Analysis of mitochondrial dynamics and function in Drosophila gastrulation

Abstract

Metazoan embryonic development relies on precise cellular and morphogenetic processes, which are highly-energy intensive. Mitochondria, the powerhouse of the cell, play vital roles in various such morphogenetic events for instance, epithelial to mesenchymal transitions, cell migration, and cell division. Regulation of mitochondrial morphology and activity is essential for embryonic survival in C. elegans, Drosophila, zebrafish and mice. Asymmetric mitochondrial activity and distribution within the embryo has been shown to govern embryonic axis specification in Xenopus, sea urchin and Drosophila. Mitochondria are fragmented and dispersed in early Drosophila blastoderm embryos, and they migrate apically during cell formation (cellularization) in all the cells throughout the embryo. We have characterized the mitochondrial organization and its function during Drosophila gastrulation, specifically focusing on the ventral furrow (VF) formation. We observe that post cellularization, a second mitochondrial migration takes place, from the basal side to the apical side of the cells, in the ventral furrow. This second migration is specific to the ventral furrow cells and is absent in the neighbouring and lateral cells, unlike the first migration during cellularization which is seen all throughout the embryo. The mitochondrial migration in the VF is found to be microtubule-dependent and is regulated by the Dorsal/Nfkb signaling pathway. Mitochondria need to be fragmented for migration, as loss of mitochondrial fission protein, Dynamin related protein 1 (Drp1), prevents the apical migration and causes mitochondrial clustering in the basal regions of the cell. Drp1 mutant embryos (Drp1SG) show reduced Myosin II levels at the apical side of ventral furrow cells resulting in a broader ventral furrow. Proteomic analysis of mitochondria isolated from Drp1SG embryos reveals decreased levels of respiratory chain complexes suggesting that the clustered mitochondria in Drp1 mutant embryos are functionally inactive. Whole mRNA sequencing coupled with proteomic analysis indicate normal expression of Dorso-ventral patterning genes but an upregulation of antioxidant pathways in Drp1SG embryos. A decrease in reactive oxygen species (ROS) is confirmed by reduced fluorescence of the dye, dihydroethidium (DHE), in staining. Strikingly, additional depletion of mitochondrial ROS scavengers and mitochondrial fusion machinery in Drp1SG embryos results in mitochondrial fragmentation rescuing the defects in mitochondrial migration and VF formation. Our study reveals a novel phenomenon of mitochondrial migration in the ventral furrow regulated by Dorsal/NF-kB, highlighting a crucial role of mitochondrial fragmentation and activity in cytoskeletal remodeling and morphogenesis in Drosophila embryo.