Cleavage and blastoderm formation in normal and experimentally deformed naked eggs of a dipteran insect

Summary The eggs of the gall midgeHeteropeza pygmaea develop parthenogenetically inside of the mother larva. They lack a chorion and remain enveloped by the follicular epithelium. After experimental elimination of the follicular epithelium naked eggs are formed, which reach the blastoderm stage but remain spherical instead of assuming an elongated shape. To analyze this peculiar egg development and the roles of egg shape and envelope during development, the ultrastructure of cleaving normal and naked eggs was investigated. It was shown that the number of elements of Golgi apparatus and endoplasmic reticulum strongly increases during early cleavage. Their association with cleavage furrows and nuclei suggests that these organelles play a dominant role in membrane production. Egg yolk consists of lipids and glycogen, wheareas no proteins are found. Cleaving eggs contain numerous vesicles with lysosomal characteristics, indicating intense autophagic processes. Cleavage furrow formation occurs independently from the positioning of cleavage nuclei. The numerous microtubules, which are associated with cleavage furrows and nuclei and located in the egg periphery, the intercellular bridges, and in the central part of the egg, suggest that the cytoskeleton has an important role in cleavage furrow formation, blastoderm layer establishment, and yolk localization. Since these processes are accurately accomplished in naked spherical eggs, they can be considered as independent of normal egg shape and the follicular epithelium.     

Oogenesis in a paedogenetic dipteran insect under normal conditions and after experimental elimination of the follicular epithelium

Summary Paedogenetically developing eggs of the gall midgeHeteropeza pygmaea are not deposited, but develop in the hemocoel of the mother larva. The nurse chamber remains present in the cleaving egg, and the follicular epithelium does not form a chorion but envelops the growing egg during embryonic development. It is possible to obtain naked eggs, i.e. eggs lacking the follicular epithelium, which are able to develop up to the blastoderm stage but remain spherical instead of assuming an elongated shape. Oogenesis of normal and naked eggs has been studied at the ultrastructural level with special reference to the nurse chamber. It is shown that the nurse chamber nuclei develop large nucleoli during oogenesis, indicating that the nurse chamber supplies the oocyte with ribosomal RNA (rRNA). The dense bodies in the nurse chamber may represent an intermediate stage in the transport of the rRNA from the nurse chamber to the oocyte; they are probably not related to the polar granules in the oocyte. It is also shown that the intercellular bridge joining the nurse chamber to the oocyte disappears shortly before cleavage initiation. During egg cleavage the follicular epithelium surrounds the nurse chamber, which degenerates and is gradually absorbed by the growing egg plasmodium. Naked cleaving eggs are never attached to a nurse chamber or to relics of it. Naked oocytenurse chamber complexes frequently aggregate, which may indicate a role of the follicular epithelium in follicle separation during normal development.     

Post-transcriptional regulation of protein synthesis in Ilyanassa embryos and isolated polar lobes

The experimental removal of the polar lobe, an anucleate cytoplasmic protrusion formed in preparation for the first cleavage, from the egg of Ilyanassa obsoleta results in grossly abnormal embryonic development. In experiments reported here normal and delobed embryos, as well as isolated polar lobes, were incubated with [³⁵S]methionine for 4 hr beginning at the completion of the first cleavage or 21 hr later during epiboly. Proteins were extracted and examined by fluorography after resolution by two-dimensional polyacrylamide gel electrophoresis. In normal embryos the synthesis of several proteins begins or ends between the two stages investigated. In isolated polar lobes a subset of these developmental changes in protein synthesis occurs, indicating that the regulation of these events is independent of concomitant nuclear activity and probably involves selective regulation of the translation of mRNA stored in the eggs. The patterns of protein synthesis in normal embryos and delobed embryos are qualitatively extremely similar, though quantitative differences are also observed. No proteins can be detected which are synthesized exclusively in polar lobes.     

