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First published online 4 March 2009
doi: 10.1242/dev.034199
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1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot,
Israel.
2 Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer
Center, Houston, TX 77030, USA.
3 Department of Orthopaedics, Kyoto University, Kyoto 606-8507, Japan.
4 Genentech, 1 DNA Way, S. San Francisco, CA 94080, USA.
* Author for correspondence (e-mail: eli.zelzer{at}weizmann.ac.il)
Accepted 16 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Skeleton, Skeletogenesis, Anti-angiogenic, Vascular patterning, Limb development, SOX9, VEGF, PRX1-Cre, SOX9-Cre, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Drake et al.,
1998; Folkman,
2003; Risau and Flamme,
1995; Sabin, 1920;
Sato and Loughna, 2002).
One of the key players in both angiogenesis and vasculogenesis is vascular
endothelial growth factor (VEGF)
(Carmeliet et al., 1996;
Ferrara et al., 1996). VEGF
controls blood vessel development by regulation of endothelial cell
proliferation, migration and differentiation
(Brown et al., 1997;
Ferrara and Henzel, 1989;
Leung et al., 1989). During
angiogenesis, VEGF binds to two tyrosine-kinase receptors, VEGFR1 (FLT1) and
VEGFR2 (FLK1), which are present predominantly on endothelial cells
(Carmeliet and Collen, 1999;
de Vries et al., 1992;
Fong et al., 1995;
Shalaby et al., 1997;
Shalaby et al., 1995;
Terman et al., 1992). In
addition, endothelial cells express the co-receptors neuropilin 1 (NRP1) and
neuropilin 2 (NRP2), which bind to VEGF and potentiate FLK1 activity
(Neufeld et al., 1999;
Soker et al., 1998).
During embryogenesis, one of the challenging tasks the forming organ is
faced with is the need to synchronize its development with that of the
vasculature, in order to ensure sufficient gas exchange and nutrient supply
(Cleaver and Melton, 2003;
Coultas et al., 2005;
Hogan et al., 2004) Limb
development constitutes a central model for the study of tissue and organ
patterning (Cohn and Tickle,
1996; Johnson and Tabin,
1997). Up until now, most of the patterning mechanisms that have
been extensively studied were related to limb skeleton. Interestingly, and in
contrast to its absolute necessity, the mechanisms that regulate the
patterning of limb vasculature have been left understudied.
During the initial stages of limb formation, angiogenesis is initiated as
sprouts from the dorsal aorta invade the limb bud and form a vascular plexus,
which is embedded within the limb mesenchymal core
(Seichert and Rychter, 1972a;
Seichert and Rychter, 1972b).
Concomitantly, vasculogenesis contributes to the forming vascular plexus, as
somite-derived angioblasts migrate and integrate into the developing plexus
(Ambler et al., 2001). Next, as
skeletogenesis is initiated, the initially unpatterned vascular plexus
undergoes major spatial changes that result in its rearrangement into a highly
branched and patterned network, which is segregated from the forming skeleton.
Most prominently, avascularized areas emerge from previously vascularized
regions as a result of vessel regression from the emerging cartilage anlage.
Concurrently, the surrounding vasculature undergoes an extensive
morphogenesis, forming a stereotypical, highly branched and enriched network
(Feinberg et al., 1986;
Hall and Miyake, 1992;
Seichert and Rychter, 1972a).
The mesenchymal cells that occupy these avascular areas aggregate and form
high cell density condensations that will eventually differentiate into
chondrocytes, thus forming cartilage models of the future bones
(Hall and Miyake, 2000).
Mesenchymal condensation is the initial step in skeleton formation and the
transcription factor SOX9 is an essential regulator of this process
(Bi et al., 1999).
Sox9 is first expressed in the limb bud between E10 and E10.5 in
chondroprogenitors and chondrocytes, preceding the formation of cartilage
(Wright et al., 1995).
Inactivation of Sox9 in limb mesenchymal and neural crest cells
results in complete absence of mesenchymal condensation and subsequent failure
in cartilage formation (Akiyama et al.,
2002; Mori-Akiyama et al.,
2003).
