Richa Rikhy

Dynamics of the Dorsal morphogen gradient

The dorsoventral (DV) patterning of the Drosophila embryo depends on the nuclear localization gradient of Dorsal (Dl), a protein related to the mammalian NF-κB transcription factors. Current understanding of how the Dl gradient works has been derived from studies of its transcriptional interpretation, but the gradient itself has not been quantified. In particular, it is not known whether the Dl gradient is stable or dynamic during the DV patterning of the embryo. To address this question, we developed a mathematical model of the Dl gradient and constrained its parameters by experimental data. Based on our computational analysis, we predict that the Dl gradient is dynamic and, to a first approximation, can be described as a concentration profile with increasing amplitude and constant shape. These time-dependent properties of the Dl gradient are different from those of the Bicoid and MAPK phosphorylation gradients, which pattern the anterior and terminal regions of the embryo. Specifically, the gradient of the nuclear levels of Bicoid is stable, whereas the pattern of MAPK phosphorylation changes in both shape and amplitude. We attribute these striking differences in the dynamics of maternal morphogen gradients to the differences in the initial conditions and chemistries of the anterior, DV, and terminal systems.

Plasma Membrane Polarity and Compartmentalization Are Established before Cellularization in the Fly Embryo

Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). We used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. We propose that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo.

DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis

Reduced Drp1-mediated mitochondrial fission decreases cell cycle exit and prevents Notch-dependent follicle cell differentiation during oogenesis.

Dynamin 2 orchestrates the global actomyosin cytoskeleton for epithelial maintenance and apical constriction

The mechanisms controlling cell shape changes within epithelial monolayers for tissue formation and reorganization remain unclear. Here, we investigate the role of dynamin, a large GTPase, in epithelial morphogenesis. Depletion of dynamin 2 (Dyn2), the only dynamin in epithelial cells, prevents establishment and maintenance of epithelial polarity, with no junctional formation and abnormal actin organization. Expression of Dyn2 mutants shifted to a non-GTP state, by contrast, causes dramatic apical constriction without disrupting polarity. This is due to Dyn2s interactions with deacetylated cortactin and downstream effectors, which cause enhanced actomyosin contraction. Neither inhibitors of endocytosis nor GTP-shifted Dyn2 mutants induce apical constriction. This suggests that GTPase-dependent changes in Dyn2 lead to interactions with different effectors that may differentially modulate endocytosis and/or actomyosin dynamics in polarized cells. We propose this enables Dyn2 to coordinate, in a GTPase-dependent manner, membrane recycling and actomyosin contractility during epithelial morphogenesis.

Fluorescence Imaging Techniques for Studying Drosophila Embryo Development

This unit describes fluorescence‐based techniques for noninvasive imaging of development in living Drosophila embryos, discussing considerations for fluorescent imaging within living embryos and providing protocols for generation of flies expressing fluorescently tagged proteins and for preparation of embryos for fluorescent imaging. The unit details time‐lapse confocal imaging of live embryos and discusses optimizing image acquisition and performing three‐dimensional imaging. Finally, the unit provides a variety of specific methods for optical highlighting of specific subsets of fluorescently tagged proteins and organelles in the embryo, including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and photoactivation techniques, permitting analysis of specific movements of fluorescently tagged proteins within cells. These protocols, together with the relative ease of generating transgenic animals and the ability to express tagged proteins in specific tissues or at specific developmental times, provide powerful means for examining in vivo behavior of any tagged protein in embryos in myriad mutant backgrounds. Curr. Protoc. Cell Biol. 39:4.18.1‐4.18.43.

Cells within a cell: Insights into cellular architecture and polarization from the organization of the early fly embryo.

Drosophila embryogenesis begins with 13 rapid nuclear divisions within a common cytoplasm. These divisions produce ~6,000 nuclei that, during the next division cycle, become encased in plasma membrane (PM) and generate the primary embryonic epithelium in the process known as cellularization. Despite the absence of PM boundaries between syncytial nuclei, the secretory membrane system is organized in functionally compartmentalized units around individual nuclei.1 We have recently used in vivo fluorescence imaging to characterize the dynamics of proteins in the PM of the embryonic syncytium. These studies revealed that the PM is polarized already before cellularization. One PM region resides above individual nuclei and has apical-like features, while PM regions lateral to nuclei have basolateral characteristics. Optical highlighting experiments showed that membrane components do not exchange between PM regions that reside above adjacent nuclei. An intact F-actin network was shown to be important for both the PM apicobasal-like polarity and the diffusion barriers within the syncytial PM. Our findings, as well as their possible implications, are further discussed in this Addendum.

