Elie El Agha | Justus-Liebig-University Giessen (original) (raw)

Fibroblast growth factor 10 (Fgf10) is a key regulator of diverse organogenetic programs during m... more Fibroblast growth factor 10 (Fgf10) is a key regulator of diverse organogenetic programs during mouse development, particularly branching morphogenesis. Fgf10-null mice suffer from lung and limb agenesis as well as cecal and colonic atresia and are thus not viable. To date, the Mlcv1v-nLacZ-24 transgenic mouse strain (referred to as Fgf10LacZ), which carries a LacZ insertion 114 kb upstream of exon 1 of Fgf10 gene, has been the only strain to allow transient lineage tracing of Fgf10-positive cells. Here, we describe a novel Fgf10Cre-ERT2 knock-in line (Fgf10iCre) in which a Cre-ERT2-IRES-YFP cassette has been introduced in frame with the ATG of exon 1 of Fgf10 gene. Our studies show that Cre-ERT2 insertion disrupts Fgf10 function. However, administration of tamoxifen to Fgf10iCre; Tomatoflox double transgenic embryos or adult mice results in specific labeling of Fgf10-positive cells, which can be lineage-traced temporally and spatially. Moreover, we show that the Fgf10iCre line can ...

microRNA-142 (miR-142) is an important regulator of many biological processes and asso-ciated sig... more microRNA-142 (miR-142) is an important regulator of many biological processes and asso-ciated signaling pathways during embryonic development, homeostasis and disease. The miR-142 hairpin gives rise to the “guide strand”miR-142-3p and the sister "passenger" strandmiR-142-5p.miR-142-3p has been shown to play critical, non-redundant functions in the development of the hematopoietic lineage. We have recently reported thatmiR-142-3p is critical for the control of Wnt signaling in the mesenchyme of the developing lung.miR-142-5p has been proposed to control adaptive growth in cardiomyocytes postnatally and its increase is associated with extensive apoptosis and cardiac dysfunction in a murine heart failure model. Using homologous recombination, we now report the generation and valida-tion ofmiR-142-null mice.miR-142-null mice show a significant decrease in th expression levels of both the 3p and 5p isoforms. The expression of Bzrap1, a gene immediately flank-ingmiR-142 is not a...

Canonical Wnt signaling plays multiple roles in lung organogenesis and repair by regulating early... more 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 na...

Lung Stem Cells in Development, Health and Disease

International Journal of Environmental Research and Public Health, 2021

Vascular remodeling is a prominent feature of pulmonary hypertension. This process involves incre... more Vascular remodeling is a prominent feature of pulmonary hypertension. This process involves increased muscularization of already muscularized vessels as well as neo-muscularization of non-muscularized vessels. The cell-of-origin of the newly formed vascular smooth muscle cells has been a subject of intense debate in recent years. Identifying these cells may have important clinical implications since it opens the door for attempts to therapeutically target the progenitor cells and/or reverse the differentiation of their progeny. In this context, the dominant model is that these cells derive from pre-existing smooth muscle cells that are activated in response to injury. In this mini review, we present the evidence that is in favor of this model and, at the same time, highlight other studies indicating that there are alternative cellular sources of vascular smooth muscle cells in pulmonary vascular remodeling.

Idiopathic lung fibrosis (IPF) is a progressive and fatal degenerative lung disease of unknown et... more Idiopathic lung fibrosis (IPF) is a progressive and fatal degenerative lung disease of unknown etiology. Although in its final stages it implicates in a reactive manner all lung cell types, the initial damage involves the alveolar epithelial compartment, in particular the alveolar epithelial type 2 cells (AEC2s). AEC2s serve dual progenitor and surfactant secreting functions, both of which are deeply impacted in IPF. Thus, we hypothesize that the size of the surfactant processing compartment, as measured by Lysotracker incorporation, allows the identification of different epithelial states in the IPF lung. Flow cytometry analysis of epithelial Lysotracker incorporation delineates two populations (Lysohigh and Lysolow) of AEC2s which behave in a compensatory manner during bleomycin injury and in the donor/IPF lung. Employing flow cytometry and transcriptomic analysis of cells isolated from donor and IPF lungs, we demonstrate that the Lysohigh population expresses all classical AEC2 m...

