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First published online 11 July 2007
doi: 10.1242/dev.002576
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1 Tumor Biology and Angiogenesis Department, Genentech Inc., 1 DNA Way, South
San Francisco, CA 94080, USA.
2 Lexicon Genetics Inc., 8800 Technology Forest Place, The Woodlands, TX
77381-1160, USA.
3 Cell Microscopy Center, Department of Cell Biology, University Medical Center
Utrecht and Institute for Biomembranes, 3584CX, Utrecht, Netherlands.
4 Translational Oncology Department, Genentech Inc., 1 DNA Way, South San
Francisco, CA 94080, USA.
5 Pathology Department, Genentech Inc., 1 DNA Way, South San Francisco, CA
94080, USA.
* Author for correspondence (e-mail: loni{at}gene.com)
Accepted 30 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: ECM, EGFL7, Migration, Morphogenesis, Vascular, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), it is more common that cells migrate in groups
(Haas and Gilmour, 2006).
Depending on the tissue architecture, a cohort of migrating cells can assume
different shapes, such as sheets, chains or tubes
(Friedl et al., 2004). Many
questions concerning collective cell migration have yet to be answered. For
example, how does a group of cells maintain a defined organization as they
move? How is the size of a group of migrating cells regulated? Does the
spatial organization of the migrating cells influence their migration speed?
Answering these questions will not only help us understand the basis of tissue
morphogenesis, but might also help us design ways to interfere with
pathological conditions that involve collective cell migration, such as tumor
angiogenesis. Using sprouting angiogenesis during embryonic and neonatal
development as a model, we attempted to answer some of these questions.
Vascular development in embryonic and postnatal life is a spatially and
temporally orchestrated process, as many steps in vasculogenesis and
angiogenesis are coordinated by factors present in the microenvironment
(Flamme et al., 1997;
Rossant and Howard, 2002;
Yancopoulos et al., 2000). For
example, endothelial cell (EC) migration is often guided by factors produced
by the target tissues (Carmeliet and
Tessier-Lavigne, 2005; Coultas
et al., 2005; Eichmann et al.,
2005). The cellular events related to EC migration in response to
external cues are most extensively documented during sprouting angiogenesis,
which is a means of creating new vessels from existing ones
(Gerhardt and Betsholtz, 2005;
Patan, 2004). It is generally
believed that tip cells, which are the specialized ECs situated at the tips of
sprouts, respond to chemoattractants and migrate actively along extracellular
matrix (ECM) rails; whereas the stalk cells - ECs that line the wall of a
nascent vessel - are pulled forward passively by the tip cells
(Davis and Senger, 2005). Stalk
cell proliferation contributes to the extension of sprouts
(Gerhardt and Betsholtz, 2005;
Gerhardt et al., 2003), but it
is not clear if stalk cells can influence the movement of a sprout in other
ways. In this study, we provide evidence to suggest that the spatial
organization of the stalk cells contributes to the collective EC movement, and
show that this process is regulated by the vascular-specific secreted factor
EGFL7.
EGFL7 is a secreted factor that is specifically expressed by ECs
(Campagnolo et al., 2005;
Fitch et al., 2004;
Parker et al., 2004;
Soncin et al., 2003) (see also
Fig. S2 in the supplementary material). Expression is high during embryonic
and neonatal development in all vessel types. As the vasculatures mature,
Egfl7 expression is downregulated in many vessels. In contexts
requiring new vessel growth, such as tumorigenesis or vascular injury,
Egfl7 levels increase again
(Campagnolo et al., 2005;
Parker et al., 2004). In
zebrafish embryos, Egfl7 is required for the midline angioblasts to disperse
along the dorsal-ventral axis, which is an essential step in axial vessel
tubulogenesis (Parker et al.,
2004). Owing to the strong defect in vasculogenesis, we were
unable to investigate the role of Egfl7 in angiogenesis using
zebrafish embryos because axial vessel assembly precedes and impacts sprouting
angiogenesis (Kidd and Weinstein,
2003). In this study, we describe a unique role that EGFL7 plays
in regulating sprouting angiogenesis and vascular patterning.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Zambrowicz et al., 1998). The
homologous recombination line was generated following standard techniques (see
Fig. S1 in the supplementary material).