The tight coordination between skeleton development and vascular
rearrangement has prompted studies that aimed to expose the regulatory role
that these two systems were presumed to play on each other's development. Some
of these studies addressed the obvious issue of which system is patterned
first, assuming that the first system to be patterned may regulate the
patterning of the other tissue (Feinberg
et al., 1986; Hallmann et al.,
1987; Wilson,
1986). Other studies concentrated on the influence of an abnormal
vasculature on limb skeleton formation and the mechanism that underlies such
an effect (Caplan and Koutroupas,
1973; Feinberg and Saunders,
1982; Fraser and Travill,
1978; Hootnick et al.,
1980; Jargiello and Caplan,
1983). However, although these studies have provided strong
indications for the possible regulatory interactions between limb vasculature
and the skeleton, they were not conclusive, mostly owing to the absence of
genetic tools. Thus, the mechanism that coordinates vascular patterning and
skeletogenesis remained unsolved.
In this study, we provide evidence for the centrality of the forming skeleton in regulating limb vascular patterning and implicate Vegf expression by condensed mesenchyme as a key component in the underlying mechanism. Blocking the expression of the VEGF receptors Flt1, Flk1, Nrp1 and Nrp2 in limb mesenchyme resulted in no apparent effect on vascular patterning, strongly suggesting that VEGF regulates limb vasculature via a long-range mechanism. Finally, we provide evidence for the involvement of SOX9 in the regulation of Vegf expression in the condensing mesenchyme. These findings establish Vegf expression in the condensing mesenchyme as the mechanism by which the skeleton patterns limb vasculature.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
floxed-Sox9 (Akiyama et al.,
2002), Vegf-IRES-lacZ
(Miquerol et al., 1999),
Sox9-Cre (Akiyama et al.,
2005), Prx1 (also known as Prrx1 - Mouse Genome
Informatics) -Cre (Logan et al.,
2002), rtTA (Belteki
et al., 2005), tetO-Vegf
(Benjamin and Keshet,
1997), Nrp2-LacZ
(Chen et al., 2000),
floxed-Nrp1 (Gu et al.,
2003), Nrp2 (Giger et
al., 2000) and Sox9 misexpressed
(Akiyama et al., 2007) mice
have been described previously; floxed-Flt1 and floxed-Flk1
will be described elsewhere. In all timed pregnancies, the day of the vaginal
plug appearance was defined as E0.5. For harvesting of embryos, timed-pregnant
female mice were sacrificed by CO2 intoxication. The gravid uterus
was dissected out and suspended in a bath of cold PBS, and the embryos were
harvested after amnionectomy and removal of the placenta. Tail genomic DNA was
used for genotyping. All experiments were performed with at least six
different control and knockout forelimbs from three different litters.
Whole-mount and section immunofluorescence and in-situ hybridization
For whole-mount immunofluorescence, freshly dissected tissue was fixed
overnight in 4% PFA, transferred to PBS, then dehydrated to methanol and
stored in -20°C until use. Samples were rehydrated to PBS and incubated
for 2 hours in blocking solution (PBS containing 10% normal goat serum and 1%
Triton X-100) and then incubated overnight at 4°C with primary antibody
rat anti-PECAM (CD31; BD Pharmingen, San Diego, CA) 1:25 diluted in blocking
solution. Samples were washed in PBS containing 1% Triton X-100 at room
temperature and then incubated overnight at 4°C with biotinylated anti-rat
secondary antibody (dilution 1:100; Vector Laboratories) and Cy2-conjugated
streptavidin (1:100; Jackson ImmunoResearch, West Grove, PA) antibodies
diluted in 1% BSA/PBS.
Immunofluorescence of cryosections was preformed as described previously
(Amarilio et al., 2007). Slides
were incubated with the primary antibodies: rat anti-CD31 (BD PharMingen;
1:100), monoclonal anti-collagen type IIa1 (Developmental Studies Hybridoma
Bank, The University of Iowa, IA; 1:100), goat anti-rat neuropilin 1 (R&D,
Minneapolis, MN; 1:100), rat anti-RAFL-1 (60 µg/ml) and biotin-labeled
peanut agglutinin (PNA, Sigma-Aldrich, St Louis, MO; 1:100). Secondary
antibodies used were: Alexa Fluor 488-labeled goat anti-rat IgG, Alexa Fluor
568-labeled goat anti-mouse IgG (Molecular Probes), goat anti-rabbit
indocarbocyanine (Cy3), goat anti-mouse Cy2, Cy2-conjugated streptavidin
(Jackson ImmunoResearch, West Grove, PA; 1:100). VEGF (Calbiochem) and CD34
(Abcam) staining was preformed using paraffin sections according to the
manufacturer's protocol. Samples were washed, mounted on glass slides and
analyzed with a LSM510 laser-scanning confocal microscope (Carl Zeiss, Jena,
Germany).
