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First published online 27 February 2008
doi: 10.1242/dev.010504

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Chick pulmonary Wnt5a directs airway and vascular tubulogenesis

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.

View larger version (113K): [in this window] [in a new window]   Fig. 1. The expression patterns of Wnt5a and Wnt3a are spatially and temporally distinct. (A,B) Whole-mount in situ hybridization at E7. No pulmonary expression is detected for Wnt5a (A, positive expression in esophagus, white arrow), whereas expression is detected for Wnt3a (white arrows, B). (C) Wnt5a expression is observed in the mesenchyme adjacent to the distal epithelium (black arrowheads) and weakly within the distal epithelium (arrow) at E11. (E) By E15, Wnt5a is expressed strongly in the epithelium (both luminal and budding, arrows). (G) At E20, Wnt5a is expressed in the epithelium surrounding the parabronchial lumen (black arrow), and in the proximal-most epithelium (red arrow). Interstitial vascular expression for Wnt5a is weakly present from E11 (red arrowhead, C); this expression is slightly stronger at E15 (red arrowhead, E; data not shown) but is not detectable at E20 (red arrowhead, G). (D) Wnt3a is strongly expressed in the interstitial vasculature (red arrowhead) and the distal epithelium (arrow) at E12. (F,H) Expression of Wnt3a is restricted to the budding epithelium (arrows, F) by E15, and is not detected after E16 (E20 section shown in H); red arrowheads in G and H highlight large interstitial vessels. P, parabronchi (distal airway); SB, secondary bronchi.

View larger version (119K): [in this window] [in a new window]   Fig. 2. Wnt5a mis/overexpressed lungs show pulmonary hypoplasia with abnormal air and vascular tubulogenesis. (A-C) Whole lungs dissected from wild-type (WT, A), RCAS-Wnt5a-infected (B), and RCAS-Wnt3a-infected (C) embryos at E14, showing pulmonary hypoplasia of the infected lungs. (D-I) H&E-stained lungs showing dramatic thinning of the airway epithelium by mis/overexpression of Wnt5a (compare brackets in E with D) but not following RCAS-Wnt3a infection (compare brackets in G with F). RCAS-Wnt3a mis/overexpressed lungs at E15 have more normal appearing airways and airway epithelial thickness (compare G with F) but develop with abnormal interstitial muscularized vessels, often with extravasated red blood cells (arrows and insert, G). At E20, Wnt5a mis/overexpressing lungs often show pulmonary lymphangiectasia (arrows in I) and disordered larger interstitial vessels (arrowheads, I; compare with wild-type lungs, H). (J-L) At E14, normal developing pulmonary endothelium is localized between airways or parabronchi, as Elderberry bark lectin shows (red arrow, J). With RCAS-Wnt5a infection, the developing vasculature clusters subepithelial to the parabronchi (red arrow, K). The number of elderberry bark lectin-stained interstitial vessels are increased by Wnt3a mis/overexpression but these vessels are localized normally in the interstitium (L), not clustered subepithelially as seen with Wnt5a mis/overexpression (K). Nuclei were counterstained with DAPI (blue). (M,N,O) Cartoon tracings of J,K,L make pattern changes of vasculature clearer. Epithelial cells are orange; endothelial cells, green.

View larger version (83K): [in this window] [in a new window]   Fig. 3. Wnt5a affects the VEGF pathway. (A) VEGF expression in E14 wild-type lungs is detected in the epithelium (arrows in inset) and in the interstitial vessels (arrowheads). (B,C) Although epithelial expression is not appreciably altered by Wnt5a mis/overexpression (B), expression is decreased in the interstitial vessels compared with that seen in wild-type (A) or in Wnt3a-infected (C) lungs. (D-F) Flk1 expression in normal E14 lungs (D) shows weak staining in most of the small vessels, which are nearly all interstitially placed (red arrowheads), and in the epithelium (red arrow). Flk1 expression is increased and the positively staining vessels are malpositioned in the Wnt5a-mis/overexpressing lungs (red arrowheads, E) but not in the Wnt3a-mis/overexpressing lungs (red arrowheads, F). No Flk1 expression is detected in Wnt5a lung epithelium (red arrow, E); Wnt3a lung epithelium shows weak staining (red arrow, F).

