作者: Wee Choo Puah , Martin Wasser
DOI: 10.1016/J.YMETH.2015.09.028
关键词: Functional genomics 、 Anatomy 、 Cell biology 、 Population 、 Biology 、 Developmental biology 、 Drosophila melanogaster 、 Time-lapse microscopy 、 Histolysis 、 Live cell imaging 、 Gene expression profiling
摘要: Time-lapse microscopy in developmental biology is an emerging tool for functional genomics. Phenotypic effects of gene perturbations can be studied non-invasively at multiple time points chronological order. During metamorphosis Drosophila melanogaster, time-lapse using fluorescent reporters allows visualization alternative fates larval muscles, which are a model the study genes related to muscle wasting. While doomed muscles enter hormone-induced programmed cell death, smaller population persistent survives adulthood and undergoes morphological remodeling that involves atrophy early, hypertrophy late pupation. We developed method combines vivo imaging, targeted perturbation image analysis identify characterize involved development. Macrozoom helps screen interesting phenotypes, while confocal locations over 4-5 days produces images used quantify changes morphology. Performing similar investigation fixed pupal tissues would too time-consuming therefore impractical. describe three applications our pipeline. First, we show how quantitative track measure throughout analyze atrophy. Second, assay help either promote or prevent histolysis abdominal muscles. Third, apply approach test new proteins as live markers mKO2 tagged Cysteine proteinase 1 (Cp1) Troponin-I (TnI) examples showing subcellular localization. Finally, discuss strategies improve throughput pipeline permit genome-wide screens future.