MTORC1 signaling facilitates differential stem cell differentiation to shape the developing murine lung and is associated with mitochondrial capacity
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE213202 | GEO | 2022/11/03
REPOSITORIES: GEO
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Project description:Formation of branched organs requires sequential differentiation of stem cells. In this work, we find that the conducting airways derived from SOX2+ progenitors in the murine lungs fail to form without mTOR complex 1 (mTORC1) signaling and are replaced by lung cysts. Proximal-distal patterning through transitioning of distal SOX9+ progenitors to the proximal SOX2+ cells is disrupted. Mitochondria number and ATP production are reduced. Compromised mitochondrial capacity results in a similar defect as that in mTORC1-deficient lungs. This suggests that mTORC1 promotes differentiation of SOX9+ progenitors to form the conducting airways by modulating mitochondrial capacity. Surprisingly, in all mutants saccules are produced from lung cysts at the proper developmental time despite defective branching. SOX9+ progenitors also differentiate into alveolar epithelial type I and type II cells within saccules. These findings highlight selective utilization of energy and regulatory programs during stem cell differentiation to produce distinct structures of the mammalian lungs.
2022-11-03 | GSE189327 | GEO
Project description:The lung is a branched tubular network with two distinct compartments — the proximal conducting airways and the peripheral gas exchange region — separated by a discrete boundary termed the bronchoalveolar duct junction (BADJ). Here we image the developing mouse lung in three-dimensions and show that two nested developmental waves demarcate the BADJ under the control of a global hormonal signal. A first wave of branching morphogenesis progresses throughout embryonic development, generating branches for both compartments. A second wave of conducting airway differentiation follows the first wave but terminates earlier, specifying the proximal compartment and setting the BADJ. The second wave is terminated by a glucocorticoid signaling: premature activation or loss of glucocorticoid signaling causes a proximal or distal shift, respectively, in BADJ location. The results demonstrate a novel mechanism of boundary formation in complex, three-dimensional organs and provide new insights into glucocorticoid therapies for lung defects in premature birth. RNAs were extracted from E14 lungs cultured in control and dexamethasone media for 24 hours using Trizol reagents and Qiagen RNeasy Micro kit. Two control and two treated samples were analyzed.
2014-05-22 | E-GEOD-56831 | biostudies-arrayexpress
Project description:During development, the proximal and distal regions of respiratory tract undergo distinct processes that ultimately give rise to conducting airways and alveoli. To gain insights into the genetic pathways differentially activated in these regions when branching morphogenesis is initiating, we characterized their transcriptional profiles in murine rudiments isolated at embryonic day 11.5.
2007-09-29 | E-GEOD-1423 | biostudies-arrayexpress
Project description:During development, the proximal and distal regions of respiratory tract undergo distinct processes that ultimately give rise to conducting airways and alveoli. To gain insights into the genetic pathways differentially activated in these regions when branching morphogenesis is initiating, we characterized their transcriptional profiles in murine rudiments isolated at embryonic day 11.5. Keywords: parallel sample
2004-05-23 | GSE1423 | GEO
Project description:The proximal-distal patterning program determines unique structural and functional properties of proximal and distal airways in the adult lung. Based on the knowledge that remod-eling of distal airways is the major pathologic feature of chronic obstructive pulmonary disease (COPD), and that small airway epithelium (SAE), which covers distal airways, is the primary site of the initial smoking-induced changes relevant to COPD pathogenesis, we hypothesized that in COPD smokers, the SAE transcriptome loses its region-specific biologic identity and takes on the transcriptional pattern of the proximal airways. By analyzing human airway epithelium col-lected by bronchoscopic brushings from proximal and distal airways of healthy smokers, proxi-mal and distal airway epithelial transcriptome signatures were identified. Dramatic smoking-dependent suppression of distal signature paralleled by acquisition of the proximal airway epithe-lial phenotype was found in the SAE of COPD smokers. Distal-proximal re-patterning observed in the SAE of smokers in vivo was reproduced in vitro by stimulating SAE basal cells (BC), the stem/progenitor cells of the SAE, with EGF, a growth factor up-regulated in airway epithelium by smoking. Together, this study identifies distal-proximal SAE re-patterning as a characteristic feature of small airway disordering in COPD smokers potentially driven by EGF/EGFR-mediated reprogramming of SAE BC stem/progenitor cells.
