Changes in the synthesis and intracellular localization of nuclear proteins during embryogenesis in the sea urchin Strongylocentrotus purpuratus

A rapid, gentle technique is described for the isolation of nuclei from sea urchin embryos. Using this technique, we have analyzed the synthesis and accumulation of nonhistone nuclear proteins during sea urchin development by two-dimensional gel electrophoresis. Most nuclear proteins fall into one of three patterns of synthesis, which are distinguished by maximal rates of accumulation at early (prior to hatching blastula), middle (hatching blastula/gastrula), or late (prism/pluteus) stages of development. Over 60% of observed nuclear proteins undergo apparent qualitative changes in synthesis and accumulation between the 64-cell and pluteus stages. Most of these changes represent appearances of new proteins. A large number of qualitative changes occur very early in development; the period of greatest change is between the 64-cell and 200-cell stages. Over half of the proteins which first appear in the nucleus subsequent to the 64-cell stage are synthesized at stages prior to the time of their initial appearance in nuclei, but are excluded from nuclei for some time.     

The influence of cell interactions and tissue mass on differentiation of sea urchin mesomeres

The developmental potential of different blastomeres of the sea urchin embryo was re-examined. We have employed a new method to isolate substantial numbers of different kinds of blastomeres from 16-cell-stage embryos, and we have used newly available molecular markers to analyze possible vegetal differentiation. We have found that, while isolated mesomere pairs behave according to the classical expectations and develop into ectodermal vesicles, there is a clear effect of reaggregating two or more mesomere pairs. They survive better in long-term culture and, after prolonged periods, they display an astonishing ability to express vegetal differentiation. We also combined mesomeres with stained micromeres or macromeres from the vegetal hemisphere. Although induction of guts and spicules was observed, there was little if any effect of varying the ratio of different blastomeres on the kinds of differentiation obtained.     

THE EFFECTS OF 5-BROMODEOXYURIDINE ON YOLK SAC ERYTHROPOIESIS IN THE CHICK EMBRYO

The effects of the thymidine analog, 5-bromodeoxyuridine (BUdR), on the formation of red cells in the yolk sac of the chick embryo were examined. The prospective area opaca vasculosa from a definitive primitive streak embryo was excised, disaggregated, and deposited into a cell clump, and the cell clump was placed in organ culture. Hemoglobin synthesis is detectable after about 16 hr in culture. The formation of erythropoietic foci and incorporation of ⁵⁵Fe into heme were used to measure the extent of erythropoiesis. Exposure to 40 µg/ml of BUdR within 6 hr after explantation almost completely eliminated red cell formation; subsequent transfer to thymidine medium showed that the inhibition was reversible, and there was no histological evidence of analog toxicity. Between 6 and 12 hr after initiation of organ culture, the tissue became completely refractory to BUdR. DNA synthesis, as monitored by thymidine-³H and BUdR-³H pulses, was extensive both during and after the period of BUdR sensitivity. Hence, during both BUdR sensitive and insensitive periods the analog was incorporated into DNA of cells which had not yet synthesized hemoglobin. It is proposed that between 6 and 12 hr a crucial regulatory event for terminal differentiation is perturbed by the presence of BUdR in the chromosomes.     

Capillary endothelial cell tropism of PVC-211 murine leukemia virus and its application for gene transduction.

PVC-211 murine leukemia virus (MuLV) causes neurodegenerative disease following inoculation of neonatal, but not adult, mice and rats. It was previously shown that tropism for brain capillary endothelial cells (CEC) was a determinant of the viral neuropathogenicity. In this study, we demonstrate that host age-dependent replication of PVC-211 MuLV in vivo occurs in CEC in the brain as well as in other organs, such as the liver, kidney, and heart. In contrast, primary explant cultures of CEC derived from brains and livers of adult and neonatal rats could be infected by PVC-211 MuLV, suggesting that the age-dependent susceptibility was abrogated in vitro. Although CEC were generally less susceptible to MuLV-mediated gene transduction than fibroblasts, treatment of CEC with 2-deoxyglucose followed by inoculation of a PVC-211 MuLV-pseudotyped vector in the absence of heparin improved the transduction efficiency. These observations support the possibility that PVC-211 MuLV may be useful for establishing models of CEC gene transduction.     