An ultrastructural study of the early cercarial development in Prosorhynchoides borealis (Digenea: Bucephalidae) with special reference to formation of the primitive epithelium

Primitive epithelium and outer tegumental layer formation during early cercarial development was studied in Prosorhynchoides borealis using electron microscopy. It demonstrated that germinal cells freely floating in the sporocyst body cavity divide to give rise to naked cell aggregates. These early embryos are highly irregular in outline and are composed of blastomeres differing in size and structure. In embryos consisting of about 12-14 cells a few (possibly only two) superficial macromeres become concave and produce thin extensions which envelop the embryonic mass before fusing to form a syncytial primitive epithelium. This primitive epithelium forms syncytial connections with underlying embryonic cells. Primordial tegumental cells become apparent in late germinal balls below the primitive epithelium. These cells expand and fuse to give rise to an embryonic nucleated tegument. The embryonic tegument is connected to peripheral embryonic cells by thin cytoplasmic bridges until the basement lamina is formed. Subsequently, the primitive epithelium is shed by the embryos and the nuclei in the embryonic tegument undergo pyknotic degeneration. These results are analysed and compared with data from studies on other trematode species and it is concluded that the primitive epithelium is derived from the embryo in at least the majority of digeneans.     

Placental Type Interactions and Evolutionary Trends of Development of Uterus in Cestodes

In Proteocephalus thymalli and P. torulosus, a contact of the placental type in uterus was shown to be formed at two different levels. At the first level an interaction occurs between outgrowths of uterine epithelium and thin capsule of embryos closely adjacent to uterine wall. The next level is formation of contact between neighboring egg capsules, which allows distributing nutrients among fetuses present in the uterine cavity. Placental interactions in Clestobothrium acheilognathi are limited in time and space. First, a relatively small number of eggs are involved in interaction of the placental type in the uterine sac, while uterine duct is filled with freely lying eggs. Second, the closest contact is observed in eggs with non-sclerosed egg shell. One of the main evolutionary tendencies in cestodes has been shown to be a modification of uterus for formation of close interrelations with embryonic membranes in the course of transition from the extrauterine to the intrauterine type of embryonic development. Uterus in parasites with a polylecital type of the egg is suggested to serve to the greater extent as a reservoir, whereas in cestodes with oligolecital eggs, uterus performs its direct function—supply of developing embryos with nutrients. As a result, modifications of uterine epithelium are formed: from the appearance of the placental type interactions formed repeatedly in phylogenetically distant groups of cestodes to formation of branched outgrowths separating the uterine space into units or disintegration to actively functioning uterine capsules.     

Axis determination in eggs of Xenopus laevis: A critical period before first cleavage,identified by the common effects of cold,pressure and ultraviolet irradiation

Exposure of eggs of Xenopus laevis to a temperature of 1.0°C for 4 min or a pressure of 8000 psi for 5 min in a critical period before first cleavage results in embryos exhibiting a reduction and loss of structures of the body axis. The deficiencies occur in a craniocaudal progression which is dose dependent. In the extreme, totally axis-deficient embryos with radial symmetry are formed. Maximum sensitivity to cold and pressure occurs at 0.6 of the time from fertilization to first cleavage and extends from approximately 0.4 to 0.8, the period between pronuclear contact and mitosis, and the approximate period of gray crescent formation. The effects of cold and pressure resemble those previously reported for uv irradiation in that (1) the types of axis-deficient embryos produced are morphologically indistinguishable; (2) sensitivity in all cases ends before 0.8; (3) cold and uv effects, although not those of pressure, can be prevented by cotreatment with D₂O; and (4) impaired eggs can be rescued by oblique orientation. We interpret these results as follows: during the 0.4–0.8 period the egg reorganizes its contents in a manner critical for subsequent development of the embryonic body axis. The reorganization process involves cytoskeletal elements, some of which are sensitive to cold, pressure, and uv, and protected by D₂O. Rescue by oblique orientation can be understood as the result of a gravity-driven reorganization of the egg's contents, supplanting the normal mechanochemical process impaired in treated eggs.     

Presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the spindle, the diastema and the cleavage furrow in the blastomere of the Xenopus embryo

The present study examines the indispensability of a nucleus or nucleus-deriving factors in the induction of cleavage in Xenopus eggs by testing cleavage in Xenopus eggs fertilized with ultraviolet (UV)-damaged sperm and deprived of the female nucleus. These eggs, which contain only one UV-damaged nucleus with one set of centrioles, undergo unique cleavages. Cleavage takes place in only one of the two blastomeres formed by the immediately preceding cleavage. Histologically, only one nucleus, which does not appear to be organized into typical chromosomes, is found in one of the two blastomeres formed by the immediately preceding cleavage. The typical bipolar spindle and the diastema, or a slit of astral rays, are formed in the blastomere that contains the nucleus. By contrast, only asters lacking the spindle and the diastema are formed in the remaining blastomeres, which do not contain a nucleus. The same results are obtained in eggs that contain two UV-damaged nuclei with one set of centrioles. In these eggs, cleavage appears to occur in one or two blastomeres that contain either or both of the nuclei and one bipolar spindle. In eggs that contain one intact and one UV-damaged nuclei, cleavage takes place quite normally with each blastomere containing one nucleus or one set of chromosomes as well as one bipolar spindle. Thus, there is a very close correlation between the presence of a nucleus and the formation of the mitotic spindle, the diastema and the cleavage furrow in the blastomeres of Xenopus embryos. We conclude that the presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the bipolar spindle, the diastema and the cleavage furrow in the blastomeres of the Xenopus embryos.     

UV irradiation of pig metaphase chromosomes: maturation-promoting factor degradation, nuclear cytology and cell cycle progression

Experiments were designed to test two hypotheses. The first was that irradiation of pig metaphase chromosomes would block the normal sequence of cytological and molecular events associated with activation; the second postulated that damaged DNA would prevent eggs from progressing through the first mitotic cleavage cycle. The experimental protocol involved selectively irradiating the metaphase II plate of pig oocytes with highly focused 254 nm ultraviolet (UV) light, followed by activation using standard electroactivation procedures. The following assessments were made of different groups of eggs: (i) nuclear membrane reassembly; (ii) chromosomal cytology; (iii) changes in maturation-promoting factor kinase (MPF kinase) activity at 1 h intervals after activation; and (iv) mitotic progression of eggs containing damaged chromosomal fragments. UV irradiation neither prevented the reassembly of nuclear membranes required for pronuclear formation nor interfered with the normal pattern of MPF kinase degradation after egg activation. UV irradiation did induce a wide range of chromatin defects, including condensation and dispersal of DNA fragments which, in turn, resulted in the formation of micronuclei in the treated eggs and embryos. The presence of damaged DNA retarded, but did not inhibit, progression through the first mitotic cycle. No evidence was obtained that the subsequent mitotic cycle was adversely affected by the presence of UV-damaged DNA. Overall, these results indicate that early cleavage divisions in pig eggs are not blocked by the presence of damaged, hypercondensed chromatin. In this respect, pig eggs are similar to Xenopus eggs, but are different from bovine eggs. On the basis of these findings it is suggested that focused UV irradiation offers a simple and rapid technique for the non-invasive enucleation of pig oocytes provided that the residual hypercondensed chromatin does not affect later developmental stages.     

Early embryonic development of the dipteran insect Heteropeza pygmaea in the presence of cytoskeleton-affecting drugs

