Evolutionary change in the process of dorsoventral axis determination in the direct developing sea urchin, Heliocidaris erythrogramma

Embryos of the indirect developing sea urchin, Heliocidaris tuberculata, and of Heliocidaris erythrogramma which develops directly without the formation of a pluteus larva, were bisected at the two- and four-cell stages. Paired half-embryos resulting from the bisection of H. tuberculata embryos along either the first or the second cleavage plane develop identically into miniature prism stage larvae. As in other indirect developing sea urchins, no differential segregation of developmental potential takes place as a result of the first and second cleavage divisions. Although half-embryos resulting from bisection along the second cleavage plane differentiate all cell types and develop equivalently in H. erythrogramma, the isolated first cleavage blastomeres do not. One of these two cells always forms significantly more mesodermal and endodermal cells. These patterns of differentiation are consistent with fate-mapping studies indicating that most mesodermal and endodermal cells are derived from the prospective ventral blastomere. Therefore, a differential segregation of developmental potential takes place at the first cleavage division in H. erythrogramma. When embryos of H. erythrogramma were bisected during the eight-cell stage, isolated tiers of animal blastomeres typically formed only ectodermal structures including the vestibule, whereas vegetal embryo halves formed all differentiated cell types. We propose that animal-vegetal cell determination and differentiation takes place along an axis which has been shifted relative to the pattern of cell cleavages in the embryos of H. erythrogramma. Vegetal morphogenetic potential for the formation of mesodermal and endodermal structures has become more closely associated with the prospective ventral side of the embryo during the evolution of direct development in Heliocidaris.     

Regional specification in the early embryo of the brittle star Ophiopholis aculeata

Early embryogenesis has been examined experimentally in several echinoderm and hemichordate classes. Although these studies suggest that the mechanisms which underlie regional specification have been highly conserved within the echinoderm + hemichordate clade, nothing is known about these mechanisms in several other echinoderm classes, including the Ophiuroidea. In this study, early embryogenesis was examined in a very little studied animal, the ophiuroid Ophiopholis aculeata. In O. aculeata, the first two cleavage planes do not coincide with the animal-vegetal axis but rather form approximately 45 degrees off this axis. A fate map of the early embryo was constructed using microinjected lineage tracers. Most significantly, this fate map indicates that there is a major segregation of ectodermal from endomesodermal fates at first cleavage. The distribution of developmental potential in the early embryo was also examined by isolating different regions of the early embryo and following these isolates though larval development. These analyses indicate that endomesodermal developmental potential segregates unequally at first, second, and third cleavage in O. aculeata. These results provide insight into the mechanisms of regional specification in O. aculeata and yield new material for the study of the evolution of echinoderm development.     

PM-2: an ECM epitope necessary for morphogenesis in embryos of the starfish, Pisaster ochraceus

Anti-PM-2 is a monoclonal antibody that has been developed against the ECM of embryo/larvae of the starfish Pisaster ochraceus. Immunofluorescent staining shows that the PM-2 epitope is present in the cortical granules of unfertilized eggs and is released into the perivitelline space on fertilization. At the blastula stage, staining is very faint and limited to the blastocoel and a few granules within the cells. Strong staining appears in the embryonic/larval body cavity shortly after gastrulation and continues to increase in both the embryonic/larval body cavity and lumen of the gut at least until the bipinnaria stage. The presence of PM-2 in the Golgi apparatus, its susceptibility to enzymes that attack carbohydrates, and inhibition of PM-2 synthesis by tunicamycin, a drug that inhibits the linkage of carbohydrate moieties to protein backbone chains, suggest that the PM-2 epitope is or contains carbohydrate. Western blots of the whole embryo homogenates show bands at molecular weights of 130, 122, 100, 70, and 50 kDa. As embryos grow, two other high molecular weight (greater than 200 kDa) bands also appear. This suggests that the epitope is present on a series of molecules and that some of the lower MW molecules are precursors of the higher MW ones. A single 24-h exposure to the antibody just posthatching appears to inhibit normal mesenchymal migration at the gastrula stage, and if development of these treated embryos/larvae is allowed to continue to the bipinnaria stage, the embryos are stunted and have a smaller oral hood and esophagus. Long-term exposure results in stunted animals with distorted shapes. Such animals develop a very small embryonic/larval body cavity or none at all and differentiation of the larval GI tract fails to occur. The results suggest that molecules exhibiting the PM-2 epitope are necessary for the proper formation of the blastocoel, for mesenchyme cell movement and for proper development of the larvae GI tract.     

