Richa Rikhy

Yolk-cytoplasm segregation is among the earliest spatial organization events in the developing embryo of many oviparous animals. The segregation process is intimately linked to early embryonic cleavage and pattern formation, and exhibits a wide range of spatial and temporal diversity. However, the underlying cytoskeletal mechanism remains largely unknown, except for a small number of species. Using quantitative live imaging, we investigated yolk segregation in the Drosophila embryo during the syncytial nuclear cycles 11–14. We find that the yolk vesicles move progressively inward in spatial and temporal coordination with the inward expanding microtubule networks that are nucleated from centrosomes positioned at the cortex, whereas cortical actin meshwork remains spatially restricted. Using the gnu RNAi embryo to decouple nuclear migration and division from cytoskeletal dynamics, we establish causality with targeted pharmacological disruption and find that microtubule dynamics is required for yolk segregation, while depolymerization of actin has no discernible effect. In support of a mechanism of growth-propelled passive displacement, microtubule plus end comets come in apparent contact with yolk vesicles, and injected, inert microbeads are displaced towards the embryo center presumably by the same pushing force. These findings identify microtubule polymerization as a predominant driver of yolk-cytoplasm segregation in Drosophila and suggest that diverse cytoskeletal mechanisms evolved to accomplish this crucial reorganization process.

The mechanisms underlying the scaling of the Bicoid morphogen gradient in Drosophila with the embryo size is not clearly understood. We propose a model with a spatially varying diffusion coefficient along the anterior–posterior axis, that is proportional to the nearly periodic spatial distribution of the nucleo-cytoplasmic domains and alternating between regions of fast and slow diffusion. We postulate that for specific interpretation of the heterogeneous environment, where the space available for free diffusion within an energid is assumed to be proportional to the embryo size, a change in the embryo size can lead to a size-dependent scaling of the gradient lengthscale. We further study a two component model with slow and fast diffusing Bicoid, and identify this model to be equivalent to the heterogeneous diffusion model further postulating that the fast-state occupancy which is a measure of the fraction of time spent in the fast diffusing state, should also scale with the embryo size via the energid size. Finally, we incorporate nuclear shuttling into our model to understand the effect of shuttling on the gradient lengthscale and scaling with embryo size. We argue that for the particular case where the degradation within the nucleus is low, nuclear shuttling does not perturb the Bicoid gradient. Our study suggests that spatial heterogeneity is able to generate scaling, independent of nuclear trapping. This mechanism may act in conjunction with other biological processes, to yield the experimentally observed scaling behavior of the Bicoid gradient.

BAR domain–containing proteins are key regulators of endocytosis and actin remodeling. Their function in morphogenesis remains to be investigated. We report that the I-BAR domain–containing protein, missing-in-metastasis (MIM) (also called MTSS1), promotes branched actin network formation and endocytosis to drive rapid, cyclical plasma membrane remodeling during syncytial divisions in Drosophila embryos. Actin-rich villous protrusions in the apical caps in interphase are depleted in metaphase, concurrent with furrow extension between adjacent nuclei. MIM depletion results in a loss of furrow extension and in longer, more abundant apical protrusions containing the formin diaphanous. Branched actin networks promoted by MIM are in balance with bundled actin networks induced by RhoGEF2 and diaphanous. Cyclical recruitment of MIM to the cortex promotes localization of active Rac, the WAVE regulatory complex, and the Arp2/3 complex to drive endocytic membrane remodeling. These findings identify MIM as an integrator of actin and endocytic dynamics that enables rapid membrane remodeling during Drosophila syncytial division cycles.

Mitochondrial dynamics are defined by the continuous processes of fusion and fission that regulate mitochondrial shape, distribution and activity. They are also involved in cellular functions of mitochondria, such as energy production, metabolic adaptation, apoptosis and cellular stress responses. Consequently, these organelle dynamics play a crucial role in development, growth, differentiation and disease. Mitochondrial morphology is controlled by Drp1 (also known as DNM1L) and Fis1, which drive fission, whereas Opa1, Mfn1 and Mfn2 mediate fusion. The transcription, activation and degradation of these proteins are often regulated by signaling cascades that are crucial for stem cell maintenance and differentiation. In turn, mitochondrial dynamics regulate key outcomes of these pathways. We explore the interplay between mitochondrial fusion and fission proteins and such signaling pathways, including Notch, receptor tyrosine kinase, JNK, Hippo and mTOR signaling, finding that stem cell renewal and differentiation states are dependent on the regulation of signaling pathways by mitochondrial morphology and activity. Overall, this Review highlights how mitochondrial morphology and activity crucially regulate stem cell division for renewal and differentiation, examining their impact across diverse systems.

The mechanisms underlying the scaling of the Bicoid morphogen gradient in Drosophila with the embryo size is not clearly understood. We propose a model with a spatially varying diffusion coefficient along the anterior-posterior axis, that is proportional to the nearly periodic spatial distribution of the nucleo-cytoplasmic domains and alternating between regions of fast and slow diffusion. We postulate that for specific interpretation of the heterogeneous environment, where the space available for free diffusion within an energid is assumed to be proportional to the embryo size, a change in the embryo size can lead to a size-dependent scaling of the gradient lengthscale. We further study a two component model with slow and fast diffusing Bicoid, and identify this model to be equivalent to the heterogeneous diffusion model further postulating that the fast-state occupancy which is a measure of the fraction of time spent in the fast diffusing state, should also scale with the embryo size via the energid size. Finally, we incorporate nuclear shuttling into our model to understand the effect of shuttling on the gradient lengthscale and scaling with embryo size. We argue that for the particular case where the degradation within the nucleus is low, nuclear shuttling does not perturb the Bicoid gradient. Our study suggests that scaling of the gradient length scale is possible due to spatial heterogeneity and does not depend on nuclear trapping as suggested previously.

