migration and division of cleavage nuclei in the gall midge,wachtliella persicariae

Wilhelm Roux's Archives 188, 65-73 (1980) Roux's Archives of Developmental Biology �9 by Springer-Verlag 1980

Migration and Division of Cleavage Nuclei in the Gall Midge, Wachtliella persicariae II. Origin and Ultrastructure of the Migration Cytaster

Rainer Wolf Zoologisches Institut der Universit~t, D-8700 Wtirzburg, Federal Republic of Germany

Summary. In the eggs of Wachtliella persicariae the cleavage nuclei move relative to the surrounding ooplasm. This 'active' migration is caused by an organelle whose ultrastructure was studied throughout the mitotic cycle. It consists of a greatly enlarged polar cytaster derived from the mitotic apparatus, linked to the nucleus by 100 ~ filaments. The microtubules of the cytaster were found only during periods of active nuclear migration, i.e., from the onset of anaphase to the early prophase of the next mitotic cycle. They are always solitary and follow the course of the astral rays, which are known to temporarily adhere to peripheral structures of the egg cell and to exert tractive forces. In contrast to the cytaster microtubules, the microtubules in the spindle are bun- dled and persist from early metaphase through late telophase.

During ontogenesis the first migration cytaster is built up between 3 and 12 rain after oviposition near the anterior egg pole, in the vicinity of the sperm nucleus. In non-inseminated eggs time lapse films show a migration cytaster to develop autonomously in a region free from nuclei, but it does not follow the normal path of the male pronucleus. In several cases the female pronucleus, which remains without a cytaster of its own, was observed to move to the cytaster generated in the absence of the male pronucleus. Whether or not it is adhering to a nucleus, the cytaster divides into two at the correct time, i.e., corresponding to the first cleavage division in fertilized eggs. In some non-inseminated eggs this type of 'pseudocleavage' has been observed to occur repeatedly, giving rise to an increasing number of anuc- leate cytasters.

Key words: Nuclear migration - Cleavage Microtu- bules - Ultrastructure - Gall midge.

Introduction

The distribution of the dividing energids (cleavage nuclei and associated cytoplasmic islands) within the ooplasmodium represents the intrinsic dynamic process of the early cleavage in insect eggs (Sander 1976). In the oblong and thin egg of Wachtliella, two inde- pendent modes of energid movement have been established: (1) a 'passive' shifting by ooplasmic flows which are sensitive to cytochalasin B, and (2) an 'active' migration, which enables the energids to move relative to the surrounding plasm by means of colchicine sensitive cytasters (Wolf 1973, 1977). Each cleavage nucleus is linked to one of these special migration organelles which are derived from the mitotic apparatus. As indicated by experimental time lapse analysis, the astral rays temporarily adhere to peripheral structures of the egg cell and exert tractive forces. These result in an extensive polarized transport of yolk particles towards the cytaster centre and create a large pigmented halo around each migrating nucleus. According to the model previously described (Wolf 1977, 1978; see Fig. 8b) the tractive forces of different astral rays combine and pull the nucleus through the ooplasm after each mitosis. Electron microscope studies indicate that the forces must be generated along solitary microtubules within the astral rays (Wolf 1978).

This model implies that there is an affinity between each daughter nucleus and its cytaster, as was experimentally shown in sea urchin eggs (Aronson 1971). The present investigation examines the mor- phological substrate by which the nucleus is linked to its cytaster. It also analyzes how the migration organelle is rebuilt during the cell cycle, when the first migration cytaster is set up during ontogenesis, and whether it is brought by the sperm.

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66 R. Wolf: Migration Cytaster of Cleavage Nuclei

Methods

For electron microscopic investigations, inseminated eggs from Wachtliella persicariae L. (for cultivation technique see Wolf 1973) were individually transferred into buffered glutaraldehyde solution (23 ~ C) and pricked with a glass needle at distinct time points before the fusion of the two pronuclei (3, 12, 19, or 30 rain after oviposition), and at various stages of the second and third cleavage mitoses. The eggs were postfixed with OsO~ after 30 min, dehy- drated, and embedded in Vestopal W or in Spurr's medium. In each test group, longitudinal sections of 5 7 eggs were made, post- stained with lead citrate (Reynolds 1963), and analyzed with the Siemens Elmis.kop IA or the EM 10 from Zeiss.

