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First published online 27 February 2008
doi: 10.1242/dev.010504
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1 Pediatric Surgical Research Laboratories, Department of Surgery, Massachusetts
General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA
02114, USA.
2 Department of Developmental Biology, Stanford University School of Medicine,
Stanford, CA 94305, USA.
3 Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur,
Boston, MA 02115, USA.
4 Department of Pathology, Massachusetts General Hospital, Harvard Medical
School, Boston, MA 02114, USA.
* Author for correspondence (e-mail: djroberts{at}partners.org)
Accepted 29 January 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Lung development, Wnt5a, Shh, Fibronectin, Ror2, VEGF, Flk1
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Their synchronization is necessary for effective gas
transfer, and maldevelopment of either is frequently lethal
(Berrocal et al., 2004;
Cullinane et al., 1992;
deMello, 2004;
Galambos and deMello, 2007).
Although there have been many studies of airway development (for reviews, see
Cardoso, 2001;
Costa et al., 2001;
Kimura and Deutsch, 2007),
relatively little is known about pulmonary vascular pattern formation or the
coordinated development of the two.
Although the anatomy of the avian lung differs from that of the mammalian
lung, both develop similarly and have anatomical functional equivalents. The
avian lung forms by a series of closed circular buds arising from the main
airway branches, which differs from the dichotomous branching morphogenesis in
mammalian lung development. In contrast to the mammalian lung, which
terminates in alveoli, the avian lung forms a looping anastomotic network of
air-vascular surfaces (parabronchi) that end in terminal air buds and air
capillaries. For all vertebrates, pulmonary vascular patterning continues in
coordination with airway development. In the chick embryo, vasculogenesis is
the main process by which the pulmonary vasculature initially forms, being
guided by the budding airways
(Anderson-Berry et al., 2005;
Hislop, 2005). This
interstitial microvasculature connects the large pulmonary vessels to the
terminal buds of the airways and the capillaries via two mechanisms, sprouting
and intussusceptive angiogenesis (Makanya
et al., 2007). Sprouting angiogenesis predominates from early to
mid-gestation (E15), and is the major mechanism for setting up the basic
hexagonal interstitial vascular pattern seen in avians. Intussusceptive
angiogenesis, a novel process involving endothelial cell extension into the
lumen of a vessel (splitting the vessel), predominates from 
E15,
producing the massive expansion of the vasculature that is necessary for vital
gas exchange (alveologenesis). Intussusceptive angiogenesis occurs
simultaneously with the rapid growth of the airway epithelium into the
mesenchyme to create the huge surface area of the mature lung. After E18, the
air capillaries and the blood capillaries are in close proximity, forming the
functional equivalent of the mammalian alveolus (for a review, see
Maina, 2006). The chick embryo
is fully air breathing by E18, 2-3 days before hatching. Airway and vascular
channels, epithelial and endothelial tubules, are the fundamental structural
units of the lung (Cardoso and Lu,
2006), and develop in response to morphogenetic growth factors,
transcription factors, extracellular matrix proteins and their receptors
(Hogan and Kolodziej, 2002).
Although much is known about the factors regulating airway development, those
controlling this coordinated air and vascular patterning are unknown.
The Wnt family of secreted signaling molecules functions in numerous key
developmental events (Wodarz and Nusse,
1998). Wnts are broadly categorized into two groups based on their
signal transduction pathway. Canonical Wnts transduce their signals through
receptors of the frizzled (Fz) family by a β-catenin-dependent pathway
(Wodarz and Nusse, 1998).
Non-canonical Wnts signal via either the planar cell polarity (PCP) pathway or
the Wnt/Ca2+ pathway (Veeman et
al., 2003; Widelitz,
2005), and may use Fz receptors or other receptors, including the
orphan tyrosine kinase Ror2 (Keeble et
al., 2006; Oishi et al.,
2003). The Wnt signaling pathway has been well described and
includes numerous regulators (Pandur et
al., 2002; Widelitz,
2005). Most described Wnt antagonists interfere with the canonical
pathway, such as the Dickkopf (Dkk) proteins, of which Dkk1 specifically
interrupts only the canonical-Wnt function
(Glinka et al., 1998;
Kawano and Kypta, 2003;
Niehrs, 2006).