Summary Embryos of the paedogenetically reproducing gall midge Heteropeza pygmaea develop floating in the haemocoel of a so-called mother larva. The egg membranes remain permeable and the embryos increase in size during embryonic development by taking up nutrients from the haemolymph. Such embryos can be cultured in vitro, i.e. in haemolymph drops obtained from mother larvae. We tested the effects of several drugs known to interact with cytoskeletal elements on different stages of embryonic development, including cleavage and gastrulation. The drugs were added to the in vitro cultures and the effects were studied with time-lapse cine-micrography. Colchicine and vinblastine blocked cleaving eggs in metaphase stage and arrested yolk globule oscillation. In spite of such a block blastoderms once formed continued development through germ band formation and extension and also increased in size. Cytochalasin B did not affect the stage of cleavage; however, it inhibited gastrulation and subsequent morphogenetic processes and also prevented size increase. We conclude that (1) the functioning of microtubules is needed for yolk globule oscillation during cleavage interphases but not for the gastrulation processes subsequent to blastoderm formation and (2) microfilaments do not play an important role in cleavage, at least not for the orderly succession of the cleavage divisions, but are essential for the morphogenetic movements associated with gastrulation. We suggest that during cleavage a limited stock of microtubules and their precursors is responsible for both transport of chromosomes during mitoses and translocation of organelles during interphase. Yolk oscillation seems to be a secondary effect and of minor or no importance for the normal course of embryonic development.Dedicated to Professor Gerhard Krause on the occasion of his 80th birthday     

The Maternal-Effect Gene cellular island Encodes Aurora B Kinase and Is Essential for Furrow Formation in the Early Zebrafish Embryo

Females homozygous for a mutation in cellular island (cei) produce embryos with defects in cytokinesis during early development. Analysis of the cytoskeletal events associated with furrow formation reveal that these defects include a general delay in furrow initiation as well as a complete failure to form furrow-associated structures in distal regions of the blastodisc. A linkage mapping-based candidate gene approach, including transgenic rescue, shows that cei encodes the zebrafish Aurora B kinase homologue. Genetic complementation analysis between the cei mutation and aurB zygotic lethal mutations corroborate gene assignment and reveal a complex nature of the maternal-effect cei allele, which appears to preferentially affect a function important for cytokinesis in the early blastomeres. Surprisingly, in cei mutant embryos a short yet otherwise normal furrow forms in the center of the blastodisc. Furrow formation is absent throughout the width of the blastodisc in cei mutant embryos additionally mutant for futile cycle, which lack a spindle apparatus, showing that the residual furrow signal present in cei mutants is derived from the mitotic spindle. Our analysis suggests that partially redundant signals derived from the spindle and astral apparatus mediate furrow formation in medial and distal regions of the early embryonic blastomeres, respectively, possibly as a spatial specialization to achieve furrow formation in these large cells. In addition, our data also suggest a role for Cei/AurB function in the reorganization of the furrow-associated microtubules in both early cleavage- and somite-stage embryos. In accordance with the requirement for cei/aurB in furrow induction in the early cleavage embryo, germ plasm recruitment to the forming furrow is also affected in embryos lacking normal cei/aurB function.     

Coelomic gonadal epithelia during the period of human embryonic morphologic sex differentiation]

The course of divergent differentiation of coelomic epithelium in the indifferent gonad has been traced up to the stage when morphological sex signs are distinctly seen. Eleven human embryos fixed in Carnoy and Bouin's fluid have been studied from 6 to 10 weeks of gestation. Paraffin sections were stained with hematoxylin, ferric hematoxylin after Heidenhain. Basal membrane fibres were revealed by silver impregnation after Karupu. PAS-reaction after McManus and additional staining with hematoxylin and light green were applied, as well as the reaction for total proteins with bromphenol blue after Miguel--Calvo. Sex differentiation of coelomic epithelium in the gonad developing according to the male type begins a little later than in the ovary. In the epithelium of the forming testis a great amount of mitotic figures is observed that might be connected with the presence of Y-chromosome stimulating cells for extra mitotic cycles. The increasing presence of Y-chromosome stimulating cells for extra mitotic cycles. The increasing number of mitotic figures in the epithelium should be considered as a sign demonstrating that differentiation according to the male type has started. Superficial epithelium of the embryonic ovary has a peculiar false pseudostratified structure. This secures cellular reserve for the ovarian cortex formation from the external epithelium. The apical surface of the cells in the external epithelium has a border which is evidently formed by microvilli and revealed by PAS-reaction and bromphenol blue. Ovarian follicular cells and Sertoli's cells (sustentocytes) in testis have the common origin in the human fetus--from coelomic epithelium of the gonad germ.     

