The role of polarity in the development of the hydrozoan planula larva

Summary These experiments were done in order to define the role that polarity plays during embryogenesis in hydrozoans.Parts of hydrozoan embryos isolated at different developmental stages from early cleavage to postgastrula will regulate to form normal planulae. During this process, the original anterior-posterior axis of the part is conserved. In normal embryos the posterior pole of the anterior-posterior axis is congruent with the site where the polar bodies are given off and with the site where the first cleavage is initiated. By centrifuging fertilized eggs, it is possible to create embryos in which the first cleavage initiation site does not correspond to the site where the polar bodies are given off. In these embryos the posterior pole of the anterior-posterior axis corresponds to the first cleavage initiation site. When parts of these embryos are isolated at different stages they also regulate to form normal planulae. The axial properties of these planulae are determined by the site of first cleavage initiation.The interactions between regions of the embryo with different axial properties were studied by grafting together parts in such a way as to create embryos with abnormal axial arrangements. Following gastrulation interactions take place between the grafted parts leading to the formation of normal planulae with a new set of axial properties.Blastula stage embryos can be dissociated into single cells and the cells can be reaggregated. These reaggregates form normal planulae. Polarity can be entrained in the reaggregates by grafting a small piece of tissue from any part of an intact blastula to the reaggregate. These cells organize the formation of an axis of symmetry with an appropriate orientation with respect to the graft.     

The role of oocyte maturation in the ontogeny of the fertilization site in the hydrozoan Hydractinia echinata

Summary In hydrozoans the sperm will fuse with the egg only at the site of polar body formation. The primary oocyte and maturing oocytes which have produced the first polar body cannot be fertilized even though maturing oocytes which have produced the first polar body attract sperm. These eggs do not acquire the ability to be fertilized until after second polar body formation. If either first or second polar body formation is inhibited or if first and second polar body formation do not take place in close proximity to each other, the fertilization site is not set up. Under normal circumstances the site of polar body formation takes place at the region on the maturing oocyte surface nearest the site where the germinal vesicle resided in the primary oocyte. When maturing oocytes are centrifuged prior to polar body formation, the site of polar body formation is frequently shifted so that it does not correspond to the site where it would be given off under normal circumstances. Under these conditions the shifted site of polar body formation is the only site where the egg can be fertilized, indicating that the fertilization site is selected during oocyte maturation.Oocyte maturation in these hydrozoans is mediated by a hormone released by the somatic cells of gonophores as a consequence of bringing dark adapted gonophores into the light. The hormone acts directly on the oocyte to induce maturation. The oocyte only has to be exposed to the hormone for the first few minutes of the maturation process in order to complete the process of maturation.Dedicated to Professor N.H. Verdonk of the Rijksuniversiteit Utrecht on his 65th birthday     

Cell polarity emerges at first cleavage in sea urchin embryos

In protostomes, cell polarity is present after fertilization whereas most deuterostome embryos show minimal polarity during the early cleavages. We now show establishment of cell polarity as early as the first cleavage division in sea urchin embryos. We find, using the apical markers G_M1, integrins, and the aPKC-PAR6 complex, that cells are polarized upon insertion of distinct basolateral membrane at the first division. This early apical-basolateral polarity, similar to that found in much larger cleaving amphibian zygotes, reflects precocious functional epithelial cell polarity. Isolated cleavage blastomeres exhibit polarized actin-dependent fluid phase endocytosis only on the G_M1, integrin, microvillus-containing apical surface. A role for a functional PAR complex in cleavage plane determination was shown with experiments interfering with aPKC activity, which results in several spindle defects and compromised blastula development. These studies suggest that cell and embryonic polarity is established at the first cleavage, mediated in part by the Par complex of proteins, and is achieved by directed insertion of basolateral membrane in the cleavage furrow.     

Organisation and assembly of the surface membrane during early cleavage of the mouse embryo

Summary An attempt was made to understand the ways in which ‘newly inserted’ membrane was organised in relation to existing membrane during early cleavage of the mouse embryo by (i) monitoring the redistribution of a variety of surface-binding ligands (applied to the embryo during the previous cell cycle) and (ii) analysing the localisation of newly synthesised lipid at defined stages during the second cell cycle. The membrane dynamics of the embryo appear similar to those of somatic cells during cytokinesis and/or motility, and are consistent with previous suggestions (Pratt 1985) that the main cytocortical domains of the polarised 8-cell blastomere may start to diverge during early cleavage as a result of localised assembly and reorganisation of the embryo cytocortex.     

