Rainer Wolf

The cytaster, a colchicine-sensitive migration organelle of cleavage nuclei in an insect egg☆

Abstract Time-lapse analyses of nuclear multiplication in the eggs of the gall midge Wachtliella persicariae L., documented in film D 1235 (available from the IWF, Gottingen), give evidence of a special migration organelle of cleavage nuclei. Each of these “migration cytasters” represents one greatly enlarged polar cytaster of the mitotic apparatus, which is connected to one nucleus. From the films it can be concluded that the astral rays temporarily adhere to peripheral egg structures and exert tractive forces toward the cytaster center. These forces combine and pull the accompanying daughter nucleus through the ooplasm after each mitosis. This “active” mode of migration, which is accompanied by extensive polarized transport of yolk particles toward the cytaster center, enables the energids (= cleavage nucleus and its associated island of cytoplasm) to move relative to the surrounding ooplasm. In addition, there is a “passive” mode of nuclear migration: The energids are moved by means of plasmic flows, thereby maintaining their position in relation to the surrounding ooplasm. Electron microscopic studies show solitary microtubules running radially toward the cytaster center. As a result of colchicine injection (1) the microtubules disintegrate, (2) the polarized transport of yolk particles cases, (3) the active nuclear migration stops and the nuclei are only passively moved by rhythmic ooplasmic flows. This inhibition of active nuclear migration gives further evidence that microtubules take an essential part in it. Control experiments with lumicolchicine show no effect on nuclear migration. Conversely, under the influence of cytochalasin B active nuclear migration is continued, while the ooplasmic flows are inhibited. Thus the mechanisms of active and passive nuclear migration can work independently of each other. The generation of tractive forces along the astral rays is discussed with respect to current models of spindle function.

Die Ooplasmabewegungen während der Furchung vonPimpla turionellae L. (Hymenoptera), eine Zeitrafferfilmanalyse

SummaryIn the eggs ofPimpla turionellae, which are characterized by a long germ anlage (“long-germ egg” type), the cleavage nuclei primarily populate the anterior part and only later appear in the posterior of the egg lumen during the intravitelline cleavage. Gastrulation and segmentation also start within this anterior region. Time-lapse motion pictures served to observe and to check quantitatively even slow movements during cleavage and blastogenesis. In motion diagrams made by means of microkymographic technics the flow within the ooplasm along the longer axis of the egg has been timed.Shortly before the first cleavage in thestrictly unfertilized male eggs a short-time“unipolar flow” sets in from a primary initial region at 90% of their length. Thus a pillar of “central plasm” between both of the poles becomes shifted towards the posterior, while its outer coating layer of “marginal-plasm” is displaced forwards by the same distance. In eggs from fertilized females two successive flows of the same “unipolar” type have been observed.At the end of the third cleavage the energids, heretofore loosely grouped together, become distributed within the central plasm to form a “nuclear column”. At the same time a fluently pulsatory “bipolar flow” sets in, within asecondary initial region at 80% of the egg length. Comparable to two mirror-image fountains, parts of the central plasm are carried towards the front pole and to the rear pole of the egg, respectively, while the marginal plasm, together with the oolemma, flows in opposite directions at times. With each pulsation the moving areas of the bipolar flow are shifted more and more towards the egg poles. The occurrence of bipolar flow pulsations, amounting to five, is correlated with the nuclear divisions in a still unknown way. In the rhythm of the bipolar flow, the energids become dispersed within the central plasm with a certain spatial lagging.After the bipolar flow has come to a halt, four further cleavages are indicated by faint local pulsations of the ooplasm. The cleavage nuclei move to the egg surface and pole cells become separatedtied off During blastoderm formation another four faint pulsations are observed, especially within the central ooplasm, all of them clearly synchronized with superficial cleavages. Occurring in mitotic waves, these cleavages indicate a third initial region, with the individual position varying between 10 and 28% of the egg length.Furthermore the technics of time-lapse motion pictures permit a local and temporal determination of extravitelline pole space formation, of a ring-shaped contracted region of slightly thickening periplasm within the secondary initial region, and the dislocation of the oosome towards the egg surface, which results from the activity of the posterior fountain during the phase of bipolar flow. Invagination and segmentation of the embryo become distinct within the secondary initial region, thus identifying this region as a differentiation centre.The correlation of plasm flow and nuclear divisions is discussed as well as the correlation of the initial regions to the different patterns of egg architecture in the longgerm egg type. The correlation between bipolar pulsations and the development of the metameric pattern including the function of the oosomal region is also discussed. The ooplasmic movements as known from egg types other thanPimpla are compared to the above observations.

