Degradation of yolk proteins in sea urchin eggs and embryos

Yolk granules isolated from unfertilized and fertilized eggs of the sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina, were incubated in acidic media, and the protein components were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By the incubation, a protein (molecular weight 180,000 in H. pulcherrimus and 178,000 in A. crassispina) most abundant in unfertilized eggs decreased, while proteins (molecular weight 61,000, 72,000, 94,000, 114,000 in H. pulcherrimus and 56,000, 70,000, 92,000, 112,000 in A. crassispina) dominant in developed embryos increased. Neither alkaline nor neutral condition resulted in such changes in the electrophoretic patterns of proteins as observed in acidic media. Experiments with various inhibitors of proteases suggested that thiol protease(s), such as cathepsin B, may be the most important enzyme(s) in the degradation of yolk proteins in embryogenesis of the sea urchin.     

Ultraweak emissions of the developing Xenopus laevis eggs and embryos

We measured ultraweak emissions of the Xenopus laevis eggs and embryos during normal development and under the influence of stress factors in a spectral range of 250 to 800 nm using a photomultiplier. The registered emissions were analyzed by several basic characteristics: mean intensity, histograms, kurtosis, linear trends, and Fourier spectra. We followed relationships between these parameters and developmental stage, as well as the number of individuals in optic contact with each other. The ultraweak emissions did not differ from the background at all developmental stages according to the mean intensity. But Fourier analysis revealed the reliable presence of a number of spectral lines of ultraweak emission, predominantly in the ranges of 10-20 and 30-40 Hz, in the embryos at developmental stages 2 to 11. The intensity of ultraweak emissions reliably decreased within the first 10 min after egg activation and fertilization, as well as in the case of optic interaction between groups of embryos. Sharp cooling, increase in osmotic medium pressure, and transfer in a Ca(2+)- and Mg(2+)-free medium induced a short term (approximately 1-5 min) increase in the mean intensity of ultraweak emission. We studied specific features of ultraweak emissions from different parts of the embryo. The intensity of emission from the animal part of early blastula exceeded those from the vegetal area and entire embryo. Separated fragments of the lateral ectoderm at the neurula stage had higher mean intensities of ultraweak emission than intact embryos at the same developmental stages.     

Changes in ribosomal proteins in developing Artemia salina embryos

Ribosomal proteins from cysts and nauplii of Artemia salina were analyzed by three kinds of two-dimensional polyacrylamide gel electrophoresis. The basic-acidic and basic-SDS gel systems were used to compare the basic ribosomal proteins, and some changes were observed between the cysts and nauplii in proteins S6, S14, and L24. The phosphorylation of protein S6 was increased in the nauplii. Basic proteins S14 and L24 in the cysts changed and none of the corresponding proteins in the nauplii were detected at the same positions on two-dimensional gels as in the cysts. The acidic-SDS gel system was used to compare the acidic proteins in ribosomes and it was revealed that an acidic protein, AX (Mr = 24,000), in the cysts was not present in the ribosomes from the nauplii. The ribosomal activities as to the formation of an 80S initiation complex with globin mRNA and poly(U)-directed polyphenylalanine synthesis were compared. There was no significant difference between the cyst and nauplius ribosomes.     

Two actin variants in developing axolotl heart

Heart development in the Mexican salamander, Ambystoma mexicanum has been studied using two-dimensional gel electrophoresis and electron microscopy. At the preheart beat stage two forms of action, α and β, are present in the heart in approximately equal quantity, however, very few definitive thin filaments can be distinguished at this stage. Once the heart beat is initiated and heart development progresses α-actin increases relative to β-actin. This increase in muscle-specific actin is coincidental with the appearance of numerous thin filaments in the myocyte.     

