PMC

Anatomic structures and the domains of Nkx2.1 (red) and Sox2 (purple) expression are depicted in diagrams in A and K, where axial levels of each row of micrographs are marked with dotted lines. The left column shows hematoxylin and eosin-stained tissue sections, with dashed lines demarcating the undivided foregut (Fo), esophagus (Es), trachea (Tr) and mainstem bronchi (Br); these dashed lines carry over into the immunohistochemical micrographs for NKX2.1 (middle) and SOX2 (right column). In each image, dorsal is on top and ventral on the bottom. The results reveal ectopic NKX2.1 (red arrowhead in S) and loss of SOX2 (purple arrowhead in T) expression in the ventral endoderm of the undivided Barx1^−/− foregut, corresponding to tissue that shows SOX2 (purple arrow in J) but no Nkx2.1 (red arrow in I) expression in Barx1^+/+ littermates. Results are representative of experiments with 3 embryos of each genotype.

Expression of the respiratory marker NKX2.1 and the stratified epithelial marker p63 in the thoracic foregut of E13.5 Barx1^+/+ (A–C) and Barx1^−/− (D–F) embryos. The sagittal tissue sections reveal an undivided rostral foregut, with NKX2.1-expressing respiratory epithelial cells (red arrowhead in D) replacing p63-expressing (blue arrow in B and blue arrowhead in E) squamous epithelial cells in the ventral esophageal endoderm. The results are interpreted in diagrams in C, F. (G–I) Histologic and immunologic demonstration of asymmetry in the lining of the distal esophagus in E19.5 Barx1^−/− pups, with a stratified squamous epithelium on the dorsal surface and a columnar respiratory epithelium along the ventral surface (arrowheads in G). Immunostaining revealed mutually exclusive NKX2.1 expression in ventral cells (H) and p63 expression in the dorsal epithelium (I). The data represent results from 2 mutant embryos.

Radioactive Barx1 in situ hybridization at E10.5 (A), E11.5 (B) and E13.5 (C). Upper panels show bright-field and lower panels show dark-field images; in each case, dorsal is on top and ventral on the bottom. Images I to III in A and C and I to IV in B represent cross-sections at the axial levels indicated in the diagrams at the left, where the green shading summarizes sites of Barx1 expression; images A-IV and A-V show sagittal sections of E10.5 embryos, with medial to the left and lateral to the right. Sense probes hybridized at the same time gave no signal. Dashed lines mark developing structures Es, esophagus; Tr, trachea; Br, bronchi. Red arrowheads point to selected sites of abundant Barx1 mRNA expression: dorsolateral mesenchyme around the laryngotracheal groove and upper esophagus (A-I, A-IV, A-V, B-I), mesenchyme separating the prospective esophagus and trachea (B-III, B-IV), and mesenchyme between the mainstem bronchi (C-III). Data were obtained on 2 embryos at each stage.

(A–K) ß-galactosidase staining (blue) in tissues from Barx1^+/+; TOPGAL (A–C, G–I) and Barx1^+/+;TOPGAL embryos (D–F, J–K) at E10.5 (A–F) and E14.5 (G–K). Wnt activity persists in the Barx1^−/− dorsal foregut (D), medial bronchial endoderm (F), and adjoining mesenchyme (E) at E10.5, as well as the undivided foregut at E14.5 (J,K), areas with clearly diminished ß-galactosidase/Wnt activity in Barx1^+/+ littermates. The domains of aberrant Wnt activity in mutant embryos correspond to sites adjacent to mesenchymal Barx1 expression shown in . Images in H, I and K show higher magnification of tubular structures shown in G and J. (L–O) In situ hybridization analysis of secreted Wnt antagonists sFRP1 (L, N) and sFRP2 (M, O) in the esophageal mesenchyme of E13.5 wild-type (L, M) and Barx1^−/− (N, O) embryos; dotted lines demarcate the esophagus. Caudal displacement of the tracheo-esophageal bifurcation is again evident in these sagittal images and persistence of signal outside the esophagus highlights the anatomic restriction of reduced sFRP expression. The data represent results from 3 (ß-galactosidase staining) or 2 (sFRP expression) embryos of each genotype. Es, esophagus; Tr, trachea; Br, bronchi; Cla, clavicle; Fo, foregut. (P) Model for the role of Barx1 and Wnt signaling in differentiation of thoracic foregut structures and epithelia. Absence of Barx1 and ensuing excessive Wnt signaling result in differentiation of NKX2.1+ respiratory epithelium at the expense of Sox2+ p63+ squamous esophageal epithelium.