Section in situ hybridization process was preformed as described previously
(Murtaugh et al., 1999;
Riddle et al., 1993). All
probes are available by request.
X-gal staining
Freshly dissected tissue was fixed in 4% PFA/PBS, rinsed in a solution
containing 5 mM EGTA, 0.01% deoxycholate, 0.02% NP40 and 2 mM
MgCl2, and then stained in a solution containing 5 mM
K3Fe(CN)6, 5 mM K4Fe(CN)6, 5 mM
EGTA, 0.01% deoxycholate, 0.02% NP40, 2 mM MgCl2 and 1 mg/ml X-gal.
The tissue was either cleared in 0.3% KOH or dehydrated and embedded in
paraffin for longitudinal sections.
Overexpression of Vegf in the condensed mesenchyme
Inducible Vegf overexpression in the condensed mesenchyme was
carried out by the reverse tetracycline transactivator
(rtTA)/tetracycline-responsive element (tetO)-driven transgene system
(Belteki et al., 2005;
Gossen et al., 1995), with
Sox9-Cre as an inducer (Akiyama et
al., 2005). Briefly, tetO-Vegf mice were crossed with
rtTA mice. Mice heterozygous for rtTA and tetO-Vegf
(rtTA-tetO-Vegf) were crossed with mice heterozygous for
Sox9-Cre transgene as an inducer. To induce Vegf expression,
doxycycline was administered to pregnant females starting at E10.5 and embryos
heterozygous for Sox9-Cre, rtTA and tetO-Vegf
(Sox9-rtTA-tetO-Vegf) were compared with embryos heterozygous for
rtTA and Sox9-Cre alleles (control).
Conditional blockage of Vegf in limb mesenchyme
Conditional blockage of Vegf in limb mesenchyme was obtained by
crossing floxed-Vegf mice with the Prx1-Cre
transgenic mouse as a deletor (Logan et
al., 2002). Embryos homozygous for floxed-Vegf
and heterozygous for Prx1-Cre alleles (Prx1-Vegf) were
compared with embryos heterozygous for floxed-Vegf and
Prx1-Cre alleles (control).
Primary cell culture preparations and viral transfer
For micromass cultures, limbs of E11.0-E11.5 floxed-Sox9 embryos
were collected, digested with 0.1% collagenase IV, 0.1% trypsin (Sigma) and 2%
FCS for 15 minutes. The cell suspension was placed in DMEM-F12, 10% FCS. Cells
were plated as 10 µl droplets at 2x107 cells/ml. Cells
were allowed to attach for 75 minutes and were then overlaid with 300 µl of
DMEM-F12, 10% FCS containing 6.5x107 viral particles/µl of
Adeno-Cre and Ad-βgal (Gene Transfer Vector Core, University of Iowa,
IA). Medium was changed daily. Cells were cultured with 20% oxygen in a
humidified atmosphere and then harvested to extract RNA.
Quantitative RT-PCR (qRT-PCR)
For qRT-PCR analysis, 1 µg total RNA was used to produce first-strand
cDNA. Reverse transcription was performed with SuperScriptII (Invitrogen,
Carlsbad, CA) according to the manufacturer's protocol. qRT-PCR was performed
using SYBR Green (Roche). Values were calculated using the second derivative
method and normalized to 18S rRNA expression. All primers are available on
request.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Skeleton formation is necessary for limb vascular patterning
The discovery of a well-orchestrated process of vascular and skeletal
patterning during limb development raised the hypothesis that the forming
skeleton regulated the patterning of limb vasculature. The transcription
factor SOX9 has been shown to be a key mediator of limb skeletal development
(Akiyama et al., 2002;
Mori-Akiyama et al., 2003). To
uncover the involvement of mesenchymal condensation in the regulation of
vascular patterning, we blocked Sox9 expression in limb mesenchymal
cells using the Prx1-Cre mouse as a deletor
(Logan et al., 2002) and
examined the vasculature. Embryos homozygous for floxed-Sox9
and heterozygous for Prx1-Cre alleles (Prx1-Sox9) were
compared with embryos heterozygous for floxed-Sox9 and
Prx1-Cre alleles (control). qRT-PCR of E12.5 control and
Prx1-Sox9 limbs demonstrate 60% decrease in Sox9 mRNA
expression in Prx1-Sox9 limbs, relative to the control.
