Lung Branching Morphogenesis Research Articles

Epidermal growth factor induced macropinocytosis directs branch formation of lung epithelial cells.

Lung branching morphogenesis is a complex system involving many molecular interactions to filling the three dimensional spaces; however, the underlying developmental mechanisms are still not fully understood. In this paper, we have investigated the effect of epidermal growth factor (EGF) on normal human bronchial epithelial cells and their three-dimensional (3D) branching pattern formation by using invitro experiments and mathematical simulation. The results show that EGF is essential for 3D branch pattern formation and its receptor is highly expressed at the tip of branches to generate the drive force for cells to migrate. Macropinocytosis induced by EGFR expression is firmly contributed to the nutrition uptake at the tip of branches. Our findings for effective branching formation of human lung cells contribute to further understanding molecular mechanisms of organogenesis, and the important mechanisms also possibly participate in related lung disease such as malformation.

Assessment of the nitrofen model of congenital diaphragmatic hernia and of the dysregulated factors involved in pulmonary hypoplasia.

To study pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH), investigators have been employing a fetal rat model based on nitrofen administration to dams. Herein, we aimed to: (1) investigate the validity of the model, and (2) synthesize the main biological pathways implicated in the development of PH associated with CDH. Using a defined strategy, we conducted a systematic review of the literature searching for studies reporting the incidence of CDH or factors involved in PH development. We also searched for PH factor interactions, relevance to lung development and to human PH. Of 335 full-text articles, 116 reported the incidence of CDH after nitrofen exposure or dysregulated factors in the lungs of nitrofen-exposed rat fetuses. CDH incidence: 54% (27-85%) fetuses developed a diaphragmatic defect, whereas the whole litter had PH in varying degrees. Downregulated signaling pathways included FGF/FGFR, BMP/BMPR, Sonic Hedgehog and retinoid acid signaling pathway, resulting in a delay in early epithelial differentiation, immature distal epithelium and dysfunctional mesenchyme. The nitrofen model effectively reproduces PH as it disrupts pathways that are critical for lung branching morphogenesis and alveolar differentiation. The low CDH rate confirms that PH is an associated phenomenon rather than the result of mechanical compression alone.

Expression of dispatched RND transporter family member 1 is decreased in the diaphragmatic and pulmonary mesenchyme of nitrofen-induced congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH) are thought to be caused by a malformation of the diaphragmatic and pulmonary mesenchyme. Dispatched RND transporter family member 1 (Disp-1) encodes a transmembrane protein that regulates the release of cholesterol and palmitoyl, which is critical for normal diaphragmatic and airway development. Disp-1 is strongly expressed in mesenchymal compartments of fetal diaphragms and lungs. Recently, Disp-1 mutations have been identified in patients with CDH. We hypothesized that diaphragmatic and pulmonary Disp-1 expression is decreased in the nitrofen-induced CDH model. Time-mated rats received nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms and lungs were microdissected on selected endpoints D13, D15 and D18; and divided into control and nitrofen-exposed specimens (n = 12 per sample, time-point and experimental group). Diaphragmatic and pulmonary Disp-1 expression was evaluated by qRT-PCR. Immunofluorescence double staining for Disp-1 was combined with diaphragmatic and pulmonary mesenchymal markers Wt-1 and Sox-9 to localize protein expression in fetal diaphragms and lungs. Relative mRNA levels of Disp-1 were significantly decreased in pleuroperitoneal folds/primordial lungs on D13 (0.18 ± 0.08 vs. 0.46 ± 0.41; p < 0.05/1.06 ± 0.27 vs. 1.34 ± 0.79; p < 0.05), developing diaphragms/lungs on D15 (0.18 ± 0.06 vs. 0.44 ± 0.23; p < 0.05/0.73 ± 0.36 vs. 1.16 ± 0.27; p < 0.05) and fully muscularized diaphragms/differentiated lungs on D18 (0.22 ± 0.18 vs. 0.32 ± 0.23; p < 0.05/0.56 ± 0.16 vs. 0.77 ± 0.14; p < 0.05) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished Disp-1 immunofluorescence predominately in the diaphragmatic and pulmonary mesenchyme of nitrofen-exposed fetuses on D13, D15 and D18, associated with a clear reduction of proliferating mesenchymal cells. Decreased Disp-1 expression during diaphragmatic development and lung branching morphogenesis may interrupt mesenchymal cell proliferation, thus leading to diaphragmatic defects and PH in the nitrofen-induced CDH model.

