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First published online January 25, 2008
doi: 10.1242/10.1242/dev.013623
1 Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake
City, UT 84112, USA.
2 Division of Cardiology, Department of Internal Medicine, University of Utah,
Salt Lake City, UT 84112, USA.
3 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT
84112, USA.
4 Department of Oncological Sciences, University of Utah, Salt Lake City, UT
84112, USA.
5 Division of Hematology, Department of Internal Medicine, University of Utah,
Salt Lake City, UT 84112, USA.
6 Brain Institute, University of Utah, Salt Lake City, UT 84112, USA.
Authors for correspondence (e-mails:
chi-bin.chien{at}neuro.utah.edu;
dean.li{at}hmbg.utah.edu)
Accepted 21 November 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Angiogenesis, Netrin, Placenta, UNC5B, Zebrafish, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Serafini et al., 1994).
Reports from our group and others have shown that netrins can also play a
similar role in the vasculature, promoting developmental and therapeutic
angiogenesis in a number of in vitro and in vivo systems
(Nguyen and Cai, 2006;
Park et al., 2004;
Wilson et al., 2006).
Netrin signaling is complex, however, and is not always
attractive/stimulatory. In mammals there are three netrins and at least eight
potential netrin receptors: DCC, neogenin, UNC5A-D, and 
6β4 and
3β4 integrins (Cirulli and
Yebra, 2007; Huber et al.,
2003; Tessier-Lavigne and
Goodman, 1996; Yebra et al.,
2003). Additional netrin receptors have been postulated, and
efforts to identify them are ongoing. Although netrins are the prototypic
neural attractants, they can also act as repulsive guidance signals under the
appropriate cellular context. One receptor, UNC5B, was identified as a
mediator of the repulsive response in neurons
(Leonardo et al., 1997), and
it is the only known netrin receptor with prominent endothelial expression
(Engelkamp, 2002;
Lu et al., 2004).
Unc5b is expressed in the endothelium of developing mouse embryos
beginning at embryonic day 8.5 (Engelkamp,
2002; Lu et al.,
2004). A published study concluded that the axon-repulsive
activity mediated by UNC5B is mirrored by an anti-angiogenic role for netrin
1-UNC5B signaling in both mice and zebrafish
(Lu et al., 2004). This
conclusion was based on observations that the mutation of Unc5b
resulted in increased sprouting within developing arterial beds, which was
interpreted as an indicator of reduced repulsion. This report also postulated
that the excessive vascular sprouting caused by the global absence of UNC5B
during murine embryogenesis precipitated greater resistance to circulation,
resulting in heart failure and fetal demise.
In contrast to the conclusions of Lu and colleagues, our previous analysis
of netrin signaling in the vasculature
(Park et al., 2004;
Wilson et al., 2006) suggested
a starkly different situation. We found that the addition of netrins
stimulated angiogenesis both in vivo and in vitro, and that the knock-down of
netrin1a in zebrafish inhibited growth of the parachordal vessel
(PAV), a blood vessel that has recently been found essential for lymphatic
development (Yaniv et al.,
2006).
In an attempt to resolve these different interpretations, we have made use of non-invasive imaging technologies to examine mice carrying a conditional mutant allele of the Unc5b gene. Although the vascular-restricted deletion of Unc5b indeed causes embryonic lethality, we could find no evidence of either low or high-output heart failure prior to abrupt death at embryonic day 12. Instead, our data showed a previously unappreciated and essential role for UNC5B in promoting placental arteriogenesis. In zebrafish we found a similar pro-angiogenic role, where knocking down unc5b, like knocking down netrin1a, prevents formation of the PAV.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
R26R1, Tie2-Cre, Vav1-Cre and Netrin1:lacZ mice were
generously provided by Phil Soriano, Masashi Yanagisawa, Matthias Stadtfeld
and Lindsay Hinck, respectively. SM22-Cre (Tagln-Cre),
Nestin-Cre and Wnt1-Cre mice were obtained from The Jackson
Laboratory. Genotypes were determined by PCR analysis of genomic DNA isolated
from either ear biopsies or yolk sacs; amplification used primers and
conditions previously described for Tie2-Cre
(Kisanuki et al., 2001),
SM22-Cre (Holtwick et al.,
2002), Nestin-Cre
(Sclafani et al., 2006),
Vav1-Cre (Stadtfeld and Graf,
2005) and Rosa26
(Soriano, 1999). For
Wnt1-Cre, the primers were 5'-AGCAACCACAGTCGTCAGAACC and
5'-AAGATAATCGCGAACATCTTCAGG with amplification of 94°C for 30
seconds, 58°C for 30 seconds, and 72°C for 40 seconds. Unc5b
mice were genotyped with primers: 5'-TAGCCTCAGGGTCTACTGTCTG;
5'-CTCTCAGACTTCTCAAAGAGATTC; and 5'-CCACTGTATGCCAGACGACATG under
conditions of 94°C for 30 seconds, 62°C for 20 seconds and 68°C
for 40 seconds.
