Wnt2/2b and beta-catenin signaling are necessary and sufficient to specify lung progenitors in the foregut

Abstract

Patterning of the primitive foregut promotes appropriate organ specification along its anterior-posterior axis. However, the molecular pathways specifying foregut endoderm progenitors are poorly understood. We show here that Wnt2/2b signaling is required to specify lung endoderm progenitors within the anterior foregut. Embryos lacking Wnt2/2b expression exhibit complete lung agenesis and do not express Nkx2.1, the earliest marker of the lung endoderm. In contrast, other foregut endoderm-derived organs, including the thyroid, liver, and pancreas, are correctly specified. The phenotype observed is recapitulated by an endoderm-restricted deletion of beta-catenin, demonstrating that Wnt2/2b signaling through the canonical Wnt pathway is required to specify lung endoderm progenitors within the foregut. Moreover, activation of canonical Wnt/beta-catenin signaling results in the reprogramming of esophagus and stomach endoderm to a lung endoderm progenitor fate. Together, these data reveal that canonical Wnt2/2b signaling is required for the specification of lung endoderm progenitors in the developing foregut.

Figure 1. Loss of Wnt2 leads to lung hypoplasia and down-regulation of critical pathways required for lung development

Wnt2^−/− null mutants exhibit severe lung hypoplasia as shown by whole mount comparison (A and B) and histological sectioning at E14.5 and E18.5 (C, D, F, G). Dilation of alveolar sacs is observed at E18.5 (E and H) and at P0 (M and N) in Wnt2^−/− null mutants. Whole mount staining with E-cadherin shows relatively normal distal branching of airways in Wnt2^−/− null mutants (I–L). Wnt2^−/− null mutants have a dilated endothelial vasculature indicating significant lung mesenchymal defects (O and P, arrows). Wnt2^−/− null mutants have a significant decrease in proliferation in both the lung endoderm and mesenchyme (Q and R). Quantitation of proliferation defects in Wnt2^−/− null mutants (S). Wnt2^−/− null mutants exhibit decreased expression of genes critical for lung development as assessed by Q-PCR at E14.5 (T). Normal proximal-distal patterning in Wnt2^−/− null mutants as assessed by SP-C and CC10 immunostainng (U–X). Scale bars: C, D, F, G=800 μm, E, H, U and V=200 μm, W and X=400 μm, M and N=600 μm, O–R=100 μm.

Figure 2. Loss of both Wnt2 and Wnt2b leads to specific loss of lung progenitor specification in the foregut endoderm and complete lung agenesis

Wnt2/2b DKO mutants exhibit complete lung agenesis as shown at E11.5 (A–F) and E14.5 (G–L). A clear separation of the esophagus (B and C, arrow) from the trachea (B and C, arrowhead) is observed in wild-type embryos. At E9.5, when the lung is initially specified, there is no detectable budding of the trachea from the anterior foregut in Wnt2/2b DKO mutants (M and Q). While wild-type embryos express Nkx2.1 in the region where the trachea will bud from the foregut (N), expression is not observed in Wnt2/2b DKO mutants (R, outline). However, Nkx2.1 expression is observed in both wild-type (O) and Wnt2/2b DKO mutants (S) in the thyroid primordium. The esophagus epithelial marker p63 is expressed in the single gut tube in Wnt2/2b DKO mutants at E11.5 (P and T, arrows). Wnt7b, which also marks early lung endoderm progenitors in the ventral aspect of the anterior foregut (arrow) versus the dorsal aspect (dashed arrow), is lost in Wnt2/2b DKO anterior foregut endoderm (U and V). E-cadherin whole mount immunostaining shows lack of tracheal budding in Wnt2/2b DKO mutants (W and X, arrows and brackets). Scale bars: A–F=200 μm, G–L=800 μm, M–V=100 μm.

Figure 3. Wnt2/2b signal through the β-catenin dependent canonical pathway to specify lung endoderm progenitors in the anterior foregut

Whole mount staining of Wnt2^−/− :BAT-GAL embryos for lacZ expression revealing a significant decrease in canonical Wnt signaling in the anterior foregut at E10.5 (A and B, arrows). Histological sections of wild-type BAT-GAL, Wnt2^−/−:BAT-GAL, Wnt2b^−/− :BAT-GAL, and Wnt2/2b:BAT-GAL DKO mutants shows a significant loss of staining in the ventral aspect of the foregut endoderm in the region where the trachea is specified (C–F, brackets). Histological sections from E10.5 wild-type (G–I) and Ctnnb1:Shh-cre (K–M) demonstrating lung agenesis upon deletion of foregut endoderm β-catenin expression. Nkx2.1 expression is observed in the ventral foregut endoderm of wild-type embryos at E10.5 (J) but not in Ctnnb1:Shh-cre mutants (N) indicating loss of lung specification in these mutants. E-cadherin whole mount immunostaining shows normal tracheal budding in wild-type embryos (O, arrow) and lack of tracheal budding in Ctnnb1:Shh-cre mutants (P, arrow). However, the esophagus is still present in the Ctnnb1:Shh-cre mutants (P′, bracket). Fgf10 expression is substantially reduced in the ventral mesoderm surrounding the foregut in Wnt2/2b DKO mutants (Q and R) while Gli3 expression is unchanged at E10.0 (S and T). Es=esophagus, Tr=trachea. Scale bars: C–F=100 μm, G–N, Q, R=200 μm, and S and T=150 μm.

Figure 4. Activation of Wnt/β-catenin signaling leads to expansion of lung endoderm progenitors into the stomach

H+E stained sections from E10.5 wild-type (A–C) and Ctnnb1:Shh-cre (E–G) mutants show that trachea-esophagus septation is disrupted in these mutants (F, arrowheads). Immunostaining for Nkx2.1 protein expression reveals expansion of Nkx2.1 positive lung endoderm progenitors into the stomach (D and H, outlined region). H+E stained sections from E11.5 wild-type (I and J) and Ctnnb1:Shh-cre (M and N) mutants. Immunostaining for Nkx2.1 protein expression at E11.5 shows expression of Nkx2.1 in esophagus of Ctnnb1:Shh-cre mutants (O, arrow) and not in wild-type littermates (K, arrow). Expression of Nkx2.1 is also extended into the stomach of E11.5 Ctnnb1:Shh-cre mutants (P, arrows). In contrast, expression of p63 is reduced in the esophagus and stomach endoderm of Ctnnb1:Shh-cre mutants at E11.5 (Q–T). Co-staining for both Nkx2.1 and p63 show that p63 positive endoderm is lost while Nkx2.1 positive endoderm is present in the esophagus (U–V). Model showing necessity and sufficiency of Wnt2/2b and β-catenin signaling for lung progenitor specification in the anterior foregut endoderm (W).