility, Zebrafish
INTRODUCTION
Both during development and in adult life, blood vessels undergo periods of
sprouting and growth, as well as periods of quiescence. During these quiescent
periods, the vascular bed remains static while blood circulates through it,
whereas, during growth, new vessels are generated by the proliferation and
migration of endothelial cells, which form sprouts from the sides of existing
vessels under the influence of vascular endothelial growth factor (VEGF) and
other signals (Carmeliet and
Tessier-Lavigne, 2005
; Coultas
et al., 2005; Goishi and
Klagsbrun, 2004; Weinstein,
2002). To create a vascular network with the right density of
branches, this motile behaviour must be strictly controlled. What, then, is
the mechanism that switches endothelial cells from motile to non-motile
behaviour at the appropriate times and places?
Many previous studies have shown that the disruption of Notch signalling
gives rise to abnormal patterns of blood vessels
(Krebs et al., 2000;
Shawber and Kitajewski, 2004;
Uyttendaele et al., 2001), and
that the Notch ligand Delta-like 4 (Dll4), in particular, is essential for
normal vascular development. Mice homozygous for a knockout mutation of Dll4
die early, at around E10, and heterozygotes show severe vascular defects
(Duarte et al., 2004;
Gale et al., 2004;
Krebs et al., 2004). However,
what type of disrupted behaviour by individual endothelial cells is
responsible for these vascular abnormalities has remained a puzzle. Some clues
have come from studies in culture, where the blockade of Notch signalling
favours vessel branching and endothelial cell proliferation
(Liu et al., 2006;
Sainson et al., 2005;
Shawber et al., 2003).
However, other evidence, obtained from tumour angiogenesis, suggests that
Notch signalling may have an opposite effect and that, by targeting Dll4, one
might be able to deprive tumours of their blood supply and stop their growth
(Patel et al., 2005;
Zeng et al., 2005).
The zebrafish embryo provides a unique opportunity to observe the behaviour
of endothelial cells in the living organism
(Weinstein, 2002). In this
study, this model is used to show that Notch signalling governs the transition
from active angiogenesis to quiescence. The Notch ligand Dll4 is expressed in
arterial endothelial cells, which are responsible for primary angiogenesis. In
the normal course of development, a subset of these cells becomes highly
motile and forms angiogenic sprouts, which eventually join up to create
vascular circuits (Childs et al.,
2002). Once the connections have been made, the endothelial cells
halt their motility. In embryos in which either Dll4 is lacking or Notch
signalling is blocked, angiogenic sprouting begins normally. Later, however,
as the sprouts join up to form vascular circuits, the cells in the defective
embryos continue to proliferate and migrate. Artificial overactivation of the
Notch pathway has opposite consequences. When Vegf signalling and Dll4-Notch
signalling are both blocked, the excessive sprouting is prevented. Thus, while
endothelial cells depend on Vegf from the environment to switch on angiogenic
behaviour, they depend on Dll4-Notch signals exchanged with other endothelial
cells to switch it off; and this `off' switch works by making the cells
unresponsive to Vegf.
MATERIALS AND METHODS
Animals
Zebrafish were raised and maintained under standard conditions.
Gene sequence and in situ hybridization
The zebrafish ortholog of mammalian Dll4 (dll4, Ensembl
Gene ENSDARG00000053401) has recently come to light through the zebrafish
genome sequencing project. Whole-mount in situ hybridization (ISH) was carried
out as previously described
(Ariza-McNaughton and Krumlauf,
2002). The DIG-labelled antisense probe for dll4
corresponded to amino acids 73-232. Probes for notch1b
(Westin and Lardelli, 1997),
notch3 (Westin and Lardelli,
1997), efnb2a (Durbin
et al., 1998) and ephb4a
(Cooke et al., 1997) were as
previously described. After ISH, embryos were processed for histology either
as whole mounts or as cryostat sections and were immunostained with anti-GFP
antibody (Molecular Probes).
Morpholino injection and heat-shock constructs
Morpholino antisense oligonucleotides (Gene Tools) were diluted in Danieau
buffer containing 0.2% phenol red. This solution (2 nl, which contained 5-10
ng oligonucleotide) was then injected into the yolk of 2- to 4-cell-stage
embryos.