Histology
Smooth muscle cells were identified by anti--smooth muscle actin
antibody (Sigma) staining of formaldehyde-fixed, paraffin-embedded tissue
sectioned at 10 µm as described previously
(Urness et al., 2000). The
number of arterioles in the labyrinth was determined by examination of all
serial sections from a placental hemisphere. Wild-type and mutant placentas
were from paired littermates. β-Galactosidase staining was performed
using a standard protocol (Soriano,
1999) with the following modification: the tissues were fixed in
PBS containing 2% formaldehyde, 0.2% glutaraldehyde, 0.02% sodium deoxycholate
and 0.01% NP-40 for 10-30 minutes. Placentas were hemisected with a razor
blade before fixation. The tissues were permeabilized in PBS containing 0.02%
sodium deoxycholate and 0.1% NP-40 overnight at 4°C before staining with
X-gal.
Staining for PECAM1 was performed on tissues fixed overnight in Dent's
fixative (4:1 MEOH: DMSO) at 4°C, followed by bleaching by addition of an
equal volume of 37%H2O2 for 6-10 hours at room
temperature, and storage at -20°C in 100% methanol. Placentas were
rehydrated to PBS, hemisected sagittally at the umbilical cord,
blocked/permeabilized for 3 hours in PBSMT (PBS+ 0.5% Triton X-100 +2% nonfat
milk). Following the addition of 5 µg/ml rat anti-mouse PECAM1 antibody
(Pharmingen), samples were incubated overnight at 4°C. Tissues were washed
six times in PBSMT and incubated overnight in PBSMT+1:50 anti-rat Ig
HRP-conjugate (Pharmingen). HRP staining was performed as previously described
(Urness et al., 2000). Tissues
were postfixed in 4% paraformaldehyde overnight at 4°C, cleared in 80%
glycerol and photographed on a Leica MZ12 stereoscope equipped with a Zeiss
Axiocam digital camera.
Hypoxia assay
Pregnant females (30 g) were injected IP with 200 ml of pimonidazole
(`Hypoxyprobe' 10 mg/ml in PBS, Chemicon). After 2 hours, mice were killed by
cervical dislocation, the embryos were dissected into 4% formaldehyde and the
yolk sacs removed for genotyping. Following 16 hours fixation, embryos were
serially dehydrated in ethanol, followed by xylene, embedded in paraffin and
stored at 4°C. Sections (10 µm) were mounted on glass slides and
stained with monoclonal antibodies directed against pimonidazole under
conditions provided by the manufacturer. Primary antibody was diluted
200-fold; peroxidase staining for the secondary antibody was for 2 minutes at
room temperature. Sections were counterstained with Mayer's hematoxylin prior
to photography.
mRNA detection
Whole-mount in situ hybridization was performed as described previously
(Urness et al., 2000). Embryos
were harvested at E11.5. Decidual tissue was trimmed away and yolk sac
fragments were collected from each embryo for genotyping. Placentas were fixed
overnight at 4°C in 4% formaldehyde, pH 7.4. Antisense digoxigenin-labeled
riboprobe was generated from mouse Unc5b DNA using a DIG RNA Labeling
Kit (Roche). In situ hybridization was performed overnight at 65°C with
rotation in a Hybaid hybridization oven. All comparisons were between sibling
pairs.
