Wnt3a regulates Lef-1 expression during airway submucosal gland morphogenesis

Abstract

Regulation of the lymphoid enhancer factor 1 (Lef-1) transcription factor is important for the inductive formation of many epithelial-derived appendages including airway submucosal glands (SMGs). Although Wnts have been linked to developmental processes involving transcriptional activation of the Lef-1 protein, there is little in vivo information directly linking Wnts with the transcriptional regulation of the Lef-1 promoter. In the present study, we hypothesized that Wnt3a directly regulates Lef-1 gene expression required for SMG morphogenesis in mice. In support of this hypothesis, TOPGAL reporter mice demonstrated activation of beta-catenin/Tcf complexes during early phases of SMG development and immunolocalization studies confirmed abundant expression of Tcf4, but not Tcf1 or Tcf3, at this stage. ChIP analysis in primary airway epithelial cells revealed that Tcf4 associates with a known Wnt Responsive Region in the Lef-1 promoter and transfection of Cos-1 cells with dominant active beta-catenin and Tcf4 synergistically activated the Lef-1 promoter. Using Wnt3a deficient and Lef-1 promoter-GFP reporter mice, we also demonstrate that Wnt3a induces Lef-1 gene expression in newly forming SMG buds of mice and is required for the maintenance of gland bud growth. These findings provide the first in vivo evidence that Wnt3a can transcriptionally regulate the Lef-1 gene.

R-Spondin1 protects mice from chemotherapy or radiation-induced oral mucositis through the canonical Wnt/β-catenin pathway

“…TOPGAL mice were previously developed to monitor canonical Wnt/␤-catenin activity by engineering the transgene containing the LacZ gene under the control of a regulatory sequence consisting of three consensus LEF/TCFbinding motifs upstream of a minimal c-fos promoter (22). The TOPGAL transgenic mouse model was extensively used to analyze the canonical Wnt signaling in vivo (23)(24)(25)(26).…”

The Glandular Stem/Progenitor Cell Niche in Airway Development and Repair

“…To date, a number of signaling pathways (including those involving wingless 1 int-1 [Wnt], bone morphogenetic protein [BMP], fibroblast growth factor [FGF] Notch, and Hedgehog) and their downstream effectors or modulators have been identified as important mediators of cell-fate specification during the morphogenesis of glandular organs. For example, the HMG-box transcriptional factor lymphoid enhancer binding factor (Lef-1) of the Wnt pathway is involved in the initiation of the development of SMGs and a wide range of other epithelial, bud-forming organs, including mammary glands, teeth, and hair follicles (37)(38)(39)(40)(41). Like Lef-1, other transcription factors, such as Pax6 and Msx1/2, have been shown to be involved in bud formation of glandular organs (42,43).…”

“…Mice lacking the Lef-1 gene are impaired in bud formation of the mammary gland, tooth, vibrissa, hair, and airway/nasal SMGs, suggesting that Lef-1 expression is required for formation of these organs (38)(39)(40)(41). In the context of airway SMG development, expression of Lef-1 at both the mRNA and protein levels is highly induced during the early bud stage, in which three to five cells aggregate within the surface epithelium before invagination (37)(38)(39)(40). During later stages of gland development, Lef-1 expression is restricted to the cells in the most distal invading tips of the tubules.…”

“…More specifically, a Lef-1 promoter lacking a WRE exhibited increased basal levels of transcription and a loss of Wnt responsiveness, suggesting that the WRE acts as a repressor in the absence of Wnt signals. Furthermore, chromatin immunoprecipitation (ChIP) assays have suggested that TCF4 can bind the WRE in airway epithelial cells (40) and other cell types (66). The analysis of mice expressing bgalactosidase or EGFP reporter transgenes under the control of the 2.5-kb Lef-1 promoter fragment has indicated that this promoter segment alone is sufficient to regulate expression in tracheal and nasal SMG buds (39,40).…”

“…Furthermore, chromatin immunoprecipitation (ChIP) assays have suggested that TCF4 can bind the WRE in airway epithelial cells (40) and other cell types (66). The analysis of mice expressing bgalactosidase or EGFP reporter transgenes under the control of the 2.5-kb Lef-1 promoter fragment has indicated that this promoter segment alone is sufficient to regulate expression in tracheal and nasal SMG buds (39,40). Furthermore, in this model system, the deletion of the 110-bp WRE from the Lef-1 promoter led to a lack of expression in gland buds (39).…”