FOXF1 transcription factor promotes lung morphogenesis by inducing cellular proliferation in fetal lung mesenchyme

Organogenesis is regulated by mesenchymal-epithelial signaling events that induce expression of cell-type specific transcription factors critical for cellular proliferation, differentiation and appropriate tissue patterning. While mesenchymal transcription factors play a key role in mesenchymal-epithelial interactions, transcriptional networks in septum transversum and splanchnic mesenchyme remain poorly characterized. Forkhead Box F1 (FOXF1) transcription factor is expressed in mesenchymal cell lineages; however, its role in organogenesis remains uncharacterized due to early embryonic lethality of Foxf1-/- mice. In the present study, we generated mesenchyme-specific Foxf1 knockout mice (Dermo1-Cre Foxf1-/-) and demonstrated that FOXF1 is required for development of respiratory, cardiovascular and gastrointestinal organ systems. Deletion of Foxf1 from mesenchyme caused embryonic lethality in the middle of gestation due to multiple developmental defects in the heart, lung, liver and esophagus. Deletion of Foxf1 inhibited mesenchyme proliferation and delayed branching lung morphogenesis. Gene expression profiling of micro-dissected distal lung mesenchyme and ChIP sequencing of fetal lung tissue identified multiple target genes activated by FOXF1, including Wnt2, Wnt11, Wnt5A and Hoxb7. FOXF1 decreased expression of the Wnt inhibitor Wif1 through direct transcriptional repression. Furthermore, using a global Foxf1 knockout mouse line (Foxf1-/-) we demonstrated that FOXF1-deficiency disrupts the formation of the lung bud in foregut tissue explants. Finally, deletion of Foxf1 from smooth muscle cell lineage (smMHC-Cre Foxf1-/-) caused hyper-extension of esophagus and trachea, loss of tracheal and esophageal muscle, mispatterning of esophageal epithelium and decreased proliferation of smooth muscle cells. Altogether, FOXF1 promotes lung morphogenesis by regulating mesenchymal-epithelial signaling and stimulating cellular proliferation in fetal lung mesenchyme.

TGF-β Signaling in Lung Health and Disease.

Transforming growth factor (TGF)-β is an evolutionarily conserved pleiotropic factor that regulates a myriad of biological processes including development, tissue regeneration, immune responses, and tumorigenesis. TGF-β is necessary for lung organogenesis and homeostasis as evidenced by genetically engineered mouse models. TGF-β is crucial for epithelial-mesenchymal interactions during lung branching morphogenesis and alveolarization. Expression and activation of the three TGF-β ligand isoforms in the lungs are temporally and spatially regulated by multiple mechanisms. The lungs are structurally exposed to extrinsic stimuli and pathogens, and are susceptible to inflammation, allergic reactions, and carcinogenesis. Upregulation of TGF-β ligands is observed in major pulmonary diseases, including pulmonary fibrosis, emphysema, bronchial asthma, and lung cancer. TGF-β regulates multiple cellular processes such as growth suppression of epithelial cells, alveolar epithelial cell differentiation, fibroblast activation, and extracellular matrix organization. These effects are closely associated with tissue remodeling in pulmonary fibrosis and emphysema. TGF-β is also central to T cell homeostasis and is deeply involved in asthmatic airway inflammation. TGF-β is the most potent inducer of epithelial-mesenchymal transition in non-small cell lung cancer cells and is pivotal to the development of tumor-promoting microenvironment in the lung cancer tissue. This review summarizes and integrates the current knowledge of TGF-β signaling relevant to lung health and disease.

Histone arginine methylation by Prmt5 is required for lung branching morphogenesis through repression of BMP signaling.

Branching morphogenesis is essential for the successful development of a functional lung to accomplish its gas exchange function. Although many studies have highlighted requirements for the bone morphogenetic protein (BMP) signaling pathway during branching morphogenesis, little is known about how BMP signaling is regulated. Here, we report that the protein arginine methyltransferase 5 (Prmt5) and symmetric dimethylation at histone H4 arginine 3 (H4R3sme2) directly associate with chromatin of Bmp4 to suppress its transcription. Inactivation of Prmt5 in the lung epithelium results in halted branching morphogenesis, altered epithelial cell differentiation and neonatal lethality. These defects are accompanied by increased apoptosis and reduced proliferation of lung epithelium, as a consequence of elevated canonical BMP-Smad1/5/9 signaling. Inhibition of BMP signaling by Noggin rescues the lung branching defects of Prmt5 mutant in vitro Taken together, our results identify a novel mechanism through which Prmt5-mediated histone arginine methylation represses canonical BMP signaling to regulate lung branching morphogenesis.