Whereas canonical Wnt signaling is known to regulate lung development early
in branching morphogenesis (Dean et al.,
2005; Mucenski et al.,
2005; Okubo and Hogan,
2004), the non-canonical Wnts (such as Wnt5a) appear to act later
(Li et al., 2005). Wnt5a
moderates many cellular events, including cell adhesion
(Jonsson and Andersson, 2001;
Torres et al., 1996;
Toyofuku et al., 2000;
Weeraratna et al., 2002),
migration (Jonsson and Andersson,
2001), proliferation (Liang et
al., 2003; Yamaguchi et al.,
1999) and differentiation (He
et al, 1997; Kuhl et al.,
2001; Kuhl et al.,
2000; Sheldahl et al.,
1999). These findings suggest that Wnt5a is a good candidate for
coordinating pulmonary air and vascular pattern formation.
One mechanism by which Wnt5a might regulate these cellular events is by
affecting the extracellular matrix, a component of which is fibronectin.
Fibronectin has been implicated in branching morphogenesis
(Roman and McDonald, 1992;
Sakai et al., 2003),
vasculogenesis (Bull et al.,
1993; Hall et al.,
2000; Jozaki et al.,
1990) and intussusceptive angiogenesis
(Makanya et al., 2007), and it
interacts with VEGF (Vascular Endothelial Growth Factor) (Goerges and Nugent,
2004), which is also implicated in lung vascular and airway branching
morphogenesis (Warburton et al.,
2005). Although it is known that canonical Wnt signaling decreases
fibronectin expression in murine lung development
(De Langhe et al., 2005;
Gradl et al., 1999), Wnt5a
signaling in fibronectin regulation has not been well studied. We hypothesized
that Wnt5a affects pulmonary vascular tubulogenesis through
interactions with fibronectin and the VEGF pathway.
Here, we show that mis/overexpressed Wnt5a in the developing avian lung acts non-canonically to induce marked pulmonary hypoplasia with dramatic alterations of vascular pattern. Wnt5a controls aspects of both distal airway and vascular tubulogenesis at mid-developmental stages via interactions with Shh, VEGF, Flk1 and fibronectin, differing from the canonical Wnt pathway. Understanding the role of Wnt5a in pulmonary air and vascular tubulogenesis may help to explain the pathogenesis of pulmonary malformations that often involve developmental errors or arrest, in both systems of the lung, as is seen in pulmonary hypoplasia with its associated pulmonary hypertension.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
or by embryonic day (E).
In ovo viral infection
We used replication-competent avian specific retrovirus (RCAS; both A and B
coats) expressing murine Wnt5a, chick Shh
(Riddle et al., 1993), chick
Wnt3a (Stevens et al.,
2003) or a dominant-negative construct of murine Ror2.
The Ror2-DAF-GPI (dnRor2) construct is composed of the
extracellular domain of murine Ror2 fused in frame with the membrane
anchoring GPI domain (Mikels and
Nusse, 2006). All were synthesized using established techniques
(Logan and Tabin, 1998;
Morgan and Fekete, 1996), and
were grown and harvested in DF1 cells
(Cepko, 1991). Control
infections were performed using RCAS-A or B constructed with green fluorescent
protein (GFP).
Embryos were injected, in ovo, at E2 (st11-15) in the right
anterior-lateral region targeting the pre-lung field, following a published
fate map (Matsushita, 1995),
with approximately 1 µl of freshly defrosted virus. Injections were carried
out under a Nikon SMZ800 dissection microscope, using a Hamilton syringe
fitted with pulled glass micropipette needles
(Morgan and Fekete, 1996).
When two viruses were used (made with different coats of RCAS to ensure
coordinated infection), equal aliquots from each were mixed before injection.
