Cell Membrane Formation During the Cellularization of the Syncytial Blastoderm Ofdrosophila (Embryonic Development/Epithelium Formation/Electron Microscopy)
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Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2199-2203, March 1995 Developmental Biology Cell membrane formation during the cellularization of the syncytial blastoderm ofDrosophila (embryonic development/epithelium formation/electron microscopy) DRAGUTIN LONCAR* AND S. J. SINGERt Department of Biology, University of California, San Diego, La Jolla, CA 92093-0322 Contributed by S. J. Singer, December 2, 1994 ABSTRACT The early blastoderm of Drosophila is a syn- to permit entry of a water-soluble fixative into the space cytium in which about 6000 nuclei become localized in the between the vitelline and plasma membranes. Permeation of peripheral cytoplasm. During cycle 14 interphase, a wave of the fixative through the plasma membrane then occurred, but membrane formation encircles each nucleus inside its own the resultant fixation of the cytoplasmic contents was not plasma membrane, thereby generating an intact epithelial reproducibly adequate in our hands. In a small but useful layer. The details of this process of cellularization have been addition to this procedure, we have microinjected fixative unclear. Using an improved method offixation ofthe embryos through the puncture. The original periplasmic contents of the for electron microscopy, we show by morphological observa- space between the punctured vitelline and intact plasma tions that a large number of membrane-bounded, electron- membranes were thus rapidly and uniformly displaced by transparent vesicles, ofdiameters ranging from 0.05 ,im to 0.5 fixative, which, after permeation of the plasma membrane, ,um, are present in the periplasm and become redistributed resulted in rapid and uniform fixation of the cytoplasmic during cellularization so as to provide the membrane mass contents. By virtue of this procedure, we believe that we have required at each phase of the process. We recognize three been able to visualize ultrastructural details of the cellular- phases. In the first two phases, the vesicles that were present ization process not previously described. in the apical periplasmic space at earlier stages become Our morphological results strongly suggest that the source concentrated and aligned between the nuclei. The vesicles then of the membranes formed throughout cellularization consists undergo concerted but not precisely synchronous fusion to predominantly of small intraembryonic membrane-bounded, form double membranes, starting at furrows in the plasma electron-transparent vesicles, which are repositioned at several membrane of the embryo and extending about 7 ,um into the stages in the process to become fused with the forming mem- periplasmic space. Subsequently, in the third phase vesicles branes. Several consecutive phases in the continuous process are recruited to the basal periplasmic space but do not become of cellularization were observed and are described. aligned between the nuclei as in the first phase. We presume that these vesicles fuse individually with the growing ends of the double membranes until encirclement of each nucleus is MATERIALS AND METHODS complete. We speculate that these vesicles are all derived from Drosophila embryos.Drosophila eggs were provided through the Golgi apparatus and are moved about in the blastoderm the kindness of James Kadonaga, employing the detailed by interactions with components of the cytoskeleton. conditions and procedures described elsewhere (5). In brief, collecting plates were kept in collecting chambers at 25°C for The development of the early embryo of insects such as a half hour and then removed from the chambers but left at Drosophila is unusual in that the zygote undergoes a series of 25°C. The embryos were then sampled every half hour for 4 hr. 14 nuclear divisions before any cellularization arises (except They were then dechorionated in 50% sodium hypochlorite for the pole cells). The first 9 nuclear divisions occur rapidly and fixed (see below), and specimens were prepared for elec- in the interior of the embryo, after which most of the nuclei tron microscopy. This procedure resulted in a collection of migrate to the periphery ofthe embryo where they divide more progressive developmental stages. Within the time interval slowly another 5 times. The syncytial blastoderm then has after mitotic division 14 (2.5-3.5 hr), more precise ordering of about 6000 somatic nuclei organized in the peripheral peri- the developmental sequence was based on morphological plasm underneath the plasma membrane. At this stage, during criteria such as nuclear positions and shapes and the state of the interphase of cycle 14, a synchronized wave of membrane membrane furrow formation as observed in the electron formation appears to descend into the periplasmic space, micrographs (3, 6). initiating at a hexagonal cytoskeletal cage surrounding each Fixation and Electron Microscopy. After dechorionation, nucleus underneath furrows in the plasma membrane, and Drosophila eggs and embryos were immobilized on double- eventually