nanos expression at the embryonic posterior pole and the medusa phase in the hydrozoan Podocoryne carnea

Summary The distinction between soma and germline is an important process in the development of animals with sexual reproduction. It is regulated by a number of germline-specific genes, most of which appear conserved in evolution and therefore can be used to study the formation of the germline in diverged animal groups. Here we report the isolation of two orthologs of one such gene, nanos (nos), in the cnidarian Podocoryne carnea, a species with representative zoological features among the hydrozoans. By studying nos gene expression throughout the Podocoryne biphasic life cycle, we find that the germline differentiates exclusively during medusa development, whereas the polyp does not contribute to the process. An early widespread nos expression in developing medusae progressively refines into a mainly germline-specific pattern at terminal stages of medusa formation. Thus, the distinction between germline and soma is a late event in hydrozoan development. Also, we show that the formation of the medusa is a de novo process that relies on active local cell proliferation and differentiation of novel cell and tissue types not present in the polyp, including nos-expressing cells. Finally, we find nos expression at the posterior pole of Podocoryne developing embryos, not related to germline formation. This second aspect of nos expression is also found in Drosophila, where nos functions as a posterior determinant essential for the formation of the fly abdomen. This raises the possibility that nos embryonic expression could play a role in establishing axial polarity in cnidarians.     

The direction of cleavage waves and the regional variation in the duration of cleavage cycles on the dorsal side of the Xenopus laevis blastula

Summary The animal and the dorsal side of five embryos of Xenopus laevis were studied in detail from the 7th to the 13th cleavage by means of time-lapse cinematography. At each cleavage the regionally ordered sequence of blastomere divisions is visible in the films as a cleavage wave, propagating about three times slower in the dorsal than in the animal view. In the dorsal view the waves run in an animal-vegetal direction, initially with a left-to-right deviation and in later cleavages converging on the region of the future blastopore. The lengthening of cleavage cycles begins at cycle 8 on the dorsal side, just above the future blastopore. From cycle 9 to 11 nearly equal lengthening occurs in each cycle at all animal-vegetal levels. In general, cycles lengthen a little more in median than in lateral sectors and a little more in right than in left sectors. Cycle 12 is longest in the sector above the future blastopore and shortest in the animal region. The results show that the initial pattern of a regionally ordered sequence of cleavage cycles of equal duration changes into a pattern of cycles of different durations as a result of gradual cycle lengthening, starting in the region just above the future blastopore and spreading in animal direction. The results are compared with data on the cleavage cycles of isolated blastomeres, and the possible relation with the induction of the mesoendoderm occurring during the stages studied is discussed.     

The mechanism of human tyrosyl-DNA phosphodiesterase 1 in the cleavage of AP site and its synthetic analogs

The mechanism of hydrolysis of the apurinic/apyrimidinic (AP) site and its synthetic analogs by using tyrosyl-DNA phosphodiesterase 1 (Tdp1) was analyzed. Tdp1 catalyzes the cleavage of AP site and the synthetic analog of the AP site, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5′ adjacent phosphate. The product of Tdp1 cleavage in the case of the AP site is unstable and is hydrolyzed with the formation of 3′- and 5′-margin phosphates. The following repair demands the ordered action of polynucleotide kinase phosphorylase, with XRCC1, DNA polymerase β, and DNA ligase. In the case of THF, Tdp1 generates break with the 5′-THF and the 3′-phosphate termini. Tdp1 is also able to effectively cleave non-nucleotide insertions in DNA, decanediol and diethyleneglycol moieties by the same mechanism as in the case of THF cleavage. The efficiency of Tdp1 catalyzed hydrolysis of AP-site analog correlates with the DNA helix distortion induced by the substituent. The following repair of 5′-THF and other AP-site analogs can be processed by the long-patch base excision repair pathway.     

Phosphorylation-regulated cleavage of the reticulon protein Nogo-B by caspase-7 at a noncanonical recognition site

Reticulons (RTNs) are a large family of transmembrane proteins present throughout the eukaryotic domain in virtually every cell type. Despite their wide distribution, their function is still mostly unknown. RTN4, also termed Nogo, comes in three isoforms, Nogo-A, -B, and -C. While Nogo-A has been described as potent inhibitor of nerve growth, Nogo-B has been implicated in vascular remodeling and regulation of apoptosis. We show here that Nogo-B gets cleaved by caspase-7, but not caspase-3, during apoptosis at a caspase nonconsensus site. By a combination of MS and site-directed mutagenesis we demonstrate that proteolytic processing of Nogo-B is regulated by phosphorylation of Ser(16) within the cleavage site. We present cyclin-dependent kinase (Cdk)1 and Cdk2 as kinases that phosphorylate Nogo-B at Ser(16) in vitro. In vivo, cleavage of Nogo-B is markedly increased in Schwann cells in a lesion model of the rat sciatic nerve. Taken together, we identified an RTN protein as one out of a selected number of caspase targets during apoptosis and as a novel substrate for Cdk1 and 2. Furthermore, our data support a functionality of caspase-7 that is distinct from closely related caspase-3.     