Heterogeneous nuclear-transferred-embryos reconstructed with camel (Camelus bactrianus) skin fibroblasts and enucleated ovine oocytes and their development H-M

This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and ovine oocytes as recipient cytoplasts for investigating the developmental potential of the reconstructed embryos. Serum-starved adult camel skin fibroblast cells were used as donor somatic cells. Ovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electrofusion. The fused oocytes were activated by 5mM/ml inomycin with 2mM/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168h. A total of 300 enucleated ovine oocytes were available for xenonuclear embryo reconstruction. The results showed that 71% of the nuclear transfer couplets were successfully fused, 55% of the fused oocytes cleaved within 48h after activation, 82% of the cleaved oocytes developed to 2-16-cell embryo stages and 18% of the cleaved nuclear transfer zygotes developed to the morula stage. This study demonstrated that the xenonuclear transfer camel embryos can undergo the first embryonic division and subsequent development to morula stage in vitro.     

Cyto-differentiation of the neuroblasts from the neural ectodermal cells in grasshopper,Chortophaga viridifasciata (De Geer) (Orthoptera: Acrididae) embryos

The first sign of neurogenesis in the embryo of grasshopper, Chortophaga viridifasciata (Orthoptera: Acrididae), is signaled by a partition of the ectodermal cells into non-neural ectodermal cells and neural eetodermal cells. The neuroblasts are differentiated from neural ectodermal cells. In the present study, we examined the pattern of mitotic activity in the developing embryo by tracing the incorporation of BrdU in S phase nuclei. The results indicate that the ectodermal cells in 6-day old embryos do not show any signs of differentiation. In 7-day old embryos, in which ectodermal cells become partitioned into 2 types, almost no neural ectodermal cells are incorporated with BrdU, whereas a constant incorporation is revealed in non-neural ectodermal cells. Among the mitotically quiescent neural ectodermal cells, which are arrested at the GI stage of the cell cycle, in 8-day old embryos, the neuroblasts are the first to resume their mitotic activity, while the other cells are then released from the mitotic quiescence. It seems that the mitotic quiescence may be an essential process to acquire a neural fate.     

3D-DIASemb: a computer-assisted system for reconstructing and motion analyzing in 4D every cell and nucleus in a developing embryo

A computer-assisted three-dimensional (3D) system, 3D-DIASemb, has been developed that allows reconstruction and motion analysis of cells and nuclei in a developing embryo. In the system, 75 optical sections through a live embryo are collected in the z axis by using differential interference contrast microscopy. Optical sections for one reconstruction are collected in a 2.5-s period, and this process is repeated every 5 s. The outer perimeter and nuclear perimeter of each cell in the embryo are outlined in each optical section, converted into beta-spline models, and then used to construct 3D faceted images of the surface and nucleus of every cell in the developing embryo. Because all individual components of the embryo (i.e., each cell surface and each nuclear surface) are individually reconstructed, 3D-DIASemb allows isolation and analysis of (1) all or select nuclei in the absence of cell surfaces, (2) any single cell lineage, and (3) any single nuclear lineage through embryogenesis. Because all reconstructions represent mathematical models, 3D-DIASemb computes over 100 motility and dynamic morphology parameters for every cell, nucleus, or group of cells in the developing embryo at time intervals as short as 5 s. Finally, 3D-DIASemb reconstructs and motion analyzes cytoplasmic flow through the generation and analysis of "vector flow plots." To demonstrate the unique capabilities of this new technology, a Caenorhabditis elegans embryo is reconstructed and motion analyzed through the 28-cell stage. Although 3D-DIASemb was developed by using the C. elegans embryo as the experimental model, it can be applied to other embryonic systems. 3D-DIASemb therefore provides a new method for reconstructing and motion analyzing in 4D every cell and nucleus in a live, developing embryo, and should provide a powerful tool for assessing the effects of drugs, environmental perturbations, and mutations on the cellular and nuclear dynamics accompanying embryogenesis.     