Examination of whole limbs and sections of E10.5-E12.5 Prx1-Sox9 revealed a failure of the vasculature to pattern normally (Fig. 2). At E10.5, the vasculature of both control and Prx1-Sox9 limbs were comparable (Fig. 2A,B). However, at E11.5 the axial artery that was observed at control limbs was missing from Prx1-Sox9 limbs (Fig. 2C,D). At E12.5, instead of the highly branched metacarpal centers that were observed in control limbs, the Prx1-Sox9 limb vessels formed a single wide center that spanned along the anterior-posterior axis of the limb (Fig. 2E,F). In addition, no avascular areas emerged as the Prx1-Sox9 vasculature was evenly distributed throughout the autopod (Fig. 2H,J) and zeugopod (Fig. 2L), similar to the pre-condensation pattern we had observed in control limbs (Fig. 2A; Fig. 1A,E,H). These results demonstrate that the forming skeleton is necessary for limb vascular patterning, suggesting that the condensing mesenchyme produces a yet undescribed angiogenic signal that regulates this process.
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Previous works on mice that expressed only the heparin non-binding isoform
of VEGF, namely VEGF120, identified abnormalities in the limb microvessel
network, raising the hypothesis that VEGF, a key angiogenic factor, could be
implicated in limb vascular patterning
(Ruhrberg et al., 2002;
Vieira et al., 2007). In order
to test this hypothesis, we analyzed Vegf expression in the
developing limb, using mice with an IRES-lacZ reporter cassette
inserted into the 3'UTR of the Vegf gene
(Vegf-lacZ) (Miquerol et
al., 1999). X-gal staining of whole limbs and sections revealed a
dynamic expression of Vegf in the developing limb
(Fig. 3A-F).
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Overexpression of Vegf in condensed mesenchyme increases limb vascularization
To strengthen our hypothesis that the forming skeleton serves as a
signaling center for the flanking vasculature by expressing Vegf, we
used a gain-of-function approach. To examine the effect of overexpressing
Vegf specifically in the forming condensation on limb vasculature, we
used a triple transgenic system, in which the expression of the reverse
tetracycline transactivator (rtTA) and the tetracycline-responsive
element (tetO-Vegf165) transgene system was induced by
Sox9-Cre (Akiyama et al.,
2005; Belteki et al.,
2005; Gossen et al.,
1995) (for more details, see Materials and methods). qRT-PCR of
E13.5 control and Vegf-overexpression forelimbs show a 1.7-fold
increase in VEGF165 mRNA levels in the mutant, relative to the control. As the
vasculature of the autopod is highly stereotypical, we concentrated on the
effect of VEGF in that region.
The vasculature of Vegf overexpressed limbs was highly enriched, with endothelial cells that created denser and more complex networks in comparison with control limbs (Fig. 4A,B). Unlike in the control limbs, the metacarpal centers of Vegf overexpressed limbs were enriched with thicker vessels that originated in the axial artery (Fig. 4C,D). Moreover, these enriched metacarpal centers were wider and split into a denser and more complex network of small capillaries that occupied the interdigital areas (Fig. 4E,F). Interestingly, overexpression of Vegf in the condensing mesenchyme did not change the avascular properties of these areas, where no vessels were detected (Fig. 4G,H). These results support the hypothesis that VEGF produced in the avascularized condensations regulates the morphogenesis of the flanking vasculature.
Lack of Vegf in limb mesenchyme results in absence of vascular morphogenesis
In order to examine directly the role of VEGF in limb vascular patterning,
we took a loss-of-function approach. Having found that Vegf was
expressed in the condensing mesenchyme as early as day 10.5, we used the
Prx1-Cre mouse to delete Vegf in limb mesenchyme
(Logan et al., 2002) (see
Materials and methods). The reduction in Vegf expression was
confirmed by qRT-PCR of E11.5 control and Prx1-Vegf limbs that
demonstrated a 70% decrease in Vegf mRNA levels in Prx1-Vegf
limbs, relative to the control. Vascular development and patterning was
examined in E10.5-E12.5 whole limbs stained for endothelial cells using
antibodies for CD31 (Fig. 5).
