Stijn De Langhe

Molecular Mechanisms of Early Lung Specification and Branching Morphogenesis

The “hard wiring” encoded within the genome that determines the emergence of the laryngotracheal groove and subsequently early lung branching morphogenesis is mediated by finely regulated, interactive growth factor signaling mechanisms that determine the automaticity of branching, interbranch length, stereotypy of branching, left-right asymmetry, and finally gas diffusion surface area. The extracellular matrix is an important regulator as well as a target for growth factor signaling in lung branching morphogenesis and alveolarization. Coordination not only of epithelial but also endothelial branching morphogenesis determines bronchial branching and the eventual alveolar-capillary interface. Improved prospects for lung protection, repair, regeneration, and engineering will depend on more detailed understanding of these processes. Herein, we concisely review the functionally integrated morphogenetic signaling network comprising the critical bone morphogenetic protein, fibroblast growth factor, Sonic hedgehog, transforming growth factor-β, vascular endothelial growth factor, and Wnt signaling pathways that specify and drive early embryonic lung morphogenesis.

Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage.

Lineage formation in the lung mesenchyme is poorly understood. Using a transgenic mouse line expressing LacZ under the control of Fgf10 regulatory sequences, we show that the pool of Fgf10-positive cells in the distal lung mesenchyme contains progenitors of the parabronchial smooth muscle cells. Fgf10 gene expression is slightly repressed in this transgenic line. This allowed us to create a hypomorphic Fgf10 phenotype by expressing the LacZ transgene in a heterozygous Fgf10 background. Hypomorphic Fgf10 mutant lungs display a decrease inβ -galactosidase-positive cells around the bronchial epithelium associated with an accumulation of β-galactosidase-expressing cells in the distal mesenchyme. This correlates with a marked reduction of α smooth muscle actin expression, thereby demonstrating that FGF10 is mostly required for the entry of mesenchymal cells into the parabronchial smooth muscle cell lineage. The failure of exogenous FGF10 to phosphorylate its known downstream targets ERK and AKT in lung mesenchymal cultures strongly suggests that FGF10 acts indirectly on the progenitor population via an epithelial intermediate. We provide support for a role of epithelial BMP4 in mediating the formation of parabronchial smooth muscle cells.

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.

Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo.

Reprogramming somatic cells into an ESC‐like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage, and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for 8–12 weeks. Differentiation was assessed by lineage‐specific morphology, gene expression, histological stain, and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS‐derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic imprinting control region (ICR) mice. Implants were harvested at 12 weeks, histological analyses of cell and mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage, and fat was confirmed by morphology and expression of lineage‐specific genes. Isolated implants of iPS cell‐derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells, which merit evaluation for their potential uses in orthopedic medicine and understanding of molecular mechanisms of orthopedic disease. STEM CELLS 2011;29:206–216

Neutrophil transmigration triggers repair of the lung epithelium via β-catenin signaling

Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. We used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiological basolateral-to-apical direction. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifested by restoration of transepithelial resistance and reepithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated β-catenin signaling, and this was verified by real-time PCR, nuclear translocation of β-catenin, and TOPFlash reporter activity. Leukocyte elastase, likely via cleavage of E-cadherin, was required for activation of β-catenin signaling in response to neutrophil transmigration. Knockdown of β-catenin using shRNA delayed epithelial repair. In mice treated with intratracheal LPS or keratinocyte chemokine, neutrophil emigration resulted in activation of β-catenin signaling in alveolar type II epithelial cells, as demonstrated by cyclin D1 expression and/or reporter activity in TOPGAL mice. Attenuation of β-catenin signaling by IQ-1 inhibited alveolar type II epithelial cell proliferation in response to neutrophil migration induced by intratracheal keratinocyte chemokine. We conclude that β-catenin signaling is activated in lung epithelial cells during neutrophil transmigration, likely via elastase-mediated cleavage of E-cadherin, and regulates epithelial repair. This pathway represents a potential therapeutic target to accelerate physiological recovery in inflammatory lung diseases.

Localized Fgf10 expression is not required for lung branching morphogenesis but prevents differentiation of epithelial progenitors

Localized 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.