Time lapse films of eggs deposited by mated or by virginal females were made and analyzed according to Wolf (1973); for a better visualization of the development some of the eggs were slightly flattened.

Results and Discussion

Development of the First Migration Cytaster Before Fertilization

Three Minutes After Oviposition. The sperm nucleus is seen near the anterior pole just below the surface of the egg cell. The micropyle, where the sperm enters the egg, is characterized by a thin region of the vitelline membrane (Fig. l a). In ultrathin sections the sperm nucleus appears as a ring shaped electron dense mass of mean diameter 6 gin. A nuclear membrane cannot be detected (Fig. lb) as is usual in this stage (Longo 1973) and yolk particles and endoplasmic membranes, i.e., parts of the ooplasm, are surrounded by the male nuclear structures.

Twelve Minutes After Oviposition. The male pronucleus derived from the sperm nucleus has penetrated the ooplasm to a depth of 35-40 gin. Yolk particles and lipid droplets have been moved aside as indicated by a broad track of clear ooplasm left behind the nucleus (Fig. 2). At 12 and 19 min after oviposition, the pronucleus is oval shaped with a maximum diameter of about 3 pro. There are no ooplasmic inclusions and it is surrounded by a compact envelope, which lacks the common two layers and is locally interrupted (Fig. 3 a, arrowheads; Fig. 7, inset a).

During these early stages the first migration cytaster is already visible. Solitary microtubules are found surrounding the male pronucleus (Fig. 3, arrows) which are commonly surrounded by a clear exclusion zone (Dustin 1978). They are aligned in the directions of the astral rays deduced from the saltatory motions of yolk particles observed in time lapse films (Wolf 1977).

The first migration cytaster develops long before pronuclear fusion takes place. However, no cytaster was found in the newly laid egg. This suggests that cytaster formation might be triggered by the sperm itself. To investigate the origin of the cytaster, time lapse films of non-inseminated eggs of Wachtliella were analyzed. Quite unexpectedly for eggs from a strictly nonparthenogenetic species, a cytaster was seen to develop within the central ooplasm at about 100 gm from the anterior pole. This was indicated by the radially orientated saltations of yolk particles, which accumulate and form a pigmented halo around the cytaster centre. In 4 eggs out of 42, the female pronucleus was seen to move to the cytaster formed in the absence of the male pronucleus. In all the 42 eggs filmed, the cytaster divided into two at about the time when the first cleavage mitosis would occur in fertilized eggs. In 7 eggs 'pseudocteavage' continued for 2-3 further cycles, each lasting 25-30 rain (cleavage cycles in fertilized eggs last 20 rain), but not all the cytasters divided in each cycle. As a result up to 12 cytasters were observed, all apparently devoid of nuclei.

Thirty Minutes After Oviposition. After completion of meiosis, the female pronucleus lies close to the middle of the egg at a considerable distance from the male pronucleus. Each nucleus is now surrounded by a typical double layered envelope with nucleo- pores. From time lapse films it has previously been concluded that an astral ray radiating from the migration organelle of the male pronucleus contacts and attracts the female pronucleus (Wolf 1973, 1977). This process was seen in time lapse films only and is so fast (less than 3 rain) that we failed to obtain histolog- ical sections of it in spite of extensive efforts.

Fig. 1. a Micropyle region where the chorion (Ch) is lifted off the thinned out vitelline membrane (V, arrows). The sperm nucleus is cut tangentially, shortly after entering the egg. 3 rain after oviposition; 8,800x. b Cross section of the same nucleus as in a with ring shaped karyoplasm enclosing protein yolk (I1) and clear ooplasm. L: lipid droplet; 8,800 x

Fig. 2. Male pronucleus on its way to the right into the interior of the egg cell. It leaves behind a broad track of clear ooplasm which is lined by protein yolk and lipid droplets (characterized in the section by their typical chatter structure). Twelve minutes after oviposition; 5,200 x

Fig. 3. Same stage as in Fig. 2. Serial transverse sections show that the nuclear envelope is compact but locally interrupted (arrowheads). Solitary microtubules (arrows) point towards the cytaster centre (C; cf. Figs. 8b and 11). The movement of the pronucleus is directed towards the right margin. 23,600 x, inset 57,000 x

R. Wolf: Migration Cytaster of Cleavage Nuclei 67

Fig. 4. Late prophase of the third cleavage mitosis, 5 min before anaphase. The nucleus with its condensing chromosomes (Chr) is surrounded by the characteristic multilayered envelope (M) connected with the endoplasmic reticulum (ER) ; 6,600 x