In control limbs, we observed the stereotypical changes in vessel branching
and complexity, namely the thickening of the axial artery and its split into
thick vascular stems that supplied the interdigital zone
(Fig. 5A,C,E). In
Prx1-Vegf limbs, however, although the uniform capillary network and
axial artery were formed, the capillary network branching was reduced, leading
to a sparse network compared with control limbs
(Fig. 5B,D,F). Longitudinal
sections of Prx1-Vegf E12.5 limbs stained for endothelial cells
demonstrated the absence of vascular stems at the interdigital areas
(Fig. 5G,H). Transverse
sections stained for endothelial cells and chondrocytes showed a decrease in
the number and diameter of vessels flanking the condensation areas of the
forming digits (Fig. 5I,J).
Similar to the control limbs, the vasculature regressed from the forming
condensations in Vegf-ablated limbs. These results demonstrate that
VEGF regulates vascular morphogenesis during limb vascular patterning.
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As Nrp1 and Nrp2 are functionality redundant, in order to directly examine the possibility that Nrp2 is involved in mediating Vegf signaling in mesenchymal cells, we blocked the expression of Nrp1 in limb mesenchyme of Nrp2-null embryos (Prx1-Nrp1, Nrp2). Examination of limb vasculature of E13.5 Prx1-Nrp1, Nrp2 embryos did not reveal any major abnormalities in vascular patterning, suggesting that in mesenchymal cells, Nrp1 and Nrp2 are not involved in the propagation of Vegf signaling to the limb vasculature (Fig. 6B). Although we failed to detect any expression of either Flt1 or Flk1 in limb mesenchyme, in order to exclude the possibility of sub-detectable, yet functionally significant, expression levels, we ablated Flt1 and Flk1 in limb mesenchyme. As expected, no major abnormalities were observed either in Prx1-Flt1 or in Prx1-Flk1 limbs (Fig. 6B). These results strongly imply that VEGF expressed by the condensation affects limb vasculature via long-range interactions.
SOX9 is involved in the regulation of Vegf expression in condensing mesenchyme
The expression of Vegf by the condensing mesenchyme raised the
hypothesis that SOX9 was involved in its regulation. This conjecture prompted
us to examine the expression of the Vegf-lacZ reporter in
Prx1-Sox9 limbs (Fig.
7). Vegf expression was indeed reduced, most prominently
at E12.5, when it could only be observed in a few cells located in the center
of the limb (Fig. 7A,B). To
further validate the possibility that SOX9 regulates Vegf in the
condensing mesenchyme, we used a high-density micromass culture as an in vitro
model (DeLise et al., 2000).
Micromass cultures derived from limb buds of floxed-Sox9
embryos were infected by either adeno-Cre virus (AdCre) to delete
Sox9, or with β-Gal-expressing adenovirus (AdβGal) as a
control. To assess the efficiency of Sox9 deletion by AdCre, we used
quantitative real-time PCR. The expression level of Sox9 in
AdCre-infected cells was reduced by 86% relative to the control cells,
suggesting an efficient blockage of Sox9
(Fig. 7C). Next, we examined
the expression of Vegf transcript, which was reduced by 70% compared
with control cells (Fig.
7D).
Finally, to determine whether or not SOX9 is sufficient to regulate the
expression of Vegf in limb mesenchyme, we used transgenic mice in
which Sox9 expression is under the control of the Prx1
regulatory sequence (Akiyama et al.,
2005), thereby ectopically expressed in limb mesenchyme. Next, we
examined the expression of Vegf in sections of E12.5
Sox9-misexpressing and control forelimbs using immunofluorescence
staining with anti-VEGF antibody. Our results show that Vegf
expression was maintained in the condensation areas of the future digits,
similar to its expression pattern in control limbs. No staining was observed
in areas of the limb that exhibited ectopic Sox9 misexpression
(Fig. 7E). In addition, we
examined vascular patterning in E12.5 Sox9-misexpressing forelimbs by
whole-mount immunofluorescence staining with anti-CD31 antibody. No major
changes were detected in vascular branching and morphogenesis of
Sox9-misexpressing limbs (Fig.
7F). These experiments emphasize SOX9 involvement in Vegf
expression; however, they also show that SOX9 is not sufficient to induce
Vegf expression in limb mesenchyme, suggesting its dependence on
other factor or factors that are localized to the condensation.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The skeleton serves as a signaling center for the patterning of limb vasculature
The coordination among bones, muscles, tendons, nerves and blood vessels in
the developing limb is needed for normal development and functionality.
Although much has been learned in recent years about the signals that
orchestrate limb patterning and morphogenesis, the mechanism that underlies
the specific patterning of limb vasculature and its coordination with other
tissues that compose the limb has remained poorly understood.