Effect of sodium dodecyl sulfate on polar lobe formation and function in Ilyanassa obsoleta embryos

Polar lobes, anucleate vegetal pole protrusions formed by Ilyanassa obsoleta embryos, serve as a mechanism for shunting morphogenetic determinants to one cell during the first two cleavages. Polar lobe material becomes segregated in the CD cell during first cleavage and in the D cell during second cleavage, resulting in a very unequal four-cell stage. Larval structures including external shell, foot, operculum, statocysts, and eyes develop only when polar lobe material is present. Treatment with the anionic detergent sodium dodecyl sulfate (SDS) before and during the first cleavage inhibited polar lobe formation and equalized cleavage, as the lobe material was distributed to two cells. No polar lobes formed during second clevage in SDS-equalized embryos, and the four-cell stage consisted of four equal cells with reduced cell contacts. SDS inrreversibly inhibited polar lobe formation without affecting cytokinesis. Although 27% of the larvae from SDS-equalized embryos had one or more lobe-dependent structures duplicated, morphogenesis was impaired: more than 40% of such larvae failed to form shell and/or statocysts. When cells were separated after equalized first cleavage and raised as pairs, the pairs of resulting larvae duplicated lobe-dependent structures with the same frequency as whole equalized embryos. Possible explanations for impaired morphogenesis in SDS-treated embryos are discussed.     

3D confocal reconstruction of gene expression in mouse

Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.     

Membrane glycoproteins immunologically related to the human insulin receptor are associated with presumptive neuronal territories and developing neurones in Drosophila melanogaster

We have generated a monoclonal antibody (Mab E1C) that recognizes the differentiated nervous system in Drosophila embryos. At the cellular blastoderm stage, Mab E1C behaves as a general ectodermal marker but, in subsequent stages, it also labels the mesoderm. As neurogenesis takes place, staining increases within the neuromeres and is almost exclusively restricted to the nervous tissue by the time neuronal differentiation is completed. In third instar larvae, Mab E1C stains the central nervous system (CNS) as well as the imaginal discs which display a staining pattern related to their degree of neuronal differentiation. No labelling can be detected in adult brains or ovaries. Western blots are consistent with this developmental profile and allow the characterization of a major glycoprotein of 135 X 10(3) Mr (135K) which cosediments with a membrane fraction prepared from embryos. Additional glycoproteins (100K and 80K) are extracted from embryo homogenates by immunoaffinity procedures. In larvae, the 100K polypeptide is not detected. The properties of the 135K and 100K components are highly reminiscent of the molecular pattern of the Drosophila insulin receptor homologue (Petruzzelli et al. (1985) J. biol. Chem. 250, 16072-16075). It is shown that a Mab directed against the human insulin receptor stains the same cells as Mab E1C in imaginal discs and in the CNS. Moreover, this Mab cross-reacts with the 135K and 100K components of the embryonic antigen E1C.     

Muscle lineage cytoplasm can change the developmental expression in epidermal lineage cells of ascidian embryos

Cytoplasm from muscle lineage blastomeres of an ascidian embryo can cause cells of a nonmuscle lineage to produce larval tail muscle acetylcholinesterase. Muscle cytoplasm was partitioned microsurgically into epidermal lineage blastomeres at the eight-cell stage. Posterior half-embryos (the two B3 cells) of Ascidia nigra were obtained first by separating the anterior and posterior blastomere pairs at the four-cell stage. At third cleavage, the two B3 cells divide into an ectodermal cell pair that gives rise solely to epidermal tissues, and a mesodermal-endodermal blastomere pair from which the tail muscle cells are derived. When the ectodermal and mesendodermal blastomere pairs were isolated from one another by microsurgery and reared as partial embryos, only cells originating from the mesendodermal blastomeres produced a histochemical acetylcholinesterase reaction. Immediately after cleavage of the isolated B3 cells into ectodermal and mesendodermal cell pairs, the cleavage furrows could be made to disappear by pressing firmly on the mesendodermal cells with a microneedle. Repeated up and down pressure with the microneedle at a new position across the mesendodermal cells caused furrows to reestablish in the new position, thereby incorporating mesodermal cytoplasm and increasing the size of the ectodermal cells. The cytoplasmically altered ectodermal blastomere pairs, which became detached from the mesendodermal cells by this microsurgical procedure, continued to divide and were reared to “larval” stages. One-third of these epidermal partial larvae produced patches of cells containing acetylcholinesterase. These results lend further support to the theory that choice of particular differentiation pathways (embryonic determination) in ascidian embryos is mediated by segregation of specific egg cytoplasmic determinants.     