Histology and immunostaining
Primary antibodies: 
SMA (Sigma), BrdU (Serotec), activated caspase 3
(BioVision), CD31 (BD), type IV collagen (CosmoBio), FAK (Calbiochem),
FAKpY397 (Upstate), FAKpY861 (BioSource), fibronectin (NeoMarkers), GFAP
(NeoMarkers), Ki67 (Chemicon), NG2 (Chemicon), VE-cadherin (cadherin 5) (BD),
VEGF (RDI), EGFL7 (Genentech) (Parker et
al., 2004) and Isolectin B4 (Sigma).
Wholemount immunohistochemistry (IHC) and immunofluorescence (IF) were
performed as described
(http://spot.colorado.edu/~klym/)
(Gerhardt et al., 2003).
For BrdU labeling, animals were injected i.p. with 100 µg BrdU/g, 2 hours prior to sacrifice. For vessel perfusion, adult animals were injected with FITC-conjugated tomato lectin (Vector) into the tail vein (150 µl at 1 mg/ml per mouse), whereas neonatal animals were injected via the intracardiac route under anesthesia.
Hypoxia was detected using the Hypoxyprobe-1 Plus Kit following manufacturer's instruction (Chemicon).
For Hematoxylin and Eosin (H&E) and IHC staining on tissue sections, tissues were fixed with Bouin's. For IF, tissues were fixed with 4% paraformaldehyde/PBS for the majority of markers, or unfixed for the anti-EGFL7. For ß-galactosidase staining, tissues were fixed in 0.25% glutaraldehyde/PBS for 15 minutes.
Angiography
Fluorescent angiography was performed as described
(Rice et al., 2004).
Electronmicroscopy analysis
Neonates were perfused under anesthesia with PBS/heparin, followed by
half-strength Karnovsky fixative. After further fixation, retinas were excised
in 0.1 M Na cacodylate buffer (pH 7.4), and processed for Epon embedding
following standard protocols. A quarter of each retina was stained with
Isolectin B4 to allow monitoring of vascular progression.
EGFL7 subcellular localization
Human umbilical vein endothelial cells (HUVECs, Cambrex) were seeded onto
chamber slides (Nunc) precoated with 5 µg/ml fibronectin (Sigma), type IV
collagen, type I collagen (BD) or laminin (Invitrogen), or co-cultured with
human dermal fibroblasts (Clonetics) in EGM2 medium (Cambrex). After 3-6 days
in culture, cells were incubated for 20 minutes in EGM2 medium with 5% BSA
(Roche) and complete protease inhibitor (Roche). EGFL7 antibody (10 µg/ml,
Genentech) was added and incubated for 1 hour. Cells were stained with
secondary antibody in detergent-free buffers.
Chicken embryonic fibroblasts (CEFs) were transfected with plasmids encoding the HA-tagged full-length human EGFL7 or GFP in the RCAS vector. CEFs were grown in 10 ml serum-free culture medium on a 10 cm plate for 2 days at confluent density. Conditioned media were harvested and concentrated to 1 ml. Cells were washed and incubated for 15 minutes at 37°C with 1 ml 1 M NaCl (high salt extract), lysed in 1 ml cold 0.5% deoxycholate (DOC) in PBS for 15 minutes on ice (cell lysate extract). Plates were washed several times with PBS to remove all cells, and 1 ml of the ECM-extraction buffer (5% SDS, 100 mM Tris, pH 6.8) was added for 15 minutes. ECM was scraped from the plate and heated at 96°C for 10 minutes. An equal volume of each fraction was analyzed by western blotting with an anti-HA antibody (Covance).
VEGF ELISA
Whole retinas were collected at P5 and P8. VEGF levels in the homogenized
tissues were quantified by ELISA following the manufacturer's instructions
(Quantikine).
Endothelial cell isolation from mouse tissue
Mice (8 to 12 weeks old) were anesthetized and perfused with 0.05%
collagenase/HBSS. ECs were isolated from the liver using biotinylated
anti-CD31 antibody (BD) and streptavidin microbeads (MACS system, Miltenyi
Biotec). An aliquot of cells was stained with anti-VE-cadherin (BD) to
evaluate purity.