European Respiratory Journal, 2020

Frontiers in Cell and Developmental Biology

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously report... more Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a knock-in Fgf10Cre–ERT2 line (with the Cre-ERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki–v1, to target FGF10Pos cells. While this line allowed fairly efficient and specific labeling of FGF10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus of our research. We report here the generation and validation of a new knock-in Fgf10Cre–ERT2 line (called thereafter Fgf10Ki–v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. Fgf10Ki−v2/+ heterozygous mice exhibited comparable Fgf10 expression levels to wild type animals. However, a mismatch between Fgf10 and Cre expression levels was observed in Fgf10Ki–v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 function...

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously report... more Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a Fgf10CreERT2 line (with the CreERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki-v1, to target Fgf10Pos cells. While this line allowed fairly efficient and specific labeling of Fgf10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus on our research. We report here the generation and validation of a new Fgf10CreERT2 (called thereafter Fgf10Ki-v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. This new Fgf10Ki-v2 line exhibited comparable Fgf10 expression level to their wild type counterpart. However, a disconnection between the Fgf10 and the Cre expression was observed in Fgf10Ki-v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf1...

The EMBO Journal

Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to stu... more Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three‐dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self‐organization of FACS‐sorted bronchioalveolar stem cells (BASCs) upon co‐culture with lung‐resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single‐cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue‐resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial–mesenchymal‐myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro.

Frontiers in Cell and Developmental Biology

Multiple cellular, biochemical, and physical factors converge to coordinate organogenesis. During... more Multiple cellular, biochemical, and physical factors converge to coordinate organogenesis. During embryonic development, several organs such as the lung, salivary glands, mammary glands, and kidneys undergo rapid, but intricate, iterative branching. This biological process not only determines the overall architecture, size and shape of such organs but is also a pre-requisite for optimal organ function. The lung, in particular, relies on a vast surface area to carry out efficient gas exchange, and it is logical to suggest that airway branching during lung development represents a rate-limiting step in this context. Against this background, the vascular network develops in parallel to the airway tree and reciprocal interaction between these two compartments is critical for their patterning, branching, and co-alignment. In this mini review, we present an overview of the branching process in the developing mouse lung and discuss whether the vasculature plays a leading role in the proces...

American Journal of Physiology-Lung Cellular and Molecular Physiology

Pulmonary fibrosis is characterized by pronounced collagen deposition and myofibroblast expansion... more Pulmonary fibrosis is characterized by pronounced collagen deposition and myofibroblast expansion, whose origin and plasticity remain elusive. We utilized a fate-mapping approach to investigate α-smooth muscle actin (αSMA)+ and platelet-derived growth factor receptor α (PDGFRα)+ cells in two lung fibrosis models, complemented by cell type-specific next-generation sequencing and investigations on human lungs. Our data revealed that αSMA+ and PDGFRα+ cells mark two distinct mesenchymal lineages with minimal transdifferentiation potential during lung fibrotic remodeling. Parenchymal and perivascular fibrotic regions were populated predominantly with PDGFRα+ cells expressing collagen, while αSMA+ cells in the parenchyma and vessel wall showed variable expression of collagen and the contractile protein desmin. The distinct gene expression profile found in normal conditions was retained during pathologic remodeling. Cumulatively, our findings identify αSMA+ and PDGFRα+ cells as two separa...

American Journal of Respiratory Cell and Molecular Biology

Nature Communications

Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of... more Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of the lung is progressively replaced by fibrotic scars. Myofibroblasts are the effector cells that excessively deposit extracellular matrix proteins thus compromising lung structure and function. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug metformin exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGFβ1 action, suppressing collagen formation, activating PPARγ signaling and inducing lipogenic differentiation in lung fibroblasts derived from IPF patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-to-lipofibroblast transdifferentiation. Detailed pathway analysis revealed a two-arm mechanism by which metformin accelerates fibrosis resolution. Our data report an antifibrotic role for metformin in the lung, thus warranting further therapeutic evaluation.

