32 research outputs found
Mechanisms of TGFĪ² inhibition of LUNG endodermal morphogenesis: The role of TĪ²RII, Smads, Nkx2.1 and Pten
AbstractTransforming 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 TGFĪ² isoforms inhibited FGF10-induced morphogenesis of mesenchyme-free embryonic lung endoderm. Inhibition of budding by TGFĪ² was partially abrogated in endodermal explants from Smad3ā/ā or conditional endodermal-specific Smad4Ī/Ī embryonic lungs. Endodermal explants from conditional TGFĪ² receptor II knockout lungs were entirely refractive to TGFĪ²-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). TGFĪ² 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 TGFĪ²'s inhibitory effect on cell proliferation, but did not restore morphogenesis. Thus, the mechanisms by which TGFĪ² 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 TĪ²RII
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
Role of Pten in lung development
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.
Objective: To test the role of Pten in lung development and injury, we conditionally deleted Pten throughout the lung epithelium by crossing Ptenflox/flox with Nkx2.1-cre driver mice and throughout the lung mesenchyme by crossing Ptenflox/flox with Nkx2.1-cre driver or Dermo1-cre driver. The resulting PtenNkx2.1-cre mutants were analyzed for lung defects and response to injury.
Results: PtenNkx2.1-cre embryonic lungs showed airway epithelial hyperplasia with no branching abnormalities. In vitro culture of mutant lungs also showed an altered responsed to TGF-? when in vivo In adult mice, PtenNkx2.1-cre lungs exhibit increased progenitor cell pools comprised of basal cells in the trachea, CGRP/CC10 double-positive neuroendocrine cells in the bronchi and CC10/SpC double positive cells in the bronchioalveolar duct junction (BADJ). Pten deletion impacted differentiation of various lung epithelial cell lineages, with decreased number of terminally differentiated cells. Over time, PtenNxk2.1-cre epithelial cells residing in the BADJ underwent proliferation, and formed uniform masses, supporting the concept that the cells residing in this distal niche may also be the source of pro-carcinogenic āstemā cells. Finally, increased progenitor cells in all the lung compartments conferred an overall selective advantage to naphthalene injury compared to wild type control mice.
PtenDermo1cre embryonic lungs, moreover, showed normal lung development but increased collagen1 and extracellular matrix production.
Conclusions: Pten has a pivotal role in lung stem cell homeostasis, cell differentiation and consequently resistance to lung injury in the epithelium, Further studies are necessary to clarify the real role of Pten in lung mesenchyme.Rationale: Pten eā un gene coinvolto nellāomeostasi delle cellule staminali e nella formazione di tumori. Nei topi, Pten inizia ad essere presente 7 giorni dopo il concepimento. Pten ha un ruolo critico nello sviluppo embrionale: gli embrioni di topo Pten-/-, infatti, muoiono molto presto durante la gestazione.
Scopo dello studio: Studiare il ruolo di Pten nello sviluppo polmonare, eliminando Pten nellāepitelio polmonare, incrociando Ptenflox/flox con topi portatori di Nkx2.1-cre; Pten eā stato anche eliminato dal mesenchima polmonare incrociando Ptenflox/flox con topi Dermo1-cre. I risultanti PtenNkx2.1cre sono stati analizzati alla ricerca di difetti nello sviluppo polmonare.