GATA6 suppression enhances lung specification from human pluripotent stem cells.

The transcription factor GATA6 has been shown to be important for lung development and branching morphogenesis in mouse models, but its role in human lung development is largely unknown. Here, we studied the role of GATA6 during lung differentiation using human pluripotent stem cells. We found that the human stem cell lines most efficient at generating NKX2.1+ lung progenitors express lower endogenous levels of GATA6 during endoderm patterning and that knockdown of GATA6 during endoderm patterning increased the generation of these cells. Complete ablation of GATA6 resulted in the generation of lung progenitors displaying increased cell proliferation with up to a 15-fold expansion compared with control cells, whereas the null cell line displayed a defect in further development into mature lung cell types. Furthermore, transgenic expression of GATA6 at the endoderm anteriorization stage skewed development toward a liver fate at the expense of lung progenitors. Our results suggest a critical dosage effect of GATA6 during human endoderm patterning and a later requirement during terminal lung differentiation. These studies offer an approach of modulating GATA6 expression to enhance the production of lung progenitors from human stem cell sources.

Tissue geometry may govern lung branching mode selection

Lung branching morphogenesis proceeds in three stereotyped modes (domain, planar, and orthogonal branching). Much is known about the molecular players, including growth factors such as fibroblast growth factor 10 but it is unknown how these signals could actuate the different branching patterns. With the aim of identifying mechanisms that may determine the different branching modes, we developed a computational model of the epithelial lung bud and its surrounding mesenchyme. We studied transport of morphogens and localization of morphogen flux at lobe surfaces and lobe edges. We find that a single simple mechanism is theoretically capable of directing an epithelial tubule to elongate, bend, flatten, or bifurcate, depending solely on geometric ratios of the tissues in the vicinity of a growing tubule tip. Furthermore, the same simple mechanism is capable of generating orthogonal or planar branching, depending only on the same geometric ratios.

Gata-6 expression is decreased in diaphragmatic and pulmonary mesenchyme of fetal rats with nitrofen-induced congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia are thought to be caused by a malformation of the underlying diaphragmatic and airway mesenchyme. GATA binding protein 6 (Gata-6) is a zinc finger-containing transcription factor that plays a crucial role during diaphragm and lung development. In the primordial diaphragm, Gata-6 expression is restricted to mesenchymal compartments of the pleuroperitoneal folds (PPFs). In addition, Gata-6 is essential for airway branching morphogenesis through upregulation of mesenchymal signaling. Recently, mutations in Gata-6 have been linked to human CDH. We hypothesized that diaphragmatic and pulmonary Gata-6 expression is decreased in the nitrofen-induced CDH model. Time-mated rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 48) were microdissected on selected timepoints D13, D15 and D18, and divided into control and nitrofen-exposed specimens (n = 12 per sample, timepoint and experimental group, respectively). Diaphragmatic and pulmonary gene expression of Gata-6 was analyzed by qRT-PCR. Immunofluorescence-double staining for Gata-6 was combined with the diaphragmatic mesenchymal marker Gata-4 and the pulmonary mesenchymal marker Fgf-10 to evaluate protein expression and localization in fetal diaphragms and lungs. Relative mRNA expression levels of Gata-6 were significantly decreased in PPFs on D13 (0.57 ± 0.21 vs. 2.27 ± 1.30; p < 0.05), developing diaphragms (0.94 ± 0.59 vs. 2.28 ± 1.89; p < 0.05) and lungs (0.56 ± 0.16 vs. 0.71 ± 0.39; p < 0.05) on D15 and fully muscularized diaphragms (1.20 ± 1.10 vs. 2.52 ± 1.86; p < 0.05) and differentiated lungs (0.56 ± 0.05 vs. 0.77 ± 0.14; p < 0.05) on D18 of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished immunofluorescence of Gata-6 mainly in diaphragmatic and pulmonary mesenchyme, which was associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared to controls. Decreased Gata-6 expression during diaphragmatic development and lung branching morphogenesis may disrupt mesenchymal cell proliferation, causing malformed PPFs and reduced airway branching, thus leading to diaphragmatic defects and pulmonary hypoplasia in the nitrofen-induced CDH model.

Human lung branching morphogenesis is orchestrated by the spatiotemporal distribution of ACTA2, SOX2, and SOX9.