Several models can account for coordinated patterning of the skeleton and its vasculature. First, the developing skeleton may regulate vascular patterning; second, the developing vasculature may regulate skeletal patterning; and, third, both tissues may respond to common signals that originate in other tissues. This study provides direct evidence for the centrality of the skeleton in limb vasculature patterning. Once skeletogenesis is initiated, the limb vasculature undergoes an extensive rearrangement that involves two seemingly opposing processes. The first process is regression of vessels from the sites of condensation, which renders them avascular. Concurrently, the surrounding vasculature undergoes extensive morphogenesis to form a stereotypical, highly complex branched network (Fig. 1). However, in the absence of mesenchymal condensation and skeleton formation, both vessel regression and morphogenesis were lost (Fig. 2). These findings strongly support the active regulatory role of the limb skeleton in patterning limb vasculature. Moreover, these results imply the existence of a previously unappreciated signal from the forming skeleton to the endothelial cells of the limb vasculature. Hence, the forming skeleton serves as a signaling center that regulates limb vasculature.
Further support for the centrality of the skeleton as a regulator of limb
vasculature comes from studies on the involvement of the musculature in limb
vasculature. Although the musculature is, like the skeleton, a central
component of the limb, its absence does not have dramatic effect on limb
vascular development (De Angelis et al.,
1999).
The evolutionary driving force that selected the skeleton as a signaling center for limb vasculature is unclear. One plausible explanation for this selection lies in the mechanism of skeleton formation. As shown in this work and unlike numerous other tissues, mesenchymal condensations develop in an avascular environment (Fig. 1F,G). Thus, the formation of the skeleton dictates its segregation from the vasculature by regression of vessels from condensation areas. Yet, to ensure sufficient supply of nutrients and oxygen while it is segregated from the vasculature, the skeleton has adopted a mechanism that compensates for vessel regression by inducing vascular morphogenesis in its vicinity.
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), regulated the
expression of Sox9 in hypoxic prechondrogenic cells
(Amarilio et al., 2007;
Provot et al., 2007).
Although our results clearly demonstrate that the skeleton regulates
vascular patterning, we cannot exclude the possibility that the vasculature
has a reciprocal role in regulating skeletogenesis; in fact, we favor this
hypothesis. Previous studies demonstrating the important role of the
vasculature in skeleton development support this hypothesis
(Yin and Pacifici, 2001;
Fraser and Travill, 1978;
Feinberg and Saunders, 1982;
Hootnick et al., 1980). The
advantage of crossregulation between the forming skeleton and its vasculature
is higher levels of flexibility and subtle coordination between the tissues,
which are required for proper limb development and functionality.
VEGF expressed in condensing mesenchyme regulates limb vascular morphogenesis
The prevailing model for limb vascularization is based on three elements:
the inherent tendency of the endothelium to continuously divide and branch,
resulting in the formation of a dense vascular network; a shared response of
the limb endothelium and the mesenchymal cells to mitotic stimuli; and the
anti-angiogenic properties of the forming skeleton, which are responsible for
the formation of avascular regions (Belteki
et al., 2005; Caplan,
1985). The predicted outcome of this model would be either poor
vascularization in the vicinity of the condensation, assuming that the
anti-angiogenic signal is mediated by a soluble molecule, or an even
distribution of vessels outside the avascular condensation, if the signal is
strictly localized to the forming skeleton. One obvious problem with such a
model is the lack of a compensating mechanism that ensures sufficient supply
of nutrients and oxygen to the segregated avascular condensation. Now, our
results challenge this model, specifically the claim that vascular
morphogenesis is solely the result of the intrinsic property of endothelial
cells. Instead, we argue that, in addition to its anti-angiogenic signal, the
condensing mesenchyme in the forming limb produces a yet undescribed
proangiogenic signal.
The expression of Vegf, a key angiogenic regulator, in the
condensing mesenchyme suggests the involvement of VEGF in limb vasculature
patterning. Previous examples of VEGF involvement in vascular patterning have
been provided by studies on avian embryos, in which loss and gain of function
of Vegf resulted in severe alteration in the patterning of the
vascular plexus (Drake, 1995;
Drake, 2000). The alterations
in limb vasculature we witnessed in both loss- and gain-of-function
experiments on Vegf in limb mesenchyme strongly supported this
hypothesis (Figs 4 and
5). Interestingly, the
expression of Vegf in the condensing mesenchyme was temporal: once
the vasculature has become patterned and the mesenchymal cells have
differentiated to chondrocytes, Vegf expression is reduced and can
only be observed in the forming perichondrium and joints
(Fig. 3C,D). The expression of
Vegf in differentiated chondrocytes will be elevated again later in
development, in order to ensure the invasion of blood vessels during
endochondral bone formation (Zelzer et
al., 2004; Zelzer et al.,
2002).
