The let-99 gene is required for proper spindle orientation during cleavage of the C. elegans embryo

The orientation of cell division is a critical aspect of development. In 2-cell C. elegans embryos, the spindle in the posterior cell is aligned along the long axis of the embryo and contributes to the unequal partitioning of cytoplasm, while the spindle in the anterior cell is oriented transverse to the long axis. Differing spindle alignments arise from blastomere-specific rotations of the nuclear-centrosome complex at prophase. We have found that mutations in the maternally expressed gene let-99 affect spindle orientation in all cells during the first three cleavages. During these divisions, the nuclear-centrosome complex appears unstable in position. In addition, in almost half of the mutant embryos, there are reversals of the normal pattern of spindle orientations at second cleavage: the spindle of the anterior cell is aligned with the long axis of the embryo and nuclear rotation fails in the posterior cell causing the spindle to form transverse to the long axis. In most of the remaining embryos, spindles in both cells are transverse at second cleavage. The distributions of several asymmetrically localized proteins, including P granules and PAR-3, are normal in early let-99 embryos, but are perturbed by the abnormal cell division orientations at second cleavage. The accumulation of actin and actin capping protein, which marks the site involved in nuclear rotation in 2-cell wild-type embryos, is abnormal but is not reversed in let-99 mutant embryos. Based on these data, we conclude that let-99(+) is required for the proper orientation of spindles after the establishment of polarity, and we postulate that let-99(+) plays a role in interactions between the astral microtubules and the cortical cytoskeleton.     

The radon therapy: radon inhalation spa in Kowary

To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis-respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90 degrees with the sperm entry point up or down with respect to the gravitational field. At the 8-cell stage, blastomeres were injected with the lineage tracers, Texas Red- or FITC-Dextran Amines. The distribution of the labeled progeny was mapped at the tail-bud stages (stages 35-38) and compared with the fate map of an 8-cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90 degrees rotation the blastomeres in the 8-cell stage embryo roughly switched their position by 90 degrees, but the fate of the blastomeres did not simply show a 90 degrees switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position: (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.     

The dynactin complex is required for cleavage plane specification in early Caenorhabditis elegans embryos

BACKGROUND: During metazoan development, cell diversity arises primarily from asymmetric cell divisions which are executed in two phases: segregation of cytoplasmic factors and positioning of the mitotic spindle - and hence the cleavage plane -relative to the axis of segregation. When polarized cells divide, spindle alignment probably occurs through the capture and subsequent shortening of astral microtubules by a site in the cortex. RESULTS: Here, we report that dynactin, the dynein-activator complex, is localized at cortical microtubule attachment sites and is necessary for mitotic spindle alignment in early Caenorhabditis elegans embryos. Using RNA interference techniques, we eliminated expression in early embryos of dnc-1 (the ortholog of the vertebrate gene for p150(Glued)) and dnc-2 (the ortholog of the vertebrate gene for p50/Dynamitin). In both cases, misalignment of mitotic spindles occurred, demonstrating that two components of the dynactin complex, DNC-1 and DNC-2, are necessary to align the spindle. CONCLUSIONS: Dynactin complexes may serve as a tether for dynein at the cortex and allow dynein to produce forces on the astral microtubules required for mitotic spindle alignment.     

Epithelial stratification in the developing chick cornea

The process of epithelial stratification was studied in the embryonic chick corneal eipthelium between 10 and 21 days of incubation. Information was collected on the DNA synthetic activity occurring in individual cell layers, on cell density changes in the basal layer, and on mitotic spindle orientation in each cell layer before, during, and upon the completion of alyer formation. Prior to the initiation of stratification, mitotic spindles are oriented parallel to the basement membrane interface. This orientation changes to predominantly vertically directed spindles as layer formation proceeds. Later, the majority of the spindles are horizontally aligned once more. This pattern was observed as each successive cell layer formed. The possible relationship between the spindle data and that obtained on DNA synthetic activity and cell density changes is discussed in terms of the role these factors might play in layer formation in the cornea, as well as in other stratified epithelia.     

mex-1 and the general partitioning of cell fate in the early C. elegans embryo

It is thought that at least some of the initial specification of the five somatic founder cells of the C. elegans embryo occurs cell-autonomously through the segregation of factors during cell divisions. It has been suggested that in embryos from mothers homozygous for mutations in the maternal-effect gene mex-1, four blastomeres of the 8-cell embryo adopt the fate of the MS blastomere. It was proposed that mex-1 functions to localise or regulate factors that determine the fate of this blastomere. Here, a detailed cell lineage analysis of 9 mex-1 mutants reveals that the fates of all somatic founder cells are affected by mutations in this gene. We propose that mex-1, like the par genes, is involved in establishing the initial polarity of the embryo.     

