Lateral mobility of plasma membrane lipids in Xenopus eggs: regional differences related to animal/vegetal polarity
J. G. Bluemink*1, W. J. A. G., Dictus, E. J. J. van Zoelen1, P. A. T. Tetteroo, L. G. J. Tertoolen1 and S. W. de Laat1
1Hubrecht Laboratory, International Embryological Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. 2Department of Biology, V. U., Workgroup Histology and Electron Microscopy, P. 0. Box 7161,1007 MC Amsterdam, The Netherlands. 3Central Laboraotry of the Blood-transfusion Service, Plesmanlaan 125,1066 CX Amsterdam, The Netherlands
Regional differences in the lateral mobility properties of plasma membrane lipids were studied in unfertilized and fertilized eggs and early embryos of Xenopus laevis by fluorescent photobleaching recovery (FPR) measurements. The aminofluorescein-labelled fatty acids HEDAF and TEDAF appear to distribute themselves in the plasma membrane under all conditions used. These molecules show partial recovery of fluorescence upon photobleaching, indicating the existence of lipidie microdomains in the membrane. In the unfertilized egg the mobile fraction of plasma membrane lipid (∼ 50 %) has a 5-fold smaller lateral diffusion coefficient (D = 1-5 x 10-8 enr.s-1) in the animal than in the vegetal polarity within the egg plasma membrane. Upon fertilization this polarity is strongly (> 100x) enhanced, leading to the formation of two distinct macrodomains within the plasma membrane. On the animal side of the egg the lipids are completely immobilized on the time scale of FPR measurements (D " 10-10 cm2.s-1), whereas on the vegetal side D is only slightly reduced (D = 2-8X10-8 cm2 s-1). The transition from the non-fluid animal to the fluid vegetal domain is sharp and lies on the equator. This animal/vegetal difference in lateral mobility properties of plasma membrane lipids is maintained through the period of cellulation, as shown by FPR measurements on the morula (st. ) and blastula (st. 8). At stage
the transition from the animal to the vegetal domain across the equator is two cells wide, lipid mobility changing stepwise from one cell to the next. Preliminary FPR measurements on the mid-gastrula (st.
) show that the dorsal and ventral blastoporal lips are within the fluid domain. Current research is aimed at investigating the morphogenetic role of the animal/vegetal difference in plasma membrane mobility characteristics, using embryos which have developed under conditions when the blastopore should appear in the non-fluid domain of the animal half (eggs turned 180 ° with respect to gravity)
Electrical phenomena and their possible significance in vitellogenic follicles of Drosophila melanogaster
J. Bohrmann1U.-R. Heinrich1A. Dorn2, K. Sander1and H. Gutzeit*1.1Institut für Biologie I (Zoologie), Albertstrasse 21a, D-7800 Freiburg, West Germany. 2Botanisches Institut I, Kaiserstrasse 2, D-7500 Karlsruhe, West Germany
We have measured extracellular currents in vitellogenic follicles of Drosophila with a vibrating probe. In stage 10 follicles current enters at the anterior half of the follicle (nurse cells) and leaves at the posterior half (oocyte). Stage 11 follicles show a variable pattern of current flow. Often a strong current (either inward or outward) was measured in the region of the centripetally migrating follicle cells. Some of these cells are specialized with respect to their ion content: when cations (in particular Ca++) are precipitated with pyroantimonate and the formed precipitates viewed in the electron microscope, some cells with a particularly high number of precipitates per unit area can be identified. These cells increase in number during stage 10B and extend from the area of the ring canals to the outer face of the follicle. These precipitate-rich cells may be part of an intra-follicular current loop whose existence in Cecropia follicles was inferred by extracellular potential measurements (Jaffe and Woodruff, 1979).
The physiological role of the electrical phenomena is still unclear. Electrophoretic migration of charged proteins (Woodruff and Telfer, 1980) in the follicle may occur but positive evidence with endogenous proteins is still lacking and this mechanisms probably does not account for the site-specific deposition of molecules within the follicle. The rapid influx of nurse cell cytoplasm into the oocyte during stage 10B to 12 appears not to be related to the electrical phenomena, since some follicles which for unknown reasons did not generate any extracellular currents continued cytoplasmic streaming in vitro. Our observation that ion asymmetries in Drosophila follicles can first be detected with the pyroantimonate method when yolk uptake begins (stage 7) and then increase up to the stage of nurse cell breakdown (stage 10B) suggests that electrical phenomena may be related to vitellogenesis.
Cytoskeletal control of endosome polarisation in mouse preimplantation embryos
Peter M. Cannon and Tom P. Fleming, Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY
The 8-cell stage of mouse development is characterized by a process of cell polarisation which includes, in the majority of cells, the clustering of endosomes from a uniform distribution into an apical cytoplasmic region, subtending the microvillous pole. The endosome pole is present in outer blastomeres following division to the 16-cell stage.
