Wnt7b stimulates embryonic lung growth by coordinately increasing the replication of epithelium and mesenchyme

Abstract

The effects of Wnt7b on lung development were examined using a conditional Wnt7b-null mouse. Wnt7b-null lungs are markedly hypoplastic, yet display largely normal patterning and cell differentiation. In contrast to findings in prior hypomorphic Wnt7b models, we find decreased replication of both developing epithelium and mesenchyme, without abnormalities of vascular smooth muscle development. We further demonstrate that Wnt7b signals to neighboring cells to activate both autocrine and paracrine canonical Wnt signaling cascades. In contrast to results from hypomorphic models, we show that Wnt7b modulates several important signaling pathways in the lung. Together, these cascades result in the coordinated proliferation of adjacent epithelial and mesenchymal cells to stimulate organ growth with few alterations in differentiation and patterning.

Fig. 1. Deletion of mouse Wnt7b results in hypoplastic lungs

(A) Strategy to remove Wnt7b from only the embryo proper. (B) Mutant lungs are 50% smaller than wild-type at E18.5. The overall mass of the embryo is unchanged. (C) Mutant lungs are 75% smaller than wild-type at E14.5. The overall mass of the embryo is unchanged. (D) Control and mutant lungs at E12.5. E-cadherin staining shows grossly normal lobulation in mutant lungs. However, the right medial lobe bronchus (*) of the Wnt7b mutant lung emanates from the cephalic lobe bronchus.

Fig. 2. Cell fate specification, timing of differentiation and tissue architecture are unchanged in Wnt7b-null lungs

(A-D′) E14.5 immunohistochemistry of heterozygous (A-D) and Wnt7b mutant (A′-D′) mouse lungs. Blood vessels appear normal in the mutant, as marked by PECAM (A,A′), as does airway smooth muscle, marked by smooth muscle myosin (B,B′). Proximal Sox2 (C,C′) and distal Sox9 (D,D′) expression are unchanged in mutants. Dotted lines encircle the epithelium. (E-I′) At E18.5, immunohistochemistry of heterozygous (E-I) and Wnt7b mutant (E′-I′) lungs demonstrates that blood vessels (E,E′), airway smooth muscle cells (F,F′), Clara cells as marked by CC10 (G,G′), and type 2 cells as marked by surfactant protein C (H,H′) are unchanged in mutant lungs. Type 1 cells marked by caveolin 1 (I,I′) are decreased in number. (J) Quantification of cell types in E18.5 lungs demonstrates that the proportions of Clara, ciliated and mucous cells are unchanged in the trachea and airways of Wnt7b mutants. In the distal saccules, the proportions of cells positive for SP-C or PECAM are also unchanged. Error bars represent 1 s.d. (K) The number of Sox9-positive cells per lung bud tip at E14.5 is unchanged in Wnt7b mutants.

Fig. 3. Deletion of Wnt7b results in proportionate decreases in replication of epithelial and mesenchymal tip cells

(A,A′) Schematic depiction (A′) of BrdU staining (brown) of E14.5 lung bud tip (A). Replication is highest in tip epithelium (A′, dark yellow) and tip mesenchyme (A′, dark purple) as compared with proximal epithelium (A′, light yellow) and mesenchyme (A′, light purple). (B,C TUNEL staining of E14.5 wild-type (B) and Wnt7b mutant (C) mouse lungs shows no increase in apoptosis in the mutant. Dotted lines encircle epithelium. (D,E) BrdU staining of E14.5 wild-type (D) and mutant (E) lungs demonstrates decreased replication in mesenchymal and epithelial cells of the mutant. Arrowheads point to the tips. Dotted lines encircle epithelium. (F) A comparison of the percentage of BrdU-labeled cells in wild-type (gray) and mutant (white) tips demonstrates proportionate drops in epithelial and mesenchymal replication (P<0.001).

Fig. 4. Preserved expression of many factors affecting growth in Wnt7b mutant lungs

(A-E′) E14.5 ISH of Wnt7b mutant (A′-E′) and control (A-E) mouse lungs. Expression of Shh (A,A′), Gli1 (B,B′), Ptch1 (C,C′) and Spry2 (E,E′) are unchanged in the mutant. The epithelial tip is encircled. Fgf10 staining (D,D′) is largely preserved in mutant lungs; there is a variable decrease in the most-distal expression of Fgf10 in mutant lungs (arrowhead). (F-G′) E14.5 immunohistochemistry for N-myc (F,F′) and fibronectin expression (G,G′) showing that these are unchanged in the mutant. ISH signal (purple) is indicated by the arrowheads.

Fig. 5. Mechanism of Wnt7b action in mouse lung development

(A) ISH showing loss of canonical Wnt targets in mesenchyme and endoderm of Wnt7b mutant lungs. Dotted lines encircle epithelium. Arrowheads point to periendodermal mesenchyme. (B) E12.5 whole-mount ISH of Axin2 and Lef1 in cultured wild-type lungs. Wnt signaling is decreased with the addition of Dkk1 (left) and enhanced by lithium (right) when compared with control cultures (middle). In lithium-treated lungs, mesenchymal staining of the canonical targets has expanded. (C) Axin2 expression in mesenchyme cultured alone and in mesenchyme recombined with epithelium, showing that mesenchymal Axin2 expression requires endoderm induction. (D) Lithium rescues Lef1 expression in E12.5 mutant lungs when compared with Wnt7b mutant lungs cultured without lithium. (E) Epithelial Wnt7b activates a canonical pathway in neighboring mesenchyme. (F) Bmp4 and Id2 expression are coincident on serial sections of E14.5 wild-type lung (left). Expression of epithelial Bmp4 and Id2 are decreased in mutant lungs (middle and right). (G) Lithium induces ectopic Id2 expression in cultured E12.5 wild-type lungs. (H) Wnt7b induces Bmp4 and Id2 production in endoderm tip cells. (I) Wnt7b coordinately activates autocrine and paracrine signaling cascades to increase cell replication.