Morpholino sequences were:
for dll4 splicing (MO[Dll4]), 5'-TAGGGTTTAGTCTTACCTTGGTCAC-3';
for MO[Dll4] mismatch control (mismatched bases in lowercase), 5'-TAcGGTTTAcTCTTAgCTTcGTgAC-3';
for sox32 (casanova) (MO[cas]),
5'-GCATCCGGTCGAGATACATGCTGTT-3'
(Sakaguchi et al., 2001);
for notch1a, 5'-GAAACGGTTCATAACTCCGCCTCGG-3'
(Lorent et al., 2004);
for notch1b, 5'-AATCTCAAACTGACCTCAAACCGAC-3'
(Milan et al., 2006);
for notch2, 5'-AGGTGAACACTTACTTCATGCCAAA-3'
(Lorent et al., 2004);
and for notch3, 5'-ATATCCAAAGGCTGTAATTCCCCAT-3'
(Lorent et al., 2004).
For the misexpression of NICD, we used fish carrying
hsp70:Gal4 and UAS:NICD
(UAS:myc-notch1a-intra) constructs, as described
(Scheer et al., 2001), and
heat-shocked them at 37°C for 30 minutes.
|
Fluorescence microscopy
fli1:EGFP embryos were fixed in 4% paraformaldehyde in PBS
overnight at 4°C and were then washed in PBS at room temperature. Embryos
were permeabilized in PBS containing 10% goat serum, 2% BSA, 0.5% Triton X-100
and 10 mM NaN3 for 1-3 hours at room temperature, washed in PBS
containing 1% Triton X-100 and then mounted in SlowFade Gold (Invitrogen)
containing 0.2 µg/ml DAPI (Sigma). Specimens were examined with a Zeiss
LSM510 confocal microscope. Endothelial cells were counted from projections of
Z-series of optical sections. The region counted in each specimen spanned two
somites [three intersegmental vessels (ISVs) and the accompanying segments of
the dorsal aorta (DA) and dorsal longitudinal anastomotic vessel (DLAV)].
Live imaging
Living fli1:EGFP embryos were anaesthetized in aquarium water
containing 320 µg/ml Tricaine (Sigma) and were then mounted on a
glass-bottom dish (MatTek) in a drop of aquarium water containing 0.2%
agarose. Images were captured with a Zeiss LSM 510 confocal microscope. Frames
for movies were collected every 30 seconds for 20 minutes. Each frame is the
projection of a Z-series of optical sections.
RESULTS AND DISCUSSION
In the zebrafish, dll4 transcript was detectable by reverse
transcriptase (RT)-PCR analysis by 8 hours post-fertilization (hpf), with
expression levels increasing through to the third day of development
(Fig. 1A). Whole-mount in situ
hybridization showed that all endothelial cells in the dorsal aorta (DA) and
intersegmental vessels (ISV) expressed dll4, but no dll4
transcript was detectable in the posterior cardinal vein (PCV) or caudal vein
(Fig. 1B-D,F-H). Thus,
zebrafish dll4, like Dll4 in the mouse
(Duarte et al., 2004;
Gale et al., 2004;
Mailhos et al., 2001;
Rao et al., 2000;
Shutter et al., 2000), is
expressed in arterial, but not venous, endothelial cells, as is another member
of the delta gene family, deltaC (dlc)
(Lawson et al., 2001;
Smithers et al., 2000).
dll4 was also detectable in a small subset of cells within the neural
tube (Fig. 1B,D) and in the two
developing sensory patches of the ear (data not shown). Endothelial expression
of dll4, like that of dlc, was lost in mind bomb
mutants (Fig. 1E), in which
arterial cells are partially converted to a venous character as a result of a
failure of Notch signalling during the initial determination of endothelial
lineages (Lawson et al.,
2001).