The role of the cytoskeleton during the formation and subsequent stabilization of endosome poles has been investigated in 8- and 16-cell embryos pre-incubated in horseradish peroxidase to label the endocytotic apparatus. The formation of endosome poles is significantly reduced if newly-formed 8-cell embryos (or natural 2/8 couplets) are incubated in cytochalasin D (CCD: 0-5 µ g/ml) during the initial period of the fourth cell cycle, demonstrating that intact microfilaments are required for endosome relocation. In contrast, pre-existing endosome poles within compacted 8-cell embryos are insensitive to CCD treatment but are significantly disrupted by colcemid (5 0 µ g/ml) incubation, indicating that microtubules, but not microfilaments, are responsible for stabilizing the asymmetric pattern of endosome cytolocation. Colcemid-induced loss of endosome polarity is blocked by CCD suggesting that relocation of endosomes is microfilament-dependent.
The occurrence of endosome poles within outer blastomeres at the 16-cell stage is significantly greater than within compacted 8-cell blastomeres and they consist of more tightly-packed clusters of endocytotic vesicles. In addition, endosome poles at the 16-cell stage are insensitive to both colcemid and CCD, demonstrating a phase of stabilization in the generation of endocytotic polarity.
Concanavalin A as a probe for the polar organization of the plasma membrane in a molluscan egg cell
M. R. Dohmen, J. E. Speksnijder and K. J. Teerds, Zoological Laboratory, University of Utrecht, Padualaan 8, Utrecht, The Netherlands
The polarity of egg cells is an important factor in early development. The determinants of the spatial organisation of cells are largely unknown. Most likely, the plasma membrane plays a central role in this respect. The results of experiments with conA support the view that a polar structure exists at the level of the plasma membrane. When fertilized uncleaved eggs of the gastropod Nassarius reticulatus are treated with conA the plasma membrane/cytoskeleton complex starts being affected at the vegetal pole of the egg and this reaction progressively spreads towards the animal pole. Irrespective of the moment of incubation with conA, the reaction of the egg begins when the first polar lobe is being formed, a process immediately preceding first cleavage. As soon as the polar lobe constriction appears, the microvilli start disappearing at the vegetal pole. Concomitantly, the vitelline membrane which is attached to the egg surface disappears at the same rate and to the same extent as the microvilli. This process stops short of the constriction of the polar lobe. The position of the polar lobe constriction may also be strongly influenced by conA treatment. At low doses the constriction is situated in its normal position, but an abnormally long and narrow stalk is formed. At high doses the position of the constriction may shift extremely towards the animal or vegetal pole of the egg. A cleavage furrow starts being formed at the animal pole, but its progression is inhibited and it soon regresses. Nuclear division is not impaired. After some time most of the eggs lyse, a process which occurs invariably at the vegetal pole.
It is concluded that conA exerts a propagated transmembrane effect on the cortical cytoskeleton via receptors present in the plasma membrane at the vegetal pole. The phenomenon that the effect of conA is triggered by the onset of cytokinesis may be due to cell cycle dependent modulation of these receptors or their relay systems.
Maturation and polarisation of the endocytotic apparatus in outer blastomeres of the preimplantation mouse embryo
TomP. Fleming, Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3 DY
During cleavage, outer blastomeres undergo a progressive maturation and polarisation of the endocytotic apparatus. These incremental phases in the generation of a polarised epithelium (trophectoderm) have been studied using various ultrastructural techniques.
Oocytes to early 8-cell stages possess apolar clusters of endosomes predominantly confined to the cortical cytoplasm. In compacted 8-cell embryos, endosomes become preferentially localised in the apical cytoplasm beneath the microvillous pole. Incubation in the endocytotic tracer, horseradish peroxidase (HRP; 5 mg/ml) results in pinocytic vesicle (PV) formation and rapid fusion with apical endosomes from 5-20 m. PV formation (expressed as PV number/unit length plasma membrane) is significantly greater at the apical (outer) PM than at the basolateral PM in both compacted and decompacted 8-cell embryos; no such distinction in pinocytic activity is evident in apolar, early 8-cell embryos.
A polar cytolocation of endosomes is also evident in outer blastomeres at the early 16-cell stage (9 h post-compaction). These cells display an increased preferential endocytotic activity at the apical PM face. During the late 16- and early 32-cell stages, outer blastomeres generate and accumulate electron dense secondary lysosomes (SLs) in the basal cytoplasm. SLs show trimataphosphatase activity, and become labelled with HRP by 30 m incubation. They are derived from autolytic digestion of cytoplasmic granules and transformation of endosomes. Following zonular tight junction formation at the 32-cell stage, outer cells (trophectoderm) possess transcellular and recycling endocytotic pathways (visualized by HRP and/or cationized ferritin labelling), which collectively appear responsible for maintaining PM asymmetry and cell polarity.
