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Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). This study 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. It is proposed that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo (Mavrakis, 2008).
To study the organization of the PM and the spatiotemporal dynamics of membrane components in living Drosophila embryos, transgenic animals were generated expressing different PM proteins tagged with Cerulean or Venus fluorescent proteins. The proteins were selected because they have different modes of membrane attachment and potentially different PM distributions. They included: (1) Venus fused to the first 20 amino acids of growth-associated protein 43 (GAP43), which contain a dual palmitoylation signal that tightly anchors the protein to the inner leaflet of the PM, (2) Cerulean fused to the pleckstrin-homology domain of phospholipase C delta 1, PH(PLCδ1), which binds specifically to the phosphoinositide PI(4,5)P2, and (3) Venus fused to full-length Toll receptor, a type I transmembrane protein that is required for dorsal-ventral embryonic polarity (Mavrakis, 2008).
This study provides evidence that the plasma membrane of the fly syncytial blastoderm exhibits a polarized, epithelial-like organization prior to cellularization. Previously, it was thought that the PM of the blastoderm had no specialized organization prior to the formation of cell boundaries at cellularization. The results show that despite the absence of cell boundaries, the PM of the syncytial blastoderm has apical- and basolateral-like domains surrounding individual cortical nuclei and that PM proteins do not exchange between PM regions surrounding adjacent nuclei. This organization is maintained throughout syncytial mitotic division cycles and is dependent on an intact F-actin network (Mavrakis, 2008).
Support for these conclusions came from live imaging and fluorescent highlighting experiments in living embryos. Using a variety of membrane markers, two distinct PM regions were distinguished. One region was above individual nuclei and had apical-like characteristics, including the presence of microvilli and an enrichment in PI(4,5)P2, a key determinant of apical PM biogenesis, as well as in GAP43, a protein that localizes to raft-like membranes, which typically compose apical PM surfaces in epithelial cells. The second PM region was lateral to nuclei, and was enriched in markers typically associated with basolateral membranes and junctions, including the cell-cell adhesion molecule E-cadherin, the multi-PDZ domain scaffolding protein DPatj. FRAP experiments showed that the molecules could freely diffuse in the PM domains surrounding individual nuclei but did not diffuse outside them, suggesting the presence of a diffusion barrier between the domains during interphase. Moreover, optical pulse-chase experiments showed that these components did not diffuse outside PM domains surrounding mitotic units throughout the time period of syncytial divisions. Thus, during mitosis, the polarized organization and restricted diffusion pattern of proteins in the PM did not change. Finally, the requirement of an intact F-actin network was supported by drug-induced actin depolymerization, which disrupted PM association of DPatj and Peanut and abolished the restricted diffusion pattern in the PM (Mavrakis, 2008).
The finding that the PM of the syncytial blastoderm is organized as a pseudoepithelium prior to cellularization has several important implications for understanding many aspects of embryo development. First, it directly impacts on how dorsal-ventral and terminal patterning are set up prior to cellularization. These are dependent on Toll and Torso membrane receptors. Toll is distributed uniformly along the syncytial PM, but is activated only ventrally. Similarly, Torso is uniformly expressed along the surface membrane of early embryos, but its activation occurs only at the anterior and posterior poles. Given that membrane receptors have the capacity to diffuse across the PM, it has been unclear why the activation zones of these receptors do not spread widely across the PM. The results revealing the compartmentalized character of the PM during interphase and syncytial nuclear divisions now provide a potential answer. Receptors diffuse locally within the PM surrounding a particular nucleus, but they do not diffuse to PM regions associated with other nuclei. Consequently, activation zones of receptors (set up by the localized spatial signal of ligands) do not spread, allowing robust downstream signaling events in particular regions of the embryo. This possibility is supported by the spreading of the Dorsal gradient to more anterior and posterior regions in embryos treated with latA. LatA-induced actin depolymerization abolished the confined diffusion pattern in the PM suggesting that an intact actin network is likely to be important for containing activated Toll diffusion and thus maintaining a robust downstream Dorsal gradient (Mavrakis, 2008).
The molecular basis for the compartmentalized diffusion in the PM of the syncytial embryo appears to be due to the presence of bona fide diffusion barriers in the PM regions directly between adjacent nuclei. The finding that septins and components of junctions are specifically enriched in this PM region raises the possibility that these molecules together with other cytoskeletal components organize a barrier to diffusion in the plane of the PM in a way similar either to the organization of septin rings at the yeast bud neck or of adherens junctions in epithelial cells. Moreover, the loss of PM association of DPatj and Peanut, as well as the abolishment of the restricted diffusion pattern in latA-treated embryos, suggest that an intact F-actin network is required both to localize and/or maintain septins and junctional components to specialized PM regions and to contain diffusion of proteins in PM units around individual syncytial nuclei. An intact F-actin network was recently shown to be required for compartmentalizing furrow canals during cellularization further supporting that F-actin organizes lateral diffusion of proteins in the PM. Future studies will need to genetically dissect the molecular machineries involved in organizing such diffusion barriers (Mavrakis, 2008).
A second implication of the observed PM dynamics during syncytial mitoses relates to the machinery driving PM invagination. It was found that the PM was organized into highly convoluted microvillous membrane buds over interphase nuclei and these flattened out as soon as nuclei entered mitosis before reorganizing again into microvillous buds upon re-entry into the next interphase. Furthermore, the rate at which PM invaginated (~1.5-2 μm/min) was twice as fast as during the fast phase of cellularization, which involves de novo membrane delivery. Although endocytosis was recently shown to accompany metaphase furrow ingression, the current observations support a mechanism for PM invagination in mitosis that involves contractile machinery which transiently redistributes PM from microvilli caps into transient furrows surrounding mitotic units rather than an internal membrane source (Mavrakis, 2008).
A final implication of these findings relates to cellularization, which produces the primary epithelial cells of the embryo. Polarization of the invaginating PM during cellularization has been reported, and it is during cellularization that PM polarity is first thought to be achieved in early fly embryogenesis. Because the data demonstrate that the PM is already polarized prior to cellularization, it is likely that the embryo uses this organization to initiate and organize the cellularization process. Consistent with this, it was found that the junctional proteins E-cadherin and DPatj, the septin protein Peanut, and Toll are all highly enriched in the PM at sites between adjacent nuclei during syncytial interphases, which reflects the PM organization between nuclei right at the onset of cellularization (first few minutes of interphase 14). Indeed, these are precisely the PM sites that become further differentiated within the first 5 min into cellularization, with the formation of an invaginating membrane front that contains Peanut and DPatj, basal adherens junctions directly adjacent to the invaginating front that contain E-cadherin, and the extension of the lateral membranes that are positive for Toll. The epithelial polarization occurring during cellularization is thus already reflected in the organization of the syncytial blastoderm PM (Mavrakis, 2008).
In summary, these findings that the syncytial blastoderm PM exhibits an epithelial-like polarization prior to cellularization, and that distinct PM domains do not significantly exchange membrane components, point to an as yet unexplored mechanism for how the embryo maintains and generates morphogen gradients at this stage. By preventing activation zones of membrane receptors on the PM from spreading, robust downstream signaling events within the cytoplasm and nuclei of the embryo can be established. This mechanism would work in conjunction with nuclear-cytoplasmic shuttling of transcription factors, and a compartmentalized secretory pathway, to generate the dorsal-ventral and terminal patterning systems of the blastoderm fly embryo (Mavrakis, 2008).
Mavrakis, M., Rikhy, R. and Lippincott-Schwartz, J. (2008). Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo. Dev. Cell 16: 93-104. PubMed Citation: 19154721
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