Background: Disorders of lung development cause death and disability in the young and old: novel insights into developmental regulators can aid therapeutic strategies. The Ca2+ATPase SERCA, already implicated in asthma and cystic fibrosis, appears to play a key role in lung development. SERCA inhibition with cyclopiazonic acid (CPA) in vitro, reduces both airway branching and peristalsis reversibly and dose dependently, whilst also halting myogenesis. It is unclear however, whether changes in branching are mediated via SERCA dependent contractility, or whether SERCA is a direct regulator of airway branching. Aims: (i) to further explore the CPA-induced embryonic lung phenotype by assaying gene expression and cell proliferation; and (ii) to determine effects of genetic perturbation of SERCA function in vivo on airway branching morphogenesis, in the absence of contractility (using a Drosophila model). Methods: Embryonic mouse (E11.5) lung explants were cultured +/- CPA at an air/fluid interface. Standard techniques were used to rear Drosophila and SERCA expression manipulated using conditional, heat-sensitive mutants and RNAi targeted to the trachea. Positively labelled, loss-of-function ‘flip-out’ RNAi and mutant clones were produced using heat-shock induced FLP-recombinase. Gene expression was assayed using real-time RT-PCR and SERCA function assessed using calcium dyes and genetic indicators. Embryonic and larval fly airways were imaged using fluorescent proteins and immunostaining, with live or fixed-sample confocal microscopy. Immunofluorescent staining was used to assess protein expression and cell proliferation. Results: SERCA inhibition with CPA significantly up or down regulated mRNA levels of key genes involved in lung branching morphogenesis, myogenesis and angiogenesis in vitro. CPA treatment also reduced cell proliferation dose-dependently in the lung epithelium and mesenchyme. In the fly embryo, neither conditional SERCA mutants nor targeted RNAi significantly affected tracheal morphology. However, residual SERCA mRNA and protein function was evident at this stage of development. Tracheal maturation, in the form of gas filling was significantly impaired though, in embryos expressing a conditional SERCA mutation. In larvae, development of the dorsal air sac primordium (ASP) was severely disrupted by targeted SERCA RNAi and this phenotype could be reproduced when sufficient numbers of loss-of–function clones were present. SERCA inhibition reduced the number of mitotic cells in the ASP and correspondingly, SERCA deficient clones comprised fewer cells than control counterparts: SERCA regulation of airway cell proliferation was therefore evident across species. Fewer SERCA deficient cells reached the tip of the ASP during morphogenesis compared to controls, whereas a greater proportion remained in the stalk, findings that indicate a cell-autonomous defect in cell migration. Changes in morphology were independent of changes in expression of the key ASP signalling pathways MAP kinase and Notch. Expression of the ASP tip-cell marker escargot was expanded in SERCA deficient larvae, with a number of positive cells being abnormally present in the stalk. This finding could be explained by a failure of these cells to migrate to the tip, alternatively by changes in cell fate. Given key roles of tip cells in morphogenetic signalling, escargot may play a role in SERCA inhibition-induced dysmorphogenesis. Conclusions: SERCA has an essential, conserved role in airway branching morphogenesis across species: this role appears independent of contractility. SERCA regulates cell migration and proliferation processes in the airway, findings that may have wider relevance, e.g. in proliferative disease, metastasis and tissue regeneration. Given evidence in plants and fungi of Ca2+ cycling regulating budding, findings here may indicate a role for SERCA as a generic regulator of iterative branching across biology, with clear implications for further research.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:617398 |
| Date | January 2013 |
| Creators | Lansdale, Nicholas |
| Contributors | Jesudason, Edwin ; Bennett, Daimark |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/12693/ |
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