The maturation of a polarised endocytotic apparatus in outer blastomeres during preimplantation development in the mouse
TomP. Fleming* and Peter M. Cannon, Department of Anatomy, University of Cambridge, Downing Street, Cambridge
Morphological studies have shown that:
(a) the endocytotic apparatus within the trophectoderm epithelium of the blastocyst is polarised with respect to organelle distribution and constituent processing pathways.
(b) the development of endocytotic asymmetry occurs incrementally in outer blastomeres during proceeding stages of cleavage.
The important phases in the generation of endocytotic polarity in outer cells can be summarised as follows:
1) Oocyte - early 8-cell stage. Endocytotic organelles consist primarily of endosomes (or multivesicular bodies) which are in apolar distribution and occur mainly in the cortical cytoplasm. No difference in pinocytic activity is evident between apical (outer) and basolateral membrane faces.
2) Compacted 8-cell stage. Endosomes become preferentially localised in the apical cytoplasm underlying the microvillous pole. Relocation is sensitive to cytochalasin D; pre-formed endosome poles are disrupted by colcemid. Pinocytic activity is significantly greater at the apical rather than the basolateral membrane face.
3) Early 16-cell stage. Endosomes remain polarised and become stable to either colcemid or cytochalasin D treatments. Preferential pinocytic activity at the apical membrane face is increased.
4) Late 16- to early 32-cell stages. Secondary lysosomes, showing trimetaphosphatase activity, accumulate in the basal cytoplasm.
5) Trophectoderm. Apical and basolateral membrane faces are separated by zonular tight junctions. Transcellular endocytotic pathways via endosomes operate in both directions; these, in conjunction with short-circuit endosome recycling pathways, are likely to preserve the asymmetry of cell membrane domains.
A three-step scheme of early grey crescent formation in the Axolotl oocyte
J. Gautier1,2and J. C. Beetshcen1 1Laboratoire de Biologie générale, Université Paul Sabatier, 31062 Toulouse, France. 2Département de Biologie moléculaire, Université Libre de Bruxelles, 1640 Rhode-Saint-Genese, Belgium
It has been shown that inhibition of protein synthesis can elicit, within a few hours, the precocious appearance of the grey crescent (GC) in Axolotl oocytes, artificially maturing in vitro (1,2). However, such a symmetry reaction does not occur when the oocyte has been enucleated before progesterone-induced maturation. The ability to form a GC is re-established in enucleated oocytes by the injection of nucleoplasm from a normal oocyte, either before or after injection of the protein synthesis inhibitor (diphtheria toxin, 10∼8M) (3). In the latter case, the GC appears even though the protein synthesis level is as low as one-tenth that of the control enucleated oocyte, and it is formed very quickly (after 30 min in 50 % of the injected oocytes). Finally, it has been established that the interactions oetween nuclear factor(s) and cytoplasm are possible only when cytoplasmic maturation has been proceeding for 10 h at least.
Our results thus suggest a three-step scheme for establishing preliminary conditions essential to Axolotl oocyte symmetrization (GC formation):
1) Cytoplasmic maturation.
2) Nucleoplasmic factor(s) mix(es) with cytoplasm after germinal vesicle breakdown.
3) Inhibition of protein synthesis.
Steps 2 and 3 can be experimentally inverted, but step 1 must always occur first. A similar sequence of events should apply to normal GC formation in the fertilized egg, since a sharp decrease of the protein synthesis level (about 50 %) was observed in uncleaved eggs before appearance of the grey crescent.
The active nuclear fraction of the normal oocyte is under investigation. It rapidly migrates out of isolated germinal vesicles but it is present in the soluble fraction from whole oocytes extracts, in the supemate (150,000xg, 3 h). It is not found in the extract of enucleated oocytes. The active fraction is heat-resistant, RNAase-resistant and trypsin-sensitive.
Displacement of cellular components during the maturation of the Xenopus oocyte
P. Hausen, Ya-Hui Wang, and C. Dreyer, Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35/V, 7400 Tübingen, West Germany
Oocyte maturation was induced in vitro by progesterone. The movements and translocations of the intracellular components in the course of the maturation process were followed first by standard histological techniques at the light microscope level. By use of monoclonal antibodies directed against various oocyte components, their fate during egg maturation was followed. Special reference has been made to proteins of the germinal vesicle and their allocation to the different regions of the egg.