Lung morphogenesis relies on a number of important processes, including proximal-distal patterning, cell proliferation, migration and differentiation, as well as epithelial-mesenchymal interactions. In mouse lung development, SOX2+ cells are localized in the proximal epithelium, whereas SOX9+ cells are present in the distal epithelium. We show that, in human lung, expression of these transcription factors differs, in that during the pseudoglandular stage distal epithelial progenitors at the tips coexpress SOX2 and SOX9. This double-positive population was no longer present by the canalicular stages of development. As in mouse, the human proximal epithelial progenitors express solely SOX2 and are surrounded by smooth muscle cells (SMCs) both in the proximal airways and at the epithelial clefts. Upon Ras-related C3 botulinum toxin substrate 1 inhibition, we noted decreased branching, as well as increased SMC differentiation, attenuated peristalsis, and a reduction in the distal double-positive SOX2/SOX9 progenitor cell population. Thus, the presence of SOX2/SOX9 double-positive progenitor cells in the distal epithelium during the pseudoglandular stage of human lung development appears to be critical to proximal-distal patterning and lung branching. Moreover, SMCs promote a SOX2 proximal phenotype and seem to suppress the SOX9+ population.

Lung branching morphogenesis, in the chicken model, is accompanied by temporal metabolic changes

Lung branching morphogenesis, in the chicken model, is accompanied by temporal metabolic changes

In Vitro Experimental Model for the Long-Term Analysis of Cellular Dynamics During Bronchial Tree Development from Lung Epithelial Cells.

Lung branching morphogenesis has been studied for decades, but the underlying developmental mechanisms are still not fully understood. Cellular movements dynamically change during the branching process, but it is difficult to observe long-term cellular dynamics by in vivo or tissue culture experiments. Therefore, developing an in vitro experimental model of bronchial tree would provide an essential tool for developmental biology, pathology, and systems biology. In this study, we succeeded in reconstructing a bronchial tree in vitro by using primary human bronchial epithelial cells. A high concentration gradient of bronchial epithelial cells was required for branching initiation, whereas homogeneously distributed endothelial cells induced the formation of successive branches. Subsequently, the branches grew in size to the order of millimeter. The developed model contains only two types of cells and it facilitates the analysis of lung branching morphogenesis. By taking advantage of our experimental model, we carried out long-term time-lapse observations, which revealed self-assembly, collective migration with leader cells, rotational motion, and spiral motion of epithelial cells in each developmental event. Mathematical simulation was also carried out to analyze the self-assembly process and it revealed simple rules that govern cellular dynamics. Our experimental model has provided many new insights into lung development and it has the potential to accelerate the study of developmental mechanisms, pattern formation, left–right asymmetry, and disease pathogenesis of the human lung.

YAP is crucial for lung branching morphogenesis.

Development Lung morphogenesis is a complex process involving rearrangements of various cell types to achieve the branched patterning necessary to maximize gaseous exchange. Lin et al. found that YAP, a component of the Hippo pathway known to be important for organ size regulation, is required for branching. YAP is expressed throughout the murine lung epithelium. Local inhibition of YAP decreases local cell proliferation, which means inadequate force is produced to move cells and form airway branches. Genomic analysis showed that YAP regulates levels of pMLC (phosphorylated myosin light chain), which is required for cells to produce mechanical force. The YAP mechanism may also be involved in branching in other tissues. eLife 10.7554/eLife.21130 (2017). ![Figure][1] Regulation of motor proteins forces developing tissues to branch. PHOTO: BIOPHOTO ASSOCIATES/SCIENCE SOURCE [1]: pending:yes

Turing mechanism underlying a branching model for lung morphogenesis.

The mammalian lung develops through branching morphogenesis. Two primary forms of branching, which occur in order, in the lung have been identified: tip bifurcation and side branching. However, the mechanisms of lung branching morphogenesis remain to be explored. In our previous study, a biological mechanism was presented for lung branching pattern formation through a branching model. Here, we provide a mathematical mechanism underlying the branching patterns. By decoupling the branching model, we demonstrated the existence of Turing instability. We performed Turing instability analysis to reveal the mathematical mechanism of the branching patterns. Our simulation results show that the Turing patterns underlying the branching patterns are spot patterns that exhibit high local morphogen concentration. The high local morphogen concentration induces the growth of branching. Furthermore, we found that the sparse spot patterns underlie the tip bifurcation patterns, while the dense spot patterns underlies the side branching patterns. The dispersion relation analysis shows that the Turing wavelength affects the branching structure. As the wavelength decreases, the spot patterns change from sparse to dense, the rate of tip bifurcation decreases and side branching eventually occurs instead. In the process of transformation, there may exists hybrid branching that mixes tip bifurcation and side branching. Since experimental studies have reported that branching mode switching from side branching to tip bifurcation in the lung is under genetic control, our simulation results suggest that genes control the switch of the branching mode by regulating the Turing wavelength. Our results provide a novel insight into and understanding of the formation of branching patterns in the lung and other biological systems.