Aortic ring explants
Thoracic aortae were excised from mice used for EC isolation (see method
above), immediately after perfusion. Aortae were cleaned of all connective
tissue and cut into 1- to 2-mm-thick rings. Rings were embedded in rat tail
type I collagen (BD) and cultured for several days in EGM2 medium (Cambrex)
plus 3% mouse serum (Taconic). Live rings were imaged using an inverted
microscope from days 4-7. Some rings were fixed at day 6 for marker
analysis.
Trans-well migration assay
Trans-well inserts (BD) were coated overnight with 8 µg/ml fibronectin
(Roche) or EGFL7 (Genentech). Mouse ECs isolated from liver were plated in EBM
medium (Cambrex), 0.1% BSA at 106 cells/well. After overnight
incubation with 10 ng/ml VEGF in the lower chamber, cells were fixed with
methanol and stained with Sytox Green (Invitrogen). Nuclei on the bottom of
the insert were counted.
HUVEC random migration stimulated by HGF
Chamber slides were coated overnight with 5 µg/ml indicated proteins.
After blocking with BSA, HUVECs were seeded sparsely and cultured in EGM2
medium containing 100 ng/ml HGF. Bright-field images were collected every 8
minutes for 24 hours. Individual cell migration over this time frame was
analyzed by object tracking (Metamorph).
Quantification methods
For a detailed explanation of the quantification methods employed, see Fig.
S4 in the supplementary material.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Vector insertion occurred in intron 2, upstream of the
translation initiation codon in exon 3 (see Fig. S1A in the supplementary
material), which leads to silencing of endogenous Egfl7 transcription
(Fig. S1B in the supplementary material), thus abolishing EGFL7 translation
(Fig. 7L,M and see Fig. S1C in
the supplementary material). We also generated an Egfl7 mutant line
by homologous recombination that removes exons 5-7 (see Fig. S1D in the
supplementary material) and introduces a premature stop codon, thereby
producing a truncated protein with an incomplete EMI domain that terminates at
Tyr90. Loss of EGFL7 protein was confirmed by immunofluorescence staining on
frozen tissues from the wild-type (Egfl7+/+) and
homozygous (Egfl7-/-) littermates (see Fig. S1G in the
supplementary material). We found that the phenotypes were consistent between
the two mutant lines, confirming that the biological activities described in
this study are attributable to the EGFL7 protein. Because of space
limitations, data presented in the main text are from the insertional line
unless specified otherwise, and most of the data from the homologous
recombination line are presented in the supplementary material.
Egfl7-/- mice are born with a substantially reduced
mendelian ratio, whereas the mendelian ratio for embryos analyzed at 9.5-11.5
dpc (E9.5-11.5) was normal (Table
1). Between E13.5 and E15.5, a significant number of
Egfl7-/- embryos had severe systemic oedema
(Fig. 1A-D,
Table 2), and a subset of the
oedematous embryos had no heartbeat at E14.5 and E15.5
(Table 2). These analyses
indicate that 
50% of the Egfl7-/- embryos die in
utero. Lethality probably occurs at around E14.5-15.5.
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E11.5, and extends downward and outward to
cover the entire epicardium by E14.5
(Lavine et al., 2006). Loss of
EGFL7 resulted in a significant delay in this sequence of events. The degrees
of coronary vascular coverage were significantly reduced in 50% of the
Egfl7-/- embryos at E12.5 and E14.5
(Fig. 2A-D,G).
The development of the retinal vasculature also follows a precise temporal
schedule (Benjamin et al.,
1998; Dorrell and Friedlander,
2006; Stone et al.,
1995). Vascularization in the retina commences at postnatal day 0
(P0), when ECs from the central retinal artery begin to migrate outward to the
periphery within the nerve fiber layer (NFL), and vascular coverage of the NFL
completes around P8-9. In 100% of Egfl7-/- mice, the
centrifugal expansion of the NFL vascular plexus across the retina was
impaired. This delay was evident as early as P2 and persisted until P11
(Fig. 2I-O and see Fig. S3C-G
in the supplementary material; data not shown). Interestingly, retinal sizes
were unaffected despite the vascular coverage defect, possibly owing to the
slow growth of this organ (Fig.