The mitochondrial cloud of Xenopus oocytes is the source of germinal granule material; its breakdown localises this material in the vegetal pole
Janet Heasman and C. C. Wylie, Department of Anatomy, St. George’s Hospital Medical School, Cranmer Terrace, London, SW17 ORE
The mitochondrial cloud is a prominent mass in the cytoplasm of previtellogenic oocytes of Xenopus laevis. We report here that it contains electron-dense granulo-fibrillar material (GFM) as well as mitochondria. Using a combination of light microscopical, fluorescence, time-lapse filming and electron microscopical techniques, the ontogeny of these components has been studied. We find that the cloud is stationary in previtellogenic stages, and fragments into islands of mitochondria and GFM at the time of onset of vitellogenesis. These islands become localized in the peripheral cytoplasm at one pole of the small oocyte. By studying successive stages, we find that GFM remains localized at one pole; in larger oocytes, where the animal/vegetal axis becomes obvious due to pigment accumulation, this is found to be the vegetal pole. Furthermore GFM bears a striking resemblance in position, appearance and association with mitochondria to the ‘germinal granules’ found in the vegetal pole of unfertilized eggs. Germinal granules have been shown by others to become incorporated into germ-line cells. We conclude that GFM is the precursor of this material and that it accumulates in the mitochondrial cloud of previtellogenic oocytes. The dispersing cloud may provide a mechanism for its localization in the vegetal pole of the oocyte and egg, and is the first indication of polarity in the Xenopus oocyte.
Segregation of informational macromolecules in Ascidian embryos
W. Jeffrey
No abstract for publication
Mechanisms for generating and stabilizing cell asymmetries
M. H. Johnson*, Department of Anatomy, Downing Street, Cambridge CB23DY
The generation of cell diversity by the differentiative division of an asymmetrically organised cell is well established in embryology. This Symposium is not concerned with describing more examples of this phenomenon but rather in probing the way in which cell asymmetries are set up, elaborated and stabilized. The Symposium will concentrate on mechanisms. Many, if not all, cell asymmetries are set up, and the orientation of the reorganisation determined, by external signals. Early, spatially-defined responses to these signals include changes in electrical permeability, local membrane properties and cytoskeletal organisation. Subsequently, major redistributions of cell organelles and of specific informational molecules such as mRNAs and proteins occur. Careful analysis of the temporal sequence in which these changes occur, and the use of specific reagents that affect individual components of the sequence, can help elucidate the nature of the primary response to the signal for asymmetry, and how this response is transmitted throughout the cell. Similarly, once asymmetry is established, manipulations on the cell, followed by observation of the immediate and medium term regulative capacity of the cell, can give clues as to whether a specific ‘memory’ of the asymmetry is present in the cell and if so, where it resides. This latter feature is of particular importance to the cell during division if non-equivalent progency are to be produced. It is the mechanisms underlying these various events that concerns this Symposium.
An EM study of germ plasm in normal and inverted eggs of Xenopus laevis
A. Jurand1and K. E. Dixon2. 1Edinburgh University. 2The Flinders University of South Australia, Bedford Park, 5042, Australia
Germ plasm in unfertilised eggs of anuran amphibians is distributed throughout the cortex in small, isolated islets containing mitochondria and germinal granules (Czolowska, 1972). During the first two cleavage divisions, the individual islets move towards the vegetal pole, coalescing as they do so to form large aggregates identified in the light microscope by their yolk-free, granular structure. In 8-cell embryos in normal orientation, the region identified as the germ plasm in the light microscope is found at the ultrastructural level to be composed of a number of organelles - numerous mitochondria, many germinal granules, vesicles, ribosomes, glycogen and some microfilaments, ER and Golgi bodies. The mitochondria adhere together to form chains which presumably provide structural cohesion (ie. a cytoskeletal function) for the whole germ plasm region which can therefore be considered largely autonomous within the cytoplasm of the cell containing it. The germinal granules are aggregated together, and intimate structural associations are observed between the aggregates and the mitochondria. When fertilised eggs were rotated through 90 ° or inverted during the first cleavage cycle and then examined at the 8-cell stage, the mitochondria in the germ plasm were more dispersed and adhesions between them were rare. The aggregates of germinal granules were less compact and large spaces due to expansion and thinning of their texture were common, resulting in their exhibiting a sponge-like appearance. The failure of the mitochondria to adhere together and the abnormal structures formed by the germinal granules may be causally related to the decreased number of germ cells which enter the genital ridges in rotated and inverted embryos (Wakahara et al., 1984; Cleine and Dixon, unpublished).
Microtubules, centrioles, and cell asymmetry
Marc Kirschner*, Eric Karsenti and Tim Mitchison, Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143, U.S.A.
Microtubules play an important role in establishing and maintaining the asymmetry of somatic cells and eggs. The organization of microtubules depends in part on the disposition and activity of microtubule organizing centers which in most animal cells consists of a pair of centrioles surrounded by osmiophillic material. These organizing centers function in both interphase and mitosis. We have recently studied the mechanics by which the organizing centers spatially organize microtubules in cells in three types of experiments: a) the function of organizing centers in anchoring microtubules in interphase somatic cells, b) the mechanisms by which microtubule arrays are converted from interphase to mitosis using microinjection into frog eggs, and c) the underlying molecular mechanisms by which organizing centers function to nucleate microtubules, by studying the assembly of pure tubulin on isolated centers in vitro. The results of the latter studies suggest that microtubules may be much more dynamic than previously thought. They also suggest that nucleation itself may be a simple process but the unusual physical and chemical properties of microtubule polymerization can provide mechanisms by which the cell can impose a pattern of microtubule organization on a nucleated array. The conversion of microtubule arrays from mitosis to interphase suggests that such unusual mechanisms operated in vivo.