View larger version (33K): [in this window] [in a new window]   Fig. 4. Wnt5a acts in the non-canonical pathway via Ror2 in lung development. (A,E) In wild-type lungs at E14, high levels of active β-catenin are detected in a patchy distribution in the buds of the most distal epithelium (black arrow, A and red arrows, inset A), whereas levels in the Wnt5a-mis/overexpressed lung are decreased (black arrow, E and red arrows, inset E). Active β-catenin is expressed in large interstitial vessels in wild type (red arrowheads, A) but is not detected in RCAS-Wnt5a-infected lungs (red arrowheads, E). (B-D,F-H) Explants grown for 5 days in organ culture demonstrate complex budding and overall organ growth in wild type (B), but lungs cultured after infection with RCAS-Wnt5a are hypoplastic with airway budding disarray and simplification (F). Treatment with the recombinant Dickkopf protein (Dkk1) impaired lung budding and growth in wild type (C) and did not rescue the Wnt5a phenotype (G, compare with F). The Wnt5a-mis/overexpressed hypoplastic phenotype (F) is partially rescued when embryos are co-infected with dnRor2 (H). Infection with the RCAS-dnRor2 construct alone showed dilated airways and mild overgrowth (hyperplasia; D).

View larger version (121K): [in this window] [in a new window]   Fig. 5. Inhibition of Wnt5a by dnRor2 affects pulmonary development. (A) Ror2 immunohistochemistry demonstrates expression at E14 in the epithelium (red arrow) and interstitial vessels (red arrowhead) of wild-type lungs. (B) At E18, Ror2 is expressed in the interstitial large muscularized vessel (red arrowhead) and surrounding the luminal epithelium (red arrow and inset). (C,D) H&E staining of RCAS-dnRor2-infected lungs (D) shows an increase in size compared with wild type (C). (E,F) Elderberry bark lectin staining highlights the decrease in endothelium with dnRor2 mis/overexpression.

View larger version (82K): [in this window] [in a new window]   Fig. 6. Wnt5a affects the expression of adhesion factors. Lungs imaged in A-G are all from in-ovo experiments; H-J are from explant (Exp) experiments. (A) L-CAM antibody localizes in the epithelium in wild-type lungs at E14 (red arrowhead, inset); arrow indicates the distal airway magnified in inset. (B) In Wnt5a-mis/overexpressing lungs, L-CAM expression is observed in the proximal epithelium (red arrow), but is reduced in the most-distal budding epithelium (red arrowhead, inset). (C) At E14 in wild-type lungs, fibronectin is principally expressed subepithelially (arrow, inset) and in the interstitial vasculature (red arrowhead, inset). (D) At this stage, fibronectin levels are increased diffusely in RCAS-Wnt5a-injected lungs as compared with wild type. (E,F) The fibronectin levels remain altered through to E20, when fibronectin is normally localized around the parabronchi lumen, surrounding the atrial muscle (red arrow, inset, E), in few distal luminal epithelial cells, and in the interstitial vasculature (red arrowhead, E). With Wnt5a mis/overexpression (F), fibronectin is overexpressed diffusely (red arrow shows a similar pattern surrounding the atrial muscle as in E, but note the diffuse expression throughout epithelium); however, almost no expression of fibronectin can be detected clustered in the interstitium between parabronchi (red arrowhead). (G) At E14, Wnt3a mis/overexpression does not significantly affect the level or pattern of fibronectin expression (compare with wild type in C). (H) In wild-type explants, normal fibronectin expression is observed subepithelially (red arrow) and in the interstitial vasculature (red arrowhead). (I) dnRor2 mis/overexpression decreases fibronectin expression overall and in the subepithelium (red arrow) and the interstitial vessels (red arrowhead). (J) Explants incubated with Dkk1 recombinant protein fail to develop vessels but show near normal subepithelial fibronectin expression (red arrow, J; compare with H).

View larger version (66K): [in this window] [in a new window]   Fig. 7. Inhibition of fibronectin function rescues the Wnt5a-mis/overexpressed phenotype. (A-F) Lung explants isolated from E8 RCAS-Wnt5a (D,E), RCAS-Wnt3a (C,F) and wild-type (A,B) embryos were grown in culture in absence or in presence of RGDS. RCAS-Wnt5a (D) and wild-type RGDS-treated (B) lung explants showed a reduction in size and a simplification of budding compared with untreated wild-type explants (A). When RGDS was added to Wnt5a mis/overexpressed lung explants (E) an improvement in budding complexity and number was observed. RCAS-Wnt3a explants are hypoplastic (C); incubation in the presence of RGDS (F) has little effect.

View larger version (99K): [in this window] [in a new window]   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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