Caterina Tiozzo

Human Amniotic Fluid Stem Cells Can Integrate and Differentiate into Epithelial Lung Lineages

A new source of stem cells has recently been isolated from amniotic fluid; these amniotic fluid stem cells have significant potential for regenerative medicine. These cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. We investigated the ability of human amniotic fluid stem cells (hAFSC) to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice, following hyperoxia injury, tail vein‐injected hAFSC localized in the distal lung and expressed both TTF1 and the type II pneumocyte marker surfactant protein C. Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions with expression of the specific Clara cell 10‐kDa protein. These results illustrate the plasticity of hAFSC to respond in different ways to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers, depending on the type of injury to recipient lung.

Parabronchial smooth muscle constitutes an airway epithelial stem cell niche in the mouse lung after injury

During lung development, parabronchial SMC (PSMC) progenitors in the distal mesenchyme secrete fibroblast growth factor 10 (Fgf10), which acts on distal epithelial progenitors to promote their proliferation. β-catenin signaling within PSMC progenitors is essential for their maintenance, proliferation, and expression of Fgf10. Here, we report that this Wnt/Fgf10 embryonic signaling cascade is reactivated in mature PSMCs after naphthalene-induced injury to airway epithelium. Furthermore, we found that this paracrine Fgf10 action was essential for activating surviving variant Clara cells (the cells in the airway epithelium from which replacement epithelial cells originate) located at the bronchoalveolar duct junctions and adjacent to neuroendocrine bodies. After naphthalene injury, PSMCs secreted Fgf10 to activate Notch signaling and induce Snai1 expression in surviving variant Clara cells, which subsequently underwent a transient epithelial to mesenchymal transition to initiate the repair process. Epithelial Snai1 expression was important for regeneration after injury. We have therefore identified PSMCs as a stem cell niche for the variant Clara cells in the lung and established that paracrine Fgf10 signaling from the niche is critical for epithelial repair after naphthalene injury. These findings also have implications for understanding the misregulation of lung repair in asthma and cancer.

miR-17 family of microRNAs controls FGF10-mediated embryonic lung epithelial branching morphogenesis through MAPK14 and STAT3 regulation of E-Cadherin distribution

The miR-17 family of microRNAs has recently been recognized for its importance during lung development. The transgenic overexpression of the entire miR-17-92 cluster in the lung epithelium led to elevated cellular proliferation and inhibition of differentiation, while targeted deletion of miR-17-92 and miR-106b-25 clusters showed embryonic or early post-natal lethality. Herein we demonstrate that miR-17 and its paralogs, miR-20a, and miR-106b, are highly expressed during the pseudoglandular stage and identify their critical functional role during embryonic lung development. Simultaneous downregulation of these three miRNAs in explants of isolated lung epithelium altered FGF10 induced budding morphogenesis, an effect that was rescued by synthetic miR-17. E-Cadherin levels were reduced, and its distribution was altered by miR-17, miR-20a and miR-106b downregulation, while conversely, beta-catenin activity was augmented, and expression of its downstream targets, including Bmp4 as well as Fgfr2b, increased. Finally, we identified Stat3 and Mapk14 as key direct targets of miR-17, miR-20a, and miR-106b and showed that simultaneous overexpression of Stat3 and Mapk14 mimics the alteration of E-Cadherin distribution observed after miR-17, miR-20a, and miR-106b downregulation. We conclude that the mir-17 family of miRNA modulates FGF10-FGFR2b downstream signaling by specifically targeting Stat3 and Mapk14, hence regulating E-Cadherin expression, which in turn modulates epithelial bud morphogenesis in response to FGF10 signaling.