To block the production of Dll4, we designed a splice-blocking morpholino antisense oligonucleotide, MO[Dll4] (see Fig. 1 in the supplementary material). We injected MO[Dll4] into fertilized eggs and followed vascular development in the morphant embryos up to 5 days post-fertilization (dpf). In normal zebrafish, blood flow in the trunk and head was readily observable by 30 hpf, and by 2.5 dpf the DA, PCV and ISVs carried robust circulation (Fig. 2A). However, in MO[Dll4]-injected embryos at this age, the flow of blood was severely reduced: circulation in the DA and PCV was slowed and blood flow was undetectable in the majority of ISVs (Fig. 2B). Embryos injected with a control morpholino generally had normal circulation (Fig. 2C).
|
;
Krebs et al., 2004;
Lawson et al., 2001;
Lawson et al., 2002;
Lawson and Weinstein, 2002a),
we examined the expression of genes that mark differences between arterial and
venous endothelial cells: notch3, efnb2a and ephb4a were all
unaffected in the MO[Dll4] morphants (data not shown). Because dlc is
also found in the embryonic vasculature
(Lawson et al., 2001;
Smithers et al., 2000) and
could function redundantly with dll4, we repeated the Dll4-knockdown
experiment in dlcbea/bea mutants, in which Dlc is
defective. Again, we found no effect on vessel identity (data not shown). This
suggests that some Notch ligand other than Dll4 or Dlc is responsible for the
control of vessel identity, although it is also possible that our morpholino
allowed some residual dll4 expression, which was sufficient to
maintain arterial identity even though other aspects of endothelial behaviour
were abnormal.
To clarify the reasons for the circulatory defects, we examined in detail
the development and maintenance of the pattern of ISVs using
fli1:EGFP zebrafish, in which all endothelial cells are labelled with
a cytosolic EGFP reporter (Lawson and
Weinstein, 2002b). In normal embryos, the ISVs originate at somite
boundaries as sprouts from the DA. These sprouts are first seen at 20-24 hpf
and grow dorsally, reaching the level of the top of the neural tube at
approximately 30 hpf. There, they bifurcate to form two branches, which
extend, in a T-shape, along the body axis
(Fig. 3A). These longitudinal
sprouts join up to form the dorsal longitudinal anastomotic vessel (DLAV),
which can be seen carrying blood by 48 hpf. Over the next 3 days of
development, the pattern of connections between the ISVs and the DLAV remain
essentially static.
In the morphant embryos, formation of the ISVs began normally, and the DLAV
was able to form as normal. However, by 2.5 dpf, striking abnormalities were
visible. Instead of a simple T-junction between each ISV and the DLAV, we
observed a network of aberrant interconnected branches
(Fig. 2D,E), which became more
profuse over the following few days (Fig.
2F,G). The abnormality was reminiscent of that seen in the
vasculature of mouse embryos lacking one or both copies of Dll4
(Duarte et al., 2004;
Gale et al., 2004;
Krebs et al., 2004), and
implies a failure of a mechanism that normally arrests the formation of new
vessels. Blocking Vegf signalling with the inhibitor SU5416
(Fong et al., 1999) both
blocked normal endothelial sprouting (data not shown) (see also
Covassin et al., 2006) and the
additional sprouting observed in embryos lacking Dll4
(Fig. 2K). Thus, Dll4-Notch
signalling affects angiogenesis by interfering with a response to Vegf.
|
;
Liu et al., 2006;
Sainson et al., 2005;
Williams et al., 2006).
Further experiments will be needed to establish whether the effect on
endothelial cell numbers in the zebrafish reflects an increase of division
rate or a decrease of cell death.
Because MO[Dll4]-injected embryos have reduced circulation, one might
suggest that the observed vessel-branching phenotype simply reflects a normal
physiological response to hypoxic conditions, to reduced blood flow or to
reduced pressure. To investigate this possibility, we blocked all trunk
circulation using a morpholino (MO[cas]) targeted to the sox32
(casanova, cas) transcript, which is required for the development of
a normal, beating heart (Sakaguchi et al.,
2001). Although MO[cas]-treated embryos showed some dilations of
their vessels, there was no sign of the ectopic sprouts observed in
Dll4-morphant and -mutant embryos (compare
Fig. 2J with
Fig. 2E,H), indicating that the
observed ISV-branching phenotype is indeed a direct effect of Dll4 knockdown
and not a secondary effect resulting from compromised circulation.