Role of nonrandomly oriented cell division in shaping and bending of the neural plate

Neural plate shaping and bending are crucial to the process of neural tube formation. Shaping requires intrinsic forces generated by alterations in neuroepithelial cell behavior, whereas bending requires similar intrinsic forces and extrinsic forces generated by alterations in cell behaviors outside the neural plate. This study evaluates the role of nonrandomly oriented cell division in neural plate shaping and epidermal ectoderm expansion during bending by examining mitotic spindle orientation in the neuroepithelium and epidermal ectoderm throughout neurulation. Neuroepithelial mitotic spindles are oriented preferentially in the rostrocaudal plane, suggesting a role for nonrandomly oriented (i.e., rostrocaudal) neuroepithelial cell division in longitudinal lengthening of the neural plate during shaping. Epidermal ectoderm mitotic spindles are oriented preferentially in both rostrocaudal and mediolateral planes, suggesting a role for nonrandomly oriented cell division in epidermal ectoderm elongation and expansion. In neural plate and epidermal ectoderm isolates separated prior to shaping and bending, mitotic spindles continued to be oriented preferentially in the rostrocaudal plane; however, a preferential mediolateral mitotic spindle orientation could not be demonstrated in the epidermal ectoderm isolates. We conclude that the nonrandom rostrocaudal orientation of cell division in the neuroepithelium and epidermal ectoderm is an autonomous process, occurring in the absence of forces from adjacent tissues, whereas the nonrandom mediolateral orientation of cell division in the epidermal ectoderm is dependent upon interactions with the neural plate.     

Cleavage planes in frog eggs are altered by strong magnetic fields

Early cleavages of Xenopus embryos were oriented in strong, static magnetic fields. Third-cleavage planes, normally horizontal, were seen to orient to a vertical plane parallel with a vertical magnetic field. Second cleavages, normally vertical, could also be oriented by applying a horizontal magnetic field. We argue that these changes in cleavage-furrow geometries result from changes in the orientation of the mitotic apparatus. We hypothesize that the magnetic field acts directly on the microtubules of the mitotic apparatus. Considerations of the length of the astral microtubules, their diamagnetic anisotropy, and flexural rigidity predict the required field strength for an effect that agrees with the data. This observation provides a clear example of a static magnetic-field effect on a fundamental cellular process, cell division.     

Disruption of polyamine modulation by a single amino acid substitution on the L3 loop of the OmpC porin channel

The development of Antechinus stuartii from the 2-cell stage to the blastocyst stage in vivo was examined by routine transmission electron microscopy. The 2-8-cell stages had a similar organization of organelles, whereas the 16- to 32-cell stages had pluriblast cells and trophoblast cells forming an epithelium closely apposed to the zona pellucida. Specialized cell-zona plugs were formed at the 8-cell stage, and primitive cell junctions appeared in later conceptuses. The cytoplasmic organelles included mitochondria, lysosomes, aggregates of smooth endoplasmic reticulum, lipid and protein yolk bodies and fibrillar arrays, possibly contractile in function. Nuclei had uniformly-dispersed dense chromatin. Nucleoli of 2-4-cell conceptuses were dense, compact and fibrillar, and those of 8-cell conceptuses and later conceptuses were finely granular and became progressively reticulated. The embryonic genome is probably not switched on before the 8-cell stage. Sperm tails were detected in cells in several early conceptuses. The yolk mass had the same organelles as cells. Centrioles were discovered for the first time in marsupial conceptuses. These were prominently situated at a spindle pole in a 32-cell blastomere and were associated with a nucleus and sperm tail at the 4-cell stage. It is very likely that the paternal centrosome is inherited at fertilization and perpetuated in Antechinus embryos during cleavage.     