Eggs were then sealed and returned to the incubator until harvesting. More
than 35 dozen embryos were injected with RCAS-Wnt5a, and 20
dozen embryos were injected with either RCAS-Wnt3a or
RCAS-dnROR2. Controls consisted of both uninjected and
RCAS-GFP-injected embryos; these controls showed normal lung
development in all cases. Injections were verified either by tissue or
whole-mount analysis using standard in situ hybridization techniques and
published riboprobes (Riddle et al.,
1993; Roberts et al.,
1995); immunohistochemistry was used to detect expression of Rcs
protein (Riddle et al., 1993;
Roberts et al., 1995) and
fluorescence microscopy for GFP.
Organ Culture
Lungs explants were isolated from wild-type and injected/infected embryos
at E8, placed in 24-mm Transwell-clear permeable support plates (Corning) and
incubated for 4 days in BGJB (Gibco) medium containing 0.2 mg/ml ascorbic acid
(Sigma, 50 units/ml penicillin and 50 units/ml streptomycin (Sigma) at
37°C with 5% CO2. Experiments involved the addition of 1 mg/ml
H-Arg-Gly-Asp-Ser-OH (RGDS; Calbiochem)
(Pozzetto et al., 2005), 250
ng/ml murine recombinant Dkk1 protein (R&D Systems), 1 µg/ml
recombinant mouse Shh (R&D Systems) or 4 µg/ml cyclopamine (Toronto
Research Chemical) (Li et al.,
2004). As a control, we incubated RCAS-Wnt5a (injected in
ovo) and wild-type explants with non-treated medium. RCAS-dnRor2(A)
was injected into E8 lungs either alone or in combination with
RCAS(A)-Wnt5a or RCAS(B)-Wnt5a. Harvested lungs were then
incubated for 4 days in treated or non-treated medium. Controls were injected
with RCAS-GFP using the same protocol. To study the interactions
between fibronectin and Shh, we cultured E8 wild-type or
Wnt5a-overexpressing lung explants in cyclopamine-treated medium for
2 days, followed by cyclopamine and RGDS combined medium for 2 days. Control
lungs were incubated with cyclopamine or RGDS alone for 4 days. When possible,
as an internal control, the contralateral lungs (completely untreated) were
cultured in wells adjacent to the experimental lung.
Tissue processing
Lungs were dissected and fixed [with 4% paraformaldehyde (PFA) or 10%
formalin in RNase-free PBS] for 2 hours at room temperature. Fixed embryonic
lungs were washed in PBS with 0.1% Tween 20 (PBT) and either taken through a
graded series of methanol/PBT washes or kept at -20°C in methanol until
used for expression studies. Hematoxylin and Eosin (H&E) or periodic
acid-Schiff (PAS) stains were prepared using standard protocols (Brancroft and
Stevens, 1990) on 5-µm paraffin sections.
In situ hybridization
In situ hybridization, on 5-µm sections from paraffin-embedded tissues,
was performed using digoxigenin-labeled riboprobes generated from chick
Wnt5a [a gift of Andrew McMahon
(Dealy et al., 1993)],
Nkx2.1 [provided by Michael Kessel
(Pera and Kessel, 1998)],
Shh, Bmp4, Fgf10, Rcs, Wnt3a [provided by Cliff Tabin
(Riddle et al., 1993;
Roberts et al., 1995;
Roberts et al., 1998)] and
quail VEGF [from E. Laufer and C. Tabin
(Flamme et al., 1995)], using
techniques minimally altered from those published
(Riddle et al., 1993) and
developed using BM Purple AP Substrate (Roche), per the manufacturer's
instructions.