The role of microtubules during compaction of the mouse 8-cell embryo
B. Maro, M. H. Johnson and S. J. Pickering, Department of Anatomy, Downing Street, Cambridge CB2 3DY
The role of microtubules during compaction of the mouse 8-cell stage embryo was investigated using the drugs Taxol (which leads to a non controlled polymerization of tubulin) and Nocodazole (which causes depolymerization of microtubules). Taxol inhibits compaction in most non-compacted embryos and reverses it in already compacted embryos. These effects were observed on cell flattening (as judged by phase contrast microscopy), on cell polarization (as judged by scanning electron microscopy and the surface binding of fluorescent concanavalin A) and on polarization of intracellular organelles (as judged by immunofluorescent staining of actin and clathrin). In contrast, Nocodazole does not inhibit cell flattening but rather accelerates its completion. Nocodazole influences the detailed organization of the surface poles and appears to reduce the incidence of surface polarization but does not reverse (to a significant extent) polarity once established. Nocodazole is also able to inhibit and reverse the redistribution of intracellular organelles which takes place during compaction. We conclude that microtubules exercise a constraining role during compaction, regulating the changes in cell shape and cell organisation, and being involved in the timing of compaction rather than constituting part of the mechanism of cell flattening and cell polarization.
Assembly and topogenesis of the spectrin-based membrane-cytoskeleton during erythroid development
Randall T. Moon* and Elias Lazarides, Division of Biology, California Institute of Technology, Pasadena, CA 91125, U.S.A.
Components of multisubunit protein complexes are frequently synthesized at one or more cytoplasmic sites, and assembled at another site. As a model for the assembly of localized multisubunit complexes we have studied the biogenesis of the membrane skeleton of erythroid cells. In erythrocytes a/3-spectrin is linked to the plasma membrane through ankyrin, which binds to both the ^-subunit of spectrin and to a subset of the transmembrane anion transporters. Development of in vivo and in vitro reconstitution assays which distinguish assembled from unassembled subunits has revealed that during chicken erythroid cell development a- and /3-spectrin and ankyrin are synthesized simultaneously but in excess of the amount of each polypeptide assembled. Assembly of all three polypeptides onto the membrane occurs rapidly after synthesis and at the stoichiometry detected at steady state. Assembly proceeds by a series of limiting steps: the availability of membrane binding sites limits the amount of ankyrin assembled, which in turn limits the amount of a-spectrin assembled. Pulse-chase experiments reveal that assembled subunits are stable while unassembled subunits turn over with a half-life of approximately 45 min. These studies support a scheme for the assembly of spectrin and ankyrin where high affinity receptors localized on the plasma membrane (presumably the anion trnasporter) mediate post-translationally the stable assembly of the three cytoskeletal proteins in their correct stoichiometry, thus also defining their spatial localization at the plasma membrane. Failure of subunits to assemble results in their degradation, suggesting that synthesis and assembly of these polypeptides although concurrent, are not tightly coupled events. These principles of the assembly of the spectrin-based membrane cytoskeleton also apply to the assembly of many other spatially segregated multisubunit complexes in both plants and animals. These principles may, therefore, reflect widespread mechanisms involved in the establishment of cell asymmetries.
Strikingly different distributions of nuclear constituents during fibroblast cell division revealed by monoclonal antibodies
E. A. Nigg, C. F. Lehner, V. KurerandH. M. Eppenberger, Institute for Cell Biology, ETH-Hoenggerberg, CH-8093 Zurich, Switzerland
Monoclonal antibodies were raised against nuclear extracts prepared from chicken embryonic tissues. According to immunofluorescent localizations in fibroblast interphase nuclei, four different classes of antigens were distinguished: i) envelope-associated lamin proteins (67-70K), ii) nucleolar constituents (90,40K), iii) nucleoplasmic proteins (mw range: 25-130K) and iv) antigens apparently confined to discrete nuclear substructures (e.g. 40K). Using immunofluorescence microscopy, antibodies against selected antigens were used to study the process of nuclear disassembly and re-formation during cell division. Following breakdown of the nuclear envelope in late prophase most nuclear antigens become diffusely distributed throughout the entire cell. Lamin proteins are detectable in partial association with condensed chromosomes up to early metaphase, but no longer in anaphase. Nucleolar proteins are the first antigens to re-associate with chromosomes in late anaphase. Concomitant with the onset of envelope re-formation in telophase, nucleoplasmic antigens begin to accumulate in the forming daughter nuclei and completely disappear from the cytoplasm within an impressively short time. Undoubtedly most interesting is the behaviour of those antigens which are located within a characteristic substructure of interphase nuclei. These antigens begin to display a ‘patchy’ distribution in anaphase, suggesting the formation of a relatively large supramolecular complex, e.g. a vesicular structure. This ‘patchy’ distribution persists in the cytoplasm of daughter cells for a considerable time into early interphase. The nature of the antigens showing this particular delayed type of re-entry and the reason for their temporary extranuclear sequestration are currently under study. (Supported by the Swiss NSF and an ETH doctoral grant to C.F.L).