2017-07-18 | GSE64614 | GEO
Project description:The lung epithelium contains multiple distinct cell types which are maintained by regionally specific progenitor cell populations in both the airways and in the alveolar compartments. Each of the progenitor populations are known to have varying roles during homeostasis and have increasingly been shown to be deranged in lung diseases. The progenitor cells of the proximal lung epithelium are the basal cells (Krt5+/Krt14+/p63+) which sit at the basement membrane of the trachea, referred to as, proximal progenitors in this study. On the other hand, the distal alveolar epithelium is maintained by alveolar type II cells (Sftpc+), referred to as distal progenitors in this study. Several cell isolation methods are available to isolate each of these populations of proximal and distal progenitors but are traditionally performed separately on mice which is especially important in the context of disease. Methods which allow the isolation of the entire epithelial population from murine lungs, while retaining the capability to perform downstream studies such as organoid assays and air-liquid-interface, would open new possibilities for studying interactions between the proximal and distal airways in lung disease. However, an easy and reproducible method that allows isolation of both cell types simultaneously from an individual mouse has not been previously described. This is in part due to the technical and surgical challenges associated with each isolation method as well as, in part, due to a lack of cell surface markers that are specific and unique to each cell type that could allow their isolation from a single cell suspension of the entire epithelium. Additionally, transgenic models have been shown to be leaky and mark populations other than the target cells. Thus, classical methods that rely on positive or negative selection, coupled by surgical isolation remain the state of the art. In order to facilitate isolation of proximal and distal progenitors from an individual mouse, we developed a 3D printable Lobe Divider (3DLD) that allows for the reproducible separation of the trachea and lung lobes while allowing for controllable and temporal instillation of cell dissociation buffers for both cell isolation procedures. The efficacy of the 3DLD in isolating the two populations and the functionality of the cells isolated with the 3DLD was validated through multiple differentiation assays. Proximal progenitors were differentiated in 2D at air-liquid interface (ALI) culture for 28 days after monolayer formation for 7days. Distal progenitors isolated with the 3DLD or with the equivalent classical tracheal ligation method were differentiated in organoid culture in Matrigel for 14 days while at ALI. Total RNA was isolated from cell pellets of all cell types before differentiation, from ALI cultures of proximal cells, and organoids of distal cells isolated with 3DLD or classic method. Qiagen RNeasy micro kit (74004, QIAGEN, Sweden) was used to isolate total RNA following the manufacturer’s protocol. RNA concentrations were measured using NanoDrop 1000 and Qubit™ RNA HS Assay Kit (Q32852, Fisher Scientific). RNA quality was evaluated by the Agilent 2100 BioAnalyzer. Library preparation was done using the Illumina Stranded mRNA Prep Ligation (20040534, Illumina) with 14 cycles under final PCR amplification and samples were indexed with IDT for Illumina RNA UD Indexes Set B, Ligation (20040554, Illumina) for multiplexing. Clean up steps were automated using the King Fisher FLEX (18-5400620, Thermo Scientific). Library concentration was checked using the QuantIT 1X dsDNA HS Assay Kit (Q33232) and library size and quality were checked using the 5200 Fragment Analyzer™ 12-capillary (M3510AA, Agilent) using the DNF-930 dsDNA Reagent Kit, 75 bp – 20,000 bp(500 Samples) (Part # DNF-930-K0500, Agilent). A library input of 1.25 nM was sequenced using NovaSeq 6000 Sequencing System (20012850, Illumina).
2022-04-30 | E-MTAB-11502 | biostudies-arrayexpress
Project description:The lung is a branched tubular network with two distinct compartments — the proximal conducting airways and the peripheral gas exchange region — separated by a discrete boundary termed the bronchoalveolar duct junction (BADJ). Here we image the developing mouse lung in three-dimensions and show that two nested developmental waves demarcate the BADJ under the control of a global hormonal signal. A first wave of branching morphogenesis progresses throughout embryonic development, generating branches for both compartments. A second wave of conducting airway differentiation follows the first wave but terminates earlier, specifying the proximal compartment and setting the BADJ. The second wave is terminated by a glucocorticoid signaling: premature activation or loss of glucocorticoid signaling causes a proximal or distal shift, respectively, in BADJ location. The results demonstrate a novel mechanism of boundary formation in complex, three-dimensional organs and provide new insights into glucocorticoid therapies for lung defects in premature birth.