Contrasting Expression of Canonical Wnt Signaling Reporters TOPGAL, BATGAL and Axin2LacZ during Murine Lung Development and Repair

Canonical Wnt signaling plays multiple roles in lung organogenesis and repair by regulating early progenitor cell fates: investigation has been enhanced by canonical Wnt reporter mice, TOPGAL, BATGAL and Axin2LacZ. Although widely used, it remains unclear whether these reporters convey the same information about canonical Wnt signaling. We therefore compared beta-galactosidase expression patterns in canonical Wnt signaling of these reporter mice in whole embryo versus isolated prenatal lungs. To determine if expression varied further during repair, we analyzed comparative pulmonary expression of beta-galactosidase after naphthalene injury. Our data show important differences between reporter mice. While TOPGAL and BATGAL lines demonstrate Wnt signaling well in early lung epithelium, BATGAL expression is markedly reduced in late embryonic and adult lungs. By contrast, Axin2LacZ expression is sustained in embryonic lung mesenchyme as well as epithelium. Three days into repair after naphthalene, BATGAL expression is induced in bronchial epithelium as well as TOPGAL expression (already strongly expressed without injury). Axin2LacZ expression is increased in bronchial epithelium of injured lungs. Interestingly, both TOPGAL and Axin2LacZ are up regulated in parabronchial smooth muscle cells during repair. Therefore the optimal choice of Wnt reporter line depends on whether up- or down-regulation of canonical Wnt signal reporting in either lung epithelium or mesenchyme is being compared.

FGF10 controls the patterning of the tracheal cartilage rings via Shh

During embryonic development, appropriate dorsoventral patterning of the trachea leads to the formation of periodic cartilage rings from the ventral mesenchyme and continuous smooth muscle from the dorsal mesenchyme. In this work, we have investigated the role of two crucial morphogens, fibroblast growth factor 10 and sonic hedgehog, in the formation of periodically alternating cartilaginous and non-cartilaginous domains in the ventral mesenchyme. Using a combination of gain- and loss-of-function approaches for FGF10 and SHH, we demonstrate that precise spatio-temporal patterns and appropriate levels of expression of these two signaling molecules in the ventral area are crucial between embryonic day 11.5 and 13.5 for the proper patterning of the cartilage rings. We conclude that the expression level of FGF10 in the mesenchyme has to be within a critical range to allow for periodic expression of Shh in the ventral epithelium, and consequently for the correct patterning of the cartilage rings. We propose that disturbed balances of Fgf10 and Shh may explain a subset of human tracheomalacia without tracheo-esophageal fistula or tracheal atresia.

Deletion of Pten expands lung epithelial progenitor pools and confers resistance to airway injury.

RATIONALE Pten is a tumor-suppressor gene involved in stem cell homeostasis and tumorigenesis. In mouse, Pten expression is ubiquitous and begins as early as 7 days of gestation. Pten(-/-) mouse embryos die early during gestation indicating a critical role for Pten in embryonic development. OBJECTIVES To test the role of Pten in lung development and injury. METHODS We conditionally deleted Pten throughout the lung epithelium by crossing Pten(flox/flox) with Nkx2.1-cre driver mice. The resulting Pten(Nkx2.1-cre) mutants were analyzed for lung defects and response to injury. MEASUREMENTS AND MAIN RESULTS Pten(Nkx2.1-cre) embryonic lungs showed airway epithelial hyperplasia with no branching abnormalities. In adult mice, Pten(Nkx2.1-cre) lungs exhibit increased progenitor cell pools composed of basal cells in the trachea, CGRP/CC10 double-positive neuroendocrine cells in the bronchi, and CC10/SPC double-positive cells at the bronchioalveolar duct junctions. Pten deletion affected differentiation of various lung epithelial cell lineages, with a decreased number of terminally differentiated cells. Over time, Pten(Nxk2.1-cre) epithelial cells residing in the bronchioalveolar duct junctions underwent proliferation and formed uniform masses, supporting the concept that the cells residing in this distal niche may also be the source of procarcinogenic stem cells. Finally, increased progenitor cells in all the lung compartments conferred an overall selective advantage to naphthalene injury compared with wild-type control mice. CONCLUSIONS Pten has a pivotal role in lung stem cell homeostasis, cell differentiation, and consequently resistance to lung injury.

