WntC-catenin signaling regulates cell fate during organ development and postnatal tissue maintenance, but its contribution to specification of distinct lung epithelial lineages is still unclear. endoderm disrupts airway patterning We initially achieved expression of an N-terminally truncated form of -catenin (N–catenin) within early distal lung endoderm using a surfactant protein C (transgene to drive efficient recombination of (supplementary material Fig. S1) and (Okubo et al., 2005). The driver transgene directs recombination of LoxP-flanked (flox) genomic substrates within endodermal lung progenitors, which generate all distal conducting airway and alveolar cell types. Variable low levels of recombination were also observed within epithelial cells of more proximal conducting airway epithelium because descendants of early lung progenitor cells contribute to these structures (supplementary material Fig. S1C,G). mice that were heterozygotes (or mice. Lungs of both embryonic and postnatal mice included large dilated airways (Fig. 1ACH), and pups dying during the early postnatal period showed labored breathing and cyanosis at the time of birth. Mice carrying only were identical to wild-type mice and displayed none of the phenotypic changes observed in mice (supplementary material Figs S1, S2). Surviving pups gained weight in parallel with littermates (wild type, or animals were fertile, enabling generation of mice that were homozygotes ((ACD, ICL) and [… Luminal distension of developing airways in embryos was detected as early as E14.5CE16.5 (compare Fig. ?Fig.1I1I and ?and1M1M for E16.5; supplementary material Fig. S1). At later stages, these malformations were confined to the distal regions of the conducting (bronchiolar) airway tree (Fig. 1JCL,NCP). Distal lung parenchyma appeared to be largely normal, with some evidence for local hypoplasia in close proximity to distended bronchiolar airways. Although the number and organization of lung lobes was normal, airway defects were seen in all lobes and in both heterozygous and homozygous mice. Given that phenotypic changes Proparacaine HCl to lungs of mice were restricted to the distal conducting airway without obvious involvement of alveolar or proximal conducting airways, we defined more precisely the spatial pattern of recombination mediated by the transgene. This was initially accomplished by evaluating (referred to as allele was reflective of the spatial domain of recombination. Lung tissue from mice was evaluated by dual immunofluorescence using primary antibodies recognizing epitopes within either the N terminus (N–cat) or C terminus (C–cat) of -catenin (supplementary material Figs S1, S3). The full-length wild-type form of -catenin was detected at the lateral membrane of all airway epithelial cells of Cre? mice (supplementary material Fig. S3ACC; and data not shown). By contrast, lungs contained epithelial cells in distal tubules that stained only for C-terminal -catenin at lateral membranes and clusters of epithelial cells with intense cytoplasmic and nuclear anti-C–cat antibody staining Proparacaine HCl but no N–cat antibody reactivity (supplementary material Fig. S1MCO, arrows; Fig. S3DCI, arrows). These patterns of C–cat staining without significant N–cat immunoreactivity confirmed that distal lung epithelium efficiently recombined the allele, leading to expression of N–catenin. Epithelial clusters showing intense immunoreactivity for N–catenin were less numerous within dilated ISG15 airways of Proparacaine HCl postnatal mice and were only rarely observed within lungs of surviving adult mice (supplementary material Fig. S3F,I). By contrast, epithelial cells lining relatively normal alveolar structures of mice showed lower levels of N–catenin and expanded postnatally in concert with alveolarization. N–catenin expressed within alveolar epithelial cells of adult lungs displayed a nuclear pattern of subcellular localization (supplementary material Fig. S3I, arrowheads). Stabilized -catenin activates transcription of Wnt target genes in developing airways We used a -galactosidase (mice. At E14.5, lungs of both and embryos showed intense, patchy, X-Gal staining (Fig. 2A,B). At Proparacaine HCl E16.5 and postnatal day 1 (P1), X-Gal staining was only infrequently observed, with a punctate and apparently random distribution within lungs of mice showed robust and broadly distributed X-Gal staining (Fig. 2D) that.