Kinematik und Feinstruktur plasmatischer Faktorenbereiche des Eies vonWachtliella persicariae L. (Diptera)

The experimental results ofGEYER-DUSZYNSKA (1959), speaking in favour of three ooplasmic factors localized in the pole plasm, in the basophilic oosome material contained therein, as well as in the periplasm of the posterior egg pole ofWachtliella persicariae, suggested to investigate for further factor regions with other technical means. Since ooplasmic factor regions may be indicated by initial regions of morphogenetic development, kinematics were used for in vivo analysis of early embryonic development by means of time-lapse motion pictures. Electron microscopic investigations added to the micro-morphological aspects of plasmic systems within the egg for a better understanding of nature and effectivity of ooplasmic factors.Cleavage nuclei do not move exclusively by means of their spindle activity during anaphase movement. The nuclear envelope of cleavage energides consists of either two unit membranes with pores or of many tube-like as well as membranous elements. The appearence of a complex multi-layered nuclear envelope coincides with the moving phase of energides, an observation which is discussed in relation to the possibility of active nuclear movement. During late preblastoderm the entoplasm contains horse-shoe-shaped and multilobed vitellophagues with dense karyoplasm. With the blastoderm formed, the nuclei may become pycnotic, their membranes fragmenting at the same time. These fragments probably are piled up to form annulated membranes.The pole plasm does not show specific structures apart from the oosome material, contained therein. It is free of yolk material and nearly exclusively consists of ground plasm. The basophilic oosome material within the pole plasm is not surrounded by any membranes. It consists of numerous ribosome-like units and is restricted to the plasm of the future pole cells. The micro structure of the oosome material is preserved at least till the germ band has reached its maximal length.The cell membranes develop by invagination of the oolemma which penetrates into the egg interior. While pole cells and blastoderm cells become tied off, the ground plasm possibly participates in the growing-in process of the cell membranes by developing fibrous differentiations at the terminal extensions of oolemma folds.There is no clear cut limitation between periplasm and entoplasm. The periplasm which is without yolk material, appears rich in ground plasm and does not contain specific ultra structures. During the process of cleavage external ooplasmic regions of the egg are shifted in rhythmical pulsation parallel to its longer axis by a maximum of about 6 % of the entire egg length. Topographic statements of certain areas concerning any anlage therefore are bound to suffer from an adequate lack of exactness. Since comparable shifting processes within the egg plasm probably are common in insects other thanWachtliella, they should be considered as a certain source of error.At 60+-3 % of egg length as measured from its posterior pole, there exists a cleavage centre, an initial region of intravitelline cleavage and of repeated mitotic waves. Adjacent to the middle of the egg follows an initial region of germ band formation (differentiation centre). By their electron microscopic appearance, both developmental centres are not characterized by specific ultra structures. The factor region at the posterior pole exclusively represents an initial region of cell wall formation during superficial cleavage.Other than any experimental marking procedure the technique of time-lapse motion pictures permits to evaluate quantitatively and without artificial interfering the shifting of presumptive segment material during morphogenetic movements of the germ band. The embryonic material of the blastoderm at the egg equator is used for building up the first abdominal segment. The prothoracal and mesothoracal material at about 60% of the egg length stays in site when the germ band becomes extended lengthwise. Closely behind the differentiation centre there is