Spatial distribution of abundant proteins in oocytes and fertilized eggs of the Mexican axolotl (Ambystoma mexicanum)

Two-dimensional protein patterns were compared from sections along the longitudinal axis of oocytes and fertilized eggs of the Mexican axolotl (Ambystoma mexicanum). Only a few differences were observed between four different sections through both oocyte and fertilized eggs. A set of proteins (14 out of 120 proteins) were found that reside only in the germinal vesicles (GV) of the fully grown oocyte. Two of these were observed exclusively in the vegetal half, and one in the animal half after GV breakdown, while other proteins were randomly distributed within the fertilized egg. One cytoplasmic protein was present only in the vegetal half of the mature oocyte and became present also in the animal half of the fertilized egg. Additional proteins were observed in all transverse sections of both mature oocyte and fertilized eggs. It is proposed that these proteins are modified rather than newly synthesized proteins.     

Messenger RNA for ribosomal proteins in dormant and developing Artemia salina embryos

Ribosomal proteins from Artemia salina have been separated in a two-dimensional acrylamide gel system and assigned to the small and large ribosomal subunits. Poly(A)-containing RNA was prepared from dormant cysts and from polysomes of 30 min, 1 h, 5 h and 12 h embryos and hatching larvae. The mRNA from different stages was translated in a wheat-germ lysate and its template activity for ribosomal proteins was analyzed. It was observed that mRNA activity for ribosomal proteins is stored in the cytoplasm of dormant cysts and that it is found associated with polysomes of 30 min and of later stages.     

Developmentally regulated lectin in dark versus white axolotl embryos

The white mutant of the Mexican axolotl, A. mexicanum, involves an ectodermal defect which prevents melanophore colonization. Endogenous lectins have been suggested to function in neural crest-derived melanophore adhesion in other animals. To determine if differences in endogenous lectins exist in dark and white axolotls during melanophore colonization, white and dark ectoderm and carcass tissues have been assayed for lectin activity at premigratory, early migratory, and late migratory neural crest stages. Lectin content (specific for D-glucosamine, N-acetyl-D-glucosamine and D-mannose) increases significantly during early migration only in dark ectoderm and white carcass tissues, whereas white ectoderm and dark carcass lectin activities remain close to premigration levels. Neural crest cells in these embryos are associated with regions of high lectin activity suggesting that the differences in endogenous lectins may be involved in establishment of the dark/white phenotype.     

Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs

We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s-1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus.     

Expression of the cardiac actin gene in axolotl embryos

Axolotis are an important model system for studying heart development. Patterning of the somitic mesoderm occurs in axolotis in a manner that is much more similar to the pattern observed in higher vertebrates than in Xenopus. For these reasons we cloned the axolotl cardiac actin gene, since this gene is expressed during the development of both somitic and cardiac muscle in other vertebrates. In this paper we characterize its expression. Expression of cardiac actin RNA is switched on during gastrula stages and appears in the somitic mesoderm when it is formed; expression is later activated in the embryonic heart. In adults the gene is expressed only in the heart. The results demonstrate that the clone encoding cardiac actin provides a useful marker for studying development of both skeletal and cardiac muscle development in axolotls.     

Analysis of proteins secreted by mouse embryos developing in vivo and in vitro

Proteins secreted by mouse blastocysts developing in vitro were compared to these from blastocysts developing in utero to determine if a simple medium supporting blastocyst development also supports secreted protein expression. In-vivo embryos were collected on days 3, 4, or 5 of pregnancy and incubated in 35S-methionine to produce conditioned medium containing released, labeled proteins. Embryos for culture were collected on day 3 and after 48 or 72 h labeled conditioned medium was produced. Labeled proteins were separated by two-dimensional electrophoresis and compared using a digital image analysis system. Day 3 embryos did not release proteins in detectable amounts, although synthesis of intracellular proteins was substantial. Day-4 and -5 blastocysts released proteins in increasing amount and complexity, consistent with previous results. When day-3 embryos were cultured in medium containing 4 mg/ml BSA for 48 h, secreted protein patterns were similar but not identical to those of day-5 uterine blastocysts. Although most of the proteins produced by uterine blastocysts were secreted by cultured embryos, differences were found in the relative quantities of certain proteins. Neither crystallized BSA nor polyvinyl alcohol at 4 mg/ml supported development of protein secretion as well as the crude fraction-V BSA. Blastocysts restricted to the oviduct also exhibited quantitative differences in protein secretion patterns compared to uterine blastocysts. Thus, although blastocyst development and the expression of many secreted proteins are supported outside the uterus, the full pattern of secretion characteristic of the peri-implantation embryo may be dependent on specific uterine influences.     

