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Fig. 8. Wnt5a acts upstream of Shh and mediates fibronectin
function via Shh. (A) Shh mRNA is normally
expressed principally in the epithelium of the airways (arrow) early in
development at E11. (B) In Wnt5a mis/overexpressing lungs at
E11, the expression of Shh is markedly diminished (arrow).
(C,D) In organ culture, lungs exposed to recombinant Shh protein
(D) develop a hyperplastic phenotype with an increase in both airway budding
and mesenchymal proliferation compared with wild type (C). (F,G)
RCAS-Wnt5a explants grown in culture exposed to Shh recombinant
protein (G) are rescued from their hypoplastic phenotype (F). (E)
Cyclopamine treatment of wild-type lungs resulted in pulmonary hypoplasia,
resembling the RCAS-Wnt5a lungs (F). (H) The
Wnt5a-mis/overexpressed hypoplastic lungs phenotype (F) is
exacerbated when cultured in the presence of cyclopamine. (I-T) H&E
staining. (I-N) Cross sections of lung explants. (O-T) Higher magnification
views of I-N. (I) Wild-type lung explants develop fairly normally in both
airway and vascular (ellipse) pattern. (O) The normal interstitial hexagonal
vascular pattern is evident (black arrowheads). (J,P) Hyperplastic Shh
explants (J, image is 1x compared with all others at 2x) with
mesenchymal hyperplasia (asterix) maintain a normal interstitial vascular
pattern where airways develop (arrowheads, P). (L,R) Wnt5a
mis/overexpressing explants are hypoplastic (L) and have an abnormal
proliferation of small vessels unassociated with the airways (arrows, L,R);
arrowhead in R shows normal position of small interstitial airway. (K,Q) This
unusual vascular phenotype is also present in cyclopamine-alone exposed
explants (arrows). (M,S) Although the hypoplastic phenotype in the
Wnt5a-mis/overexpressed explants is rescued with exogenous Shh
exposure (M, see also G), the lungs are still maldeveloped with excess
mesenchyme (asterisks, M); however, they show more normal vascular development
(arrowheads, S), without the atypical non-airway associated vessels seen in L
and R (arrows). (N,T) Cyclopamine exacerbates the airway phenotype and
hypoplasia in the RCAS-Wnt5a-infected explants (H,N,T) and fails to
rescue the vascular proliferation (arrow, N). (U-W) Fibronectin is
expressed normally in a subepithelial (red arrow, U) and interstitial vascular
(red arrowheads, U) pattern, and is not altered significantly in pattern or
levels with Shh (V) or cyclopamine (W; red arrowhead on vessel and arrows on
airways; no normal vessels are present with cyclopamine exposure). (X)
Fibronectin levels are markedly increased with RCAS-Wnt5a infection.
(Y,Z) Addition of Shh fails to reduce the increase in the
subepithelially clustered expression (red arrows in Y), but cyclopamine
exposure does (Z; red bracket is tangential epithelium, compare with black
bracket in T; red asterisk highlights abnormal vessels).
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