a region of maximal extension as well as of shortening of the germ band. No proliferous growth of segments (segment formation zone) has been found.SummaryThe experimental results ofGeyer-Duszynska (1959), speaking in favour of three ooplasmic factors localized in the pole plasm, in the basophilic oosome material contained therein, as well as in the periplasm of the posterior egg pole ofWachtliella persicariae, suggested to investigate for further factor regions with other technical means. Since ooplasmic factor regions may be indicated by initial regions of morphogenetic development, kinematics were used for in vivo analysis of early embryonic development by means of time-lapse motion pictures. Electron microscopic investigations added to the micro-morphological aspects of plasmic systems within the egg for a better understanding of nature and effectivity of ooplasmic factors.Cleavage nuclei do not move exclusively by means of their spindle activity during anaphase movement. The nuclear envelope of cleavage energides consists of either two unit membranes with pores or of many tube-like as well as membranous elements. The appearence of a complex multi-layered nuclear envelope coincides with the moving phase of energides, an observation which is discussed in relation to the possibility of active nuclear movement. During late preblastoderm the entoplasm contains horse-shoe-shaped and multilobed vitellophagues with dense karyoplasm. With the blastoderm formed, the nuclei may become pycnotic, their membranes fragmenting at the same time. These fragments probably are piled up to form annulated membranes.The pole plasm does not show specific structures apart from the oosome material, contained therein. It is free of yolk material and nearly exclusively consists of ground plasm. The basophilic oosome material within the pole plasm is not surrounded by any membranes. It consists of numerous ribosome-like units and is restricted to the plasm of the future pole cells. The micro structure of the oosome material is preserved at least till the germ band has reached its maximal length.The cell membranes develop by invagination of the oolemma which penetrates into the egg interior. While pole cells and blastoderm cells become tied off, the ground plasm possibly participates in the growing-in process of the cell membranes by developing fibrous differentiations at the terminal extensions of oolemma folds.There is no clear cut limitation between periplasm and entoplasm. The periplasm which is without yolk material, appears rich in ground plasm and does not contain specific ultra structures. During the process of cleavage external ooplasmic regions of the egg are shifted in rhythmical pulsation parallel to its longer axis by a maximum of about 6 % of the entire egg length. Topographic statements of certain areas concerning any anlage therefore are bound to suffer from an adequate lack of exactness. Since comparable shifting processes within the egg plasm probably are common in insects other thanWachtliella, they should be considered as a certain source of error.At 60+−3 % of egg length as measured from its posterior pole, there exists a cleavage centre, an initial region of intravitelline cleavage and of repeated mitotic waves. Adjacent to the middle of the egg follows an initial region of germ band formation (differentiation centre). By their electron microscopic appearance, both developmental centres are not characterized by specific ultra structures. The factor region at the posterior pole exclusively represents an initial region of cell wall formation during superficial cleavage.Other than any experimental marking procedure the technique of time-lapse motion pictures permits to evaluate quantitatively and without artificial interfering the shifting of presumptive segment material during morphogenetic movements of the germ band. The embryonic material of the blastoderm at the egg equator is used for building up the first abdominal segment. The prothoracal and mesothoracal material at about 60% of the egg length stays in site when the germ band becomes extended lengthwise. Closely