Dynamin regulates metaphase furrow formation and plasma membrane compartmentalization in the syncytial Drosophila embryo.

ABSTRACT The successive nuclear division cycles in the syncytial Drosophila embryo are accompanied by ingression and regression of plasma membrane furrows, which surround individual nuclei at the embryo periphery, playing a central role in embryo compartmentalization prior to cellularization. Here, we demonstrate that cell cycle changes in dynamin localization and activity at the plasma membrane (PM) regulate metaphase furrow formation and PM organization in the syncytial embryo. Dynamin was localized on short PM furrows during interphase, mediating endocytosis of PM components. Dynamin redistributed off ingressed PM furrows in metaphase, correlating with stabilized PM components and the associated actin regulatory machinery on long furrows. Acute inhibition of dynamin in the temperature sensitive shibire mutant embryo resulted in morphogenetic consequences in the syncytial division cycle. These included inhibition of metaphase furrow ingression, randomization of proteins normally polarized to intercap PM and disruption of the diffusion barrier separating PM domains above nuclei. Based on these findings, we propose that cell cycle changes in dynamin orchestrate recruitment of actin regulatory machinery for PM furrow dynamics during the early mitotic cycles in the Drosophila embryo.

Multiscale diffusion in the mitotic Drosophila melanogaster syncytial blastoderm

Despite the fundamental importance of diffusion for embryonic morphogen gradient formation in the early Drosophila melanogaster embryo, there remains controversy regarding both the extent and the rate of diffusion of well-characterized morphogens. Furthermore, the recent observation of diffusional “compartmentalization” has suggested that diffusion may in fact be nonideal and mediated by an as-yet-unidentified mechanism. Here, we characterize the effects of the geometry of the early syncytial Drosophila embryo on the effective diffusivity of cytoplasmic proteins. Our results demonstrate that the presence of transient mitotic membrane furrows results in a multiscale diffusion effect that has a significant impact on effective diffusion rates across the embryo. Using a combination of live-cell experiments and computational modeling, we characterize these effects and relate effective bulk diffusion rates to instantaneous diffusion coefficients throughout the syncytial blastoderm nuclear cycle phase of the early embryo. This multiscale effect may be related to the effect of interphase nuclei on effective diffusion, and thus we propose that an as-yet-unidentified role of syncytial membrane furrows is to temporally regulate bulk embryonic diffusion rates to balance the multiscale effect of interphase nuclei, which ultimately stabilizes the shapes of various morphogen gradients.

Syndapin bridges the membrane-cytoskeleton divide during furrow extension

ABSTRACT BAR domain proteins can regulate ‘membrane reservoirs’ that provide surface area and buffer membrane tension. Syndapin is an F-BAR and SH3 domain containing protein involved in cytoskeletal remodelling and endocytosis. The Syndapin F-BAR domain is uniquely versatile compared to others in the family and can bend phospholipid membranes into tubules of various diameters and directly bind actin. The Syndapin SH3 domain can also interact with actin remodelling proteins and modulate cytoskeletal contractility. Pseudocleavage furrow extension in the syncytial division cycles of Drosophila embryos requires the homeostatic control of conserved processes that control plasma membrane tension and actin contractility. We find that Syndapin plays an important role in promoting pseudocleavage furrow extension. We propose a model involving roles for Syndapin in membrane dynamics and direct or indirect effect on the cytoskeleton to explain how it affects pseudocleavage furrow growth, independent of its role in endocytosis.

Ras/ERK dependent increase in mitochondrial membrane potential in fission deficient Drosophila follicle cells leads to loss of differentiation

GATA family transcription regulators and their co-factors control the cell fate and differentiation of several tissues. Gata2 and Gata3 and their partner Tal1 are also expressed in central nervous system where they regulate development of GABAergic and serotonergic neurons. Without these proteins subtypes of these neurons are lost as their precursors change their fate to other neuron types. Here we analyse the role of two GATA co-factors Friend of Gata 1 and 2 (Fog1 and Fog2) in development of GABAergic and serotonergic neurons. Both Fog1 and Fog2 are expressed early in the development of GABAergic precursors in midbrain and rhombomere 1, while only Fog1 is expressed in the serotonergic neurons in the anterior hindbrain. Similarly to GATA factors and Tal1, the Fog proteins regulate the heterogeneity of GABAergic and serotonergic neurons, but the inactivation of these factors in the midbrain and rhombomere 1 does not lead to fate change that is characteristic to Gata2; Gata3 and Tal1 mutants. Instead, Fog1 and Fog2 work redundantly to generate the different subtypes of GABAergic neurons in the midbrain, while Fog1 is needed to create a specific population of serotonergic neurons.