细胞壁在细胞极性建立和胚胎发生中的作用

植物细胞壁是一个活性的动态结构,其结构层次与组分随着发育进程而发生变化,且广泛参与细胞的各项生命活动,特别是在参与细胞命运决定、充当细胞发育信使、调控植物胚胎早期极性建立以及模式建成等方面发挥重要作用。     

Localization and segregation of lineage-specific cleavage potential in embryos of Caenorhabditis elegans

Summary Early embryogenesis of the nematode Caenorhabditis elegans is characterized by the continuous visibility of a germline and the stepwise separation of all somatic cells from it. Germline and somatic cells exhibit different cleavage patterns. Typical for the germline is a series of stemcell-like, unequal cleavages generating blastomeres, which differ in size, cell cycle periods, and fate. Typical for members of somatic cell lineages during early development are their equal and synchronous cleavages generating cells of similar appearance. Using a laser microbeam various experiments have been carried out to investigate the conditions that lead to the two different types of cleavage. Development of partial embryos demonstrates that the potential for germline-like cleavage is localized in the posterior region of the fertilized egg prior to both the formation of pronuclei and the posterior aggregation of germline-specific granules. Experimental alteration of the cleavage plane can result in a switch from unequal to equal cleavage, with an apparent correlation between the orientation of the mitotic spindle and the type of cleavage. Nuclear transfer experiments indicate that nuclei and centrioles are not involved in the decision as to which type of cleavage will be executed. Cytoplasmic transfer from soma-like to germline-like cleaving cells and vice versa does not alter the cleavage type in the recipient cell. But if separation of germline from soma is delayed after the removal of a centrosome, germline-like cleavage may be completely suppressed, all cells thereafter dividing soma-like.Dedicated to Professor A. Egelhaaf on the occasion of his retirement     

Biogenesis of the posterior pole is mediated by the exosome/microvesicle protein-sorting pathway

Biogenesis of the posterior pole is critical to directed cell migration and other polarity-dependent processes. We show here that proteins are targeted to the posterior pole on the basis of higher order oligomerization and plasma membrane binding, the same elements that target proteins to exosomes/microvesicles (EMVs), HIV, and other retrovirus particles. We also demonstrate that the polarization of the EMV protein-sorting pathway can occur in morphologically non-polarized cells, defines the site of uropod formation, is induced by increased expression of EMV cargo proteins, and is evolutionarily conserved between humans and the protozoan Dictyostelium discoideum. Based on these results, we propose a mechanism of posterior pole biogenesis in which elevated levels of EMV cargoes (i) polarize the EMV protein-sorting pathway, (ii) generate a nascent posterior pole, and (iii) prime cells for signal-induced biogenesis of a uropod. This model also offers a mechanistic explanation for the polarized budding of EMVs and retroviruses, including HIV.     

The actin cytoskeleton is required for maintenance of posterior pole plasm components in the Drosophila embryo.

Localization of mRNAs is one of many aspects of cellular organization that requires the cytoskeleton. In Drosophila, microtubules are known to be required for correct localization of developmentally important mRNAs and proteins during oogenesis; however, the role of the actin cytoskeleton in localization is less clear. Furthermore, it is not known whether either of these cytoskeletal systems are necessary for maintenance of RNA localization in the early embryo. We have examined the contribution of the actin and microtubule cytoskeletons to maintenance of RNA and protein localization in the early Drosophila embryo. We have found that while microtubules are not necessary, the actin cytoskeleton is needed for stable association of nanos, oskar, germ cell-less and cyclin B mRNAs and Oskar and Vasa proteins at the posterior pole in the early embryo. In contrast, bicoid RNA, which is located at the anterior pole, does not require either cytoskeletal system to remain at the anterior.     

