(A,E) Hematoxylin and eosin-stained sections of wild-type and Yapf/f; ShhCre/+ embryos at 10.5 dpc. No apparent difference in the morphology of lung buds was observed between wild-type and Yap mutants. (B,C,F,G) Ventral view of dissected lungs from wild-type and Yapf/f; ShhCre/+ mice at 11.5 and 18.5 dpc. Five buds were produced in both control and Yap-deficient lungs at 11.5 dpc; defective lung branching along the entire lung epithelium could already be detected at this stage in Yap mutants. As lung development proceeded, failure to execute a stereotyped program of branching in the absence of Yap resulted in lungs consisting only of multiple cysts at 18.5 dpc. R, right; L, left; Tr, trachea; Cr, cranial; Md. middle; Cd, caudal; Ac, accessory. (D,H) Immunostaining of lung sections collected from wild-type and Yapf/f; ShhCre/+ mice at 18.5 dpc. Cell types in the proximal airway of Yapf/f; ShhCre/+ mice failed to be specified. For instance, expression of markers for Clara [club] cells (CC10+), ciliated cells (acetylated-tubulin [Ac-tub]+) and pulmonary neuroendocrine cells (CGRP+) were barely detectable (not shown). Reduction in the expression of distal lung cell markers, such as SPC (type II cells) and T1α (type I cells), in the cysts of Yapf/f; ShhCre/+ lungs was also noted. (I,N) Whole-mount immunostaining of wild-type and Yapf/f; ShhCre/+ lungs at 11.5 dpc by two-photon microscopy. Lung epithelium was identified by E-cadherin (E-cad). (J,O) Hematoxylin and eosin-stained sections of wild-type and Yapf/f; ShhCre/+ embryos at 11.5 dpc. The arrow points to ‘evagination’ of epithelial cells in Yap-deficient lung buds. (K–M, P–Q) Ventral view of dissected lungs from wild-type and Yapf/f; ShhCre/+ embryos at the developmental stages indicated. Epithelial ‘evagination’ (arrow in Q) could be seen in Yap-deficient lung buds but they failed to produce new buds subsequently. All views are ventral. Scale bar = 200 μm for C,G; 50 μm for D,H; 100 μm for I,N.