Risultati: I polmoni PtenNkx2.1-cre hanno evidenziato in vitro una alterata risposta al TGF-?. In vivo non presentavano nessuna alterazione nel branching bensi una iperplasia polmonare nelle vie aerre. Nei topi adulti, I polmoni PtenNkx2.1-cre presentavano un aumentato pool di cellule progenitori in tutti i distretti: nella trachea, le cellule basali, nei bronchi le cellule neuroepiteliali, positive per CGRP/CC10 ed infine, nella giunzione tra gli alveoli e I bronchi terminali (BADJ), le cellule positive per Spc/CC10. Lāassenza di Pten ha un impatto nella differenziazione cellulare, con un diminuito aumento delle cellule allāultimo stadio di differenziazione. Nel tempo, le cellule epiteliali PtenNxk2.1-cre residenti a livello del BADJ proliferano e formano delle masse di tipo tumorale; questi dati supportano lāidea che le cellule presenti in questa niche possano essere lāorigine delle cosidette āpro-carcinogenic stem cellsā. Lāaumento delle cellule progenitrici, inoltre, conferisce un selettivo vantaggio dopo danno polmonare. I topi con Pten eliminato nellāepitelio, invece, non evidenziavano ne uno sviluppo polmonare alterato ne una alterata differenziazione delle cellule mesenchimali; tuttavia, dimostravano un aumentata deposizione di collagene1 e di matrice extracellulare. Conclusioni: Pten ha un ruolo importante nellāomeostasi delle cellule progenitori del polmone, nella differenziazione epiteliale polmonare e nella resistenza dopo danno. Ulteriori studi sono necessary per chiarire lāesatto ruolo di Pten nel mesenchima polmonare
Lung organogenesis
Developmental lung biology is a field that has the potential for significant human impact: lung disease at the extremes of age continues to cause major morbidity and mortality worldwide. Understanding how the lung develops holds the promise that investigators can use this knowledge to aid lung repair and regeneration. In the decade since the āmolecular embryologyā of the lung was first comprehensively reviewed, new challenges have emergedāand it is on these that we focus the current review. Firstly, there is a critical need to understand the progenitor cell biology of the lung in order to exploit the potential of stem cells for the treatment of lung disease. Secondly, the current familiar descriptions of lung morphogenesis governed by growth and transcription factors need to be elaborated upon with the reinclusion and reconsideration of other factors, such as mechanics, in lung growth. Thirdly, efforts to parse the finer detail of lung bud signaling may need to be combined with broader consideration of overarching mechanisms that may be therapeutically easier to target: in this arena, we advance the proposal that looking at the lung in general (and branching in particular) in terms of clocks may yield unexpected benefits
FGF9āPitx2āFGF10 signaling controls cecal formation in mice
AbstractFibroblast growth factor (FGF) signaling to the epithelium and mesenchyme mediated by FGF10 and FGF9, respectively, controls cecal formation during embryonic development. In particular, mesenchymal FGF10 signals to the epithelium via FGFR2b to induce epithelial cecal progenitor cell proliferation. Yet the precise upstream mechanisms controlling mesenchymal FGF10 signaling are unknown. Complete deletion of Fgf9 as well as of Pitx2, a gene encoding a homeobox transcription factor, both lead to cecal agenesis. Herein, we used mouse genetic approaches to determine the precise contribution of the epithelium and/or mesenchyme tissue compartments in this process. Using tissue compartment specific Fgf9 versus Pitx2 loss of function approaches in the gut epithelium and/or mesenchyme, we determined that FGF9 signals to the mesenchyme via Pitx2 to induce mesenchymal Fgf10 expression, which in turn leads to epithelial cecal bud formation
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. Ī²-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
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. Ptenā/ā mouse embryos die early during gestation indicating a critical role for Pten in embryonic development
Children accessing accident and emergency department for non-urgent consultations: A cross-sectional study about parentsā use of primary care services
Introduction: Parents often take their children to the Paediatric Accident and Emergency Department (A&E) for non-urgent consultations rather than using community-based primary care services. This study describes the use of primary care services in parents taking their children to the A&E for non-urgent consultations. Methods: A cross-sectional study was conducted from July 2018 to June 2019, in a second-level Italian paediatric A&E of a tertiary-level children's academic research and hub hospital. Parents of children aged between 3 months and 6 years assigned with a white code at the triage were asked to complete a paper-and-pencil 40-item questionnaire after accessing the A&E for a non-urgent consultation. Results: The questionnaire was completed by the parents of 237 children (males 58 %; median age = 2.3 years). Overall, 48.1 % (n = 114) of the parents reported consulting 'often/always' the primary care paediatrician, mainly when their child was sick and for check-ups (n = 182, 76.8 %). However, only 7.2 % (n = 17) of the parents 'often/always' used any other health service in the community. Most of them (n = 191, 82 %) did not even know where the community health centre was located. Conclusion: Parents accessing the A&E for non-urgent consultations should be better informed/educated on how to use community health services