2P and see Fig. S3H in the supplementary material).
Cranial vascular development is also easy to follow in the mouse embryo. At
E10, the superficial cranial vessels develop by sprouting from the
cardinal vein. The subsequent rostral migration and branching of these vessels
give rise to an elaborate vasculature that covers a significant portion of the
lateral midbrain by E11.5 (Fiore et al.,
2005). The development of the cranial vascular bed was delayed in
the absence of EGFL7, as vascular complexity was reduced in the heads of the
Egfl7-/- embryos (Fig.
2E,F,H and see Fig. S3A,B in the supplementary material).
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Loss of Egfl7 causes vascular morphological alterations in the surviving mice
Despite the significant delay in vascular development in many organs,
50% of the Egfl7-/- mice survived
(Table 1), and all of the
surviving mice had vascular coverage in all the organs that we examined
(Fig. 3 and data not shown).
However, the morphology and organization of these vasculatures were altered in
more than 80% of the Egfl7-/- mice. The most prominent
vascular phenotypes found in these mice were: tortuosity
(Fig. 3C,N and see Fig. S3K,O,R
in the supplementary material); irregular diameters
(Fig. 3G-K and see Fig. S3P,S
in the supplementary material); and clustering of multiple vessels (arrows in
Fig. 3 and Fig. S3K,P-Q in the
supplementary material).
The presence of tissue hypoxia (Fig. 1) and vascular morphological defects suggest that the vasculatures in the mutant adult mice might not function optimally.
The vascular development phenotype is a primary defect in the endothelium
In order to understand the cellular mechanism of the phenotypes described
above, we addressed several questions:
First, we investigated whether the vascular phenotypes are caused by defects outside of the vasculature. We found no obvious defects in a number of cell types closely associated with the vasculature, and the expression of VEGFA and fibronectin - two factors that are relevant to retinal vascular development - was unchanged in the Egfl7-/- tissues (see Figs S5, S6 in the supplementary material). Together with the fact that EGFL7 expression has not been found outside of the vascular endothelium, we conclude that the vascular defects are primary.
Second, we examined whether the reduced vascular coverage in the neonatal retinas is due to a reduction in EC proliferation by carrying out in vivo BrdU labeling and Ki67 staining. Our results indicated that EC proliferation is not altered in the Egfl7-/- retina at any of the postnatal stages examined (see Fig. S7A-C in the supplementary material; data not shown). This conclusion is further supported by the analysis of primary ECs isolated from the Egfl7-/- and Egfl7+/+ littermates and cultured in vitro (see Fig. S7F in the supplementary material).
Third, we asked whether reduced vascular coverage in the neonatal retinas is due to altered EC apoptosis during postnatal development. Immunostaining of activated caspase 3 on retinal sections (see Fig. S7D,E in the supplementary material) revealed minimal retinal EC apoptosis at P2, regardless of the Egfl7 genotype.
EGFL7 regulates the collective movement of ECs during sprouting angiogenesis
The results described in the previous section prompted us to investigate
whether EC migration is altered in the Egfl7-/- tissues.
Since delayed sprouting angiogenesis is evident in multiple mutant organs, we
decided to use aortic ring culture, which is a sprouting angiogenesis model
that can be followed temporally. Using live imaging, we monitored numerous
sprouts individually over a period of 4 days in rings isolated from
Egfl7+/+ and Egfl7-/- littermates, and
saw a significant reduction in sprout extension velocity in the knockout rings
(Fig. 4A-D).
We also sought additional, but less direct, evidence for the alteration of
EC motility. Focal adhesion kinase (FAK; also known as PTK2 - Mouse Genome
Informatics) phosphorylation was chosen because this is an important event
required for cells to adhere to and migrate on ECM substrates
(Parsons, 2003). We found that
phosphorylation on tyrosine 861 (pY861) is the predominant form in ECs in the
wild-type neonatal retinas and aortic rings (see Fig. S8A,B,I,J in the
supplementary material), whereas significant reduction of FAK-pY861 occurred
in the Egfl7-deficient stalk cells in these two tissues (see Fig.