We obtained further confirmation of the role of Dll4 in angiogenesis from a dll4 mutant, j16e1. This was discovered through its dominant, haplo-insufficient phenotype in a study of fin-morphology mutants, and subsequent mapping and positional cloning revealed a glycine to serine (G270S) substitution at a conserved site in an EGF-like domain of the zebrafish dll4 gene on LG20. Homozygous embryos survived for up to 2 weeks and showed a vascular phenotype similar to, but less severe than, that seen in Dll4 morphants (Fig. 2H). At 11 dpf, a stage that cannot be studied by morpholino knockdown, mutant embryos showed excessive angiogenesis in other sites in addition to in the ISVs (Fig. 2L). Thus, Dll4-Notch signalling is required to keep angiogenesis under proper control, not only in the embryo, but also at later stages and in multiple tissues.
To discover what kind of disrupted endothelial cell behaviour underlies this aberrant pattern of vessels in Dll4 morphants and mutants, we examined living embryos at high resolution using time-lapse confocal microscopy. During ISV sprouting and migration, we could see the exploratory endothelial cells at the tips of growing ISVs rapidly extending and retracting filopodia; this behaviour was similar in morphants and control embryos (Fig. 3A-C). However, differences became apparent later in development. In normal embryos, the leading cells of adjacent ISVs joined up and formed a DLAV with a lumen, the endothelial cells ceased their exploratory behaviour, and filopodia became few and far between (Fig. 3A,D and see Movie 1 in the supplementary material). By striking contrast, we found that endothelial cells comprising the DLAV and dorsal ISVs of embryos lacking Dll4 continued to extend and retract filopodia, and readily advanced into ectopic locations (Fig. 3E and see Movie 2 in the supplementary material).
Evidently, Dll4 is required for endothelial cells to make the transition from exploratory behaviour to quiescence. This could reflect a cell-autonomous influence of Dll4 on filopodium formation and motility, or could depend on cell-cell signalling via Notch. To shed light on this issue, we blocked Notch activation using the gamma-secretase inhibitor DAPT, starting at 33 hpf (when ISVs begin to connect), and examined the behaviour of endothelial cells at 54 hpf. As in embryos lacking Dll4, endothelial cells in DAPT-treated embryos had greatly increased filopodial activity and exploratory behaviour compared with DMSO-treated control siblings (Fig. 3F,G and see Movies 3,4 in the supplementary material). They also showed increased endothelial cell numbers (Fig. 2I). An increase of filopodial activity was also observed in 54-hpf embryos in which DAPT treatment was initiated at 48 hpf (after the DLAV has formed; Fig. 3H,I).
These findings strongly suggest that Dll4 acts via Notch. To identify the relevant receptor, we tested the effects of morpholinos targeted against each of the four known zebrafish notch genes. Notch1b morphants showed a clear phenotype resembling that of Dll4 morphants, Dll4 mutants and DAPT-treated embryos (Fig. 3J); we were unable to detect any such effect in Notch3 morphants. Thus, it appears that signalling by Dll4 via Notch1b is required to restrict the exploratory behaviour of endothelial cells and to reduce their proliferation once the capillary sprouts have connected to form a vascular circuit.
To test whether ectopic activation of Notch would, conversely, block the
normal exploratory behaviour of endothelial cells, we used a GAL4-UAS
technique to trigger the overproduction of the active intracellular fragment
of Notch (NICD) via heat shock
(Scheer et al., 2001). Heat
shocking during the early stages of ISV formation severely reduced the
filopodial activity of the pioneering endothelial cells and inhibited the
extension of the ISV sprouts (Fig.
3K,L). The strong suggestion, therefore, is that Notch signalling
acts as a switch to control the transition between motility and quiescence in
endothelial cells exposed to Vegf. Previous work has shown that expression of
the vegfr3 gene (VEGF receptor 3, also called flt4)
is required for normal extension of ISVs
(Covassin et al., 2006) and is
downregulated in response to Notch activation
(Lawson et al., 2001). It
seems likely, therefore, that Notch signalling inhibits motility by blocking
the production of the receptors needed for a response to Vegf to occur.