Microtubule assembly in developing mammalian brain.

Microtubule protein was measured in mouse brain homogenates by quantitative colchicine binding. Neonatal animals contained more than twice the amount of brain tubulin as adult mice. The percentage of colchicine-binding protein which was polymerized was determined by extracting brain at room temperature into a medium designed to stabilize intact microtubules. Under identical conditions and tubulin concentrations, neonatal brain tubulin (colchicine-binding activity) had a greater proportion of the total extracted in an apparently polymerized state (pelletable by centrifugation) than did adult brain. A slight variation in the ratio of assembled to unassembled tubulin was observed with varying protein concentration (volume of extract), indicating that the values obtained may not reflect exactly the in vivo situation, because a rapid equilibration takes place upon homogenization. At all protein concentrations, the neonatal brain extracts contained a significantly greater proportion of assembled tubulin than did adult brain. This proportion began to fall at 5 days postnatal and reached the adult level at 30 days. The tubulin assembled/not assembled ratios were not altered by addition of nucleoside triphosphates, additional EGTA, or sulfhydryl protecting agents, and did not vary with preparation times of 30–90 min. The colchicine-binding reaction and decay of colchicine-binding activity with time were similar in extracts of different aged mouse brains, with neonatal slightly more stable than adult. Pools of tubulin from any age which were soluble at room temperature (unpolymerized) could not repolymerize well in vitro when incubated with GTP at 37 °C, whereas pools of tubulin which were sedimentable at room temperature (polymerized) could be redissolved at 0 °C and readily reassembled at 37 °C. The neonatal extract tubulin was thus more polymerization competent than the adult extracts; this correlates with a greater proportion of assembled tubulin in extracts at room temperature and possibly in vivo.     

Elongation of axolotl tailbud embryos requires GPI-linked proteins and organizer-induced, active, ventral trunk endoderm cell rearrangements

Application of phosphatidylinositol-specific phospholipase C to early tailbud stage axolotl embryos reveals that a specific subset of morphogenetic movements requires glycosylphosphatidylinositol (GPI)-linked cell-surface proteins. These include pronephric duct extension, "gill bulge" formation, and embryonic elongation along the anteroposterior axis. The work of Kitchin (1949, J. Exp. Zool. 112, 393-416) led to the conclusion that extension of the notochord provided the motive force driving anteroposterior stretching in axolotl embryos, elongation of other tissues being a passive response. We therefore conjectured that axial mesoderm cells might display the GPI-linked proteins required for elongation of the embryo. However, we show here that removal of most of the neural plate and axial and paraxial mesoderm prior to neural tube closure does not prevent elongation of ventrolateral tissues. Tissue-extirpation and tissue-marking experiments indicate that elongation of the ventral trunk occurs via active, directed tissue rearrangements within the endoderm, directed by signals emanating from the blastopore region. Extension of both dorsal and ventral tissues requires GPI-linked proteins. We conclude that elongation of axolotl embryos requires active cell rearrangements within ventral as well as axial tissues. The fact that both types of elongation are prevented by removal of GPI-linked proteins implies that they share a common molecular mechanism.     