Fig. 5. a Metaphase nucleus of the third cleavage mitosis, 2 min before anaphase, with a multilayered envelope (M) in its polar regions. The centre (C) of the developing cytaster is free from yolk particles (11); 11,500x. b Two daughter chromosomes with bundles of microtubules attached to the electron dense kinetochores (K) ; 44,000 x


The small number o f microtubules present in the sections is p robably no t caused by the fixation, because large numbers were apparent in the mitotic appara tus o f the cleavage energids. But small numbers of microtubules should be sufficient to account for the saltatory mot ions observed (and consequently for the migrat ion process), because only a few yolk particles are involved simultaneously. This corresponds to earlier observations that the astral rays repeatedly adhere to yolk particles and peripheral structures for only 10 40 s (Wolf 1973). These short times are probably the reason why no direct contacts between microtubules and yolk particles have been demonst ra ted in sections.

The process o f pseudocleavage in non- inseminated eggs indicates that in the anterior egg pole there is the potency to generate a single cytaster which is able to divide autonomously . In inseminated eggs this cytaster is seen to adhere to the male pronucleus, whereas the female pronucleus remains without a cytaster o f its own (Wolf 1973). This agrees with the finding of Zissler and Sander (1973) that a single large cytaster is found near the anterior egg pole of the par thenogenet ic midge Smittia.

Migration Cytasters and Cell Cycle

The ul trastructure of the energids and their cytasters was analyzed during the second and third cell cycle, i.e., when the daughter energids migrate over the larg- est distances observed (up to 130 gm in 5 rain). In late prophase, 5 min before the onset o f anaphase, the preceding nuclear migrat ion has come to an end and no microtubules can be detected a round the nu-

clei. Large amounts of membraneous vesicular mate- rial are assembled as a multi layered nuclear envelope which is connected to the endoplasmic ret iculum (Figs. 4 and 7, inset c). This type of nuclear envelope is found up to and including late anaphase. Inside the nucleus the ch romosomes begin to condense.

Dur ing metaphase numerous spindle microtubules are a t tached to the kinetochores o f the highly con- densed chromosomes (Fig. 5a and b). Similar large bundles were found only in the interzonal region during anaphase and telophase. However , in the region of the spindle poles no microtubules could be detected.

In the filmed eggs radially orientated sal tatory mot ions o f yolk particles were seen to begin simultaneously with the onset o f anaphase. They indicate the activation o f the cytasters (Wolf 1973). Ultrathin sections reveal that in the region o f the cytasters the electron dense yolk particles, mi tochondr ia and endoplasmic membranes are lined up radially, thus indi- cating the course of the astral rays. All these configu- rations are orientated towards the cytaster centre (Fig. 6; for compar i son see filmed energid on the left in Fig. 8b) which consists o f an electron dense mass similar to the centriole-equivalent structures as described in other cells (Fuge 1977). Solitary microtubules running in the directions of the astral rays are comparat ively rare, just as in the migrat ing postmitotic energids (Fig. 11).

In the early telophase, 2 min after metaphase, the chromosomes are already decondensed (Fig. 7). In Wachtliella no karyomeres are segregated, in contrast to the kelp fly (Schwalm and Bender 1973). The multilayered nuclear envelope is replaced by the typical

Fig. 6. Late anaphase of the second cleavage mitosis, 1 min after metaphase. The chromosomes (Chr) are close to the poles, where the nuclear envelope is highly multilayered (M, cf. Fig. 7, inset c) as compared to the equatorial region. C: cytaster centre, F: Flemming body ; 5,700 x

Fig. 7. Telophase of the second cleavage mitosis, 2 min after metaphase. The double layered envelope of the nucleus (N) is still open on the side facing the former equatorial region; 9,400x. Insets'." nuclear membranes in early development, 33,000• a Early sperm nucleus; local interruption (arrowhead). b Double layered membrane with pores (arrowheads), interphase, c Multilayered membrane, prophase

Fig. 8a and b. Early postmitotic daughter nuclei and their cytasters fixed during active migration, 3 min after metaphase of the third cleavage mitosis, a 2,300 x ; b frame copy from a time lapse film of a partially flattened living egg, 6 min after metaphase; interference contrast, 1,320 • On the right energid the migration cytaster is superimposed according to our model: The astral rays point to the cytaster centre (C) which is connected to the nucleus by microfilaments (dotted lines, cf. Figs. 10 and 11)