Although studies in mice have shown that the loss of Dll4 produces
a malformation of the vascular bed (Duarte
et al., 2004; Gale et al.,
2004; Krebs et al.,
2004), ours is the first account to show directly in a living
organism how Notch signalling affects endothelial cell behaviour and how this
relates to vascular malformations. We have demonstrated that the removal of
Dll4 results in a persistence of angiogenesis at a time when endothelial cells
should normally make a transition to quiescence. Our findings agree with in
vitro studies demonstrating that Notch signalling suppresses endothelial
proliferation and branching (Leong et al.,
2002; Liu et al.,
2006; Sainson et al.,
2005; Shawber et al.,
2003; Williams et al.,
2006). Given that Dll4 is strongly expressed in
developing vasculature, both in embryos and in tumours
(Claxton and Fruttiger, 2004;
Mailhos et al., 2001;
Rao et al., 2000;
Shutter et al., 2000), it
seems that it forms part of an essential mechanism to keep angiogenesis under
control at sites where the angiogenic program has been activated. Although
Notch in mammalian endothelial cells may also be activated by Notch ligands of
the Jagged family in neighbouring non-endothelial cells, with a stimulatory
effect on angiogenesis (Patel et al.,
2005; Zeng et al.,
2005), our data clearly indicate that, in our system, the more
important signal is from Dll4 expressed in endothelial cells and that its
effect is to inhibit angiogenesis.
Our findings help to show how the functions of VEGF and Notch signalling
are interwoven in the control of angiogenesis. In the continued presence of
VEGF, angiogenesis may proceed or halt according to the level of activation of
the Notch pathway, and this effect appears to be exerted through the
regulation of VEGF-receptor expression by Notch signalling. In mammalian cells
(Liu et al., 2003;
Patel et al., 2005;
Williams et al., 2006), VEGF
has been shown to induce dll4 expression, which, in turn, apparently
by activating Notch, inhibits the expression of VEGF receptors
(Liu et al., 2003;
Patel et al., 2005;
Williams et al., 2006). In
this way, VEGF acting on a given cell, while inducing dll4 in that
cell, will tend to reduce VEGF-receptor expression in its neighbours and
thereby downregulate their expression of dll4, creating a form of
lateral inhibition that could serve to single out a subset of the endothelial
population as motile tip cells
(Hellström et al., 2007).
The same effect could, however, also be achieved more directly by the standard
Notch-dependent lateral-inhibition mechanism: that is, a cell strongly
expressing dll4 will activate Notch in its neighbours, and the
activated Notch could directly inhibit their expression of dll4,
enabling the first cell to escape Notch activation.
In the fish, we have not found evidence of any influence of the Vegf signalling pathway on the expression of dll4: blocking Vegf signalling with SU5416 had no visible effect on dll4 expression (data not shown). Neither have we detected any obvious difference in dll4 expression between the leading cells of the ISV sprouts and the cells that follow behind them. Moreover, both classes of cells normally show intense filopodial activity. When filopodial activity ceases upon formation of the DLAV, it ceases throughout the population of intersegmental endothelial cells. It is tempting to suggest that the trigger is the contact between the leading endothelial cells of the sprouts that meet, which could lead to mutual activation of Notch; however, it remains to be seen how the switch in behaviour of the leading endothelial cells leads to quiescence throughout the cell population - this might be coordinated by signals via the Notch pathway, or by some other means.
However this may be, our data indicate that Dll4-Notch signalling provides
a way to halt endothelial motility and to downregulate endothelial
proliferation in a coordinated way once the endothelial cells have become
linked together into a vascular circuit. Thus, VEGF- and Notch-signalling may
represent two distinct controls of endothelial cell behaviour, with VEGF
signalling [and perhaps Jagged-Notch signalling
(Zeng et al., 2005)] conveying
information from the tissues that require a blood supply, and Dll4-Notch1b
signalling conveying information about the relationship of an endothelial cell
to its neighbours.
Note added in proof
Shortly before submission of this manuscript, two related papers were
published (Noguera-Troise et al.,
2006; Ridgway et al.,
2006).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/839/DC1
ACKNOWLEDGMENTS
We thank Peter Jordan and Mike Mitchell for expert help with time-lapse confocal microscopy and bioinformatics; Brant Weinstein for the gift of fli1:EGFP fish; Arndt Siekmann and Nathan Lawson for sharing unpublished data; Corinne Houart and Roger Patient for Nrp1b and VEGFR2 probes; Ralf Adams, Erik Sahai, the members of the Vertebrate Development Laboratoryand especially Holger Gerhardt for advice and suggestions; and Phil Taylor for fish care. S.L.J. is supported by NIH grant GM056988. This work was funded by Cancer Research UK.
