Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a knock-in Fgf10 Cre – ERT 2 line (with the Cre-ERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10 Ki – v 1 , to target FGF10 Pos cells. While this line allowed fairly efficient and specific labeling of FGF10 Pos 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 Fgf10 Cre – ERT 2 line (called thereafter Fgf10 Ki – v 2 ) with the insertion of the expression cassette in frame with the stop codon of exon 3. Fgf10 Ki − v 2/+ heterozygous mice exhibited comparable Fgf10 expression levels to wild type animals. However, a mismatch between Fgf10 and Cre expression levels was observed in Fgf10 Ki – v 2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10 Ki – v 2 mice. Bioinformatic analysis shows that the 3′UTR, where the Cre-ERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Importantly, Fgf10 Ki – v 2 mouse is capable of significantly targeting FGF10 Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10 Ki – v 2 line opens the way for future mechanistic experiments involving the postnatal lung.
The journal is retracting ‘Eya1 controls cell polarity, spindle orientation, cell fate and Notch signalling in distal embryonic lung epithelium’ by Ahmed HK El-Hashash, Gianluca Turcatel, Denise Al Alam, Sue Buckley, Hiroshi Tokumitsu, Saverio Bellusci and David Warburton (2011). [Development
Organ regeneration requires a proper balance between self-renewal and differentiation of tissue specific progenitor cells. Progenitor expansion and differentiation recapitulate many of the mechanisms regulating embryonic development. Here, we uncovered the role of the high mobility group AT-hook protein 2 (HMGA2) as a key regulator of epithelial differentiation during embryonic lung development. Hmga2 loss-of-function increased WNT signaling resulting not only in defective epithelial differentiation but also expansion of progenitors in the developing lung. We found that HMGA2 direct regulation of Gata6 is crucial in fine-tuning WNT signaling in airway epithelium. Furthermore, we combined proteomic, ChIP-seq, and transcriptome data to show that HMGA2-induced transcription requires phosphorylation of the histone variant H2AX at S139 (H2AXS139ph; γ-H2AX) mediated by the protein kinase ataxia telangiectasia mutated (ATM). The interplay between HMGA2, ATM, and H2AX is a novel mechanism of transcription initiation. Our results also link H2AXS139ph to transcription, assigning a new function for this DNA damage marker. Together, our data demonstrate that HMGA2-mediated changes in chromatin structure regulate WNT signaling and control the balance between progenitor expansion and differentiation required for proper lung development. Ref: Singh I, et al., (2015) Cell Research; Jul;25(7):837-50 Singh I, et al., (2014) BMC Biol; Mar 24;12:21 Ozturk N, Singh I, et al., (2014) Front Cell Dev Biol.; Mar 6;2:5
Abstract The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and 2) progenitors during embryonic lung development remains mostly elusive. In this paper, we build upon our previously published work on the regulation of airway epithelial progenitors by fibroblast growth factor receptor 2b (Fgfr2b) signalling during early (E12.5) and mid (E14.5) pseudoglandular lung development. Here, we looked at the regulation by Fgfr2b signalling on alveolar progenitors during late pseudoglandular/early canalicular (E14.5-E16.5) development. Using a dominant negative mouse model to conditionally inhibit Fgfr2b ligands at E16.5, we used gene array analyses to characterize a set of potential direct targets of Fgfr2b signalling. By mining published single-cell RNA sequence (scRNAseq) datasets, we showed that these Fgfr2b signature genes narrow on a discreet subset of AT2 cells at E17.5 and in adult lungs. Furthermore, we demonstrated that Fgfr2b signalling is lost in AT2 cells in their transition to AT1 cells during repair after injury. We also used CreERT2-based mouse models to conditionally knock-out the Fgfr2b gene in AT2 and in AT1 progenitors, as well as lineage label these cells. We found, using immunofluorescence, that in wildtype controls AT1 progenitors labeled at E14.5-E15.5 contribute a significant proportion to AT2 cells at E18.5; while AT2 progenitors labeled at the same time contribute significantly to the AT1 lineage. We show, using immunofluorescence and FACS-based analysis, that knocking out of Fgfr2b at E14.5-E15.5 in AT2 progenitors leads to an increase in lineage-labeled AT1 cells at E18.5; while the reverse is true in AT1 progenitors. Furthermore, we demonstrate that increased Fgfr signalling in AT2 progenitors reduces their contribution to the AT1 pool. Taken together, our results suggest that a significant proportion of AT2 and AT1 progenitors are cross-lineage committed during late pseudoglandular development, and that lineage commitment is regulated in part by Fgfr2b signalling. We have characterized a set of direct Fgfr2b targets at E16.5 which are likely involved in alveolar lineage formation. These signature genes concentrate on a subpopulation of AT2 cells later in development, and are downregulated in AT2 cells transitioning to the AT1 lineage during repair after injury in adults. Our findings highlight the extensive heterogeneity of alveolar cells by elucidating the role of Fgfr2b signalling in these cells during early alveolar lineage formation, as well as during repair after injury.