Feasibility study of repeated fluorescent in-situ hybridization in the same human blastomeres for preimplantation genetic diagnosis

In order to increase the number of chromosomes examined in each blastomere, we have developed a repeated fluorescent in-situ hybridization (FISH) procedure by which six or more chromosomes can be analysed per blastomere of a human embryo. Three consecutive FISH procedures with directly-labelled fluorescent Vysis DNA probes were carried out for examination of chromosomes X, Y, 11, 13, 18 and 21 in the same blastomeres (n = 126) and lymphocytes (n = 164). Based on the initial number of nuclei, the percentages of nuclear loss and presence of signals were 3 and 92% respectively in blastomeres; 6 and 91% respectively in lymphocytes after the first FISH; 7 and 87% respectively in blastomeres and 10 and 86% respectively in lymphocytes, after the second FISH. These percentages were 13 and 78% respectively in blastomeres and 14 and 81% respectively in lymphocytes after the third FISH. The FISH procedure was repeated successfully in a couple for preimplantation genetic diagnosis of chromosomal aneuploidies in biopsied blastomeres of their embryos in our clinic. In conclusion, it is feasible to carry out repeated FISH procedures in the same blastomeres. Six or more chromosomes of a single blastomere may be examined using this procedure.     

Mitosis in the human embryo: the vital role of the sperm centrosome (centriole)

The pattern of sperm centrosomal (centriolar) inheritance, centrosomal replication and perpetuation during mitosis of the human embryo is reviewed with a series of electron micrographs. Embryonic cleavage involves repeated mitoses, a convenient sequence to study centriolar behaviour during cell division. After the paternal inheritance of centrioles in the human was reported (Sathananthan et al., 1991a), there has been an upsurge of centrosomal research in mammals, which largely follow the human pattern. The human egg has an inactive non-functional centrosome. The paternal centrosome contains a prominent centriole (proximal) associated with pericentriolar material which is transmitted to the embryo at fertilization and persists during sperm incorporation. Centriolar duplication occurs at the pronuclear stage (interphase) and the centrosome initially organizes a sperm aster when male and female pronuclei breakdown (prometaphase). The astral centrosome containing diplosomes (two typical centrioles) splits and relocates at opposite poles of a bipolar spindle to establish bipolarization, a prerequisite to normal cell division. Single or double centrioles occupy pivotal positions on spindle poles and paternal and maternal chromosomes organize on the equator of a metaphase spindle, at syngamy. Bipolarization occurs in all monospermic and in most dispermic ova. Dispermic embryos occasionally form two sperm asters initially and produce tripolar spindles (tripolarization). Anaphase and telophase follows producing two or three cells respectively, completing the first cell cycle. Descendants of the sperm centriole were found at every stage of perimplantation embryo development and were traced from fertilization through cleavage (first four cell cycles) to the morula and hatching blastocyst stage. Centrioles were associated with nuclei at interphase, when they were often replicating and occupied pivotal positions on spindle poles during mitosis. Sperm remnants were associated with centrioles and were found at most stages of cleavage. Centrioles were found in trophoblast, embryoblast and endoderm cells in hatching blastocysts. Pericentriolar, centrosomal material nucleated astral and spindle microtubules. Abnormal nuclear configurations observed in embryos reflect mitotic aberrations. The bovine embryo closely resembles the human embryo in centriolar behaviour during mitosis. It is concluded that the sperm centrosome is the functional active centrosome in humans and is likely the ancestor of centrioles within centrosomes in foetal and adult somatic cells. The role of the sperm centrosome in embryogenesis and male infertility is discussed, since it is of clinical importance in assisted reproduction.     