REFERENCES
Ariza-McNaughton, L. and Krumlauf, R. (2002). Non-radioactive in situ hybridization: simplified procedures for use in whole-mounts of mouse and chick embryos. Int. Rev. Neurobiol. 47,239 -250.[Medline]
Carmeliet, P. and Tessier-Lavigne, M. (2005). Common mechanisms of nerve and blood vessel wiring. Nature 436,193 -200.[CrossRef][Medline]
Childs, S., Chen, J. N., Garrity, D. M. and Fishman, M. C. (2002). Patterning of angiogenesis in the zebrafish embryo. Development 129,973 -982.
Claxton, S. and Fruttiger, M. (2004). Periodic Delta-like 4 expression in developing retinal arteries. Gene Expr. Patterns 5,123 -127.[CrossRef][Medline]
Cooke, J. E., Xu, Q. L., Wilson, S. W. and Holder, N. (1997). Characterisation of five novel zebrafish Eph-related receptor tyrosine kinases suggests roles in patterning the neural plate. Dev. Genes Evol. 206,515 -531.[CrossRef]
Coultas, L., Chawengsaksophak, K. and Rossant, J. (2005). Endothelial cells and VEGF in vascular development. Nature 438,937 -945.[CrossRef][Medline]
Covassin, L. D., Villefranc, J. A., Kacergis, M. C., Weinstein,
B. M. and Lawson, N. D. (2006). Distinct genetic interactions
between multiple Vegf receptors are required for development of different
blood vessel types in zebrafish. Proc. Natl. Acad. Sci.
USA 103,6554
-6559.
Duarte, A., Hirashima, M., Benedito, R., Trindade, A., Diniz,
P., Bekman, E., Costa, L., Henrique, D. and Rossant, J.
(2004). Dosage-sensitive requirement for mouse Dll4 in artery
development. Genes Dev.
18,2474
-2478.
Durbin, L., Brennan, C., Shiomi, K., Cooke, J., Barrios, A.,
Shanmugalingam, S., Guthrie, B., Lindberg, R. and Holder, N.
(1998). Eph signaling is required for segmentation and
differentiation of the somites. Genes Dev.
12,3096
-3109.
Fong, T. A., Shawver, L. K., Sun, L., Tang, C., App, H., Powell,
T. J., Kim, Y. H., Schreck, R., Wang, X., Risau, W. et al.
(1999). SU5416 is a potent and selective inhibitor of the
vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine
kinase catalysis, tumor vascularization, and growth of multiple tumor types.
Cancer Res. 59,99
-106.
Gale, N. W., Dominguez, M. G., Noguera, I., Pan, L., Hughes, V.,
Valenzuela, D. M., Murphy, A. J., Adams, N. C., Lin, H. C., Holash, J. et
al. (2004). Haploinsufficiency of delta-like 4 ligand results
in embryonic lethality due to major defects in arterial and vascular
development. Proc. Natl. Acad. Sci. USA
101,15949
-15954.
Goishi, K. and Klagsbrun, M. (2004). Vascular endothelial growth factor and its receptors in embryonic zebrafish blood vessel development. Curr. Top. Dev. Biol. 62,127 -152.[Medline]
Hellström, M., Phng, L., Hofmann, J. J., Wallgard, E., Coultas, L., Lindblom, P., Alva, J., Nilsson, A., Karlsson, L., Gaiano, N. et al. (2007). Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature (in press).
Krebs, L. T., Xue, Y., Norton, C. R., Shutter, J. R., Maguire,
M., Sundberg, J. P., Gallahan, D., Closson, V., Kitajewski, J., Callahan, R.
et al. (2000). Notch signaling is essential for vascular
morphogenesis in mice. Genes Dev.
14,1343
-1352.
Krebs, L. T., Shutter, J. R., Tanigaki, K., Honjo, T., Stark, K.
L. and Gridley, T. (2004). Haploinsufficient lethality and
formation of arteriovenous malformations in Notch pathway mutants.
Genes Dev. 18,2469
-2473.