Immunohistochemistry
Immunohistochemical staining on paraffin sections was performed using
standard techniques. For antigen retrieval, sections were heat treated in a
microwave for 20 minutes at medium power in 0.01 M sodium citrate buffer (pH
6). Before antibody incubation, the peroxidase was quenched with
H2O2. Biotinylated secondary antibody (Vector
Laboratories) was used to localize antibody-antigen complexes in tissue, using
the ABComplex/HRP Detection System (Dakocytomation Antibodies), following the
manufacturer's directions. Antigen detection was enhanced with
3,3'-Diaminobenzidine (DAB). The following antibodies were used in the
study: mouse anti-active-β-catenin (1:150; Upstate), rabbit anti-Ror2
antibody (1:50, Cell Signaling), chick anti-L-CAM (no dilutions; Developmental
Studies Hybridoma Bank), rabbit anti-sheep Surfactant Protein B (SP-B; 1:50;
US Biological), mouse anti-PCNA (1:150; NeoMarkers), rabbit anti-rat Clara
Cell (CC16; 1:250; US Biological), mouse anti-actin smooth muscle Ab-1 (1:300;
NeoMarkers, clone 1A4), mouse anti-fibronectin (1:500; BD transduction
laboratories) and mouse Flk1 A-3 (1:1000; Santa Cruz Biotechnology).
Fluorescein elderberry bark lectin (E. lectin; Fluorescein labeled Sambucus
Nigra Lectin; 1:400; Vector Laboratories) was used to mark endothelial cells.
Sections were permeabilized using 0.1% Triton X-100 for one hour at room
temperature. E. lectin was incubated overnight (18 hours).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Mis/overexpression of Wnt5a disrupts lung growth and vascular pattern
From E10 onwards, Wnt5a mis/overexpression produced a striking
pulmonary hypoplastic phenotype (Fig.
2B). RCAS-Wnt5a expression was detected from the first
lung buds, at E3, and continued throughout development, with strong expression
observed primarily in the epithelium and endothelium, in the atria muscle
subjacent to the epithelium, and in the vascular muscle walls (data not
shown). Control injected embryos (sham injections and RCAS-GFP
injections) showed no phenotype (despite strong GFP expression being observed
following the RCAS-GFP injections; data not shown).
Histological features of Wnt5a mis/overexpressing lungs included a decreased numbers of airways, dilatation of the airways, and a thinner epithelial-vascular layer in the mature parabronchi and terminal bud with less complex budding (Fig. 2E). By E20, the infected lungs showed pulmonary lymphangiectasia and many showed pulmonary airway proteinaceous secretions (Fig. 2I; data not shown). Wnt5a mis/overexpressing parabronchial epithelium, although much thinner than wild type, differentiates normally into Clara cells (CC16 positive) and type 2 pneumocytes (surfactant B positive), and expresses the epithelial lung marker Nkx2.1 (see Fig. S1 in the supplementary material). There was no demonstrable difference in the number and distribution of these specific cell types.
Pulmonary hypoplasia also occurred following Wnt3a mis/overexpression (Fig. 2C), but it was not as striking as that produced by Wnt5a mis/overexpression (Fig. 2B). The histology was different as well, with less parabronchi and more terminal budding, in addition the parabronchi formed were abnormally shaped (Fig. 2F).
In the mis/overexpressing Wnt5a lung, there was a slight increase
in the number of muscularized arterioles expressing smooth muscle actin;
however, they were thinner and less muscularized than their wild-type
counterparts (see Fig. S1 in the supplementary material). The increase in
number of the smaller caliber vessels was more dramatic, as demonstrated by
endothelial-specific elderberry bark lectin staining
(Hagedorn et al., 2004) (Fig.
2K,N, compare with
2J,M). Pulmonary interstitial
vessels typically cluster centrally, between the airways, forming a distinct
hexagonal pattern (Fig. 2J,M);
this pattern was lost in the Wnt5a mis/overexpressing lungs, where
vessels were densely clustered subjacent to the parabronchial airways
(Fig. 2K,N). The Wnt5a
mis/overexpressed vascular phenotype was distinct from that of
RCAS-Wnt3a infected lungs, which developed with the larger,
earlier-formed, interstitial blood vessels normally patterned but
dysfunctional, with extravasated red blood cells spilling into the mesenchyme
surrounding them (Fig. 2F; data
not shown). The smaller, later-formed angiogenic interstitial blood vessels
were more numerous than in wild type (Fig.