Human Molecular Genetics

Bronchopulmonary dysplasia (BPD), characterized by alveoli simplification and dysmorphic pulmonar... more Bronchopulmonary dysplasia (BPD), characterized by alveoli simplification and dysmorphic pulmonary microvasculature, is a chronic lung disease affecting prematurely born infants. Pulmonary hypertension (PH) is an important BPD feature associated with morbidity and mortality. In human BPD, inflammation leads to decreased fibroblast growth factor 10 (FGF10) expression but the impact on the vasculature is so far unknown. We used lungs from Fgf10+/- versus Fgf10+/+ pups to investigate the effect of Fgf10 deficiency on vascular development in normoxia (NOX) and hyperoxia (HOX, BPD mouse model). To assess the role of fibroblast growth factor receptor 2b (Fgfr2b) ligands independently of early developmentaldefects, we used an inducible double transgenic system in mice allowing inhibition of Fgfr2b ligands activity. Using vascular morphometry, we quantified the pathological changes. Finally, we evaluated changes in FGF10, surfactant protein C (SFTPC), platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin 2 (α-SMA) expression in human lung samples from patients suffering from BPD. In NOX, no major difference in the lung vasculature between Fgf10+/- and control pups was detected. In HOX, a greater loss of blood vessels in Fgf10+/- lungs is associated with an increase of poorly muscularized vessels. Fgfr2b ligands inhibition postnatally in HOX is sufficient to decrease the number of blood vessels while increasing the level of muscularization, suggesting a PH phenotype. BPD lungs exhibited decreased FGF10, SFTPC and PECAM but increased α-SMA. Fgf10 deficiency-associated vascular defects are enhanced in HOX and could represent an additional cause of morbidity in human patients with BPD.

The Journal of pathology, 2018

Pulmonary vascular remodeling is the main pathological hallmark of pulmonary hypertension disease... more Pulmonary vascular remodeling is the main pathological hallmark of pulmonary hypertension disease. We undertook a comprehensive and multilevel approach to investigate the origin of smooth muscle actin-expressing cells in remodeled vessels. Transgenic mice that allow for specific, inducible, and permanent labeling of endothelial (Cdh5-tdTomato), smooth muscle (Acta2-, Myh11-tdTomato), pericyte (Cspg4-tdTomato), and fibroblast (Pdgfra-tdTomato) lineages were used to delineate the cellular origins of pulmonary vascular remodeling. Mapping the fate of major lung resident cell types revealed smooth muscle cells (SMCs) as the predominant source of cells that populate remodeled pulmonary vessels in chronic hypoxia and allergen-induced murine models. Combining in vivo cell type-specific, time-controlled labeling of proliferating cells with a pulmonary artery phenotypic explant assay, we identified proliferation of SMCs as an underlying remodeling pathomechanism. Multicolor immunofluorescenc...

American journal of physiology. Lung cellular and molecular physiology, Jan 3, 2018

Idiopathic pulmonary arterial hypertension (IPAH), pulmonary hypertension (PH) due to lung diseas... more Idiopathic pulmonary arterial hypertension (IPAH), pulmonary hypertension (PH) due to lung disease and/or hypoxia and idiopathic pulmonary fibrosis (IPF) are increasingly recognized as important contributors to mortality and morbidity worldwide. Among others, the current treatment paradigm considers broad inhibition of receptor tyrosine kinases, a strategy that likely leads to collateral inhibition of signaling pathways that are critical for lung repair and regeneration. Fibroblast growth factor 7 (FGF7) and FGF10 signaling in the lung through FGF receptor 2 (FGFR2) are involved in epithelial cell protection and renewal, and mutations in their corresponding genes in humans are linked to increased susceptibility to lung pathologies such as chronic obstructive pulmonary disease and bronchopulmonary dysplasia. In this report, we present data demonstrating significant upregulation of FGF7, FGF10 and FGFR2 in IPF and IPAH lungs compared to donor lungs. These ligands and their cognate rec...

Cells

Lung Stem Cells in Development, Health and Disease

International Journal of Environmental Research and Public Health, 2021

European Respiratory Journal, 2020

Frontiers in Cell and Developmental Biology

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously report... more Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a Fgf10CreERT2 line (with the CreERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki-v1, to target Fgf10Pos cells. While this line allowed fairly efficient and specific labeling of Fgf10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus on our research. We report here the generation and validation of a new Fgf10CreERT2 (called thereafter Fgf10Ki-v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. This new Fgf10Ki-v2 line exhibited comparable Fgf10 expression level to their wild type counterpart. However, a disconnection between the Fgf10 and the Cre expression was observed in Fgf10Ki-v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf1...

The EMBO Journal

Frontiers in Cell and Developmental Biology

American Journal of Physiology-Lung Cellular and Molecular Physiology

American Journal of Respiratory Cell and Molecular Biology

Nature Communications

Human Molecular Genetics

The Journal of pathology, 2018

American journal of physiology. Lung cellular and molecular physiology, Jan 3, 2018