Pattern formation during early amphibian development: Embryogenesis in inverted anuran and urodele eggs

Early embryogenesis was monitored in Xenopus, Rana (anurans), and Ambystoma (urodele) eggs which were inverted at various times between fertilization and first cleavage. The pattern of cleavage furrow formation, site of involution, and extent of organogenesis were observed. In several instances, pattern formation was dramatically altered. The small/large blastomere pattern was, for example, reversed in some inverted embryos. Developmental arrest at early organogenesis usually followed pattern reversal. By employing a series of tissue transplantations, it was possible to establish that the activity of the primary embryonic organizer of inverted embryos was diminished drastically. The developmental competence of the prospective ectoderm of inverted embryos was, however, reversed. Incomplete organogenesis in inverted embryos is therefore probably due to either abnormal mesoderm formation or defective tissue interactions.     

Follicular placenta and embryonic growth of the viviparous four-eyed fish (Anableps)

In the four-eyed fish, Anableps (Atheriniformes, Anablepidae), eggs are fertilized and embryos develop to term within the ovarian follicles. Development is highly matrotrophic. During gestation, the largest term embryo of A. anableps examined had grown to a total length of 51 mm and attained a dry weight of 149 mg. The postfertilization weight increase is 298,000%. The largest term embryo of A. dowi examined had grown to a total length of 77 mm and attained a dry weight of 910 mg. The postfertilization weight increase is 843,000%. Embryonic weight increases result from nutrient transfer across the follicular placenta. This structure is formed by apposition of the maternal follicular epithelium to absorptive surface cells of the embryo's pericardial trophoderm. The latter, a ventral ramification of the pericardial somatopleure, replaces the yolk sac during early gestation. The external surface of the pericardial trophoderm develops hemispherical projections, termed vascular bulbs. Within each bulb, the vascular plexus of the trophoderm expands to form a blood sinus. Cells of the external surface of the bulbs possess microplicae. Microvilli are absent. During middle to late gestation, the juxtaembryonic follicular epithelium differentiates into two regions. One region consists of shallow, pitlike depressions within which vascular bulbs interdigitate in a “ball and socket” arrangement. Follicular pits are formed by the curvilinear distortion of the apical surfaces of follicle cells. The second region in contact with the dorsal and lateral surfaces of the embryo, is comprised of villous extensions of the hypertrophied follicular epithelium. In both regions, follicle cells appear to constitute a transporting rather than a secretory epithlium. In terms of percentage of weight increase, the follicular placenta of Anableps appears to be the most efficient adaptation for maternal-embryonic nutrient transfer in teleost fishes and closely approaches the efficiency (1.2 × 10⁶%) of oophagy and embryonic cannibalism in lamnoid sharks.     

Roles for focal adhesion kinase (FAK) in blastomere abscission and vesicle trafficking during cleavage in the sea urchin embryo

Is focal adhesion kinase (FAK) needed for embryonic cleavage? We find that FAK is expressed during early cleavage divisions of sea urchin embryos as determined by polyclonal antibodies to the Lytechinus variegatus protein. FAK is absent in eggs and zygotes and then cycles in abundance during the first cleavages after fertilization. It is maximal at anaphase, similar to the destruction and synthesis of cyclin proteins. To investigate whether FAK is needed during early cleavage, we interfered with its function by microinjecting eggs with anti-FAK antibodies or with FAK antisense morpholino oligonucleotides. Both treatments led to regression of the cleavage furrow. FAK knockdown with antibodies or morpholino oligonucleotides also resulted in an over-accumulation of endocytic vesicles. Thus, FAK could be restricting endocytosis or increasing exocytosis in localized areas important for abscission. FAK appears to be necessary for successful cleavage. These results are the first to document a functional role for FAK during embryonic cleavage.     