The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster , most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider ( http://pim.hybrigenics.com ), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.
Non-coding RNAs have recently gained widespread attention given their broad implication in the initiation and progression of various complex diseases. Whereas previous studies on fibrotic lung disorders, especially the idiopathic form (IPF), have mainly focused on miRNAs, the precise contribution of other class of non-coding RNAs in lung fibrogenesis is unclear. Given the paucity of effective treatment in IPF, new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. Here we identify a long non-coding RNA, termed DNM3OS, which tightly controls the various cellular and molecular phenotypic changes occurring during lung fibroblast activation in response to TGF-β. Notably, DNM3OS regulates these processes in trans by giving rise to 3 distinct profibrotic miRNAs (i.e. miR-199a-5p/3p and miR-214-3p), which repress distinct but functionally related targets within TGF-β signaling cascade. Finally, we provide in vivo evidence that interfering with DNM3OS function may be a promising strategy for the treatment of lethal fibrotic diseases such as IPF.
The lung alveolus is lined with alveolar type 1 (AT1) and type 2 (AT2) epithelial cells. During alveologenesis, increasing demand associated with expanding alveolar numbers is met by proliferating progenitor AT2s (pAT2). Little information exists regarding the identity of this population and their niche microenvironment. We show that during alveologenesis, Hedgehog-responsive PDGFRa(+) progenitors (also known as SCMFs) are a source of secreted trophic molecules that maintain a unique pAT2 population. SCMFs are in turn maintained by TGFβ signaling. Compound inactivation of Alk5 TβR2 in SCMFs reduced their numbers and depleted the pAT2 pool without impacting differentiation of daughter cells. In lungs of preterm infants who died with bronchopulmonary dysplasia, PDGFRa is reduced and the number of proliferative AT2s is diminished, indicating that an evolutionarily conserved mechanism governs pAT2 behavior during alveologenesis. SCMFs are a transient cell population, active only during alveologenesis, making them a unique stage-specific niche mesodermal cell type in mammalian organs.
Pulmonary smooth muscle cells (SMC) are extremely important for normal respiratory function. Impaired formation or function of SMCs lead to organ failure. The origin of lung airway and vascular SMCs is recently emerging. Using Fgf10LacZ enhancer trap line, we were the first to show that Fgf10-expressing cells could serve as progenitors for the airway SMCs. Our recent lineage tracing data using the Fgf10CreERT2 knock in line showed that only around 3 – 18% of the Fgf10-expressing cells labeled before E13.5 contribute to the SMC (both vascular and airway). However, after E14.5, these progenitor cells are no longer committed to the SMC lineage. The final contribution of Fgf10-positive cells to the SMC lineage is very likely to be low compared to other pools of progenitors. Several studies show that there are other progenitor pools that participate to the SMC lineage such as – mesothelial Wt1+ cells, SHH-responding Gli1+ cells, Acta2+ cells, but the data are controversial and there is a need of more careful and precise investigation. Another key-player in SMCs development is Wnt signaling – conditional inactivation of Wnt/b-catenin signaling in the embryonic lung mesenchyme led to decreased SMCs formation. There is therefore a need in better characterizing Wnt-positive SMC progenitors and their contribution to the SMCs lineage. Using inducible driver lines Fgf10-CreERT2, Wt1-Cre-ERT2, Gli1-Cre-ERT2, SMA-Cre-ERT2, Axin2-Cre-ERT2 (to capture cells undergoing Wnt-signaling), we will be able to characterize the impact of each pool of progenitor cells on SMCs formation. We will use Tomatoflox reporter line to visualize specific progenitor cells, which underwent Cre activation. We will also use reporter lines to monitor Wnt signaling (Topgal and Axin2LacZ lines) and localize the cells which are undergoing positive Wnt-signaling. Unbiased quantification of the lineage commitment of the labeled cells will be also carried by FACS for Acta2 (Sma) and LipidTox (Lipofibroblasts).
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