behind the differentiation centre there is a region of maximal extension as well as of shortening of the germ band. No proliferous growth of segments (segment formation zone) has been found.Zusammenfassung1.Die aus den ExperimentenGeyer-Duszynskas (1959) gefolgerte Existenz dreier plasmatischer Faktoren, die im Polplasma, im darin eingelagerten basophilen Oosommaterial und im Periplasma am Hinterpol des Eies vonWachtliella persicariae liegen, hat den Anstoß gegeben, nach weiteren Faktorenbereichen zu suchen. Die Initialbereiche morphogenetischer Prozesse können Indikatoren für ooplasmatische Faktorenbereiche sein. Deshalb ist die Kinematik der frühen Embryonalentwick-lung anhand von Zeitraffer-Filmaufnahmenin vivo analysiert worden. Elektronenoptische Untersuchungen fügen ergänzend den mikromorphologischen Aspekt ooplasmatischer Teilsysteme hinzu mit dem Ziel, Aussagen über Natur und Wirkungsweise plasmatischer Faktoren machen zu können.2.Die Furchungskerne werden nicht allein mit Hilfe ihrer Spindel bewegt. Die Kernhülle der Furchungsenergiden besteht entweder aus zwei porentragenden Elementarmembranen oder aber aus einer Vielzahl tubulärer und membranöser Elemente. Das Auftreten der vielschichtig-komplexen Kernwand fällt in die Wanderphase der Energiden und wird im Zusammenhang mit der Möglichkeit einer aktiven Kernwanderung diskutiert.3.Das Polplasma zeichnet sich, von den Oosompartikeln abgesehen, nicht durch spezifische Feinstrukturen aus. Es ist dotterfrei und besteht fast ausschließlich aus Grundplasma.4.Das basophile Oosommaterial liegt frei im Polplasma am Hinterpol des Eies. Es besteht aus zahlreichen ribosomenähnlichen Einheiten und wird auf das Plasma der künftigen Polzellen beschränkt. Sein Feinbau bleibt mindestens bis zum Stadium des maximal langen Keimstreifs erhalten.5.Die Zellwände entstehen aus Einstülpungen des Oolemmas, die in das Ei-Innere vordringen. Bei der Abschnürung von Pol- und Blastodermzellen ist das Grundplasma möglicherweise aktiv am Einwachsen der Zellwände beteiligt, und zwar durch die Ausbildung fädiger Differenzierungen an den terminalen Erweiterungen der Oolemmafalten.6.Das dotterfreie Periplasma ist nicht deutlich vom Entoplasma abgegrenzt. Es ist reich an Grundplasma und zeichnet sich in keinem Stadium durch spezifische Feinstrukturen aus.7.BeiWachtliella verschieben sich äußere Ooplasmabereiche während der Furchung um maximal +−6% der gesamten Eilänge pulsierend parallel zur Längsachse. Bei topographischen Angaben bestimmter Anlagebereiche muß daher mit einer entsprechenden Ungenauigkeit gerechnet werden. Ähnliche Plasmaverlagerungen sind auch in den Eiern anderer Insekten zu erwarten und gegebenenfalls als Fehlerquelle zu berücksichtigen.8.Zur Zeit des späten Präblastoderms treten im Entoplasma hufeisenförmige sowie vielgelappte Dotterkerne mit dichtem Karyoplasma auf. Im Anschluß an die Blastodermbildung werden sie pyknotisch, und die Kernwand fragmentiert. Die Bruckstücke treten vermutlich zu Stapeln von annulated membranes zusammen.9.Ein Furchungszentrum, Initialbereich der intravitellinen Furchung und Ausgangspunkt der folgenden Mitosewellen, liegt bei 60 +−3 % der Eilänge. Dicht hinter der Eimitte liegt ein Initialbereich für die Bildung des Keimstreifs (Differenzierungszentrum). Beide Entwicklungszentren zeichnen sich elektronenoptisch nicht durch spezifische Feinstrukturen aus. Der Faktorenbereich am Hinterpol ist nur Initialbereich für die Zellwandbildung bei der superfiziellen Furchung.10.Die Zeitraffertechnik ermöglicht es, im Gegensatz zu experimentellen Markierungsverfahren, ohne äußere Eingriffe die Verlagerung des präsumptiven Segmentmaterials während der Gestaltungsbewegungen des Keimstreifs quantitativ auszuwerten. Das embryonale Blastodermmaterial am Äquator des Eies wird für das erste Abdomensegment verwendet. Das Material für Pro- und Meso-Thorax bei ca. 60% der Eilänge bleibt während der Streckung des Keimstreifs am Ort liegen. Dicht hinter dem Differenzierungszentrum liegt ein Intensitätsmaximum der Streckung wie auch der Verkürzung des Keimstreifs. Eine Segmentbildungszone ist nicht vorhanden.