S8C,D,K,L in the supplementary material). The reduced FAK phosphorylation was
not due to decreased expression of FAK because total FAK staining was not
altered in the Egfl7-/- tissues (see Fig. S8E-H,M-P in the
supplementary material).
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We then investigated whether the inclusion of EGFL7 in the ECM can alter the migration speed of individual ECs. To this end, we cultured HUVECs on fibronectin alone, EGFL7 alone, or a mixture of fibronectin and EGFL7, and monitored their migration using time-lapse microscopy. The migration speeds of individual ECs did not differ on these substrates (Fig. 4F).
Taken together, our data suggest that EGFL7 does not influence the migratory capacity of individual ECs, nor does it affect the ability of the ECM to support individual EC migration. Instead, EGFL7 is required for the effective movement of a cohort of ECs that migrate together.
EGFL7 regulates EC organization in angiogenic sprouts
In an attempt to understand why collective EC movement is impaired in
sprouting angiogenesis, we took a closer look at the structures of angiogenic
sprouts in the Egfl7-knockout tissues.
Sprouting is achieved through the coordinated activities of two subtypes of
ECs: the chemosensing activity and motility of tip cells determine the
direction of migration, whereas proliferation and unidirectional movement of
the stalk cells contribute to the extension of a sprout
(Gerhardt and Betsholtz,
2005). We first examined these two cell populations on wholemount
retinas stained for EC and nuclear markers
(Fig. 5A-F). Tip cells were
present at the vascular migration front in the Egfl7-/-
retinas, and the morphology of their filopodia was similar to that of the
wild-type cells. In addition, the overall direction of the tip cell filopodia
was similar in the Egfl7-/- and
Egfl7+/+ tissues (Fig.
5A,D), implying that they are sensing the chemoattractant
appropriately and are motile. However, when we examined the spatial
distribution of the tip and stalk cells within a single sprout, we saw a
striking difference between the Egfl7-/- and
Egfl7+/+ littermates. In the wild-type retinas, most
sprouts were of similar size, with each sprout consisting of 1-2 stalk cells
following a single tip cell (Fig.
5B,G,H). When examined by confocal optical sectioning along the
vitreal-choroidal axis, almost all tip cells and the majority of stalk cells
were always arranged in a single cell layer
(Fig. 5C). In the
Egfl7-/- retina, however, some tip cells and the majority
of stalk cells were seen to line up in multiple cell layers along the
vitreal-choroidal axis (Fig.
5E,F), producing markedly enlarged sprouts with increased tip and
stalk cell numbers (Fig. 5G,H).
This phenotype was most profound at P2, and partially resolved by P8.
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As mentioned above, similar aberrant EC clusters were seen in many
Egfl7-/- tissues (arrowheads in
Fig. 2 and Fig. S3 in the
supplementary material). These aggregates are reminiscent of the midline
angioblast aggregates seen in the egfl7-knockdown zebrafish embryos
(Parker et al., 2004).
These observations led us to propose that EGFL7 plays a role in regulating the spatial distribution of ECs, thereby limiting the sprout size. This activity is necessary for the efficient outgrowth of nascent vessels.
The Egfl7 mutant ECs fail to respect the boundary of nascent sprouts
We went on to investigate how EGFL7 regulates sprout size. The
multicellular nature of the aggregates suggests that ECs might fail to
recognize the boundary of nascent sprouts. To explore this possibility, we
stained neonatal retinas and cultured aortic rings for collagen IV, which is a
component of the vascular basement membrane.
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These observations suggest that Egfl7-/- ECs are unable to position themselves properly because they fail to recognize the orientation of their endothelial neighbors. However, based on the following observations, we believe that these mutant ECs can establish proper basal-luminal polarity: (1) in adult tissues, although many aberrantly associated vessels are present (Fig. 3, arrows), these vessels have lumen as indicated by fluorescein perfusion (Fig. 3C); (2) collagen IV is localized to the basal surface even in abnormally juxtaposed vessels (Fig. 3L). Therefore, EGFL7 is probably not essential for establishing polarity, but may play a role in reinforcing it.