2014-05-22 | GSE56831 | GEO
Project description:The cAMP response element binding protein (Creb) is a member of a leucine zipper transcription factor family that regulates gene expression primarily in response to the intracellular cAMP signalling pathway. Previous studies have shown Creb1-null mice suffer respiratory failure with lung atelectasis and a large reduction in Sftpd mRNA. Using a new line of Creb1-null mice we have further investigated Creb function in the developing mouse lung, focussing on differentiation of the airway epithelium. The lungs of Creb1-null fetal mice showed normal respiratory development until E17.5 when proximal and distal airways fail to inflate. Subsequent ultrastructural analysis of the lungs of E17.5 Creb1-null fetal mice revealed a defect in AEC differentiation with a reduction in proportions of type-II AECs and in particular, a very large reduction in type-I AECs. Furthermore, immunostaining for the proximal epithelial cell markers Scgb1a1 (also known as CC10) (Clara cells), Foxj1 (Ciliated cells), and CGRP (Neuroendocrine cells) showed delayed or defective proximal epithelial differentiation in Creb1-null fetal lungs. Quantitative real time PCR (qRT-PCR) analysis at E17.5 in Creb1-null fetal lungs showed differential expression of mRNAs for Creb/Atf1 subfamily members, surfactant-associated proteins, type-I AEC markers and proximal epithelial markers. Furthermore, whole-genome microarray analysis at E17.5 in Creb1-null fetal lungs has provided novel genes which will prove useful to further investigate Creb-mediated signalling in lung development. Together these results demonstrate that Creb plays a key role in determining cell lineages and differentiation of the developing lung epithelium.
2009-04-30 | E-MEXP-1295 | biostudies-arrayexpress
Project description:Fibroblast Growth Factor (FGF) signaling plays an important role in lung organogenesis. Over recent decades, FGF signaling in lung development has been extensively studied in animal models. However, little is known about the expression, localization and functional roles of FGF ligands during human fetal lung development. Therefore, we aimed to determine the expression and function of several FGF ligands and receptors in human lung development. Using in situ hybridization (ISH) and RNA-sequencing, we assessed their expression and distribution in native human fetal lung. Human fetal lung explants were treated with recombinant FGF7, FGF9 or FGF10 in air-liquid interface culture. Explants were analyzed grossly, to observe differences in branching pattern, as well as at the cellular and molecular level. ISH demonstrated that FGF7 is expressed in both the epithelium and mesenchyme; FGF9 is mainly localized in the distal epithelium, whereas FGF10 demonstrated diffuse expression throughout the parenchyma with some expression in the smooth muscle cells (SMCs). FGFR2 expression was high in both proximal and distal epithelial cells as well as the SMCs. FGFR3 was expressed mostly in the epithelial cells, with lower expression in the mesenchyme, while FGFR4 was highly expressed throughout the mesenchyme and in the distal epithelium. Using recombinant FGFs, we demonstrated that FGF7 and FGF9 had similar effects on human fetal lung as on mouse; however, FGF10 caused the human explants to expand and form cysts as opposed to inducing epithelial branching as seen in the mouse. In conjunction with decreased branching, treatment with recombinant FGF7, FGF9 and FGF10 also resulted in decreased double-positive SOX2/SOX9 progenitor cells, which are exclusively present in the distal epithelial tips in early human fetal lung. Although FGF ligand localization may be somewhat comparable between developing mouse and human lungs, their functional roles may differ substantially.
2018-10-16 | GSE121238 | GEO
Project description:Fresh lung tissue specimens from distal lungs (n=3) and airways (n=3) were obtained through bronchoscopy, and primary fibroblasts were isolated and cultured. IRB approval was obtained from the University of Pittsburgh Institutional Review Board.
2010-02-20 | GSE15359 | GEO