Dynein and dynactin are localized to astral microtubules and at cortical sites in mitotic epithelial cells

The mitotic spindle is often positioned in a characteristic location during development, for example to enable the proper segregation of developmental determinants [1,2]. When epithelial cells divide, the mitotic spindle is often positioned parallel to the plane of the epithelium, so that both daughter cells contribute to the epithelium [3]. The mechanisms by which mitotic spindles are positioned have not been characterized in great detail, but evidence is accumulating that in some systems the dynein-dynactin microtubule motor complex plays a role [4-6]. Dynein has yet not been localized to cortical sites where it could bind to microtubules and exert a force that might orient the mitotic spindle, however [7,8]. Here, we report that in mitotic polarized epithelial cells, the dynein-dynactin complex accumulates, from prometaphase onwards, along astral microtubules and at cortical spots, into which many of the astral microtubules dock. The spots are assembled at the lateral plasma membrane, in the region below the tight junctions. Their formation is inhibited by cytochalasin D, and under these conditions the spindles do not orient properly. This novel localization of the dynein-dynactin complex is consistent with a role for the complex in the positioning of the mitotic spindle. We also show that, during prophase, the motor complex colocalizes with the nuclear envelope, consistent with it having a role in separating the centrosomes that are associated with the nuclear envelope.     

Identification of 4(-) states in the 14C(p,n)14N reaction at 135 MeV

The tadpole larva of solitary ascidians has 40 notochord cells in its tail. Of these cells, 32 in the anterior and middle part of the tail are derived from the A-line blastomeres, while 8 in the posterior part of the tail originate from the B-line blastomeres. Previous experiments involving continuous dissociation of daughter blastomeres from the first cleavage to the 110-cell stage suggested that cellular interactions may be involved in the formation of notochord cells. In the present study, the presumptive-notochord blastomeres isolated from the 32-cell embryos did not develop features of notochord. These results suggest that cellular interactions may be required for the fate specification of notochord, that is to say, notochord formation occurs as a result of inductive interaction between blastomeres. In order to confirm the involvement of induction in the determination of notochord and to identify the inducer blastomeres, the presumptive-notochord blastomeres at the 32-cell stage were coisolated or recombined with one of the surrounding blastomeres in a series of experiments. The results suggested that, for the A-line precursors, notochord differentiation occurs as the result of an inductive influence from vegetal blastomeres that include the presumptive-endoderm blastomeres and the presumptive-notochord blastomeres themselves. It was also suggested that induction of notochord is complete by the 64-cell stage and that inductive interactions have to be initiated before the decompaction of blastomeres during the 32-cell stage. Ascidians are Urochordata and are closely related to vertebrates. In vertebrates, it is well known that inductive interactions play a crucial role in the determination of notochord. It appears, therefore, that induction of notochord is common throughout the phylum Chordata.     

DNA-drug interaction measurements using surface plasmon resonance

Formation of the blastocoel in early Xenopus embryos was studied with a novel biotin-permeability assay and newly generated tight junction markers. The blastocoel forms at the first cleavage division since functional tight junctions which excluded biotin and established a segregated intraembryonic compartment were found at the 2-cell and all subsequent developmental stages. Unexpectedly, tight junctions before the 64-cell stage were not at their normal apical positions, but were found deep in the embryos, up to 200 micron from the apical surface. In these positions, the tight junctions left large areas of ion permeable lateral membranes exposed to the extraembryonic environment, explaining why electrophysiological experiments record a decrease in embryonic input resistances concomitant with early cleavage stages. Immunohistochemistry revealed that the recessed tight junctions did not influence the distribution of C-cadherin and Na+,K+ATPase. Both markers were present apical to recessed tight junctions, indicating that the maintenance of polarization of these basolateral markers does not require tight junctions. With further development, tight junctions assumed an increasingly apical location until, by the 2000-cell stage, they occupied their conventional positions between the blastomeres at the apical/lateral membrane boundaries.     

Apoptosis in the pre-implantation embryo

It has been well shown that apoptosis occurs in mammalian embryos as early as the blastocyst stage, in order to regulate the importance of the inner cell mass. We have looked for apoptosis at the cleavage stage, in human embryos that could not be transferred because of a high degree of fragmentation (grade IV) or a blockage in embryo development. Most of these embryos had blastomeres with condensed or fragmented chromatin, evocating apoptosis. Two markers of programmed cell death, detecting either early (Annexin V) or late (TUNEL technique) apoptosis events, were positive in our study: 100% and 30% of embryos were marked by Annexin V and TUNEL, respectively. Therefore, it seems that apoptosis occurs very early in human embryos conceived in vitro; this could represent a response to suboptimal culture conditions.     