EGFL7 is associated with the interstitial ECM
In order to understand the molecular mechanism of the phenotypes described
above, we analyzed the biochemical properties of EGFL7 in vitro. When
overexpressed in fibroblasts, abundant EGFL7 was detected in the ECM and cell
lysates, but very little could be detected in the conditioned media even when
cells were treated with high salt (Fig.
7A), indicating that EGFL7 is tightly associated with the ECM and
therefore does not diffuse freely.
Surprisingly, overexpression in fibroblasts resulted in significantly more
efficient deposition of EGFL7 into the ECM than in epithelial cells (data not
shown). This prompted us to examine whether factor(s) produced by the
fibroblasts facilitate the deposition of EGFL7 into the ECM. We cultured
HUVECs alone, or in direct contact with fibroblasts. In a pure HUVEC culture,
EGFL7 deposition into the ECM was a slow process, as only very low levels of
EGFL7 could be detected at day 3 (Fig.
7C), and moderate levels of EGFL7 in a fibrous network could only
be detected at day 6 (data not shown). This temporal profile coincided with
the deposition of other ECM proteins, such as fibronectin, by HUVECs (data not
shown). However, when HUVECs were cultured with human dermal fibroblasts - a
cell line negative for EGFL7 (Fig.
7E) - abundant extracellular EGFL7 was detected within 3 days
(Fig. 7B), indicating that
fibroblasts expedite the deposition of EGFL7. We then asked whether the
fibroblast activity requires direct cell-cell contact by performing co-culture
of HUVECs with fibroblasts in a trans-well setting in which the two cell types
are separated. We found that the kinetics of EGFL7 deposition was unchanged in
the trans-well co-culture (Fig.
7D) compared with HUVEC alone
(Fig. 7C), suggesting that the
activity of fibroblasts requires direct contact. Similar observations were
made with other interstitial cell types such as astrocytes and vascular smooth
muscle cells (data not shown). Based on these observations, and the fact that
fibroblasts and other interstitial cell types usually produce a greater
diversity and quantity of ECM proteins than epithelial or endothelial cells,
we went on to investigate whether certain ECM proteins can facilitate the
deposition of EGFL7. Interestingly, we found that fibronectin and type I
collagen, which belong to the subtype of matrix proteins encountered by
nascent vessels when they invade new tissues
(Kalluri and Zeisberg, 2006),
facilitate EGFL7 deposition (Fig.
7F and data not shown), whereas laminin and type IV collagen,
components of the basement membrane
(Kalluri and Zeisberg, 2006),
do not (Fig. 7G and data not
shown).
These experiments indicate that EGFL7 is a matrix-associated protein, and its secretion and/or incorporation into the matrix depends on interstitial ECM component(s). In agreement with this, we found that EGFL7 always localizes on the basal side of ECs, and has significant overlap with fibronectin (Fig. 7H-J). Interestingly, a continuous layer of EGFL7 was found surrounding stalk cells in an angiogenic sprout (Fig. 7J), whereas only patchy EGFL7 signal was seen around tip cells (Fig. 7K). The difference in EGFL7 protein levels between tip and stalk cells might be a consequence of accumulative secretion, as tip cells are the first ECs in a sprout to secret EGFL7 and they are constantly moving into new environments devoid of EGFL7, whereas stalk cells move into a space in which EGFL7 has already been deposited by the previous ECs migrating along the same route.
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). This
claim is supported by the finding that Egfl7 expression is very low
in mature vascular beds, and is upregulated during angiogenesis and vascular
remodeling (Campagnolo et al.,
2005; Parker et al.,
2004).
EGFL7 defines an optimal path for the migrating ECs
Integrating all our findings, we propose the following model to explain how
EGFL7 acts. When a nascent sprout migrates into a microenvironment surrounded
by interstitial cell types capable of secreting fibrillar matrix, EGFL7 is
deposited at the interface between ECs and the interstitial cells, forming a
unique ECM coat on the outer surface of the sprouts. This coat delineates the
boundary of a new sprout and defines an efficient migratory path for the ECs
(Fig. 8A). In the absence of
EGFL7, ECs fail to respect the boundary of a sprout. As a result, new ECs can
attach to the basal sides of other ECs within a single sprout, or multiple
sprouts can adhere to each other, thereby forming larger sprouts that cannot
migrate efficiently (Fig.