Aggregation and movement of germ plasm, the cytoplasmic determinant for germ cells, in early embryos of Xenopus laevis
R. E. RessomandK. E. Dixon, School of Biological Sciences, The Flinders Unversity of South Australia, Bedford Park, 5042, Australia
Early embryos of X. laevis contain a microscopically visualisable region of cytoplasm which is segregated by cleavage divisions to a small number of cells (4 in a 5000-cell blastula) which are the progenitors of the gametes. Examination of sections of early embryos showed that the germ plasm can first be distinguished about 2 h after fertilisation as small islets in the subcortical region of the vegetal hemisphere. As development proceeds, the islets aggregate together forming an average 3-4 patches per embryo at 4 h postfertilisation (stage 6). The mechanisms controlling aggregation and movement of germ plasm were investigated in fertilised eggs and eggs artificially activated with calcium ionophore A23187. Three processes were distinguished: (i) aggregation of the islets into large masses, a process dependent on microtubules but independent of cleavage; (ii) internalisation of these aggregates as part of a general cytoplasmic flow from the vegetal pole region towards the interior; (iii) capture of the aggregates by cleavage asters. The result of the first of these processes is that the germ plasm is partitioned between the first four blastomeres. Movement internally permits capture of the germ plasm by the cleavage asters and in turn this immobilises the germ plasm and ensures that it is segregated unequally at each subsequent cleavage division.
Animal vegetal polarity and the sea urchin cortex
Christian Sardet*, ER 250 CNRS, Station Marine, F06230 Villefranche-sur-mer, France
Sea urchin eggs have animal and vegetal poles. Successive cleavages are oriented with respect to these poles and development proceeds according to this animal/vegetal polarity.
In the egg of the sea urchin Paracentrotus lividus, polarity is apparent in the cortex before fertilization as a subequatonal band of pigment granules. This structure is preserved as part of the isolated cortex. I will present evidence that similar vesicular bodies (acidic vesicles) are present in all sea urchin eggs and exhibit animal/vegetal polarity as well as apical/basal polarity in blastomeres.
I will describe other components that remain as parts of the isolated cortex and in particular an intricate network of rough endoplasmic reticulum, that is tightly anchored to the plasma membrane. I will discuss the functions of these components and their relationship to the animal/vegetal polarity.
Cytoplasmic control of cell-cycle timing in early embryos of Caenorhabditis elegans
Einhard Schierenberg*1and William B. Wood, Department of Molecular, Cellular and Developmental Biology, Boulder, Colorado, 80309, USA. 1Present address: Max-Planck-Institute for experimental Medicine, H. Rein-Str. 3, D-3400 Goettingen, West Germany
During early cleavage in the C. elegans embryo, a series of asymmetric divisions of the germ line cell generates five somatic founder cells. The cell lineages derived from each of these founder cells, including the fate of each cell are known (1). They are invariant. Lineage-specific differences in cell cycle timing appear to be important in establishing the correct spatial patterns of cells as indicated by mutants with altered cell cycle rhythms (2) and laser-induced retardations of cell cycle timing within specific cell fines (3). To examine the role of nuclei and cytoplasm in controlling cell cycle timing in C. elegans embryos, we have developed a technique for extruding either substantial amounts of cytoplasm without nuclei or nuclei with small amounts of cytoplasm from individual blastomeres. This allows us to observe the cycling behavior of blastomeres with altered nuclear/cytoplasmic ratios and of enucleated cytoplasts. By laser-induced fusion (4) of a cytoplast with an adjacent blastomere, we have been able to observe effects of exposing a nucleated cell from one lineage to cytoplasm of another, as well as effects of changing the nuclear/cytoplasmic ratio within the same lineage. We find that (a) nuclei in a common cytoplasm divide synchronously; (b) enucleated blastomeres retain a cycling period characteristic of their lineage; (c) cycling period is not substantially affected by changes in the ratio of nuclear to cytoplasmic volumes or the DNA content per cell; (d) the cycling period of a cell from one lineage can be substantially altered by introduction of cytoplasm from a cell of another lineage with a different period; and (e) short-term effects of foreign cytoplasm on the timing of the subsequent mitosis differ with time in the cell cycle of the donor cell. Based on our results we suggest a model for the action of cytoplasmic factors in controlling cell-cycle timing.