Migration and division of cleavage nuclei in the gall midge,Wachtliella persicariae

SummaryThe control of nuclear division and migration was studied in time-lapse films of the multinucleate egg cell of a gall midge by experimental alterations of the mitotic pattern. During each cleavage cycle, a wave of randomly oriented saltations of yolk particles (WROS) is seen to travel through the ooplasm. This wave proved to be an indispensable prerequisite for the accompanying anaphase wave and for the activation of the nuclear migration cytasters: WROS cycles can occur autonomously without cleavage nuclei being present, but there is no anaphase without a WROS passing the dividing nucleus. WROSs and mitotic waves can be inverted, and the WROS cycles and the cleavage cycles can be desynchronized by temperature grandients or by locally impaired gas exchange. If a nucleus is not ready for anaphase when met by a WROS, it will only divide in the course of the next WROS. WROSs thus indicate autonomous anaphase-triggering waves governing the cleavage divisions. Rhythmic ooplasmic movements continue even if the WROSs as well as the nuclear divisions are inhibited by colchinine. The characteristics of the WROSs support the hypothesis that each of them is the visible effect of a wave of calcium release (similar to that established in vertebrate eggs) which acts locally on the microtubular system and may continue even if the WROSs are suppressed. The correlations between a possible calcium release, WROS activity, microtubule disassembly and nuclear cycle are discussed.

Artifical rearrangements of insect ooplasm caused by fixation, and their microkymographic recording

SummaryTime-lapse photomicrographs of eggs from the Ichneumonid waspPimpla and from the gall midgeWachtliella reveal that during fixation the ooplasm performs excessive streaming movements when conventional fluids such as Bouins or Dubosq-Brasils are used. InWachtliella, intravitelline cleavage may continue for two mitotic cycles during fixation. The cytoplasm of individual egg regions may become displaced up to 45% of the eggs length inPimpla, and up to 14% of the eggs length inWachtliella. Therefore fixed preparations may grossly misrepresent the distribution of egg components in the living state. The changes can be traced back by microkymographic registration of movements during fixation. Microkymograms for this purpose were obtained by projecting a time-lapse film through a slit onto photosensitive material mounted on a rotating drum.The shifting of egg components and the mitotic activity of nuclei fail to occur when the egg is treated with a buffered solution of OsO4 (1%, 1 min). Subsequent fixation with glutaraldehyde results in satisfactory preservation of microtubules.

Experimental changes of the cleavage pattern in the eggs of a gall midge (Wachtliella persicariae L.) after local ultrasonic treatment