Tetraploid aggregation
Eight-week old C57Bl6/J mice were superovulated with 5 IU pregnant mares'
serum gonadotropin followed after 48 hours with 5 IU human chorionic
gonatotropin (hCG); mating with C57Bl6/J males immediately followed the hCG
injection. Two-cell stage embryos were flushed at 1.5 days post plug, washed
through two drops of M2 medium, four drops of mannitol and fused in mannitol
using a Biological Laboratory Equipment BLS CF-150/B Cell Fusion apparatus.
Settings were as follows: 40 V, 40 mseconds, enable 1.0. Following fusion,
embryos were washed through three drops M2, three drops KSOM (Chemicon) and
incubated in a 37°C CO2 incubator. Most two-cell embryos fused
to one cell within 10 minutes. Fused embryos were incubated in KSOM under oil
overnight. Morulae (2.5-day old) were flushed from superovulated
Unc5b+/- mice (mated with Unc5b+/-
males), washed through Tyrode's (Sigma) to remove the zona pellucida, then
washed through three drops M2 medium and three drops KSOM medium. One morula
and one fused tetraploid embryo (now four-cell stage) were transferred into
each miniwell of KSOM under oil, and incubated overnight. After 16 hours, all
miniwells contained one fused blastocyst, which was implanted into 2.5-day
pseudopregnant females (C57Bl6/J x CBA F1). Embryos were dissected and
analyzed at E13.5.
Echocardiography
Embryos were analyzed and imaged noninvasively in utero using ultrasound
biomicroscopy (UBS, Visualsonic Vevo 660) with a 40 MHz transducer and
image-guided 23 MHz spectral pulsed-wave (PW) Doppler. Heart rate, blood flow
velocities and blood flow volumes were determined from pulsed Doppler
waveforms. During scanning, maternal body temperature and heart rate were
maintained within normal range, and the duration of anesthesia was less than
1.5 hours (Mu and Adamson,
2006). The hemodynamics of embryos were measured every 6 hours for
36 hours, by which time all of the Unc5b-deficient embryos would have
died. During each scan, the bladder was used as a reference for the midpoint
between the left horn and the right horn of the fallopian uterus, and the
relative location of embryos was mapped within the abdomen. After scanning at
the final timepoint, a laparotomy was performed and the embryos were scanned a
final time to correlate the order of embryo location by ultrasound with
anatomic position. The genotype of embryos from previous scans was deduced
from the location of embryos relative to each other, especially relative to
the location of dead embryos. To confirm correlation between flow reversal and
mutant genotype, a number of litters were scanned only until embryos with
reversed diastolic flow in the umbilical artery were identified, at which time
laparotomy was performed, anatomic correlation was established, and the
embryos genotyped.
Umbilical vessel angiogenesis assays
Quantitative three-dimensional umbilical vessel angiogenesis assays were
performed according to published methods
(Nicosia et al., 2005) with
minor modifications. Umbilical cord sections from murine embryos from
Unc5b+/- x Unc5b+/- matings were
harvested at E10.5. Immediately prior to harvest, three-dimensional collagen
gels (0.25 ml/well) consisting of 2 mg/ml purified rat tail collagen I
(Trevigen), 2.34 mg/ml NaHCO3 in 2x concentrated EBM-2
(Lonza) medium supplemented with 1 µg/ml ascorbic acid and 0.2 µg/ml
hydrocortisone with or without 100 ng/ml rmNetrin-1 (R&D Systems) were
cast into the center wells of four-well chambered coverglasses (Nunclon) and
stored on blue ice blocks to delay polymerization. Each embryo was sterilely
dissected in the supplemented EBM-2 medium. A 0.5 mm fragment of yolk sac was
retained in lysis buffer (50 mM KCl, 2.5 mM MgCl2, 10 mM Tris-HCl
pH 8.5, 0.005% NP-40, 0.05% Tween 20, 0.01% gelatin) for PCR genotyping, and a
1.5 mm section of umbilical vessel (equidistant from placenta and embryo) was
dissected and washed in EBM-2 (supplemented as above) prior to
plating/polymerizing into the collagen gel. Gels with umbilical vessel
explants were polymerized for 30 minutes at 37°C/5% CO2, and
subsequently overlaid with 0.25 ml of the supplemented EBM-2 medium. The
following morning the medium was exchanged, and the explants photographed in
phase-contrast on a Zeiss Axiovert inverted microscope equipped with a Zeiss
Axiocam digital camera to confirm location and condition of the umbilical
vessel explants. Thereafter, the medium was exchanged every 3-4 days, and the
cultures were re-photographed on the tenth day of culture. The capillary
outgrowths observed around the perimeter of each umbilical vessel photograph
were counted, and data presented as the average number of capillary outgrowths
per umbilical vessel perimeter. The experiment was performed six times on a
total of 36 wild-type and 32 mutant siblings.
