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Quantitative Analysis of Wild Type and scrib mutant Drosophila wing imaginal discs Transcriptomes at different time points

ABSTRACT: Purpose: There are two goals of this study. Firstly, we want to figure out the transcriptomic differences between control and scrib¹ mutant wing imaginal discs. Secondly, we aim to investigate the transcriptomic changes as the scrib¹ mutant tumor develop over time. Methods: Total RNA was used to generate cDNA libraries and 150bp paired-end sequencing on Illumina HiSeq platform were performed by Novogene. Cleaned raw reads were mapped to the reference genome using STAR and counts are generated by featuresCounts available in Subread package. Count normalization was performed using edgeR.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE130243 | GEO | 2019/08/14

REPOSITORIES: GEO

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Project description: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.

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Quantitative Analysis of Wild Type and scrib mutant Drosophila wing imaginal discs Transcriptomes at different time points

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