Identification of a SOX2/SOX9 Double Positive Cells Progenitor Cell Population Required for Branching Morphogenesis in Human Lung

BackgroundTo date, the proximal‐distal (PD) axis of the lung has been distinguished by the localization of two main transcription factors: Sox2 and Sox9. In the developing mouse lung, distal tip epithelial progenitors express Sox9, whereas proximal progenitors in the stalk express Sox2. Moreover, in the mouse lung, Fgf10 controls the PD axis by preventing the differentiation of epithelial Sox9+ progenitors into Sox2‐expressing airway cells. Whereas lung branching has been extensively studied in animal models little is known about the mechanisms underlying human lung branching and whether findings from mouse apply to human. We aimed to determine the role of FGF signaling in human fetal lung.MethodsFresh human fetal lung tissue between gestational ages 10–16 weeks were collected and either stained or used for culture. Distal lung segments (1–3 mm3) with intact edges were cultured in air‐liquid interface for up to 72 hours. Explants were treated with different fibroblast growth factors (FGFs) known to alter PD epithelial cell differentiation as well as the tyrosine kinase inhibitor (SU5402).ResultsCareful examination of the human fetal lung showed that, similar to mouse, proximal progenitors express SOX2. However, the distal tip progenitors co‐expressed SOX2 and SOX9, a pattern never seen in mouse. This SOX2/SOX9+ progenitor population persists during the pseudoglandular stage up to around 16 weeks gestation. Moreover, we showed that in response to FGF7 and FGF9, branching was impaired accompanied by cysting of distal buds, decreased SOX2 expression, expansion of SOX9 with decreased of SOX2/SOX9 double positive cells. FGF10 did not promote branching in whole explant cultures or in epithelial bud cultures. A decline in SOX2/SOX9 double positive cells was also observed. In contrast, treatment of human lung explants with SU5402, a tyrosine kinase inhibitor, resulted in expansion of SOX2 and decrease of SOX9, resulting in the decline of SOX2/SOX9+ progenitor cell population.ConclusionIn conclusion, we identified the presence of a new progenitor cell population in the human lung co‐expressing SOX2 and SOX9, never seen in mouse. While FGF7 and FGF9 have similar effects on human and mouse lung branching, the effect of Fgf10 was different in that it failed to induce branching of human lung. We believe that this is mainly due to the alteration of the cells co‐expressing SOX2/SOX9+. We therefore conclude that the maintenance of this progenitor population is required for proper branching morphogenesis in human lung. However, we still need to identify the signals required for the maintenance of SOX2/SOX9 progenitor cell identity. Understanding the mechanisms driving airway branching will transform our concepts of human lung development and may allow for the discovery of possible therapeutic avenues to restore or enhance lung development for severely premature infants at increased risk for lung diseases.Support or Funding InformationAmerican Heart Association, Saban Research Institute

YAP is essential for mechanical force production and epithelial cell proliferation during lung branching morphogenesis.

Branching morphogenesis is a fundamental program for tissue patterning. We show that active YAP, a key mediator of Hippo signaling, is distributed throughout the murine lung epithelium and loss of epithelial YAP severely disrupts branching. Failure to branch is restricted to regions where YAP activity is removed. This suggests that YAP controls local epithelial cell properties. In support of this model, mechanical force production is compromised and cell proliferation is reduced in Yap mutant lungs. We propose that defective force generation and insufficient epithelial cell number underlie the branching defects. Through genomic analysis, we also uncovered a feedback control of pMLC levels, which is critical for mechanical force production, likely through the direct induction of multiple regulators by YAP. Our work provides a molecular pathway that could control epithelial cell properties required for proper morphogenetic movement and pattern formation.

Expression of T-box transcription factors 2, 4 and 5 is decreased in the branching airway mesenchyme of nitrofen-induced hypoplastic lungs.