2L,O), but remained in the interstitium, without clustering
subepithelially (Fig. 2L,O;
Fig. 3C,F).
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), and the
altered placement of the Flk1-expressing cells (black arrows,
Fig. 3D,E). Flk1 was expressed
in wild-type airway epithelium but not in RCAS-Wnt5a-infected lungs
(compare Fig. 3D and
3E). These findings were
present from E12 to E16, afterwards expression of both VEGF and Flk1 decreased
(data not shown).
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Wnt5a overexpression affects cell proliferation in epithelial and mesenchymal cells differently than in endothelial cells
The abnormally increased and malpatterned interstitial blood vessels and
the thinned epithelium in the Wnt5a-mis/overexpressing lungs were
examined with proliferation and apoptosis markers. PCNA expression tends to
cluster in the normal parabronchial epithelium and the subepithelial
mesenchyme at E14 (see Fig. S2 in the supplementary material). In the
Wnt5a-mis/overexpressing lungs, proliferation was found to be reduced
in epithelial cells and overall (i.e. total number of cells), but increased in
the subepithelial mesenchyme-endothelial cell compartment (see Fig. S2 in the
supplementary material). By E20, PCNA expression was absent in the
Wnt5a-mis/overexpressing distal epithelium, but remained strong in
the proximal epithelium (see Fig. S2 in the supplementary material). These
differences are statistically significant. There was no difference in
apoptotic markers between wild-type and Wnt5a mis/overexpressing
lungs in any compartment (epithelial, stromal or vascular; data not shown),
and there was no histologically demonstrable necrosis.
Wnt5a in the developing avian lung appears to act through Ror2 to activate a non-canonical Wnt pathway
On sections of E14 wild-type lungs, activated β-catenin was expressed
in vessels (red arrowheads, Fig.
4A) and in airway epithelium with a distinct epithelial pattern:
weak/absent in the luminal airway epithelium (black arrow in
Fig. 4A) but strong in the
apical/budding epithelium of the budding terminal airways (red arrows in
inset, Fig. 4A). In
Wnt5a-mis/overexpressed lungs, β-catenin staining was weaker
overall than in wild type, with almost no vascular staining (red arrowheads,
Fig. 4E) and with an epithelial
staining pattern that was markedly different from wild type. We detected the
strongest staining in the luminal epithelium (black arrow,
Fig. 4F), with much weaker
staining in the budding apical epithelium (red arrows in inset,
Fig. 4F), the inverse of wild
type.
No upregulation of activated β-catenin levels was detected with
Wnt5a mis/overexpression, suggesting that a non-canonical pathway
must be used. To investigate which pathway Wnt5a uses in the lung, we
preferentially inhibited the canonical pathway with soluble Dkk1 protein
(Glinka et al., 1998;
Niehrs, 2006). In organ
culture, Dkk1 exposure resulted in mild pulmonary hypoplasia (compare Fig.
4B and
4C) but, importantly, failed to
rescue the Wnt5a-mis/overexpression phenotype (compare Fig.
4G and
4H). When we used a
dominant-negative construct of Ror2 (RCAS-dnRor2) to inhibit the
non-canonical pathway (Mikels and Nusse,
2006), explants developed enlarged, hyperplastic lungs that were
in most part due to relatively normally formed, but dilated airways
(Fig. 4D). Co-expression of
dnRor2 and Wnt5a (via RCAS) resulted in a partial rescue of
the hypoplastic phenotype (Fig.
4H, compare with
4G).