The mechanism that regulates the spatial and temporal expression of Vegf in the condensing mesenchyme remains unidentified. Although it is clear that SOX9 plays a role in Vegf expression, the fact that Sox9 expression, in contrast to Vegf expression, is maintained in differentiated chondrocytes clearly indicates the involvement of additional transcriptional components in Vegf regulation. The ability of SOX9 to drive the expression of Vegf only in the condensing mesenchyme (Fig. 7) further supports this notion. Nevertheless, our finding that SOX9, an essential factor of mesenchymal condensation, is involved in the regulation of Vegf expression implies that the genetic program that controls the initial stage in skeletogenesis also regulates vascular development, thus ensuring a tight coordination in the development of both systems.
During organogenesis, the adaptation of the vasculature to the growing
demands of the developing organ for oxygen and nutrient supply is crucial.
Organs such as lung, liver, kidney and pancreas develop with an embedded
vasculature (Cleaver and Melton,
2003; Coultas et al.,
2005), suggesting that the mechanism that synchronizes their
development with the vasculature is based on intimate cellular interaction.
Vegf expression in the avascularized condensing mesenchyme represents
a different mode of vascular regulation by VEGF. Based on the well-established
ability of VEGF to induce the formation and recruitment of blood vessels
(Carmeliet, 2005;
Ferrara, 2004), it was
expected that sites with the highest level of Vegf expression would
be mostly enriched with blood vessels. The expression of Vegf in
condensed mesenchyme, several rows of cells away from the flanking
vasculature, implies that VEGF-mediated regulation of vascular morphogenesis
may operate either via a relay mechanism, or as a direct long-range mechanism.
The apparently patterned vasculature in limbs where the expression of VEGF
receptors was blocked in mesenchyme (Fig.
6B) rules out the possibility of a relay mechanism and favors a
long-range direct regulation of VEGF on limb vasculature.
Long-range regulation of vascular patterning by VEGF has previously been
demonstrated in the formation of the perineural vascular plexus (PNVP) that
encompasses the neural tube. Vegf expression in the neural tube
induced the migration and assembly of presomitic mesoderm angioblasts of the
PNVP (Hogan et al., 2004).
Another example for a long-range regulation by VEGF was given by experiments
that used VEGF-coated beads, in which vessel formation was also observed in
the vicinity of the planted beads (Bates et
al., 2003; Finkelstein and
Poole, 2003). A possible mechanistic explanation for the
long-range effect of VEGF is based on the formation of several isoforms of
VEGF, which exhibit different diffusion properties (VEGF120, VEGF164 and
VEGF188) (Ferrara and Davis-Smyth,
1997; Ferrara et al.,
1992; Park et al.,
1993; Shima et al.,
1996). Vascular abnormalities in limbs of mice expressing only the
VEGF120 isoform (Ruhrberg et al.,
2002; Vieira et al.,
2007), as well as our observation that all three isoforms of VEGF
are expressed in E12.5 limbs (data not shown) raise the hypothesis that some
aspects of VEGF regulation of limb vasculature are mediated by the different
isoforms.
The mechanism of vessel regression and segregation from mesenchymal condensations remains largely unknown. Our finding that this process proceeded normally in limbs where Vegf was either depleted (Fig. 5) or overexpressed (Fig. 4) indicates that VEGF has no role in the mechanism that underlies this phenomenon. Moreover, these experiments indicate that the process of vessel regression and segregation is not coupled with vessel morphogenesis.
During organogenesis, it is cardinal that the vasculature accommodates the growing metabolic needs of the developing organ. Interestingly and contra-intuitively, several organs, including the skeleton, develop in the absence of embedded vasculature. The segregation between the forming skeleton and its vasculature requires the existence of a genetic program that would synchronize organ development with its non-embedded vasculature. In this manuscript, we suggest a paradigm for a mechanism that allows for the coordination of skeleton development and vascular patterning by establishing the skeleton as a signaling center that regulates limb vasculature.
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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