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Immunolocalization of sprouty-1 and sprouty-2 in developing rat lung
Shuichi Hashimoto1, Hiroshi Nakano, Yuko Suguta
1Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. sh6749@my.email.ne.jp
Objective:
Sprouty, a common antagonist of fibroblast growth factor (FGF) and epidermal growth factor signaling, is a key player regulating tracheal branching and eye development in Drosophila. Four Sprouty homologs have been identified in vertebrates and all share a cysteine-rich region. However, the physiological function(s) of the individual Sprouty homologs is unknown. mRNA of Sprouty homologs is expressed during mouse lung development. In the present study, we investigated the immunolocalization of Sprouty proteins in rat lung at different stages of development.
Methods:
Rabbit antibodies were raised against peptides derived from rat Sprouty-1 and Sprouty-2 and were used in Western blot analysis to determine Sprouty distribution in subcellular fractions (pellets and supernatant centrifuged at 5,000 and 20,000 g) and bronchoalveolar lavage fluid (BAL) from adult rat lungs or used in immunohistochemistry.
Results:
Western blot analysis revealed a 30-kDa Sprouty-1 band and a 34-kDa Sprouty-2 band in the supernatant and pellet fractions centrifuged at 20,000 g. BAL contained a band of approximately 16 kDa with Sprouty-1 antibody derived from proteolytic fragmentation of Sprouty-1. In embryonic day (E) 14 and E16 lungs, Sprouty-1 and Sprouty-2 were expressed both in epithelial and peripheral mesenchymal cells. In adult rat lung, bronchiolar and alveolar type II epithelial cells showed staining for both Sprouty-1 and Sprouty-2. Sprouty-1 expression was also seen in alveolar type I epithelial cells.
Conclusion:
In light of the proximity of the distribution of Sprouty to that of FGF-10 (peripheral mesenchyme) and its receptor FGFR2IIIb (distal tubular epithelium) in lung development, and the finding that FGF-9, which is expressed in mesothelial cells, upregulates FGF-10, it appears that Sprouty expression in epithelial and mesenchymal cells during branching morphogenesis is closely related to signaling by FGF-9 and FGF-10.
Insights
Sprouty proteins are present in developing rat lungs, particularly in epithelial and mesenchymal cells. Their expression correlates with fibroblast growth factor signaling, suggesting a role in lung development.
Area of Science:
- Developmental Biology
- Cell Signaling
Background:
- Sprouty proteins are known antagonists of growth factor signaling pathways.
- Vertebrates have four Sprouty homologs, but their specific functions are largely unknown.
- Sprouty homologs are expressed during mouse lung development.
Purpose of the Study:
- To investigate the immunolocalization of Sprouty-1 and Sprouty-2 proteins in rat lung tissue during development.
- To understand the distribution and potential role of Sprouty proteins in lung morphogenesis.
Main Methods:
- Raised rabbit antibodies against rat Sprouty-1 and Sprouty-2 peptides.
- Utilized Western blot analysis to detect Sprouty proteins in subcellular fractions and bronchoalveolar lavage fluid.
- Performed immunohistochemistry to determine Sprouty protein localization in developing and adult rat lungs.
Main Results:
- Identified specific molecular weights for Sprouty-1 (30 kDa) and Sprouty-2 (34 kDa) in Western blots.
- Detected Sprouty-1 in bronchoalveolar lavage fluid, likely due to proteolytic fragmentation.
- Showed co-expression of Sprouty-1 and Sprouty-2 in both epithelial and mesenchymal cells during embryonic lung development (E14, E16).
- Observed Sprouty-1 and Sprouty-2 staining in adult rat lung bronchiolar and alveolar epithelial cells, with Sprouty-1 also in alveolar type I cells.
Conclusions:
- Sprouty protein distribution in lung development is spatially related to fibroblast growth factor (FGF) signaling components like FGF-10 and its receptor FGFR2IIIb.
- FGF-9 signaling appears to influence FGF-10, further implicating Sprouty in this pathway.
- Sprouty expression in lung epithelial and mesenchymal cells is likely integral to branching morphogenesis, mediated by FGF signaling.
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