Fibroblast growth factor 10 plays a causative role in the tracheal cartilage defects in a mouse model of Apert syndrome.

Patients with Apert syndrome (AS) display a wide range of congenital malformations including tracheal stenosis, which is a disease characterized by a uniform cartilaginous sleeve in place of a normally ribbed cartilagenous trachea. We have studied the cellular and molecular basis of this phenotype in a mouse model of AS (Fgfr2c+/Δ mice), which shows ectopic expression of Fgfr2b in mesenchymal tissues. Here we report that tracheal stenosis is associated with increased proliferation of mesenchymal cells, where the expression of Fgf10 and its upstream regulators Tbx4 and Tbx5 are abnormally elevated. We show that Fgf10 has a critical inductive role in tracheal stenosis, as genetic knockdown of Fgf10 in Fgfr2c+/Δ mice rescues this phenotype. These novel findings demonstrate a regulatory role for Fgf10 in tracheal development and shed more light on the underlying cause of tracheal defects in AS.

NF-kB induces lung maturation during mouse lung morphogenesis.

Lung maturation is hallmarked by the appearance of surfactant‐producing alveoli during transition from the saccular to alveolar stage of lung development. Inflammation can disrupt this process and accelerate lung maturity following intrauterine amniotic infection (chorioamnionitis). Nuclear factor kB (NF‐kB) is a transcription factor central to multiple inflammatory and developmental pathways, including dorsal–ventral patterning in fruit flies, limb and mammary and submandibular gland development in mice, and branching morphogenesis in chick lungs. Given its shared role in inflammation and developmental signaling, we hypothesized that overexpression of NF‐kB targeted to the lung epithelium would exert maturational effects on alveolar development. We generated transgenic mice with lung‐specific overexpression of the RelA subunit of NF‐kB using a surfactant protein C promoter construct. Our results showed that RelA overexpression in the lung yields increased alveolar type I and type II cells. These findings are consistent with a model whereby NF‐kB may induce maturation of lung development through decreased apoptosis of epithelial cells. Developmental Dynamics 237:328–338, 2008.

Mechanisms of TGFβ Inhibition of Lung Endodermal Morphogenesis: The role of TβRII, Smads, Nkx2.1 and Pten

Transforming growth factor-beta is a multifunctional growth factor with roles in normal development and disease pathogenesis. One such role is in inhibition of lung branching morphogenesis, although the precise mechanism remains unknown. In an explant model, all three TGFbeta isoforms inhibited FGF10-induced morphogenesis of mesenchyme-free embryonic lung endoderm. Inhibition of budding by TGFbeta was partially abrogated in endodermal explants from Smad3(-/-) or conditional endodermal-specific Smad4(Delta/Delta) embryonic lungs. Endodermal explants from conditional TGFbeta receptor II knockout lungs were entirely refractive to TGFbeta-induced inhibition. Inhibition of morphogenesis was associated with dedifferentiation of endodermal cells as documented by a decrease in key transcriptional factor, NKX2.1 protein, and its downstream target, surfactant protein C (SpC). TGFbeta reduced the proliferation of wild-type endodermal cells within the explants as assessed by BrdU labeling. Gene expression analysis showed increased levels of mRNA for Pten, a key regulator of cell proliferation. Conditional, endodermal-specific deletion of Pten overcame TGFbetas inhibitory effect on cell proliferation, but did not restore morphogenesis. Thus, the mechanisms by which TGFbeta inhibits FGF10-induced lung endodermal morphogenesis may entail both inhibition of cell proliferation, through increased Pten, as well as inhibition or interference with morphogenetic mediators such as Nkx2.1. Both of the latter are dependent on signaling through TbetaRII.

Evidence for the involvement of Fibroblast Growth Factor 10 in lipofibroblast formation during embryonic lung development

Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10+ progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development. Summary: During lung development in mice, Fgf10 signaling plays an essential role in the formation of lipofibroblasts, which are required for the growth and survival of adult lung epithelial stem cells.