Ror2 is normally expressed in the large muscular (proximal) pulmonary interstitial vessels (red arrowheads, Fig. 5A,B) and in the early pulmonary epithelium, prominently in the luminal epithelium (red arrow, Fig. 5A) and weakly in the budding terminal epithelium (red arrow, Fig. 5A). Expression continues in the large vessels (red arrowhead, Fig. 5B), and becomes restricted to the budding epithelium (red arrow, Fig. 5B) later in development. Mis/overexpression of dnRor2 in the lungs, which were allowed to develop in ovo, resulted in large lungs (pulmonary hyperplasia) with a relatively normal airway pattern (Fig. 5D, compare with 5C), but with a markedly decreased number of interstitial blood vessels (compare Fig. 5F with 5E). RCAS-dnRor2 infection appears to limit the vascular pattern to the interstitium as no E. Lectin was detected either within or below the epithelium (compare Fig. 5F with 5E).
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), and, of
the many members of the cadherin family, E-cadherin is most predominantly
expressed in epithelial tissues. L-CAM is its avian homolog
(Gallin et al., 1987;
Pecina-Slaus, 2003), and we
find its expression exclusively in the epithelium of the chick lung
(Fig. 6A). With
mis/overexpression of Wnt5a, L-CAM expression was reduced in distal
(red arrowhead, inset, Fig. 6B)
but not proximal (red arrow, Fig.
6B) epithelium. In fact, the epithelial bud expression is
diminished and nearly absent in Wnt5a-mis/overexpressing lungs
(compare red arrowheads, insets, Fig.
6A,B). Fibronectin is normally concentrated adjacent to the airway epithelium in the subepithelium of the terminal airways (red arrow, inset, Fig. 6C) and in the interstitium, with a hexagonal perivascular pattern (red arrowhead, inset, Fig. 6C). Wnt5a mis/overexpression increases the expression of fibronectin (Fig. 6D), and the biphasic pattern of expression observed in wild-type lungs is masked by a diffuse increase in the interstitial expression. Some fibronectin expression was still noted in the subepithelium of the terminal buds (red arrow, inset, Fig. 6D). Late in lung development, fibronectin was strongly expressed in the interstitium (red arrowhead, Fig. 6E) and surrounding the luminal-most epithelial cells (red arrow, inset, Fig. 6E). By contrast, the inverse expression was observed following RCAS-Wnt5a infection, with a marked increase in epithelial/subepithelial fibronectin expression, and expression in the vascular interstitium (Fig. 6F, red arrowhead) or luminal-most epithelium (red arrowhead, inset, Fig. 6F) either decreased or undetectable.
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Inhibition of fibronectin function rescues Wnt5a-induced branching abnormalities in the lung
We used the soluble peptide RGDS to inhibit fibronectin function
(Yamada and Kennedy, 1984).
Although RGDS markedly inhibits lung growth and airway development (compare
Fig. 7A with
7B), the
RCAS-Wnt5a-injected lung explant phenotype
(Fig. 7D) was effectively
rescued with RGDS treatment (Fig.
7E). The Wnt3a-mis/overexpressing lung explants, which
show hypoplasia with a more normal airway development
(Fig. 7C), were not rescued by
RGDS (Fig. 7E).
Wnt5a mis/overexpression affects other known pulmonary pattern formation signaling pathways
We studied whether the mis/overexpression of Wnt5a affects other
known developmentally important pathways and factors in the lung. At E10,
Shh expression, which is normally strongly detected in the proximal
and distal epithelium, was decreased in Wnt5a-mis/overexpressed lungs
(Fig. 8A,B). Epithelial
Bmp4 and Fgf10 expression were also markedly reduced by
Wnt5a mis/overexpression, but no changes in the expression of
Nkx2.1 were detected (see Fig. S3 in the supplementary material).