Embryo raising and MO injection experiments
Heterozygous Tg(fli1:egfp)y1 transgenic carriers
(Lawson and Weinstein, 2002)
were incrossed, and embryos were raised at 28.5°C in E2/GN embryo medium
with 0.003% phenylthiourea to inhibit pigment formation and staged by time and
overall morphology (Kimmel, 1995). Unc5b morphants typically reached
the 48 hpf stage 1-2 hours after controls. Three MOs were obtained from Gene
Tools: control MO (`standard control'),
5'-CCTCTTACCTCAGTTACAATTTATA-3'; unc5bSBMO1
(Lu et al., 2004),
5'-CATTTAACCGGCTCGTACCTGCATG-3', which binds to 7 bp of exon 1 and
18 bp of intron 1; and unc5bSBMO2,
5'-AGGAAGACAATACAGCACCTCAGCA-3', which binds to 7 bp of exon 4 and
18 bp of intron 4. MOs were diluted, stored and injected at 1 nl nominal
volume as described previously (Wilson et
al., 2006). RT-PCR was carried out as described previously
(Wilson et al., 2006); primer
sequences available upon request.
Microscopy and image analysis
fli:egfp-positive embryos were either imaged live or fixed and
stained with anti-GFP before imaging
(Wilson et al., 2006).
Briefly, embryos were mounted laterally in agarose, right side down and
confocal z-stacks were taken at the level of somites 7-12.
Z-stacks were used to score the presence of PAVs in four to six
hemisegments. We modified our analysis slightly by scoring only the near
(right) side of the trunk, which could be imaged most clearly; and by
calculating in each embryo the fraction of hemisegments with absent, partial
or complete PAVs, then averaging these fractions across embryos. Statistical
analysis used two-tailed Mann-Whitney tests (Instat3, Graphpad).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) that can
be inactivated, conditionally, through tissue-restricted expression of CRE
recombinase. This allele, Unc5bflox, contains
loxP sites within introns 3 and 13. CRE-mediated deletion of the
intervening sequences removes much of the ligand-binding domain, the entire
transmembrane domain, and most of the cytoplasmic signaling domain of the
mature protein, generating a presumed null allele. To reduce the chance of
cis-acting effects, the selectable marker gene used in construction of this
allele was removed prior to analysis. We also engineered mice that transmit
the deletion allele, Unc5b-, through the germline. Mice
homozygous for this mutation die in utero between 12 and 13.5 days
post-fertilization (E12-E13.5). Our preliminary phenotypic characterization of
Unc5b-/- embryos has been described
(Wilson et al., 2006) and,
surprisingly, it revealed none of the excessive vascular sprouting described
by Lu et al. (Lu et al.,
2004).
To systematically inactivate Unc5b in a tissue-specific fashion,
animals homozygous for the conditional allele
(Unc5bflox/flox) were mated with animals heterozygous for
the Unc5b null allele (Unc5b+/-) and carrying one
of a variety of Cre genes under tissue-restricted control. Deletion
of Unc5b in neural crest-derived tissues [Wnt1-Cre
(Jiang et al., 2000)], smooth
and cardiac muscle [Tag1n-Cre
(Holtwick et al., 2002)] in the
nervous system [Nestin1-Cre
(Sclafani et al., 2006)] or in
hematopoietic lineages [Vav1-Cre
(Stadtfeld and Graf, 2005)]
resulted in mice that survived to adulthood and that appeared normal
(Table 1 and data not shown).