The origins of primitive blood in Xenopus: implications for axial patterning

The marginal zone in Xenopus laevis is proposed to be patterned with dorsal mesoderm situated near the upper blastoporal lip and ventral mesoderm near the lower blastoporal lip. We determined the origins of the ventralmost mesoderm, primitive blood, and show it arises from all vegetal blastomeres at the 32-cell stage, including blastomere C1, a progenitor of Spemann's organizer. This demonstrates that cells located at the upper blastoporal lip become ventral mesoderm, not solely dorsal mesoderm as previously believed. Reassessment of extant fate maps shows dorsal mesoderm and dorsal endoderm descend from the animal region of the marginal zone, whereas ventral mesoderm descends from the vegetal region of the marginal zone, and ventral endoderm descends from cells located vegetal of the bottle cells. Thus, the orientation of the dorsal-ventral axis of the mesoderm and endoderm is rotated 90( degrees) from its current portrayal in fate maps. This reassessment leads us to propose revisions in the nomenclature of the marginal zone and the orientation of the axes in pre-gastrula Xenopus embryos.     

Sustainable development and quality of life: expected effects of prospective changes in economic and environmental conditions

The herbicide CIPC [N-(3-chlorophenyl) carbamate] has been shown to disrupt microtubule organization in plants, apparently by interfering with the functioning of the microtubule organizing center. Very few studies have examined the effects of CIPC on animal cell microtubules and centrosomes, however, and the effects of this cytoskeletal disrupting agent on fertilization and early development have not been studied in detail. To address these questions, fertilized sea urchin eggs were cultured in the presence of CIPC until the prism stage, and perturbations in the cytoskeleton and development were examined. It was found that Lytechinus pictus embryos are sensitive to micromolar amounts of CIPC, and that a characteristic set of cytoskeletal and developmental deficits is produced as a result of exposure to this herbicide. Mitotic spindles were truncated and randomly oriented within zygotes and blastomeres, and cytokinesis was compromised, resulting in the production of blastomeres of various sizes and ploidy. Interestingly, in spite of these cytoskeletal and nuclear alterations, spindle poles at fourth cleavage retained their ability to interact with the plasma membrane in a manner similar to that normally characterizing the unequal division of macromeres and micromeres. CIPC treatment resulted in unequal cell divisions at atypical times, and skeletal spicule formation in these embryos was abnormal. These results indicate that CIPC may pose a significant health risk during mammalian embryogenesis; in addition, it may be a useful tool with which to study microtubule and centrosomal functioning during animal cell division-especially in those cell types that exhibit stereotypic patterns of cell division during early development.     

Maternally expressed gamma Tub37CD in Drosophila is differentially required for female meiosis and embryonic mitosis

We report functional analysis of gamma Tub37CD, a maternally synthesized gamma-tubulin that is highly expressed during oogenesis and utilized at centrosomes in precellular embryos. Two gamma Tub37CD mutants contained missense mutations that altered residues conserved in all gamma-tubulins and alpha- and/or beta-tubulins. A third gamma Tub37CD missense mutant identified a conserved motif unique to gamma-tubulins. A fourth gamma Tub37CD mutant contained a nonsense mutation and the corresponding premature stop codon generated a protein null allele. Immunofluorescence analysis of laid eggs and activated oocytes derived from the mutants revealed microtubules and meiotic spindles that were close to normal even in the absence of gamma Tub37CD. Eggs lacking the maternal gamma-tubulin were arrested in meiosis, indicative of a deficiency in activation. Analysis of meiosis with in vitro activation techniques showed that the cortical microtubule cytoskeleton of mature wild-type eggs was reorganized upon activation and expressed as transient assembly of cortical asters, and this cortical reorganization was altered in gamma Tub37CD mutants. In precellular embryos of partial loss of function mutants, spindles were frequently abnormal and cell cycle progression was inhibited. Thus, gamma Tub37CD functions differentially in female meiosis and in the early embryo; while involved in oocyte activation, it is apparently not required or plays a subtle role in formation of the female meiotic spindle which is acentriolar, but is essential for assembly of a discrete bipolar mitotic spindle which is directed by centrosomes organized about centrioles.