Abstract
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.
Highlights
In the dipteran fly Drosophila melanogaster, wings develop during the larval and pupal stages from a pair of imaginal discs that are set aside during embryogenesis as small pockets of around 40 epithelial cells [1,2]
Not a complete solution, we report that medium supplementation with high levels of insulin in combination with a rapid and gentle partial dissection of wing imaginal discs is more effective in delaying the cell proliferation arrest in culture than previously described approaches
Our results demonstrate that moderate levels of Drosophila insulin-like peptide 2 (Dilp2) (0.5 mM) are effective as high levels of bovine insulin (35 mM, i.e., 200 mg/ml) in preventing a rapid cell proliferation arrest during disc culture (Fig. 8B)
Summary
In the dipteran fly Drosophila melanogaster, wings develop during the larval and pupal stages from a pair of imaginal discs that are set aside during embryogenesis as small pockets of around 40 epithelial cells [1,2]. Research on Drosophila wing development has provided pioneering insight into mechanisms controlling pattern formation (including morphogen activity and compartment boundaries), cell interactions, growth and proliferation [3,4,5,6]. Cells in wing imaginal discs proliferate exponentially, fostered by nutrients obtained from the hemolymph and gas exchange enhanced by the tracheal system. Progression through the cell cycle appears to slow down about twofold during the larval stages [9]. Accompanying disc cell proliferation a series of folds develops in a stereotypic pattern
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