FGF10/FGFR2b signaling is essential for cardiac fibroblast development and growth of the myocardium

The epicardium serves as a source of growth factors that regulate myocardial proliferation and as a source of epicardial-derived cells (EPDC), which give rise to interstitial cardiac fibroblasts and perivascular cells. These progenitors populate the compact myocardium to become part of the mature coronary vasculature and fibrous skeleton of the heart. Little is known about the mechanisms that regulate EPDC migration into the myocardium or the functions carried out by these cells once they enter the myocardium. However, it has been proposed that cardiac fibroblasts are important for growth of the heart during late gestation and are a source of homeostatic factors in the adult. Here, we identify a myocardial to epicardial fibroblast growth factor (FGF) signal, mediated by FGF10 and FGFR2b, that is essential for movement of cardiac fibroblasts into the compact myocardium. Inactivation of this signaling pathway results in fewer epicardial derived cells within the compact myocardium, decreased myocardial proliferation and a resulting smaller thin-walled heart.

Lung epithelial stem cells and their niches: Fgf10 takes center stage

Throughout life adult animals crucially depend on stem cell populations to maintain and repair their tissues to ensure life-long organ function. Stem cells are characterized by their capacity to extensively self-renew and give rise to one or more differentiated cell types. These powerful stem cell properties are key to meet the changing demand for tissue replacement during normal lung homeostasis and regeneration after lung injury. Great strides have been made over the last few years to identify and characterize lung epithelial stem cells as well as their lineage relationships. Unfortunately, knowledge on what regulates the behavior and fate specification of lung epithelial stem cells is still limited, but involves communication with their microenvironment or niche, a local tissue environment that hosts and influences the behaviors or characteristics of stem cells and that comprises other cell types and extracellular matrix. As such, an intimate and dynamic epithelial-mesenchymal cross-talk, which is also essential during lung development, is required for normal homeostasis and to mount an appropriate regenerative response after lung injury. Fibroblast growth factor 10 (Fgf10) signaling in particular seems to be a well-conserved signaling pathway governing epithelial-mesenchymal interactions during lung development as well as between different adult lung epithelial stem cells and their niches. On the other hand, disruption of these reciprocal interactions leads to a dysfunctional epithelial stem cell-niche unit, which may culminate in chronic lung diseases such as chronic obstructive pulmonary disease (COPD), chronic asthma and idiopathic pulmonary fibrosis (IPF).

Wnt signaling in lung organogenesis

Reporter transgene, knockout, and misexpression studies support the notion that Wnt/β-catenin signaling regulates aspects of branching morphogenesis, regional specialization of the epithelium and mesenchyme, and establishment of progenitor cell pools. As demonstrated for other foregut endoderm-derived organs, β-catenin and the Wnt/β-catenin signaling pathway contribute to control of cellular proliferation, differentiation and migration. However, the contribution of Wnt/β-catenin signaling to these processes is shaped by other signals impinging on target tissues. In this review, we will concentrate on roles for Wnt/β-catenin in respiratory system development, including segregation of the conducting airway and alveolar compartments, specialization of the mesencyme, and establishment of tracheal asymmetries and tracheal glands.

Two-Way Conversion between Lipogenic and Myogenic Fibroblastic Phenotypes Marks the Progression and Resolution of Lung Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a form of progressive interstitial lung disease with unknown etiology. Due to a lack of effective treatment, IPF is associated with a high mortality rate. The hallmark feature of this disease is the accumulation of activated myofibroblasts that excessively deposit extracellular matrix proteins, thus compromising lung architecture and function and hindering gas exchange. Here we investigated the origin of activated myofibroblasts and the molecular mechanisms governing fibrosis formation and resolution. Genetic engineering in mice enables the time-controlled labeling and monitoring of lipogenic or myogenic populations of lung fibroblasts during fibrosis formation and resolution. Our data demonstrate a lipogenic-to-myogenic switch in fibroblastic phenotype during fibrosis formation. Conversely, we observed a myogenic-to-lipogenic switch during fibrosis resolution. Analysis of human lung tissues and primary human lung fibroblasts indicates that this fate switching is involved in IPF pathogenesis, opening potential therapeutic avenues to treat patients.