Lawson, N. D. and Weinstein, B. M. (2002a). Arteries and veins: making a difference with zebrafish. Nat. Rev. Genet. 3,674 -682.[CrossRef][Medline]
Lawson, N. D. and Weinstein, B. M. (2002b). In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248,307 -318.[CrossRef][Medline]
Lawson, N. D., Scheer, N., Pham, V. N., Kim, C. H., Chitnis, A.
B., Campos- Ortega, J. A. and Weinstein, B. M. (2001).
Notch signaling is required for arterial-venous differentiation during
embryonic vascular development. Development
128,3675
-3683.
Lawson, N. D., Vogel, A. M. and Weinstein, B. M. (2002). sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev. Cell 3,127 -136.[CrossRef][Medline]
Leong, K. G., Hu, X., Li, L., Noseda, M., Larrivee, B., Hull,
C., Hood, L., Wong, F. and Karsan, A. (2002). Activated
Notch4 inhibits angiogenesis: role of beta 1-integrin activation.
Mol. Cell. Biol. 22,2830
-2841.
Liu, Z. J., Shirakawa, T., Li, Y., Soma, A., Oka, M., Dotto, G.
P., Fairman, R. M., Velazquez, O. C. and Herlyn, M. (2003).
Regulation of Notch1 and Dll4 by vascular endothelial growth factor in
arterial endothelial cells: implications for modulating arteriogenesis and
angiogenesis. Mol. Cell. Biol.
23, 14-25.
Liu, Z. J., Xiao, M., Balint, K., Soma, A., Pinnix, C. C.,
Capobianco, A. J., Velazquez, O. C. and Herlyn, M. (2006).
Inhibition of endothelial cell proliferation by Notch1 signaling is mediated
by repressing MAPK and PI3K/Akt pathways and requires MAML1. FASEB
J. 20,1009
-1011.
Lorent, K., Yeo, S. Y., Oda, T., Chandrasekharappa, S., Chitnis,
A., Matthews, R. P. and Pack, M. (2004). Inhibition of
Jagged-mediated Notch signaling disrupts zebrafish biliary development and
generates multi-organ defects compatible with an Alagille syndrome phenocopy.
Development 131,5753
-5766.
Mailhos, C., Modlich, U., Lewis, J., Harris, A., Bicknell, R. and Ish-Horowicz, D. (2001). Delta4, an endothelial specific Notch ligand expressed at sites of physiological and tumor angiogenesis. Differentiation 69,135 -144.[CrossRef][Medline]
Milan, D. J., Giokas, A. C., Serluca, F. C., Peterson, R. T. and
MacRae, C. A. (2006). Notch1b and neuregulin are required for
specification of central cardiac conduction tissue.
Development 133,1125
-1132.
Noguera-Troise, I., Daly, C., Papadopoulos, N. J., Coetzee, S., Boland, P., Gale, N. W., Lin, H. C., Yancopoulos, G. D. and Thurston, G. (2006). Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Nature 444,1032 -1037.[CrossRef][Medline]
Patel, N. S., Li, J. L., Generali, D., Poulsom, R., Cranston, D.
W. and Harris, A. L. (2005). Up-regulation of delta-like 4
ligand in human tumor vasculature and the role of basal expression in
endothelial cell function. Cancer Res.
65,8690
-8697.
Rao, P. K., Dorsch, M., Chickering, T., Zheng, G., Jiang, C., Goodearl, A., Kadesch, T. and McCarthy, S. (2000). Isolation and characterization of the notch ligand delta4. Exp. Cell Res. 260,379 -386.[CrossRef][Medline]
Ridgway, J., Zhang, G., Wu, Y., Stawicki, S., Liang, W. C., Chanthery, Y., Kowalski, J., Watts, R. J., Callahan, C., Kasman, I. et al. (2006). Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 444,1083 -1087.[CrossRef][Medline]
Sainson, R. C., Aoto, J., Nakatsu, M. N., Holderfield, M., Conn,
E., Koller, E. and Hughes, C. C. (2005). Cell-autonomous
notch signaling regulates endothelial cell branching and proliferation during
vascular tubulogenesis. FASEB J.
19,1027
-1029.