Seed storage proteins in developing somatic embryos of alfalfa: defects in accumulation compared to zygotic embryos

Somatic embryos of alfalfa (Medicago sativa L.) synthesizedall of the major storage proteins of zygotic embryos; an 11Sglobulin (medicagin), a 7S globulin (alfin), and a 2S albumin(LMW). In zygotic embryos (cotyledons and/or axis) these storageproteins accounted for 30%, 10%, and 20%, respectively, of thetotal extractable protein. In somatic embryos the 7S proteinwas predominant while the 11S (particularly subfamily I) and2S proteins were present in lower amounts. Analysis of cultivarsand selfed seed of the embryogenic clone (RL34) demonstratedthat these differences were predominantly physiologically, ratherthan genetically, based. The accumulated 7S and 11S storageproteins of somatic embryos were processed normally, aggregatedas oligomers, and were deposited in protein bodies. This wasnot the case for the 2S storage protein. In somatic embryosthat protein was localized in the cytoplasm rather than in proteinbodies, the site of deposition in zygotic embryos. Key words: Medicago (alfalfa), zygotic/somatic embryos (seeds), storage proteins, immunolocalization     

Microtubule flux and sliding in mitotic spindles of Drosophila embryos

We proposed that spindle morphogenesis in Drosophila embryos involves progression through four transient isometric structures in which a constant spacing of the spindle poles is maintained by a balance of forces generated by multiple microtubule (MT) motors and that tipping this balance drives pole-pole separation. Here we used fluorescent speckle microscopy to evaluate the influence of MT dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation in anaphase B. During metaphase and anaphase A, fluorescent punctae on kinetochore and interpolar MTs flux toward the poles at 0.03 microm/s, too slow to drive chromatid-to-pole motion at 0.11 microm/s, and during anaphase B, fluorescent punctae on interpolar MTs move away from the spindle equator at the same rate as the poles, consistent with MT-MT sliding. Loss of Ncd, a candidate flux motor or brake, did not affect flux in the metaphase/anaphase A isometric state or MT sliding in anaphase B but decreased the duration of the isometric state. Our results suggest that, throughout this isometric state, an outward force exerted on the spindle poles by MT sliding motors is balanced by flux, and that suppression of flux could tip the balance of forces at the onset of anaphase B, allowing MT sliding and polymerization to push the poles apart.     

Expression of Otx homeodomain proteins induces cell aggregation in developing zebrafish embryos

In the zebrafish embryo, cells fated to give rise to the rostral brain move in a concerted fashion and retain tissue coherence during morphogenesis. We demonstrate here that Otx proteins have a dramatic effect on cell-cell interactions when expressed ectopically in the zebrafish embryo. Injection of zebrafish Otx1 or Drosophila otd RNAs into a single cell at the 16-cell stage results in aggregation of descendants of the injected cell. The Otx/Otd homeodomain is necessary for aggregation and appears to be sufficient for the effect when substituted for the homeodomain of an unrelated homeodomain protein. When cells containing injected zOtx1 RNA are limited to the area that is normally fated to become the anterior brain and neural retina, the induced aggregates contribute to anterior brain and retina tissues. In many other embryonic regions, which do not express endogenous zOtx1, the aggregates appear to be incompatible with normal development and do not integrate into developing tissues. By using an activatable Otx1-glutocorticoid receptor fusion protein that results in the stimulation of cell association, we demonstrate that cell aggregates can form as a result of Otx1 activity even after gastrulation is completed. Time-lapse analysis of cell movements show that cell aggregation occurs with only a slight inhibition of the rate of convergence. These results suggest that promotion of cell adhesion or mediation of cell repulsion may be one of the normal functions of the Otx proteins in the establishment of the anterior brain.     

Glycine transport in mouse eggs and preimplantation embryos

In pre-compaction embryos glycine was taken up by the glycine-specific gly-system, which is concentrative, weakly exchangeable and dependent on Na+. After compaction glycine uptake increased, apparently due to the expression of the A-transport system and its reactivity with glycine. Studies of the metabolic fate of carbon from glycine indicated conversion to serine and alanine. These changes are interpreted to show that glycine could provide carbon for intermediary energy metabolism, resulting in CO2, as well as for macromolecular synthesis.