When Unc5b was deleted by an endothelial-expressed Cre
transgene, Tie2-Cre (Kisanuki et
al., 2001), however, no animals of the genotype
Unc5b-/flox; +/Tg(Tie2-Cre), were ever recovered
at birth. Embryos of this genotype died in mid-gestation between 12 and 13.5
days post-fertilization and were indistinguishable from homozygous null
Unc5b-/- embryos (data not shown). The implication from
these results is that vascular expression, and only vascular expression, of
Unc5b is required for viability.
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), and Tie2-Cre is expressed in these relevant
tissues (Kisanuki et al.,
2001). Histological analyses of E12.5 embryos revealed no
differences in heart structure or in the morphology of coronary vessels
between Unc5b+/flox; +/Tg(Tie2-Cre) and
Unc5b-/flox; +/Tg(Tie2-Cre) embryos; blood counts
were equivalent, as was the profile of circulating cells; and the vasculature
of the somites, neural tube, yolk sac and hindbrain were indistinguishable
(data not shown). The only discernible difference between the two genotypes
was within the placental labyrinth: fetal-derived arterioles, visualized
either by smooth muscle or endothelial specific stains, were less numerous in
embryos lacking vascular Unc5b
(Fig. 1).
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E12.0, significant and reversed blood flow in diastole within the
umbilical arteries of mutant embryos could be detected in some embryos
(Fig. 2A,
Table 2A). As pregnancy
proceeded, the number of embryos with abnormal flow increased, as did the
degree of flow reversal (e.g. -22% to -91%,
Fig. 2A). By E13.5, all embryos
that had exhibited reversed flow were dead. In another set of identical
crosses, embryos were probed at a single time point, E12.5, and subsequently
killed and genotyped (Fig.
2B,C). There was a strong genotype/phenotype correlation between
the vascular deletion of Unc5b and flow reversal. Umbilical artery
diastolic flow reversal is a clinical sign of fetal distress and is consistent
with an abnormally high resistance within the placenta, a plausible
consequence of the reduced number of arterioles. Consistent with this
interpretation, physiological and anatomical changes in cardiac function, such
as bradycardia and pericardial effusion, were only detected after the onset of
umbilical arterial flow reversal and immediately prior to death
(Fig. 2A) but not earlier
(Fig. 2C).
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). At
12.5 days after fertilization, pregnant females were injected with
pimonidazole. Two hours later, embryos were removed and apparently viable,
outwardly normal, embryos were fixed and sectioned. Bound pimonidazole was
then assessed by immunostaining. At E12.5, the majority (4/5) of
Unc5b-/- embryos exhibited a higher level of pimonidazole
immunoreactivity than did their wild-type counterparts
(Fig. 2D,E), suggestive of a
hypoxic environment and implying a direct link between altered umbilical flow
and ultimate death.
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;
Cross et al., 2003;
Rinkenberger and Werb, 2000;
Rossant and Cross, 2001). To
understand more specifically the requirement for Unc5b, we examined
its gene expression in the placenta. Sections of E12.0 placentas of genotypes
Unc5b+/flox; +/Tg(Tie2-Cre); and
Unc5b-/flox; +/Tg(Tie2-Cre) were probed with RNA
complementary to Unc5b mRNA. As shown in
Fig. 3A,B, binding of this
probe was restricted to the labyrinth but could not be detected in placentas
in which Unc5b had been deleted by endothelial-expressed CRE
(Fig. 3A,B). Thus, placental
expression of UNC5B is limited to the fetal-derived endothelium. These data
suggest a model whereby UNC5B-expressing endothelial cells within the
labyrinth respond to signals emanating from the trophectoderm and direct
proper vascular development. Consistent with this idea, expression of the
secreted guidance factor, netrin 1, an UNC5B ligand, can be detected in the
trophoblast giant cells (Fig.