Pulmonary hypoplasia (PH), characterized by smaller lung size and reduced airway branching, remains a major therapeutic challenge in newborns with congenital diaphragmatic hernia (CDH). T-box transcription factors (Tbx) have been identified as key components of the gene network that regulates fetal lung development. Tbx2, Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung, regulating the process of lung branching morphogenesis. Furthermore, lungs of Tbx2-, Tbx4- and Tbx5-deficient mice are hypoplastic and exhibit decreased lung branching, similar to PH in human CDH. We hypothesized that the expression of Tbx2, Tbx4 and Tbx5 is decreased in the branching airway mesenchyme of hypoplastic rat lungs with nitrofen-induced CDH. Time-mated rats received either nitrofen or vehicle on gestational day 9 (D9). Fetuses were killed on D15, D18 and D21, and dissected lungs were divided into control and nitrofen-exposed specimens. Pulmonary gene expression of Tbx2, Tbx4 and Tbx5 was investigated by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for Tbx2, Tbx4 and Tbx5 was combined with the mesenchymal marker Fgf10 to assess protein expression and localization in branching airway tissue. Relative mRNA levels of Tbx2, Tbx4 and Tbx5 were significantly reduced in lungs of nitrofen-exposed fetuses on D15, D18 and D21 compared to controls. Confocal laser scanning microscopy showed markedly diminished immunofluorescence of Tbx2, Tbx4 and Tbx5 in mesenchymal cells surrounding branching airways of nitrofen-exposed fetuses on D15, D18 and D21 compared to controls. Decreased expression of Tbx2, Tbx4 and Tbx5 in the pulmonary mesenchyme during fetal lung development may lead to a decrease or arrest of airway branching, thus contributing to PH in the nitrofen-induced CDH model.

Rac1 modulates mammalian lung branching morphogenesis in part through canonical Wnt signaling

Lung branching morphogenesis relies on a number of factors, including proper epithelial cell proliferation and differentiation, cell polarity, and migration. Rac1, a small Rho GTPase, orchestrates a number of these cellular processes, including cell proliferation and differentiation, cellular alignment, and polarization. Furthermore, Rac1 modulates both noncanonical and canonical Wnt signaling, important pathways in lung branching morphogenesis. Culture of embryonic mouse lung explants in the presence of the Rac1 inhibitor (NSC23766) resulted in a dose-dependent decrease in branching. Increased cell death and BrdU uptake were notably seen in the mesenchyme, while no direct effect on the epithelium was observed. Moreover, vasculogenesis was impaired following Rac1 inhibition as shown by decreased Vegfa expression and impaired LacZ staining in Flk1-Lacz reporter mice. Rac1 inhibition decreased Fgf10 expression in conjunction with many of its associated factors. Moreover, using the reporter lines TOPGAL and Axin2-LacZ, there was an evident decrease in canonical Wnt signaling in the explants treated with the Rac1 inhibitor. Activation of canonical Wnt pathway using WNT3a or WNT7b only partially rescued the branching inhibition. Moreover, these results were validated on human explants, where Rac1 inhibition resulted in impaired branching and decreased AXIN2 and FGFR2b expression. We therefore conclude that Rac1 regulates lung branching morphogenesis, in part through canonical Wnt signaling. However, the exact mechanisms by which Rac1 interacts with canonical Wnt in human and mouse lung requires further investigation.

Tbx2 and Tbx3 Act Downstream of Shh to Maintain Canonical Wnt Signaling during Branching Morphogenesis of the Murine Lung

Numerous signals drive the proliferative expansion of the distal endoderm and the underlying mesenchyme during lung branching morphogenesis, but little is known about how these signals are integrated. Here, we show by analysis of conditional double mutants that the two T-box transcription factor genes Tbx2 and Tbx3 act together in the lung mesenchyme to maintain branching morphogenesis. Expression ofboth genes depends on epithelially derived Shh signaling, with additional modulation by Bmp, Wnt, and Tgfβ signaling. Genetic rescue experiments reveal that Tbx2 and Tbx3 function downstream ofShh to maintain pro-proliferative mesenchymal Wnt signaling, in part by direct repression of the Wnt antagonists Frzb and Shisa3. In combination with our previous finding that Tbx2 and Tbx3 repress thecell-cycle inhibitors Cdkn1a and Cdkn1b, we conclude that Tbx2 and Tbx3 maintain proliferation of the lung mesenchyme by way of at least two molecular mechanisms: regulating cell-cycle regulation and integrating the activity of multiple signaling pathways.

E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors

The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis.