NKX2.1 is a homeodomain transcriptional factor expressed in thyroid, lung, and parts of the brain. We demonstrate that septation of the anterior foregut along the dorsoventral axis, into distinct tracheal and esophageal structures, is blocked in mouse embryos carrying a homozygous targeted disruption of the Nkx2.1 locus. This is consistent with the loss of Nkx2.1 expression, which defines the dorsoventral boundary within the anterior foregut in wild-type E9 embryos. Failure in septation between the trachea and the esophagus in Nkx2.1(-/-) mice leads to the formation of a common lumen that connects the pharynx to the stomach, serving both as trachea and as esophagus, similar in phenotype to a human pathologic condition termed tracheoesophageal fistula. The main-stem bronchi bifurcate from this common structure and connect to profoundly hypoplastic lungs. The mutant lungs fail to undergo normal branching embryogenesis, consist of highly dilated sacs that are not capable of sustaining normal gas exchange functions, and lead to immediate postnatal death. In situ hybridization suggests reduced Bmp-4 expression in the mutant lung epithelium, providing a possible mechanistic clue for impaired branching. Functional deletion of Nkx2. 1 blocks pulmonary-specific epithelial cell differentiation marked by the absence of pulmonary surfactant protein gene expression. Altered expression of temporally regulated genes such as Vegf demonstrates that the lung in Nkx2.1(-/-) mutant embryos is arrested at early pseudoglandular (E11-E15) stage. These results demonstrate a critical role for Nkx2.1 in morphogenesis of the anterior foregut and the lung as well as in differentiation of pulmonary epithelial cells.

Operational parallels in overall mechanisms of three-dimensional patterning of vertebrate organs are becoming increasingly apparent. Many key mediators, such as FGFs, BMPs, and sonic hedgehog, participate in organization of a number of organs, including the lungs, which exhibit a defined proximodistal (P-D) polarity. Recently, Wnt5a a member of the wingless family of signaling molecules involved in cell proliferation, differentiation, and organogenesis, was shown to underlie the outgrowth and P-D morphogenesis of the vertebrate limb. In the current study, we show that Wnt5a is expressed in the mouse lung and plays an important role in lung distal morphogenesis. Analysis of the mutant phenotype in mice carrying a targeted disruption of the Wnt5a locus shows distinct abnormalities in distal lung morphogenesis as manifested by distinct truncation of the trachea and overexpansion of the distal respiratory airways. In the face of deleted WNT5a activity, both epithelial and mesenchymal cell compartments of the Wnt5a(-/-) lungs exhibit increased cell proliferation. The overall architecture of the mutant lungs is characterized by overexpansion of the distal airways and inhibition of lung maturation as reflected by persistence of thickened intersaccular interstitium. Absence of WNT5a activity in the mutant lungs leads to increased expression of Fgf-10, Bmp4, Shh, and its receptor Ptc, raising the possibility that WNT5a, FGF-10, BMP4, and SHH signaling pathways are functionally interactive.

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m2 UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

SUMMARYLocalized Fgf10 expression in the distal mesenchyme adjacent to sites of lung bud formation has long been thought to drive stereotypic branching morphogenesis even though isolated lung epithelium branches in the presence of non-directional exogenous Fgf10 in Matrigel. Here, we show that lung agenesis in Fgf10 knockout mice can be rescued by ubiquitous overexpression of Fgf10, indicating that precisely localized Fgf10 expression is not required for lung branching morphogenesis in vivo. Fgf10 expression in the mesenchyme itself is regulated by Wnt signaling. Nevertheless, we found that during lung initiation simultaneous overexpression of Fgf10 is not sufficient to rescue the absence of primary lung field specification in embryos overexpressing Dkk1, a secreted inhibitor of Wnt signaling. However, after lung initiation, simultaneous overexpression of Fgf10 in lungs overexpressing Dkk1 is able to rescue defects in branching and proximal-distal differentiation. We also show that Fgf10 prevents the differentiation of distal epithelial progenitors into Sox2-expressing airway epithelial cells in part by activating epithelial β-catenin signaling, which negatively regulates Sox2 expression. As such, these findings support a model in which the main function of Fgf10 during lung development is to regulate proximal-distal differentiation. As the lung buds grow out, proximal epithelial cells become further and further displaced from the distal source of Fgf10 and differentiate into bronchial epithelial cells. Interestingly, our data presented here show that once epithelial cells are committed to the Sox2-positive airway epithelial cell fate, Fgf10 prevents ciliated cell differentiation and promotes basal cell differentiation. KEY WORDS: Basal cells, Branching, Dkk1, Fgf10, Lung development, Wnt signaling, MouseLocalized Fgf10 expression is not required for lung branching morphogenesis but prevents differentiation of epithelial progenitors progenitors from differentiating into airway epithelial cells by initially inhibiting Sox2 expression (Park et al., 1998;Que et al., 2007;Ramasamy et al., 2007;Nyeng et al., 2008; Hashimoto et al., 2012). β-Catenin is not only a downstream transcriptional target of epithelial Fgf10 signaling (Lü et al., 2005), but increasing data also indicate that Fgf10 is able to increase nuclear β-catenin directly, via phosphorylation of β-catenin on Ser552 and inhibition of Gsk3β, through the PI3K/AKT pathway (He et al., 2007;Ramasamy et al., 2007;Volckaert et al., 2011). In addition, FGF signaling via Erk/MAPK phosphorylates the Wnt co-receptor Lrp6 on Ser1490 and Thr1572 and phosphorylates β-catenin directly on Tyr142, thereby releasing it from cadherin complexes (Krejci et al., 2012). In turn, epithelial β-catenin activation participates in the induction of Fgfr2b expression to increase Fgf10 signaling further (Shu et al., 2005). Epithelial β-catenin signaling, mediated primarily through Fgf10 signaling, is a regulator of branching morphogenesis and functions to maintain the distal epithelial ...