My previous work on Dm ime4 showed its importance for oogenesis (Hongay and Orr-Weaver, PNAS 2011). Recent work in my laboratory demonstrates that this gene is crucial during embryogenesis and continues to be essential in adults for fertility. We used a balanced stock where ime4+ can be traced with GFP, and the ime4(hypomorph) allele is unmarked. We scored under several microscope magnifications, using DAPI and other stainings, GFP(+) and GFP(-) embryos in all stages of development and found statistically significant decreases -measured as departures from the expected Mendelian ratios of GFP(-) to GFP (+) and analyzed by Chi square tests-starting at gastrulation. Furthermore, GFP(-) embryos display high levels of apoptosis, aberrant DAPI stainings, and other severe malformations. In addition, we localized Dm ime4 mRNA by in situ hybridization and Dm IME4 by antibody staining in wild-type embryos and found very informative and specific localizations that strongly support its role en embryogenesis.
For our gamatogenesis analyses in adult gonads, we used the "live or dye" staining system together with other stem/germ/somatic markers to determine the role of Dm IME4 in germ and somatic cell differentiation. Our findings are in line with recent reports on the role of Dm ime4 orthologs (e.g., mttl3 mutants are embryonic lethal in mice), but with the use of hypomorphic mutants and RU-486 inducible RNAi, we are able to get detailed and valuable information on its role in Drosophila development.
Given the evolutionary conservation of Dm ime4, our findings will inform future studies in vertebrate systems.