3C,D). Although no placental phenotype has been described in mice
deficient for netrin 1 (Salminen et al.,
2000; Serafini et al.,
1996), the mutations analyzed in those studies were hypomorphic
alleles that synthesize detectable levels of wild-type transcript.
This model also predicts that wild-type trophoblast cells should be unable
to rescue embryos deficient in Unc5b
(Nagy et al., 1993). To test
this prediction, we aggregated diploid morulae from an
Unc5b+/- intercross with tetraploid, wild-type, two-cell
stage embryos that can contribute only to the extra-embryonic tissues
(Nagy et al., 1993). Fused
embryos were implanted into foster mothers, harvested at E13.5 and examined
for viability and genotype. As summarized in
Table 2B, all wild-type and
heterozygous embryos were viable, whereas 11/12 Unc5b-/-
embryos were dead. Examination by PCR of genomic DNA isolated from
extra-embryonic tissue of the Unc5b-/- embryos showed a
significant contribution from wild-type (tetraploid) cells (data not shown).
The fact that these cells were incapable of supporting an Unc5b
mutant embryo implies that the deficiencies resulting from this genotype are
within the embryo proper.
Evidence that UNC5B is active within the fetal-placental vasculature was provided by examining the growth of isolated umbilical arteries in vitro. When cultured on a collagen matrix in the absence of serum, umbilical arterial explants isolated from Unc5b+/+ embryos support a more vigorous netrin 1-dependent outgrowth than do those isolated from their Unc5b-/- littermates (Fig. 3E,F).
Knock-down of Unc5b inhibits PAV formation in zebrafish
The role of netrin signaling in zebrafish vascular development is not
without controversy. During development of the embryonic trunk vasculature,
the zebrafish netrin 1 ortholog, netrin1a, is expressed at the
horizontal myoseptum (Lauderdale et al.,
1997; Wilson et al.,
2006), precisely where secondary sprouts growing dorsally from the
posterior cardinal vein (PCV) grow laterally and turn anteroposteriorly to
form the parachordal vessel (PAV). It has been reported that knocking down
either netrin1a or unc5b using antisense morpholino
oligonucleotides (MOs) disrupts intersomitic vessel (ISV) formation and leads
to excess vessel branching (Lu et al.,
2004). We found, however, that a carefully titrated dose of a
splice-blocking MO against netrin1a led to a highly penetrant
phenotype in which the PAV failed to form
(Wilson et al., 2006). ISVs
are only very rarely affected at this dose, and overall trunk morphology
appears normal, unlike that seen at higher doses (A.S. and C.-B.C.,
unpublished).
We therefore reexamined formation of the trunk vasculature after
unc5b knockdown, injecting two different MOs against unc5b:
unc5bSBMO1 [identical to that used by Lu et al.
(Lu et al., 2004)] and
unc5bSBMO2, and using the fli:egfp transgene to visualize
endothelial cells (Fig. 4). In
preliminary dose-response experiments, we chose MO doses (1 ng
unc5bSBMO1 or 4 ng unc5bSBMO2) that yielded embryos with
normal trunk morphology (Fig.
4A-D). This was crucial as, even at moderate doses (1.5 ng),
unc5bSBMO1 caused gross morphological defects (strongly curved tails,
hindbrain edema and small eyes), which may reflect off-target effects. At
these doses, both MOs were effective, as shown by significant reductions in
wild-type unc5b mRNA detected by RT-PCR (see Fig. S1 in the
supplementary material). Although development of the overall vasculature was
normal, including the dorsal aorta, PCV and ISVs, we saw a specific, highly
penetrant effect on PAV formation (Fig.