In lung explants, we found that the addition of exogenous Shh protein in wild-type lungs resulted in pulmonary hyperplasia (enlarged lungs, Fig. 8D), mainly due to a marked expansion of mesenchyme (asterisk in 8J). The airway and vascular pattern, where present, was normal, with preservation of the hexagonal vascular pattern (upper half of explant in Fig. 8J, magnified in 8P). Cyclopamine inhibition resulted in marked pulmonary hypoplasia (Fig. 8E), with an abnormal collection of interstitial vessels (black arrows in Fig. 8K,Q). RCAS-Wnt5a-infected explants showed, as expected, marked pulmonary hypoplasia with an abnormal vascular and airway pattern. The collection of misplaced vessels, unassociated with airways, was prominent and similar to that seen in cyclopamine-treated explants (see black arrows in Fig. 8K,L). Exogenous Shh protein rescues the Wnt5a-mis/overexpression pulmonary hypoplasia (Fig. 8G, compare with 8F), mainly because of an increase in the interstitial mesenchyme (asterisks, Fig. 8M), but the hexagonal vascular pattern is only focally restored (black arrows, Fig. 8M; arrowheads, 8S). Inhibition of the Hh pathway by cyclopamine in the RCAS-Wnt5a-infected explants resulted in an exacerbation of the hyperplastic phenotype (Fig. 8H), drastically altering the airway pattern (Fig. 8N,T). The abnormal collection of small vessels present in the Wnt5a-mis/overexpressing explants was rescued by Shh and worsened by cyclopamine (black arrows in Fig. 8L,M,N).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Avian pulmonary expression of Wnt5a is present in the epithelial and subepithelial tissues, and in the interstitial vasculature, but becomes exclusively epithelial by late stages. The expression is strongest in mid-embryonic stages when it includes all airway epithelium - parabronchi to terminal buds. By near hatching stages, the expression is patchy and present only in the few epithelial cells near and at the apical lumen of the distal airways. The significance of the discontinuous epithelial expression pattern is unknown; it does not appear to be related to the expression of the epithelial cell type markers SpB and CC16. However, the apical bias in expression may be important in regulating other factors.
In the chick lung, once the branching process is complete (by E14), the
epithelium from the parabronchi bud into the surrounding mesenchyme, becoming
adjacent to the distal pulmonary vasculature to form the gas-exchange surface.
Our results show that the over/misexpression of Wnt5a results in
budding dysfunction, which is likely to be due to altered extracellular matrix
proteins and abnormal cell adhesion. We find an increase in fibronectin levels
and a decrease in L-CAM levels following Wnt5a mis/overexpression.
Spatially controlled fibronectin levels are clearly necessary for normal
pulmonary development, as its inhibition in wild-type lungs results in
dramatic branching dysfunction. This role appears to be conserved in the
mammalian lung, as fibronectin is strongly expressed and its expression is
maximal where branching morphogenesis occurs
(Roman and McDonald, 1992). De
Langhe and colleagues (De Langhe et al.,
2005) showed that canonical Wnt signaling in mouse affects (at
least) the proximal larger pulmonary vasculature by the downregulation of
fibronectin. As both the canonical and non-canonical pathway target
fibronectin, it must be an important protein to be regulated for normal
pulmonary development.
L-CAM and β-catenin expression patterns are altered by Wnt5a
mis/overexpression; their general expression levels decreased and the new
`invading' epithelial buds have lost nearly all expression. This location, at
the interface of where the migrating and budding epithelium mingles with the
migrating endothelium, must affect the coordination that needs to occur to
form a normally functioning air capillary unit. The reduction of
β-catenin and L-CAM in the epithelial buds may also affect the epithelium
in an autocrine manner, as we find the airbud epithelium to be decreased
(thinned) in the Wnt5a-mis/overexpressing lungs. The interference
with these extracellular proteins mediating epithelial and endothelial growth
and migration processes, has been shown in other systems as well
(Smith et al., 2006).