Cell culture of renal medullary, (thick ascending limb of Henle’s loop) cells
D. M. Scott, K. Zierold, E. Kinne-Saffran and R. Kinne, Max-Planck-Institut fuer systemphysiologie, 4600 Dortmund 1, West Germany
The nephron of the kidney is made up of a variety of epithelial cells that vary in their morphology, function and location. To date the majority of in vitro cell culture studies of kidney cell function have been carried out with established cell lines of unknown derivation. Consequently, in order to overcome the obvious limitations of such systems we have attempted to establish in culture cells of defined origin. Such homogeneous cell populations were derived from the thick ascending limb of Henle’s loop (TALK) of rabbit kidney and were obtained by initial collagenase/hyaluronidase digestion of medullary fragments, followed by sequential trypsin digestion and cell separation by density gradient centrifugation (Eveloff, J., Haase, W., and Kinne, R., (1980) J. Cell. Biol. 87, 672-681). These cells were then resuspended in Dulbecco’s Modified Essential Medium supplemented with 10 % foetal calf serum, non-essential amino acids, pyruvate and antibiotics, and were maintained at 37 °C in a 5 % CO2/95 % air atmosphere. Medium was initially changed at 24 h and subsequently every 48 h. Primary cultures exhibited typical epithelial morphology (as demonstrated by fight and electron microscopy) and expressed biochemical and functional characteristics of TALK cells. Cultures routinely showed high levels of Na-K-ATPase activity as would be expected of such cells and their respiration was markedly inhibited (ca. 60 %) by Furosemide (10-3M). This diuretic inhibits salt transport in TALH cells and has been shown to be a specific inhibitor of these cells (Eveloff et al., 1980). Only low levels of brush border enzymes such as alkaline phosphatase were observed. Na-K-ATPase activity was shown to increase in activity during the period in primary culture, although this activity was markedly reduced (by up to 85 %) by subculture of these cells, even when performed in the absence of proteases. These studies demonstrate the feasibility of the growth in culture of defined kidney epithelial cell populations and their subsequent detailed study should provide us with an important system for the study of the expression and regulation of TALH cell specific functions.
Enveloped viruses as tools to study cell surface polarity in epithelial cells
K. Simons* andK. Matlin, European Molecular Biology Làboratory, Postfach 10.2209, D-6900 Heidelberg, West Germany
Our present studies are directed towards elucidating the mechanisms of establishment of cell surface polarity in epithelial cells using enveloped viruses as tools. We are studying the Madin Darby Canine kidney (MDCK) cell line which grows in culture as an epithelium. In MDCK cells infected with influenza, the viral hemagglutinin behaves as an apical plasma membrane glycoprotein. To find out where the hemagglutinin was sorted, the domain of the plasma membrane, apical or basolateral, where newly synthesized hemagglutinin first appears was studied by a novel approach. Cells were cultured on Millipore filters to make cell surface domains independently accessible and infected with influenza virus. Hemagglutinin was pulse-labeled, chased and detected on the plasma membrane with a sensitive trypsin assay (Matlin, K. and K. Simons (1983) Cell 34, 233-243). Under all conditions tested, newly made hemagglutinin appeared in large excess on the apical domain, but there was always a fraction on the basolateral domain. Trypsin continously present on the basolateral surface during the chase failed to cleave an amount of hemagglutin equivalent to that transported apically. In addition specific antibodies against the hemagglutinin placed basolaterally had no effect on transport to the apical domain. These observations suggested that apically destined hemagglutinin did not transiently appear on the basolateral surface and that sorting, therefore, occurs intracellularly prior to arrival of the glycoprotein on the cell surface.
Lateral mobility of plasma membrane lipids during early molluscan development
J. E. Speksnijder, M. R. Dohmen, E. J. J. van Zoelen, L. G. J. Tertoolen, J. G. Bluemink and S. W. de Laat, Zoological Laboratory, University of Utrecht, Padualaan 8, 3508 TB Utrecht and the Hubrecht Laboratory, Uppsalalaan 8,3584 CT Utrecht, The Netherlands
The lateral diffusion of the lipid analog C14-dil (3’,3’-dihexadecylindocarbocyanine iodide) was measured in the plasma membrane of early embryos of the mollusc Nassarius reticulatus using the FPR-(Fluorescence Photobleaching Recovery) method. At almost all stages measured (from fertilized egg up to 8-cell stage) the diffusion coefficient (D) of the mobile fraction (MF) of Ci4-dil is significantly higher in the plasma membrane of the vegetal pole area as compared to the plasma membrane of the animal half of the embryo. These results demonstrate the presence of an animal/vegetal polarity in the plasma membrane of the embryo of Nassarius, possibly related with the polar localization of morphogenetic factors.