BEHAVIOR OF INTERPHASE EMBRYONIC NUCLEI TRANSPLANTED IN NUCLEAR MULTIPLICATION STAGE EMBRYOS OF DROSOPHILA MELANOGASTER

Interphase nuclei were transplanted from syncytial blastoderm into early cleavage embryos of Drosophila melanogaster. The transplanted nuclei, when exposed to host cytoplasm, were initiated to mitosis. During the period from 10 to 50 min after transplantation, the implanted nuclei and host nuclei were found not synchronous in their mitotic cycles. Synchrony was restored usually by the blastoderm stage.
About 5% of eggs with transplanted nuclei developed significantly faster than control eggs, resulting in premature blastoderm formation. This finding is discussed in relation to chimera formation and to embryonic development of grandchildless mutants.     

Cytological and biochemical analyses of the maternal-effect mutant embryos with abnormal cleavage furrow formation in Xenopus laevis

We describe an embryonic lethal mutation in Xenopus laevis that provokes regression of cleavage furrow formation. The mutant females (designated as af) were obtained by the back-cross of a female with one of her sons. All the fertilized eggs laid by the mutant females, regardless of the wild-type male used in the mating, failed to cleave although each furrow ran at a proper position superficially. Light and electron microscopic observations of the embryos revealed that the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions did. Two-dimensional gel-electrophoretic comparisons of af and wild-type embryos demonstrated that two proteins, having estimated molecular masses of about 38 kDa (pI 6.6) and 78 kDa (pI 7.6), were missing in af embryos. Microinjection of clear cytoplasm from a wild-type egg into fertilized af eggs provoked partial surface contraction and cleavage furrow formation in recipient af eggs. The results showed that the af females carry a lethal maternal-effect mutation which causes cleavage furrow regression by being deficient in a few proteins, and that cytoplasm of wild-type eggs can partially rescue the cleavage furrow formation of af eggs by furnishing the corrective material, presumably a product of the normal allele of af.     

Morphology and ultrastructure of the uterus of Lineolepis scutigera (Dujardin, 1845) Karpenko, 1985 (Cestoda,Cyclophyllidea, Hymenolepididae) in formation of uterine capsules

The microanatomy and ultrastructure of the uterus in different stages of ontogenesis were studied in a cyclophyllidean cestode Lineolepis scutigera. In the early stages, developing embryos lie freely in the cavity of a single unbroken uterus, which is later fragmented into separate compartments. As a result, numerous spherical uterine capsules are formed; each of them contains one formed egg. Capsules represent a fragmented but actively functioning uterus. Muscular cells containing numerous lipid inclusions are located around them. In the final stages, placenta-like relationships are formed between eggs and the epithelium of capsules. A comparative morphofunctional analysis of uterine capsules in cestodes is presented. Attention is paid to the formation of close interactions between the uterine epithelium and developing eggs.     

Nek2B, a novel maternal form of Nek2 kinase, is essential for the assembly or maintenance of centrosomes in early Xenopus embryos

Nek2, a NIMA-related kinase, has been postulated to play a role in both the meiotic and mitotic cell cycles in vertebrates. Xenopus has two Nek2 splice variants, Nek2A and Nek2B, which are zygotic and maternal forms, respectively. Here we have examined the role of Nek2B in oocyte meiosis and early embryonic mitosis. Specific inhibition of Nek2B function does not interfere with the oscillation of Cdc2 activity in either the meiotic or mitotic cell cycles; however, it does cause abortive cleavage of early embryos, in which bipolar spindle formation is severely impaired due to fragmentation or dispersal of the centrosomes, to which endogenous Nek2B protein localizes. In contrast, inhibition of Nek2B function does not affect meiotic spindle formation in oocytes, in which functional centrosomes are absent. Thus, strikingly, Nek2B is specifically required for centrosome assembly and/or maintenance (and hence for normal bipolar spindle formation and cleavage) in early Xenopus embryos. Finally, (ectopic) Nek2A but not Nek2B is very labile in cleaving embryos, suggesting that Nek2A cannot replace the centrosomal function of Nek2B in early embryos.