Isolation and characterisation of 26 microsatellite loci from a widespread tropical hydrozoan,Macrorhynchia phoenicea (Leptothecata,Aglaopheniidae), and cross-amplification in closely related species

We isolated and characterized 26 microsatellite loci for Macrorhynchia phoenicea (Busk, 1852), a rather common tropical hydrozoan from the Indo-Pacific. The number of alleles per locus ranged from 4 to 24. The observed heterozygosity ranged from 0.000 to 0.970 and the expected heterozygosity from 0.029 to 0.833. Ten loci were at Hardy–Weinberg equilibrium. No pair of loci presented linkage disequilibrium. Transferability of up to 18 loci was positive across four other Aglaopheniidae species from different genera. These loci will be used in studying reef population connectivity for these particular species at the scale of the Indo-Pacific, a promising but little explored research field.     

A posterior centre establishes and maintains polarity of the Caenorhabditis elegans embryo by a Wnt-dependent relay mechanism

Cellular polarity is a general feature of animal development. However, the mechanisms that establish and maintain polarity in a field of cells or even in the whole embryo remain elusive. Here we provide evidence that in the Caenorhabditis elegans embryo, the descendants of P₁, the posterior blastomere of the 2-cell stage, constitute a polarising centre that orients the cell divisions of most of the embryo. This polarisation depends on a MOM-2/Wnt signal originating from the P₁ descendants. Furthermore, we show that the MOM-2/Wnt signal is transduced from cell to cell by a relay mechanism. Our findings suggest how polarity is first established and then maintained in a field of cells. According to this model, the relay mechanism constantly orients the polarity of all cells towards the polarising centre, thus organising the whole embryo. This model may also apply to other systems such as Drosophila and vertebrates.     

The cleavage rate of digynic triploid mouse embryos during the preimplantation period

Triploidy is a lethal condition in mammals, with most dying at some stage between implantation and term. In humans, however, a very small proportion of triploids are liveborn but display a wide range of congenital abnormalities. In particular, the placentas of human diandric triploid embryos consistently display “partial” hydatidiform molar degeneration, while those of digynic triploids generally do not show these histopathological features. In mice, the postimplantation development of diandric and digynic triploid embryos also differs. While both classes are capable of developing to the forelimb bud stage, no specific degenerative features of their placentas have been reported. Diandric triploid mouse embryos are morphologically normal while digynic triploid mouse embryos consistently display neural tube and occasionally cardiac abnormalities. Previously it was shown that the preimplantation development of micromanipulated diandric triploid mouse embryos was similar to developmentally matched diploid control embryos. In this study, the preimplantation development of micromanipulated digynic triploid mouse embryos is analysed and compared with that of diandric triploid mouse embryos in order to determine whether there is any difference in cleavage rate between these two classes of triploids. Standard micromanipulatory procedures were used to insert a female or a male pronucleus into a recipient diploid 1-cell stage embryo. The karyoplast was fused to the cytoplasm of the embryo by electrofusion. These tripronucleate 1-cell stage embryos were then transferred to pseudopregnant recipients and, at specific times after the HCG injection to induce ovulation, the embryos were recovered and total cell counts made. These results were plotted and regression lines drawn. An additional control group of embryos was subjected to similar micromanipulatory procedures to those used in the experimental study. These embryos had a single pronucleus removed and this was then reinserted into the perivitelline space. Diploidy was immediately restored by electrofusion. These embryos were transferred to recipients and at specific times after the HCG injection the embryos were recovered and total cell counts made. These results were also plotted and regression lines drawn. The results show that the cell doubling time of the digynic triploid embryos was 14.84 h (± 1.19). This was not significantly different from that of the diandric triploid embryos (13.55 h ± 0.86; P > 0.05) or of the manipulated diploid controls (12.12 h ± 0.79; P > 0.05). © 1993 Wiley-Liss, Inc.     

The importance of the posterior midline region for axis initiation at early stages of the avian embryo

The avian blastoderm acts during its early stages of development as an integrative system programmed to form a single embryonic axis. Here, I report the results of a variety of transplantation experiments of the midline region at stages X-XII, which were carried out to study their relevance for axis initiation. The results of the experimental series discussed herein emphasizes the importance of the posterior midline region (including the marginal zone and Koller's sickle) for axis initiation. This ability resides mainly at stage X in the posterior side of a narrow midline region, while at stages XI-XII it is exhibited at the region which is located more anterior and lateral to the posterior midline region. This posterior midline region has developmental abilities which allow it to initiate a single embryonic axis and at the same time to prevent other regions that also have such abilities to do so. Therefore, in normal development only one embryonic axis develops in the avian blastoderm. It is proposed that the cells which are important to initiate the avian embryonic axis are concentrated mainly at the region of the posterior midline region. These cells may have organizer properties which determine the initiation site of the axis in the avian embryo.     

Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo

 The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking. Isolated wheat (Triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured in a hormone-free, maltose-containing medium in the presence of ovaries serving as a conditioning factor. Embryogenesis was followed in microspores isolated from immature anthers of freshly cut tillers or from heat- and starvation-treated, excised anthers. Three types of microspore were identified on the basis of their cytological features at the start of culture. Type-1 microspores had a big central vacuole and a nucleus close to the microspore wall, usually opposite to the germ pore. This type was identical to the late microspore stage in anthers developing in vivo. Microspores with a fragmented vacuole and a peripheral cytoplasmic pocket containing the nucleus were defined as type 2. In type-3 microspores the nucleus was positioned in a cytoplasmic pocket in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores first developed into type 2 and then into type-3 microspores. After a few more days, type-3 microspores absorbed their vacuoles and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures. Apparently the three types of microspore represent stages in a continuous process and not, as previously assumed, distinct classes of responding and non-responding microspores. The first cell division of the embryogenic microspores was always symmetric. Cell tracking also revealed that the original microspore wall opened opposite to a region in the multicellular microspore which consisted of cells containing starch grains while the remaining cells were starch grain-free. The starch-containing cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detected at the root pole of the embryo. Received: 27 December 1999 / Accepted: 11 May 2000     

Unusual cleavage and gastrulation in a freshwater nematode: developmental and phylogenetic implications

Early embryogenesis in nematodes as seen in Caenorhabditis elegans and many other species of this phylum features several characteristic events. These include the visible presence of a germline from the very beginning generating different somatic lineages via asymmetric cleavages, the absence of a coeloblastula and a unique type of gastrulation with immigration of just two gut precursor cells. Here it is shown by using Nomarski optics that development of the freshwater nematode Tobrilus diversipapillatus differs from this pattern in two prominent aspects. (1) No asymmetric cleavages and no distinct cell lineages are generated; (2) in contrast to all other nematodes studied so far, a prominent coeloblastula is formed and gastrulation resembles the classical pattern found all over the animal kingdom. These developmental peculiarities are considered to be plesiomorphic and thus the order Triplonchida, to which Tobrilus belongs, may occupy a phylogenetic position at the base of the nematode phylum. The findings reported here allow us to reject a number of conceivable correlations between the type of gastrulation and other developmental parameters.Edited by D. TautzThis revised version was published online in December 2004 with corrections to typographical errors in the abstract.     

The cleavage pattern of the axolotl egg studied by cinematography and cell counting

Summary The temporal pattern of cleavage in the egg of the axolotl,Ambystoma mexicanum, was studied 1. by time-lapse microcinematography, and 2. by counting the total number of blastomeres dissociated at successive stages.Eggs were filmed from the one-cell stage till the early gastrula either (A) simultaneously from above and below with a double-camera assembly, or (B) from the side with a single camera.The animal blastomeres divide synchronously from the 2nd up to and including the 10th cleavage. The cycle length is roughly constant from the 3rd till the 10th cleavage. The cycle from the 2nd to the 3rd cleavage is slightly longer, while that from the 1st to the 2nd cleavage is about 20% longer. After the 10th cleavage the synchrony of divisions is lost owing to variable lengthening of cell cycles in individual blastomeres. Gastrulation starts around the onset of the 15th cleavage in the animal blastomeres.The analysis of films taken in side view reveals seven recurring cleavage waves, from the 5th till the 11th cleavage. Cells in the animal, equatorial and vegetative regions in sequence repeatedly pass through the three successive phases of the cleavage cycle—rounding-up, division, and relaxation—but with a shift in phase. The start of the 10th cleavage division of the slowest vegetative cells more or less coincides with that of the 11th division of the animal cells; from then on the cleavage waves become increasingly obscured.Morulae and blastulae were dissociated by placing them in 1/15 M phosphate buffer (pH 7.8) for the duration of 2–3 cleavage cycles and then removing the vitelline membrane. In this solution cell divisions continued without disturbance of the temporal cleavage pattern. The dissociated cells were fixed either just prior to the onset of the next cleavage (up to the 10th cleavage) or at those times when cleavageswould have been expected, had there been no lenthening of cleavage cycles (beyond the 10th cleavage). The total cell number was counted, dividing cells being scored as two.Prior to the 11th cleavage the total cell number increased exponentially. Beyond the 10th cleavage the rate of increase was considerably lower. At the time when gastrulation would have started if the egg had not been dissociated, the total cell counts were 13,000–15,000, whereas the number anticipated without lengthening of cleavage cycles would be of the order of 130,000 (2¹⁷).The application of Balfour's rule to amphibian eggs is criticized.