Our results point to an important role of Wnt5a in directing pulmonary
vascular development. The number of vascular spaces is increased in
Wnt5a-mis/overexpressing lungs, suggesting an angiogenic effect of
Wnt5a, a property that has been suggested by others as well
(Cheng et al., 2008;
Masckauchan et al., 2006;
Zhang et al., 2006), but we
also show that the affected vessels are malpatterned. Results presented herein
suggest that sprouting angiogenesis (responsible for the hexagonal pattern) is
interrupted and intussusceptive angiogenesis (important in expansion and
`alveolarization') is stimulated. This appears to be a Wnt5a/non-canonical
pathway-specific function, as neither of these vascular patterns/growths is
affected by our manipulations of the canonical pathway. Wnt5a may
direct pulmonary vascular patterning by regulating the VEGF pathway via its
effect on fibronectin, as fibronectin contains VEGF-binding domains
(Flamme et al., 1995).
Wnt5a mis/overexpression results in a near loss of detectable VEGF
expression in interstitial vessels that is associated with strongly
upregulated Flk1 in the vessels, which are malpositioned
subepithelially (adjacent to the VEGF-expressing epithelium and where the
highest concentration of fibronectin is present). Also, we show that
Wnt5a inhibits Shh, which is known to regulate VEGFA
(White et al., 2007).
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Many systems important in body pattern formation are also known to play
roles in pulmonary development, including the Hedgehog, Bmp, Nk and Fgf
pathways (Bellusci et al.,
1997a; Bellusci et al.,
1997b; Bellusci et al.,
1996; Pepicelli et al.,
1998; Yuan et al.,
2000). The interaction of these principal developmental systems,
however, varies in a tissue-specific manner. Our results show that
non-canonical Wnt signaling, by Wnt5a, functions as an inhibitor of these
pathways. These results are supported by the results of others. Mice null for
Wnt5a develop hyperplastic lungs in which Shh, Bmp4 and
Fgf10 are upregulated (Li et al.,
2002). Overexpressing Wnt5a with the surfactant-B promoter
demonstrated that Wnt5a acts upstream of Fgf10, even though it acts as an
activator of Fgf10 expression (Li
et al., 2005). The difference in these and our results is likely
to be due to the timing of the mis/overexpression; the surfactant B promoter
directs expression very late in lung development. Thus, our results support a
function of Wnt5a as an important upstream modulator of the secreted
morphogens known to be involved in early-mid lung development. The interaction
of Wnt5a, Shh and fibronectin shown here is intriguing. Shh, by affecting
specifically the pattern and not the level of fibronectin, rescues the
vascular part of the Wnt5a-mis/overexpressing phenotype. These
results show the importance of the spatial pattern of expression of
fibronectin in directing the normal vascular pattern in the lung. The specific
pathway (Wnt5a-Shh-fibronectin) is a novel and important finding.
We also describe for the first time, to our knowledge, a pulmonary
hyperplastic phenotype in explants incubated with excess Shh, and in those in
which the non-canonical pathway is inhibited via RCAS-dnRor2. This
phenotype is reminiscent of the human congenital cystic adenomatoid
malformation (CCAM) (Wilson et al.,
2006), a rare malformation associated with infectious and
neoplastic sequellae that, to date, remains unexplained molecularly.
An interesting corollary to our results is that, although there are
differences in anatomy and development between the mammalian and avian lung,
there is a marked conservation of the molecular mechanisms controlling their
pattern formation. Thus, budding and branching morphogenesis may be more
homologous that has been previously thought. Our findings of abnormal pattern
formation of both the airways and the vascular system are novel. Congenital
pulmonary hypoplasia in humans is often associated with vascular dysfunction
(Vacanti et al., 1988) and
poor oxygenation (Beals et al.,
1992). Our study suggests that some of the molecular factors that
may be involved in generating pulmonary hypoplasia are likely to affect
vascular development, exacerbating clinical hypoxemia, the lethal common
denominator in this disorder. Other human congenital malformations of the lung
that involve pulmonary vascular anomalies (for example alveolar-capillary
dysplasia) (Cullaine et al., 1992) are poorly understood molecularly
(Groenman et al., 2005). The
findings described here may provide an insight into the etiology of these
often lethal, congenital diseases. In addition, they emphasize the complex
relationships of pathways during lung development, in which survival requires
interactions that are carefully choreographed by factors such as Wnt5a.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/7/1365/DC1
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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