Sakaguchi, T., Kuroiwa, A. and Takeda, H. (2001). A novel sox gene, 226D7, acts downstream of Nodal signaling to specify endoderm precursors in zebrafish. Mech. Dev. 107,25 -38.[CrossRef][Medline]
Scheer, N., Groth, A., Hans, S. and Campos-Ortega, J. A. (2001). An instructive function for Notch in promoting gliogenesis in the zebrafish retina. Development 128,1099 -1107.[Abstract]
Shawber, C. J. and Kitajewski, J. (2004). Notch function in the vasculature: insights from zebrafish, mouse and man. BioEssays 26,225 -234.[CrossRef][Medline]
Shawber, C. J., Das, I., Francisco, E. and Kitajewski, J.
(2003). Notch signaling in primary endothelial cells.
Ann. N. Y. Acad. Sci.
995,162
-170.
Shutter, J. R., Scully, S., Fan, W., Richards, W. G.,
Kitajewski, J., Deblandre, G. A., Kintner, C. R. and Stark, K. L.
(2000). Dll4, a novel Notch ligand expressed in arterial
endothelium. Genes Dev.
14,1313
-1318.
Smithers, L., Haddon, C., Jiang, Y. J. and Lewis, J. (2000). Sequence and embryonic expression of deltaC in the zebrafish. Mech. Dev. 90,119 -123.[CrossRef][Medline]
Uyttendaele, H., Ho, J., Rossant, J. and Kitajewski, J.
(2001). Vascular patterning defects associated with expression of
activated Notch4 in embryonic endothelium. Proc. Natl. Acad. Sci.
USA 98,5643
-5648.
Weinstein, B. M. (2002). Plumbing the mysteries of vascular development using the zebrafish. Semin. Cell Dev. Biol. 13,515 -522.[CrossRef][Medline]
Westin, J. and Lardelli, M. (1997). Three novel Notch genes in zebrafish: implications for vertebrate Notch gene evolution and function. Dev. Genes Evol. 207, 51-63.[CrossRef]
Williams, C. K., Li, J. L., Murga, M., Harris, A. L. and Tosato,
G. (2006). Up-regulation of the Notch ligand Delta-like 4
inhibits VEGF-induced endothelial cell function. Blood
107,931
-939.
Zeng, Q., Li, S., Chepeha, D. B., Giordano, T. J., Li, J., Zhang, H., Polverini, P. J., Nor, J., Kitajewski, J. and Wang, C. Y. (2005). Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling. Cancer Cell 8,13 -23.[CrossRef][Medline]
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is a control. (B) Side view of a
wild-type 25-hour-old embryo stained by in situ hybridization. dll4
is expressed in the dorsal aorta (DA) and intersegmental vessels (ISV), and in
a subset of neurons in the neural tube (arrowhead). (C) Cross section
through the trunk of a similar specimen showing dll4 expression in
endothelial cells of the DA and ISVs but not in those of the posterior
cardinal vein (PCV). (D) Similar section at a slightly different level
relative to somite boundaries does not pass through any ISVs but reveals cells
expressing dll4 in the ventrolateral neural tube (NT), as well as in
the DA. (E) Cross section through the trunk of a 25-hour-old
mib-mutant embryo. dll4 expression is increased and extended
in the neural tube, but is undetectable in the endothelial cells. The expanded
expression in the neural tube (and ear, for which data not shown) indicates
that, in these tissues, dll4, like other delta genes, is regulated by
lateral inhibition, so that its expression increases when Notch signalling
fails. The loss of expression in the endothelial cells probably reflects their
partial conversion to a venous character as a result of the failure of Notch
signalling at an early stage (Lawson et
al., 2001
6 specimens for each treatment; error bars
represent s.e.m.). (J) fli1:EGFP embryo at 2.5 dpf injected
with 10 ng of a morpholino targeted against casanova (sox32)
to block circulation in the trunk. (K) fli1:EGFP-positive
dll4-mutant embryo at 2.5 dpf treated from 48 hpf with 2 µM SU5416
to block Vegf signalling. (L) The gut and pectoral fin vasculature of
an fli1:EGFP-positive dll4 mutant and a wild-type (normal)
sibling embryo, both at 11 dpf. Scale bar: 50 µm in D-H,J,K; 40 µm in
L.