Fig. 9. Early prophase of the third cleavage mitosis, 8 rain after the previous metaphase. During the migration the nucleus has characteristic indentations on the side facing the cytaster (centre, C); 8,600 x

Fig. 10. 10nm filaments (arrows) between the nucleus and its cytaster centre (C; cf. Fig. 9). 4 nuclei stage, 8 min after metaphase; 20,900 x

Fig. 11. Postmitotic nucleus (N) fixed in active migration 6 min after metaphase; oblique section of the nuclear membrane. All m~icrotubules in the section (each marked by a parallel black line) point to the cytaster centre (arrow) which contains a centriole-like body of 9 fold symmetry (lower inset). In the upper inset its symmetric structure was intensified by 9 fold superposition of the projected image, each time rotated by 40~ 14,300 x, insets 62,000 x


Figs. 6-8. (for legends see p. 69)


Figs. 9-11. (for legends see p. 69)


double layered structure (Fig. 7, inset b) which, as seen from serial sections, is completed last at the side facing the former mitotic equator (Fig. 7, cf. Fig. 8 a). The daughter nuclei round up and move from one another throughout interphase (Fig. 8 a, b). The nuclear envelope is slightly indented towards the cytaster (Figs. 8, 9). Time lapse and electron microscopic pic- tures show that the distances between the nuclei and their cytaster centres can vary. They measure 2-3 gm during metaphase and anaphase, increase to 10 gm during high migration activity, and are reduced to 3 ~tm again in the early prophase of the next mitotic cycle, shortly before the cytaster disappears.

Special attention was given to the structures of the cytaster centre and to the connection of this centre with its nucleus. No microtubules were found there; however, microfilaments with diameters of about 10 nm run from the cytaster centre to the close vicinity of the nuclear envelope (Figs. 9, 10). Two methods were employed to further characterize the microfila- mentous and microtubular structures of migrating energids but gave inconclusive results. Treatment of telophase eggs with glycerin (6 days at - 8 ~ C; Ka- miya and Kuroda 1965) followed by treatment with ATP (3 gM, with 26 mM KC1 and 3 mM MgC12 in 10 mM Tris-buffer pH 7,1) before fixation led to a complete extraction of the region between the cytaster centre and the nucleus. Indirect immunofluorescence (Osborn et al. 1978) using monospecific antibodies (anti actin or anti tubulin) resulted in a homogeneous labelling of the whole ooplasm probably due to an unspecific binding of FITC.

In two eggs a centriole like body could be detected in the cytaster centre. It shows a 9 fold symmetry which can be intensified using the method of Mark- ham et al. (1963 ; Fig. 11, arrow and insets). However, a clear microtubular substructure was not seen. All the microtu.bules were apparently running radially towards this body: To our knowledge this is the first case of centrioles found in gall midges.

It was shown previously that in eggs injected with 30 pl of 250 gM colchicine during early telophase and fixed 10 min later, no microtubules can be found around the nuclei, even though they are present in untreated eggs. However, the ooplasmic flows remain unaffected by this drug (Wolf 1978). The central and marginal ooplasmic layers continue to oscillate in op- posite directions. Recently, the following supplemen- tary observations were made. I f a cleavage nucleus accidently gets between the two ooplasmic layers, the yolk particles surrounding it are gradually carried away, and the pigmented halo is seen to dissolve. Therefore, it can be assumed that in untreated eggs the yolk particles are held in place by the repeated insertions of cytaster microtubules.

Cytochalasin B was shown to specifically inhibit the passive shifting of nuclei by ooplasmic flows, but it does not impair the active nuclear migration (Wolf 1978). Consequently, the connecting link postulated between a cleavage nucleus and its cytaster must be unaffected by this drug. This agrees with findings that cytochalasin B preferably impairs intracellular movements caused by microfilaments which contact cell membranes, but it does not disintegrate the f-actin in the cytoplasm (Goldman et al. 1973 ; Wessells et al. 1971; Sanger and Sanger 1976), which is supposed to serve as the linking structure in Wachtliella. In contrast to this, another mode of energid migration, caused by the 'radial system' in dragon flies which is reportedly based on contractile plasmic filaments adhering to the egg surface (Hujer 1975), should be expected to be sensitive to cytochalasin B.