Adenosine induces dormancy in starfish blastulae

External application of 50 micrograms ml-1 adenosine inhibits development of the starfish Asterina pectinifera at the 256-cell stage when all the embryonic cells differentiate to epithelial cells. Intracellular concentration of adenosine in the adenosine-treated embryo is 2.7 times higher than those of the normal embryo whereas the contents of ATP, ADP, AMP and adenosine 3',5'-monophosphate are the same for both embryos. Adenosine causes more than 95% reduction in the rate of protein, DNA and RNA syntheses. By returning the embryo to normal sea water, macromolecular synthesis restarts and the embryo develops to the bipinnaria stage.     

The role of unequal cleavage and the polar lobe in the segregation of developmental potential during first cleavage in the embryo of Chætopterus variopedatus

Summary The inequality of the first cleavage division of the Chætopterus embryo is caused by the production of a small polar lobe and the internal shifting of the first cleavage spindle. This division produces a two-celled embryo containing a small AB and a large CD blastomere. These blastomeres have different morphogenetic potentials. Only the larvae resulting from isolated CD blastomeres are able to form bioluminescent photocytes, eyes and lateral hooked bristles. The removal of the polar lobe during first cleavage does not have a great effect on development. These lobeless embryos display a normal pattern of cleavages through the time of mesentoblast formation. The resulting larvae are essentially normal, however they do not form functional photocytes. If the CD cell is isolated after the removal of the first polar lobe, the resulting larva is virtually identical to those formed by the intact CD cell except it lacks the photocyte cells. These results indicate that two separate pathways are involved in the segregation of developmental or morphogenetic potential which takes place during first cleavage. One set of factors, which are necessary for photocyte formation, are associated with the first polar lobe. Other factors that are necessary for the formation of the eyes and lateral hooked bristles are segregated by the unequal cleavage which results from an internal shifting of the cleavage spindle. The removal of a large portion of the vegetal region of the embryo during first cleavage leads to the production of larvae which display a decreased ability to form eyes and lateral hooked bristles. These embryos frequently display an abnormal pattern of cleavages. They do not form the primary somatoblast or the mesentoblast. These results indicate that the vegetal region of the CD cell of Chætopterus is analogous to polar lobes which have been studied in other species, and is therefore important in the specification of the D quadrant. These features of the first cleavage of Chætopterus are a combination of those displayed by forms with direct unequal cleavage and other forms which cleave unequally through the production of large polar lobes. The significance of these findings is discussed relative to the origins of these different types of unequal cleavage.     

Induction of neuron-specific glycosylation by Tollo/Toll-8, a Drosophila Toll-like receptor expressed in non-neural cells

Specific glycan expression is an essential characteristic of developing tissues. Our molecular characterization of a mutation that abolishes neural-specific glycosylation in the Drosophila embryo demonstrates that cellular interactions influence glycan expression. The HRP epitope is an N-linked oligosaccharide expressed on a subset of neuronal glycoproteins. Embryos homozygous for the TM3 balancer chromosome lack neural HRP-epitope expression. Genetic and molecular mapping of the relevant locus reveals that Tollo/Toll-8, a member of the Toll-like receptor family, is altered on the TM3 chromosome. In wild-type embryos, Tollo/Toll-8 is expressed by ectodermal cells that surround differentiating neurons and precedes HRP-epitope appearance. Re-introduction of Tollo/Toll-8 into null embryos rescues neural-specific glycan expression. Thus, loss of an ectodermal cell surface protein alters glycosylation in juxtaposed differentiating neurons. The portfolio of expressed oligosaccharides in a cell reflects its identity and also influences its interactions with other cells and with pathogens. Therefore, the ability to induce specific glycan expression complements the previously identified developmental and innate immune functions of Toll-like receptors.     