4E-H). PAVs normally form by 36 hpf. To avoid potential
confounding effects of mild developmental delay, we scored PAVs at 48 hpf. The
fraction of hemisegments that completely lacked a PAV increased from
1±1% to 46±6% (mean±s.e.m., P<0.0001) with
unc5bSBMO1, and from 2±2% to 70±7%
(P<0.0001) with unc5bSBMO2
(Fig. 4I,J). The concordant
results with two nonoverlapping MOs strongly suggests that the lack of PAVs is
specifically due to loss of unc5b function, rather than an off-target
effect. Given the expression of netrin1a at the horizontal myoseptum,
the most parsimonious explanation is that Ntn1a-Unc5b signaling is
pro-angiogenic in this system.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), we did not analyze higher doses because of morphological
defects that were potentially nonspecific. This issue would be best addressed
by isolating mutant alleles for unc5b. Furthermore, we note that the
morphant images of Lu et al. (Lu et al.,
2004) appear to show missing PAVs in some segments; in other
segments, secondary sprouts appear to turn normally to form the PAV, but have
been interpreted as increased vessel branching. Together with the observations
that the PAV forms along the muscle pioneer cells that line the horizontal
myoseptum and express netrin1a, and that netrin1a morphants
have very similar phenotypes to unc5b morphants
(Wilson et al., 2006), these
data support an important pro-angiogenic role for Ntn1a/Unc5b signaling in
zebrafish lymphatic development.
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), guidance cues in general and UNC5B specifically, may
provide an additional level of regulation to coordinate the spatial and
temporal organization of tissue-specific vascular beds during embryogenesis.
Because the first vital requirement for UNC5B is during mid-gestation, the
reagents used in this study were unable to ascertain a function in later
stages of development or within the adult. Such an assessment requires the use
of alternative mutagenesis schemes, which we are vigorously pursuing.
A role for UNC5B in embryonic angiogenesis was originally postulated
because of its vascular-specific expression pattern and was supported by the
characterization of a mouse mutant in which exons 3 and 4 of Unc5b
were replaced with 8 kb of exogenous DNA encoding the lacZ and
alkaline phosphatase genes (Lu et al.,
2004). Although embryos homozygous for that allele died at the
same time as those containing the exon3-13 deletion allele described in our
previous work (Wilson et al.,
2006) and in this manuscript, they were characterized as having
increased capillary branching in the embryo proper, which the authors
hypothesized would raise vascular resistance and precipitate heart failure.
However, this study did not report physiological measurements of embryonic
cardiac function or any analyses - functional or morphological - of the
placenta. It would be interesting to re-examine this allele in the light of
our present findings.
The placenta is one of the more complex vascularized tissues in mammals. It
is derived from three distinct sources: maternal tissue, extra-embryonic fetal
tissue, and arteries and veins originating from the embryo proper. The
fetal-placental vascular system circulates fetal blood and interdigitates with
the trophoblast sinuses filled with maternal blood
(Cross, 2005;
Red-Horse et al., 2004).
Assembly of the vasculature within the placental labyrinth requires signaling
between the extra-embryonic tissue and the fetal-placental vessels. Mutational
analysis has revealed dozens of genes required for this communication, with
virtually all reports concluding that these genes exert their effects through
the extra-embryonic trophectoderm (Cross,
2005; Rossant and Cross,
2001). In fact, patterning by the trophoblast cells is deemed an
essential guide for proper growth of the fetal vessels into and within the
labyrinth layer. The specific and autonomous role of Unc5b in
promoting fetal-placental arteriogenesis emphasizes that vascular signaling
pathways on the fetal-placental side of the equation should not be ignored. As
the UNC5B-deficient phenotype could not be rescued by a wild-type
trophectoderm, we propose that UNC5B-mediated signaling is a specific and
autonomous component of fetal-placental angiogenesis, and that Unc5b
disruption represents a rare, if not the first, example of a mutation acting
solely with the fetal placental vasculature.
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;
Mayhew et al., 2007;
Redman and Sargent, 2003).
Recent studies have assigned a role for maternal angiogenic signaling pathways
in pre-eclampsia (Levine et al.,
2006; Wallner et al.,
2007). The findings presented herein provide genetic evidence that
defects in netrin/UNC5B signaling, which disrupts the fetal contribution to
the placental vasculature, should be considered in the pathogenesis of
clinical syndromes of uteroplacental insufficiency
(Cross, 2003;
Levine et al., 2006;
Wallner et al., 2007).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/4/659/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Pathology and Laboratories Medicine, UCLA,
Los Angeles, CA 90095, USA
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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