beta-Catenin promotes respiratory progenitor identity in mouse foregut

Abstract

The mammalian respiratory system, consisting of both trachea and lung, initiates from the foregut endoderm. The molecular program that instructs endodermal cells to adopt the respiratory fate is not fully understood. Here we show that conditional inactivation of beta-Catenin (also termed Ctnnb1) in foregut endoderm leads to absence of both the trachea and lung due to a failure in maintaining the respiratory fate. In converse, conditional expression of an activated form of beta-Catenin leads to expansion of Nkx2.1, an early marker for the trachea and lung, into adjacent endoderm including the stomach epithelium. Analyses of these mutants show that the loss or gain of trachea/lung progenitor identity is accompanied by an expansion or contraction of esophagus/stomach progenitor identity, respectively. Our findings reveal an early role for beta-Catenin in the establishment of respiratory progenitors in mouse foregut endoderm.

Fig. 1.

WNT/β-Catenin signaling and β-Catenin inactivation in the foregut using Shh^cre. (A–C) Wnt2 expression as determined by RNA in situ hybridization in E10.25 embryos. Bracketed region in A is magnified in B, and line in B indicates the approximate level of transverse section shown in C. Axin2 is expressed in the ventral foregut mesenchyme adjacent to nascent lung buds. (D–M) β-galactosidase (β-gal) staining in Axin2-lacZ (D–F) or Shh^cre;R26R embryos (G–M) at stages indicated. Bracketed regions in D, G, and I are enlarged in E, H, and J, respectively. Lines in E and J indicate approximate level of transverse sections shown in F and K–M. Bracketed regions in the left panels of K–M are magnified in the corresponding right panels. Arrowhead in E indicates Axin2-lacZ activity in the prospective respiratory region. Shh^cre activity in the foregut is first detected at the 16-so stage (≈E8.75), around the time of specification. By E9.75 (after lung budding), its activity is detected in the primary lung buds, trachea, ventral esophagus, ventral stomach, intestine and isolated cells in the liver primordium. (N and O) Anti-β-Catenin antibody staining in transverse sections of 19-so stage embryos at the foregut level. Arrow in O indicates diminished β-Catenin staining in the ventral foregut epithelium of the β-Cat^cko mutant lung. For transverse sections shown in all figures, dorsal is up and ventral is down. Abbreviations: eso, esophagus; fg, foregut; fp, floor plate; he, heart; lb, limb bud; li, liver; lu, lung; nc, notochord; sto, stomach; tr, trachea.

Fig. 2.

Inactivation of β-Catenin leads to a failure in maintaining the respiratory fate. (A and B) Trachea and lung are absent in β-Cat^cko mutant at E15.5. (C, D, and G–N) Nkx2.1 expression as detected by RNA in situ hybridization. Ventral (C and D) or lateral (G–N) views are shown. Arrowheads in G–N indicate regions that are enlarged in the insets of each panel, with filled arrowheads indicate presence of expression and open arrowheads indicate absence of expression. Arrows indicate Nkx2.1 expression in the thyroid as a control that the staining intensity is equivalent in all embryos. Nkx2.1 is not detected in the prospective trachea/lung region in either the control or mutant at the 16-so stage. Later, at the 18- and 20-so stages, its expression is detected in both genotypes, although at a lower intensity in the mutant. This expression is absent by the 22-so stage in the mutant. (E and F) Foxa1 expression as detected by RNA in situ hybridization outlines the endoderm in E10.5 embryos. Lateral views are shown. Lines indicate the approximate level of transverse section shown in the insets. In the β-Cat^cko mutant, there is a common foregut tube and an aberrant bud is observed along the ventral midline of the presumptive trachea/lung region (arrowhead in F). Abbreviations: same as Fig. 1 with the addition of pha, pharyngeal pouch; thm, thymus.

Fig. 3.

Inactivation of β-Catenin leads to a defect in foregut patterning. (A–D) Double immunofluorescence staining with anti-cleaved Caspase 3 antibody that labels cells undergoing apoptosis (red) and anti-E-Cadherin antibody that labels foregut epithelium (green) at the 21-so stage (specification) or 28-so stage (budding morpohogenesis). Lateral views are shown. Brackets in A and B indicate prospective trachea/lung region. There is no detectable increase in cell death in the mutants compared to controls. Asterisks in A and B indicate cleaved Caspase 3-positive cells just posterior to the liver in both the mutant and the control to show that the assay is working. (E and F) Assays for BrdU incorporation in transverse sections of the foregut at the 22-so stage. No difference in the percentage of positive cells is detected. (G and H) Double immunofluorescence staining with anti-NKX2.1 antibody (red) and anti-SOX2 antibody (green) in transverse sections of the common trachea/esophageal tube at E10.5. In the β-Cat^cko mutant, NKX2.1 expression is downregulated, and SOX2 expression is expanded to the ventral epithelium. (I and J) Fgf10 expression as detected by RNA in situ hybridization in E10.5 foregut. Ventral views are shown. Filled arrowheads indicate presence of gene expression. Lines indicate approximate level of transverse sections shown in respective insets. (K and L) FGFR2 expression as detected by an anti-FGFR2 antibody in transverse sections of trachea/esophageal region at E10.5. In the β-Cat^cko mutant, FGFR2 remains expressed in both dorsal and ventral foregut endoderm as indicated by arrows. Abbreviations: same as Fig. 1 with the addition of CASP3, cleaved Caspase 3; ECAD, E-Cadherin.

Fig. 4.

Ectopic activation of β-Catenin leads to expansion of a lung progenitor marker. (A–F) Nkx2.1 or Hex expression as detected by RNA in situ hybridization in E10.5 embryos. Boxed region in A and C are magnified in B and D, respectively. Similar magnified views are shown in E and F. In β-Cat^act embryos, expression of the activated form of β-Catenin leads to expansion of Nkx2.1 expression from the trachea/lung region (black dashed line) into the anterior ventral portion of the stomach (red dashed line). However, Hex expression remains not altered. (G and H) Double immunofluorescence staining with anti-NKX2.1 and anti-SOX2 antibodies in transverse sections of the anterior stomach region at E10.5. In β-Cat^act embryos, ectopic expression of NKX2.1 is accompanied by a down-regulation of SOX2 expression.