Towards long term cultivation of Drosophila wing imaginal discs in vitro
| Autor: | Christian F. Lehner, János Szabad, Peter V. Lidsky, Björn Handke, Ernst Hafen |
|---|---|
| Přispěvatelé: | University of Zurich |
| Rok vydání: | 2014 |
| Předmět: |
Organogenesis
Morphogenesis lcsh:Medicine 1100 General Agricultural and Biological Sciences In Vitro Techniques Cell Growth 1300 General Biochemistry Genetics and Molecular Biology Live cell imaging Hemolymph Animals Drosophila Proteins Wings Animal Cell Cycle and Cell Division lcsh:Science Cell Proliferation 1000 Multidisciplinary Multidisciplinary biology Cell growth lcsh:R Cell Cycle Gene Expression Regulation Developmental Biology and Life Sciences Cell Biology Cell cycle biology.organism_classification 10124 Institute of Molecular Life Sciences Coculture Techniques Cell biology Culture Media Transplantation Imaginal disc Drosophila melanogaster Imaginal Discs Cell Processes Larva 570 Life sciences lcsh:Q Developmental biology Organism Development Developmental Biology Research Article Signal Transduction Insulin-Dependent Signal Transduction |
| Zdroj: | PLoS ONE PLoS ONE, Vol 9, Iss 9, p e107333 (2014) PLoS ONE, 9 (9) |
| ISSN: | 1932-6203 |
| Popis: | The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs. PLoS ONE, 9 (9) ISSN:1932-6203 |
| Databáze: | OpenAIRE |
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