The present results are consistent with the structure of the nuclear migration cytaster proposed above and shown on the right hand side of Fig. 8 b. Further evidence that microtubules are involved in the func- tion of this organelle has been summarized elsewhere (Wolf 1978). The ultrastructural findings indicate that the microtubules of the cytaster appear only during the periods of active nuclear migration. In late prophase and metaphase, when no active migration oc- curs, no microtubules other than spindle microtubules could be detected. The mode of generation of the tractive forces established along solitary microtubules, however, remains an open question as it does for chromosome movement.

Acknowledgements. These investigations were supported by the Deutsche Forschungsgemeinschaft. I wish to thank Dr. B.M. Jockusch for kindly providirtg me with the monospecific antibodies, and Dorothea Wolf for excellent technical assistance.

References

Aronson JF (197i) Demonstration of a colcemid-sensitive attrac- tive force between the nucleus and a center. J Cell Biol 51 : 579- 583

Dustin P (1978) Microtubules. Springer, Berlin Heidelberg New York

Fuge H (1977) Ultrastructure of the mitotic spindle. Int Rev Cytol (Suppl) 6 : 1-52

Goldman RD, Berg G, Bushnell A, Chang ChM, Dickerman L, Hopkins N, Miller ML, Pollak R, Wang E (1973) Fibrillar systems in cell motility. In: Locomotion of tissue cells. Ciba Found Symp 14:83 107, Associated Scientific Publishers, American Elsevier, Amsterdam

Hujer H (1975) Dokumentationen zur Funktionsstruktur der Ener- giden im EntwicklungsprozeB der Eier der Libellen Platycnemis nnd Ischnura. Verh Dtsch Zool Ges 1974:178 183

Kamiya N, Kuroda K (1965) Movement of the myxomycete plas- modium. I. A study of glycerinated models. Proc Jpn Acad 41:837 841


Longo FJ (1973) Fertilization: A comparative ultrastructural re- view. Biol Reprod 9:149-215

Markham R, Frey S, Hills GJ (1963) Methods of the enhancement of image detail and accentuation of structure in electron microscopy. Virology 20 : 88-102

Osborn M, Webster RE, Weber K (1978) Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell. J Cell Biol 77:R27 R34

Reynolds ES (1963) The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J Cell Biol 17 : 208- 211

Sander K (1976) Morphogenetic movements in insect embryogene- sis. In: Lawrence PA (ed) Insect development. BlackwelI Scien- tific PuN, Oxford London Edinburgh Melbourne, pp 35-52

Sanger JW, Sanger JM (1976) Actin localization during cell division. In : Goldman R, Pollard T, Rosenbanm J (eds) Cell motility. Cold Spring Harbor Conf on cell proliferation, Vol. 3. Cold Spring Harbor, New York, pp 1295-1316

Schwalm FE, Bender HA (1973) Early development of the kelp fly, Coelopafrigida (Diptera). lI. Morphology of cleavage and blastoderm formation. J Morphol 141 : 325-356

Wessells NK, Spooner BS, Ash JF, Bradley MO, Luduena MA, Taylor EL, Wrenn JT, Yamada KM (1971) Microfilaments in cellular and developmental processes. Science 171:135-143

Wolf R (1973) Kausalmechanismen der Kernbewegung und -tei- lung w~ihrend der frfihen Furchung im Eider Gallm/icke Wacht- liella persicariae L. I. Kinematische Darstellung des ,Migra- tionsasters ~ wandernder Energiden und Steuerung seiner Aktivi- tat durch den Initialbereich der Furchung. Wilhelm Roux' Arch Entwicklungsmech Org 172 : 28-57

Wolf R (1977) Embryonic development of the gall midge Wacht- liella. Sound film D 1235 (Inst f Wiss Film, G6ttingen/FRG); accompanying text: Publ Wiss Film, Sekt Biol, Ser 10 Nr 34/D 1235:3--24

Wolf R (1978) The cytaster, a colchicine-sensitive migration organelle of cleavage nuclei in an insect egg. Dev Biol 62:464-472

Zissler D, Sander K (1973) The cytoplasmic architecture of the egg cell of Smitlia spec. (Diptera, Chironomidae). I. Anterior and posterior pole regions. Wilhelm Roux' Arch Entwick- lungsmech Org 172:175 186

Received May 19, 1979 ; Accepted in revised form January 15, 1980

migration and division of cleavage nuclei in the gall midge,wachtliella persicariae

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