The lateral diffusion of the lipid probe in the plasma membrane of the vegetal pole area shows a cell cycle-dependent modulation; the highest mean values for D are reached during S-phase (ranging from 7-0 to 7-8xl0-^ cm2/sec in the three cycles measured), while at the end of G2-phase mean values for D have decreased to a range of (5-0-5-9)xl0”9 cm2/sec. Diffusion rates in the animal membrane of the embryo are rather constant (D ranging from 4-4 to 5-0xl0∼9 cm2/sec), except for a peak during the S-phase of the first cycle (D = 6-0 xlO-9 cm’vsec).
At third cleavage, four small micromeres are formed at the animal pole. Both D and MF decrease considerably in these cells (D = 2-9 ± 0-2X10-9 cm2/sec, MF = 0-51 ± 0.02) as compared to the corresponding macromeres (D = 4-9 ± 0-3X10-9 cm2/sec, MF = 0-78 ± 0-02). The significance of this sudden change in lateral diffusion of membrane lipids in the micromeres is unknown.
Control of morphological and physiological cell asymmetries in early chick development
Claudio D. Stern*, Dept. Anatomy & Embryology, University College London, Gower Street, London WC1E6BT
Using a combination of ultrastructural, histochemical and physiological techniques it was shown that the epiblast of the chick embryo at primitive streak stages has defined dorso-ventral polarity. This polarity manifests itself in the position of nuclei, intercellular junctions, microfilament bundles and the basal lamina associated with the cells. Physiologically, cells are capable of uni-directional sodium and fluid transport, and the sodium pumps (NaK-ATPase) are localized towards the basal surfaces of the tissue. As a result of uni-directional sodium transport, the epiblast maintains a potential across itself amounting to about 20 mV (ventral side positive).
Experimental application of a potential of similar magnitude but reverse polarity to that measured (about 25 mV, dorsal side positive) leads to rapid and stable reversal of all the morphological and physiological markers studied. This is not due to simple electrophoresis of these markers across the tissue.
Based on these results, a simple model will be outlined to explain the formation and maintenance of the embryonic axis, the main factors are postulated to be sodium, calcium and fluid homeostasis by the epiblast and interactions between the forming mesoderm layer and the overlying epiblast.
Compartmentation of the nucleotides pool in eggs of the polar lobe forming mollusc Nassarius reticulatus
C. A. M. van Dongen, H. Goedemans, J. Wes, Department of Experimental Embryology, Zoological Laboratory, State University of Utrecht, Padualaan 8,3508 TB Utrecht, The Netherlands
Early determinative events in differentiation processes during development are controlled by extranuclear maternally derived morphogenetic determinants. These as a rule are localized in particular regions of the egg and are parcelled out to particular cell lines during cleavage. In molluscs, in particular the vegetal region of the egg is an important morphogenetic compartment. In polar lobe forming species, e.g. Nassarius, this compartment is temporarily set apart during fist cleavage in the form of a cytoplasmic protuberance, the so-called polar lobe, which can be removed from the egg either by surgical or cnemical methods. In lobeless embryos, differentiation in a number of specific cell lines follows an aberrant, although as such highly typical, course. The effects of polar lobe removal are strictly reproducible and at present are well defined. The restricted pattern of developmental déficiences in lobeless embryos is analogous to the phenotypic expressions of a maternal effect genetic mutant. Polar lobe forming species hence are an excellent model for studying maternal control of early developmental processes. In an attempt to characterize the molecular contents of the lobe compartment, we have analyzed the low molecular weight constituents by means of capillary isotachophoresis (an ultrasensitive micromethod). We have found, that the lobe is highly enriched in its nucleotides contents, and that its nucleotides spectrum is charcterized by several prominent compounds in particular (e.g. GTP).
Pole cell formation in Drosophila melanogaster
R. M. Warn*, L. Smith and A. Warn, School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ
The mode of pole cell formation was studied in whole mounts stained with rhodaminyl-lysine-phallotoxin (RLP), a specific stain for F-actin, and the nuclear fluorescent stain 4-6 diamidine-2-phenylindole dihydrochloride (DAPI). Polar surface caps were found to emerge first after the eighth cleavage, one cycle ahead of the somatic caps which form elsewhere, and to divide twice before pole cells formed. The caps, syncytial surface protrusions, had an F-actin rich cortex, distinct from that surrounding them and an interior somewhat less brightly stained. The first division cycle of the polar caps was similar to that previously described for the somatic caps (Warn et al. 1984). Caps were found to swell out and then flatten whilst continuing to expand. They then split and new caps bubbled out from both ends. Prior to and during splitting the region of cleavage did not show a distinctive contractile ring structure but became depleted in F-actin as compared with the forming daughter caps. The second division cycle of the polar caps followed the pattern of the first until the last stages of mitosis, when a distinctive hoop of RLP staining formed in the centre of each cap. This moved down with the plasmalemmas. ‘Haloes’ of RLP staining were then found at the bases of each forming cell. These became smaller and disappeared. Possible mechanisms of cleavage are discussed as is the role of cytoplasmic determinants in controlling the organization of regionally localized F-actin microfilament structures.