8B).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sprout size and collective EC migration
Our study suggests that sprout size impacts the speed of migration. To
understand why, we should look at the roles played by tip and stalk cells. It
is generally believed that tip cell migration and stalk cell proliferation
both contribute to the extension of a sprout, and stalk cells are pulled
forward by the tip cells (Gerhardt and
Betsholtz, 2005; Gerhardt et
al., 2003; Kurz et al.,
1996; Ruhrberg et al.,
2002). Our study adds an additional layer to this paradigm: under
normal conditions, stalk cell proliferation may directly contribute to the
forward movement of a sprout if they are lined up in a linear fashion, because
the addition of a new cell will push its neighbor forward
(Fig. 8A). In the
Egfl7 mutant tissues, a newly formed EC may attach to the basal side
of other ECs (Fig. 8B) instead
of situating side-by-side with its neighboring cells along the migratory path
of the sprout (Fig. 8A);
therefore, it fails to contribute to the overall forward migration.
EGFL7 and the spatial arrangement of ECs
How does EGFL7 prevent inappropriate EC association? In a previous study,
we showed that EGFL7 supports EC adhesion, but their adhesive strength on
EGFL7 is weaker than other matrix proteins
(Parker et al., 2004). This
molecular characteristic leads to our first hypothesis: as a new sprout forms,
ECs within or outside of a new sprout could encounter the basal surfaces of
other ECs and transiently adhere to it. However, this type of adhesion can be
resolved quickly because EGFL7 modulates the adhesion strength. Without EGFL7,
the adhesive strength of this inappropriate attachment might be enhanced,
leading to the persistence of EC aggregation and the subsequent formation of
enlarged sprouts.
Alternatively, EGFL7 could bind to a signaling molecule such as VEGF, and
modulate the signaling events elicited by the signaling factor(s). This is an
attractive possibility because the ECM is known to modulate growth
factor/cytokine signaling (Boudreau and
Bissell, 1998). Future efforts will focus on identifying factor(s)
that are potentially modulated by EGFL7.
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). (3) Delayed
vascular development might trigger the production of a strong migration factor
that stimulates the aggregated ECs to migrate away. The presence of abnormal
structures in adult vasculatures indicates that the recovery is not a complete
correction of the early problems.
Embryonic lethality
In this study, although we have not identified the exact cause of embryonic
lethality in the Egfl7 mutant lines, we believe that the vascular
developmental defects probably contribute to the cause. The percentages of
embryos with delayed coronary vascular development at E14.5
(Fig. 2G) and with systemic
oedema (Table 2) are both
50%, suggesting that the coronary vascular defect might compromise
circulation and lead to severe oedema and death.
Although defective blood circulation is a much more prevalent cause of oedema than lymphatic problems, we cannot exclude the possibility of lymphatic failure in the knockout embryos. We will investigate this possibility in future work.
Partial penetrance
The partial penetrance in embryonic lethality and some other phenotypic
aspects is puzzling. So far, we have not identified a plausible compensatory
factor. It is possible that a genetic modifier exists, but it is probably a
weak modifier based on the following observation: in the N10 C57Bl/6 mice,
there is a trend toward further reduction of viable
Egfl7-/- mice compared with N5 C57Bl/6 mice, but the
difference is not yet statistically significant (P=0.0921,
Table 1). An alternative
possibility is that the partial penetrance is due to a threshold effect and is
stochastic in nature.
Implications
The ECM influences many steps in vascular development
(Brooke et al., 2003;
Davis and Senger, 2005;
Whelan and Senger, 2003);
however, the specific mechanism through which each matrix component influences
the behavior of ECs is only partially understood. In this report, we have used
EGFL7 as an example to demonstrate that a single ECM component can play a
unique role in angiogenesis. Our study illustrates that vascular development
is a complex process orchestrated by many molecular players, including each
constituent of the ECM.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/16/2913/DC1
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ACKNOWLEDGMENTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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