Remodeling of Centrosomes in Intraspecies and Interspecies Nuclear Transfer Porcine Embryos

Remodeling of donor cell centrosomes and the centrosome-associated cytoskeleton is crucially important for nuclear cloning as centrosomes are the main microtubule organizing centers that play a significant role in cell division and embryo development. Centrosome dysfunctions have been implicated in various diseases including cancer and metabolic disorders and may also play a role in developmental abnormalities that are frequently seen in cloned animals. In the present studies we investigated microtubule organization and the reorganization and fate of the integral centrosome protein γ-tubulin and the centrosome-associated protein centrin in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using antibodies to γ-tubulin or GFP-centrin transfected mouse fibroblasts as donor cells. Microtubules were stained with antibodies to α-tubulin. In-vitro-fertilized oocytes and nuclear transfer (NT) reconstructed oocytes were sequentially analyzed at different developmental stages. Epi-fluorescence results revealed mitotic spindle abnormalities in NT embryos during the first cell cycle (39.4%, 13/33) which were significantly higher than those in IVF embryos (17.0%, 7/41). The abnormalities in IVF embryos are due to polyspermy while the abnormalities in NT embryos are due to donor cell centrosome dysfunctions. In the NT embryos with abnormal microtubule and centrosome organization, γ-tubulin staining revealed multipolar centrosome foci while DAPI staining showed misalignment of chromosomes. In intraspecies and interspecies embryos the GFP-centrin signal was detected until 3 hrs after fusion. GFP-centrin was not detected at 8 hrs after NT which is consistent with previous results using anti-centrin antibody staining in intraspecies NT porcine embryos. These data indicate that 1) abnormalities in microtubule and centrosome organization are associated with nuclear cloning at a higher rate than observed in IVF embryos; 2) centrosome and cytoskeletal abnormalities in IVF embryos are due to polyspermy while centrosome and cytoskeletal abnormalities in NT embryos are due to donor cell centrosome dysfunctions; and 3) GFP-centrin of the donor cell centrosome provides a reliable marker to follow its fate in intraspecies reconstructed embryos.     

Early inductive interactions are involved in restricting cell fates of mesomeres in sea urchin embryos

Isolated intact caps of animal blastomeres, obtained from either 8- or 16-cell embryos, differentiate as swollen ectodermal vesicles. These findings agree with earlier studies demonstrating that mesomeres contribute only to larval ectoderm during normal development. In contrast, we find that pairs of mesomeres isolated from 16-cell embryos can differentiate endodermal and mesenchymal cells in a substantial number of cases (23%). Thus, mesomeres have a greater developmental potential than is realized during normal development. Further results support hypotheses that graded distributions of morphogenetic determinants exist within these embryos, since the extent of differentiation of isolated mesomeres is related to the relative position of the third cleavage plane along the animal-vegetal axis. When the third cleavage plane is subequatorial and the resulting animal blastomeres inherit a fraction of the vegetal hemisphere, more cases (39%) differentiate endodermal and mesenchymal cell types. A significant number of mesomere pairs (9-14%), however, can still differentiate endodermal and mesenchymal cells when the mesomeres are formed within the animal hemisphere. Thus, putative vegetal morphogenetic determinants may extend into the animal hemisphere in some cases. Further results indicate a temporal restriction in the developmental potential of mesomeres or mesomere progenitor cells since their differentiative capability is greater if they are isolated earlier during development. Aggregates of isolated mesomere pairs also display a decreased developmental potential when compared to isolated mesomere pairs. These results suggest that associations with adjacent cells (vegetal cells as well as adjacent mesomeres) restrict the development of mesomeres between third and sixth cleavages.