Mitochondrial dynamics are vital for embryogenesis, but their role in morphogenesis is largely unexplored. In this study, we elucidate the role of mitochondrial dynamics and function in regulating apical constriction at the ventral furrow in Drosophila gastrulation. We find that spatiotemporal redistribution of mitochondria from basal to apically constricting regions is coincident with Myosin II accumulation in ventral epithelial cells. This apical enrichment depends on intact microtubules and is regulated by Dorsal/NFkb signaling pathway. Embryos expressing mitochondrial fission protein, Drp1 mutants, contain larger, basally accumulated mitochondria, decreased apical constriction and ventral furrow defects. Transcriptomic and proteomic profiling of Drp1 mutants shows no change in Dorsal signaling, but an upregulation of antioxidant enzymes, decreased respiratory complexes, and reduced reactive oxygen species (ROS), confirming loss of mitochondrial activity and a reduced environment. Depleting mitochondrial SOD2 in Drp1 mutant embryos restores levels of antioxidant proteins and increases ROS, leading to an oxidized environment. These embryos also show rescue in apical mitochondrial migration and Myosin II accumulation, thereby enabling proper ventral furrow formation. Our study reveals the functional importance of localized mitochondrial dynamics, regulated by the Dorsal signaling pathway, and mitochondrial activity in a spatiotemporal manner for apical constriction during gastrulation.

Mitochondrial activity and dynamics play crucial roles in regulating neuronal differentiation. Neural stem cells (NSCs) or neuroblasts in Drosophila require the regulation of mitochondrial fusion, along with the activity of the electron transport chain (ETC), to meet the metabolic demands of differentiation. However, the mechanisms by which mitochondrial fusion and activity together regulate NSC differentiation are not well understood. We investigated the relationship between mitochondrial fusion, and ETC complex I activity during Drosophila neuroblast differentiation. We found that depletion of complex I subunits did not affect the number of type II neuroblasts but reduced their proliferation, thereby decreasing the numbers of mature intermediate precursor cells (mINPs), ganglion mother cells (GMCs), and neurons in each lineage. Complex I depletion decreased the mitochondrial membrane potential and cristae numbers, and increased mitochondrial fragmentation and ROS. Increased ROS resulting from the depletion of antioxidant enzymes also led to a decrease in mINPs, GMCs, and neurons in each type II neuroblast lineage. Both complex I and antioxidant protein depletion led to delayed G1/S transition and decreased nuclear cyclin E levels. Interestingly, the defects in proliferation, differentiation, and ROS in complex I and anti-oxidant protein depleted neuroblasts could be restored by fused mitochondrial morphology obtained through additional depletion of the fission protein Drp1. Further, overexpression of anti-oxidant proteins could alleviate the ROS and rescue the differentiation defect in complex I depleted type II neuroblasts. Together, this study reveals a role for complex I and mitochondrial fusion in restricting ROS for differentiation in Drosophila neuroblasts. Significance statement: The electron transport chain complex I regulates differentiation in the Drosophila type II neuroblast lineage. Complex I and anti-oxidant scavenger protein depletion lead to mitochondrial fragmentation and an increase in ROS, thereby affecting the G1/S phase transition and decreasing the formation of lineage cells. Mitochondrial fusion induced by depletion of mitochondrial fission protein Drp1 in complex I and anti-oxidant protein depleted type II neuroblasts leads to suppression of the differentiation defects. This study confirms a crucial role of mitochondrial fusion in restricting ROS for appropriate division and differentiation in the type II neuroblast lineage in Drosophila .

Mitochondrial morphology dynamics regulate signaling pathways during epithelial cell formation and differentiation. The mitochondrial fission protein Drp1 affects the appropriate activation of EGFR and Notch signaling-driven differentiation of posterior follicle cells in Drosophila oogenesis. The mechanisms by which Drp1 regulates epithelial polarity during differentiation are not known. In this study, we show that Drp1-depleted follicle cells are constricted in early stages and present in multiple layers at later stages with decreased levels of apical polarity protein aPKC. These defects are suppressed by additional depletion of mitochondrial fusion protein Opa1. Opa1 depletion leads to mitochondrial fragmentation and increased reactive oxygen species (ROS) in follicle cells. We find that increasing ROS by depleting the ROS scavengers, mitochondrial SOD2 and catalase also leads to mitochondrial fragmentation. Further, the loss of Opa1, SOD2 and catalase partially restores the defects in epithelial polarity and aPKC, along with EGFR and Notch signaling in Drp1-depleted follicle cells. Our results show a crucial interaction between mitochondrial morphology, ROS generation and epithelial cell polarity formation during the differentiation of follicle epithelial cells in Drosophila oogenesis.