SummaryTo affirm the hypothesis that during the early cleavage inWachtliella persicariae L., central and marginal ooplasm do not only differ by their kinematic aspects (Wolf, 1969; Wolf and Krause, 1971) but also physiologically, the radial symmetry of the ooplasm has been destroyed experimentally. For this purpose a device has been developed permitting a local ultrasonic treatment of insect eggs limited to well defined regions of various sizes (Fig. 1). The changes in the egg architecture can be observed directly while the treatment is going on and their effects on embryogenesis are analyzed by time-lapse motion pictures.Cleavage nuclei which have been transferred prematurely to the egg surface during low-intensity ultrasonic radiation of the whole egg length (Fig. 2 a), move back into the central ooplasm after treatment; the marginal plasm becomes populated only later at the same time as in untreated eggs. Local high-intensity ultrasonic treatment, too (Fig. 2 c), obviously does not cause any damage to the nuclei, while the ooplasm within the treated area becomes completely mixed up. After exposure the untreated region may become partially coated by a sheath of mixed up ooplasm. The populating of the egg surface by the nuclei is restricted to those sites where the untreated marginal ooplasm remains at the surface. These are the only areas for the later formation of preblastoderm and blastoderm. For the normal distribution of the cleavage energides a certain ooplasmic component sensitive to ultrasonic treatment seems to be responsible, the ultrastructure of which will be subjected to further investigations.ZusammenfassungUm am Beispiel der GallmückeWachtliella persicariae L. die Hypothese zu erhärten, daß sich zentrales und marginales Ooplasma während der frühen Furchung nicht nur kinematisch (Wolf, 1969; Wolf und Krause, 1971), sondern auch physiologisch unterscheiden, ist experimentell die radiäre Symmetrie des Eies zerstört worden. Hierzu ist eine Versuchsanordnung entwickelt worden, die eine gezielte Ultra-Beschallung eines mehr oder weniger großen, scharf begrenzten Eibereichs ermöglicht (Abb. 1). Die Veränderungen der Eiarchitektur werden während der Behandlung direkt beobachtet und ihre Auswirkungen auf die Embryogenese mit Hilfe von Zeitrafferfilmen analysiert.Furchungskerne, die infolge schwacher Ultrabeschallung des ganzen Eies vorzeitig an die Eioberfläche verlagert worden sind (Abb. 2a), wandern nach der Behandlung wieder in dazentrale Ooplasma zurück; das Marginalplasma wird erst später, zur selben Zeit wie in uns behandelten Eiern, besiedelt. Nach starker lokaler Ultrabeschallung (Abb. 2 c) werden die Kerne offensichtlich ebenfalls nicht geschädigt, aber das Plasma wird im beschallten Eibereich völlig durchmischt. Nach der Behandlung kann ein Teil der unbeschallten Eiregion oberflächlich von durchmischtem Ooplasma schlauchartig überzogen werden. Die Kerne können die Eioberfläche nur dort besiedeln, wo noch unbeschalltes Marginalplasma an der Oberfläche liegen geblieben ist; nur in diesen Gebieten ist die Bildung eines Präblastoderms und später eines Blastoderms möglich. Für die normale Verteilung der Furchungsenergiden im Eiraum muß eine besonders schallempfindliche Komponente des Ooplasmas verantwortlich sein, deren Feinstruktur noch elektronenmikroskopisch untersucht werden soll.

Parabiotic development of fused eggs from the Hymenopteron, Pimpla turionellae, and of eggs injected with energids

Explanted oocytes and eggs of different developmental stages from the Hymenopteron Pimpla were fused in pairs as “parabiotic tandems”. Interactions within the tandem were analysed by time lapse films. Except for the exchange of nuclei, no joint development was observed, and each partner followed its own time pattern. The rapid cell cycles of the cleavage energids switched over to longer cycles according to the local developmental stage of the different egg regions, although all of the nuclei were still contained in a single plasmodium. In a second experimental series, nuclei in newly deposited eggs were X-rayed and replaced by cleavage energids from later stages injected into the wrong (=posterior) egg pole. Even injected blastoderm nuclei immediately took up mitotic activities and underwent rapid cell cycles characteristic of early cleavage. Normal embryos could be formed, although the nuclei had populated the egg in a reverse direction.

Pattern formation fails after blastoderm formation by rapid cell cycles in an artificially activated insect egg

Oocytes explanted from adult ovaries of the arrhenotokous Hymenopteron Pimpla turionellae remain in an inactive state, because development has not been initiated by mechanical deformation during natural oviposition. However, they could be induced to enter development by injecting cleavage energids into the posterior pole. After lag phases of up to 32 h, the implanted nuclei initiated a normal cleavage process, except that the polarity of its progress was reversed. In other oocytes, the injected energids congregated in a ring-shaped region at the egg surface to form a superficial “nuclear front”, which slowly advanced towards the anterior egg pole, thereby successively stimulating portions of the quiescent ooplasm to take part in development. Up to 41 rapid cell cycles started from that front, each of them with an anaphase wave running backwards into the region already peripherally occupied by nuclei. Thus, the blastoderm was formed extremely metachronously and by rapid obviously biphasic cell cycles, which never occur at the egg surface during normal cleavage. A germ band, however, was only formed under the following conditions: (1) that cleavage did not follow the nuclear front mode, and (2) that ooplasm from the donors posterior pole was co-injected with the graft nuclei. We conclude that embryonic differentiation requires some of the events which had been omitted in eggs where development failed, especially the exponential increase of the cell cycle length, and the activity of some posterior factor(s) during egg activation.