The polyadenylated RNA of zoospores and growth phase cells of the aquatic fungus,Blastocladiella

The stored poly(A) + RNA from zoospores of the aquatic fungus Blastocladiella emersonii represents 2.5% of the total RNA and has a model MW of 425,000 daltons and an average poly(A) isostich of 32 bases. The poly(A) + RNA also represents 2.5% of the total RNA from early growth phase cells and has a modal MW of 360,000 daltons and an average poly(A) isostich of 38 bases. The poly(A) + RNA from spores and 2-hr plants contains a structure resistant to RNases T₁, T₂, and A, which can be labeled with ³²PO₄ and which will bind to DBAE-cellulose. These characteristics strongly suggest that both the zoospore poly(A) + RNA and the 2-hr cell poly(A) + RNA are capped at the 5′ end; and, hence, it is unlikely that capping is involved in the control of protein synthesis during germination.Approximately 80% of the poly(A) + RNA of the spore is located in the membrane-enclosed ribosomal nuclear cap, and more than 90% of the poly(A) + RNA within the cap is found in the 80S monoribosome and heavier fractions.Synthesis of new poly(A) + RNA occurs very early during zoospore germination, and the labeled poly(A) + RNA rapidly enters the newly organized polysomes. The labeling data for early germination also suggest that cytoplasmic polyadenylation occurs.     

The stability and translation of sea urchin histone messenger RNA molecules injected into Xenopus laevis eggs and developing embryos

Embryonic sea urchin histone mRNA was injected into eggs and developing zygotes of Xenopus. The functional stability of the mRNA was monitored by separating newly synthesized sea urchin histones from those of Xenopus. Just as when injected into Xenopus oocytes, sea urchin H1, H2A, and H2B mRNA molecules have a functional half-life of about 3 hr in the developing embryo. This suggests that the endogenous Xenopus histone mRNA is also unstable and has a number of implications for the amount of histone mRNA that is stored in the oocyte and the time at which histone genes should become active in development. The injected mRNA is translated with little, if any, greater efficiency in the egg than in the oocyte. However, Xenopus histone synthesis increases about 20- to 50-fold during the transition from oocyte to egg. The injection experiments therefore suggest that this increase is brought about primarily by the mobilization of stored mRNA, rather than an increase in the efficiency of histone synthesis.     

Interactions of different vegetal cells with mesomeres during early stages of sea urchin development

It has been known from results obtained in the classical experiments on sea urchin embryos that cell isolation and transplantation showed extensive interactions between the early blastomeres and/or their descendants. In the experiments reported here a systematic reexamination of recombination of mesomeres and their progeny (which come from the animal hemisphere) with various vegetal cells derived from blastomeres of the 32- and 64-cell stage was carried out. Cells were marked with lineage tracers to follow which cell gave rise to what structures, and newly available molecular markers have been used to analyze different structures characteristic of regional differentiation. Large micromeres form spicules and induce gut and pigment cells in mesomeres, conforming to previous results. Small micromeres, a cell type not heretofore examined, gave rise to no recognizable structure and had very limited ability to evoke poorly differentiated gut tissue in mesomeres. Macromeres and their descendants, Veg 1 and Veg 2, form primarily what their normal fate dictated, though both did have some capacity to form spicules, presumably by formation from secondary mesenchyme. Macromeres and their descendants were not potent inducers of vegetal structures in animal cells, but they suppress the latent ability of mesomeres to form vegetal structures. The results lead us to propose that the significant interactions during normal development may be principally suppressive effects of mesomeres on one another and of adjacent vegetal cells on mesomeres.