encircling and compartmentalizing each nucleus. sided tape and covered by a drop of fixative (mixture of 2% of This membrane encirclement produces the cellular blasto- glutaraldehyde and 2% paraformaldehyde in 0.15 M NaCl/0.01 derm. (For reviews, see refs. 1 and 2.) M phosphate, pH 7.4). The viteiline membrane was gently The process of membrane formation during cellulariza- punctured in the fluid-filled pockets (3, 4, 7) in the pole of the tion-e.g., the source of the membrane components, and the eggs by a fine micropipette, and then a small amount of fixative mechanisms underlying membrane formation-is not well ("10 ,u) was injected into the pocket between the vitelline and understood. In large part, this stems from the fact that plasma membranes. This process replaced the fluid in the space ultrastructural studies of the process by electron microscopy between the vitelline and plasma membranes with fixative, the have been impeded by technical problems. In several previous excess fluids flowing out past the micropipette puncture. Eggs studies (e.g., refs. 3 and 4) the impermeable vitelline layer of and embryos with such perforated vitelline membranes but intact the dechorionated embryo was punctured once by a fine needle plasma membranes were kept in fixative for another 5 hr. During The publication costs of this article were defrayed in part by page charge *Present address: Department of Cell Biology, School of Medicine, payment. This article must therefore be hereby marked "advertisement" in University of California, Davis, CA 95616. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2199 Downloaded by guest on September 26, 2021 2200 Developmental Biology: Loncar and Singer Proc Natl. Acad Sci. USA 92 (1995) that time, both the vitelline and plasma membranes became rigid plasma membrane to a distance of about 7 ,tm into the peri- and fixed. Manually devitellinized eggs and embryos were then plasm at this time. Each forming double membrane is still kept in fixative overnight, washed in 0.1 M phosphate buffer (pH connected at its basal end by an enlarged furrow canal situated 7.4) and then postfixed in S04 (1% solution in 0.1 M phosphate about halfway down the length of the nuclei. Transparent buffer) for 1 hr. They were then washed in phosphate buffer, vesicles are again accumulated in the apical periplasm above dehydrated through ascending concentrations of alcohol and the nuclei (Fig. 1 C), occupying about 19% of the volume of propylene oxide, and embedded in Epon by a standard proce- that space (Table 1). No significant accumulations of vesicles dure. Epon-embedded material was cut in sections of 70-nm are observed between the nuclei or just beyond the furrow thickness, contrasted with uranyl acetate and lead citrate, and canals. The basal periplasm exhibits only a few vesicles at this examined in a JEOL 1200 EX electron microscope at 80 keV. phase. Morphometry. The volume density (%) of vesicles was mea- (iii) Phase III (3-3.5 hr). The space between neighboring sured in the periplasm of zygotes and blastoderms by the nuclei now contains a fully formed double membrane (Fig. procedures developed by Weibel and coworkers (8, 9). The 1D), extending about 10 ,tm from the plasma membrane and photographs were enlarged to a final magnification of x 4800. ending in a looped-out furrow canal. The apical periplasm is A multipurpose test system after Weibel (M 168) was modified now largely depleted of the transparent vesicles seen in Fig. and used for morphometric estimation. Volumetric density of 1 C, and the basal periplasm shows a marked accumulation of vesicles (%) in Table 1 refers to the density of vesicles in the such vesicles (Fig. 1D; Table 1). The double membranes periplasm of the zygotes and blastoderms excluding nuclei. continue to elongate, extending to distances of 20-25 ,tm from the plasma membrane (Fig. 1E). The furrow canals then RESULTS become enlarged roughly perpendicular to the internuclear double membranes, enveloping each nucleus. Eventual fusion Electron Microscopic Ultrastructural Preservation. The of two extending furrow canals occurs (cells labeled BC in Fig. method of fixation described in Materials and Methods that was 1E) to complete the process of cellularization. During this used in this study has successfully preserved ultrastructural time, large numbers of transparent vesicles occupy about 21% detail and substructure in the embryonic cytoplasm, including of the volume of the basal periplasm of the forming cells (Fig. mitochondria, nuclei, yolk granules, vesicles, and membranes 1E; Table 1). There are now very few vesicles in the apical (Figs. 1 and 2). These results validate the adequacy of our cytoplasm. fixation procedure. Embryos at the next stage of development, around 3.5-4 hr, Vesicles and Membrane Formation During Cellularization. enter gastrulation and develop several cell layers (Fig. 1F). Overview oftheprocess at low magnification. We first survey the Transparent vesicles are now almost entirely absent from the results at low magnification in Fig. 1 and then examine certain outermost layer of cells but are seen in the cells of the features in more detail at higher magnification in Fig.