Mathematical model for early development of the sea urchin embryo

In Xenopus and Drosophila, the nucleocytoplasmic ratio controls many aspects of cell-cycle remodeling during the transitory period that leads from fast and synchronous cell divisions of early development to the slow, carefully regulated growth and divisions of somatic cells. After the fifth cleavage in sea urchin embryos, there are four populations of differently sized blastomeres, whose interdivision times are inversely related to size. The inverse relation suggests nucleocytoplasmic control of cell division during sea urchin development as well. To investigate this possibility, we developed a mathematical model based on molecular interactions underlying early embryonic cell-cycle control. Introducing the nucleocytoplasmic ratio explicitly into the molecular mechanism, we are able to reproduce many physiological features of sea urchin development.     

Cell junctions during the early development of the sea urchin embryo (Paracentrotus lividus)

Thin sections, lanthanum tracer and the freeze-fracture technique revealed the presence of different types of cell junctions in early sea urchin (Paracentrotus lividus) embryos. During the first four cleavage cycles, which are characterized by synchrony of cell division, sister blastomeres were connected only by intercellular bridges, formed as a result of incomplete cytokinesis; no trace of other junctions was found at these stages. From the 16-cell stage onwards, septate junctions and gap junctions began to appear between blastomeres. It is postulated that cell-cell interactions may provide a mechanism for the propagation of signals necessary for the coordination of cell proliferation and differentiation.     

Animal-vegetal axis patterning mechanisms in the early sea urchin embryo

During mouse fertilization the spermatozoon induces a series of low-frequency long-lasting Ca(2+) oscillations. It is generally accepted that these oscillations are due to Ca(2+) release through the inositol 1,4,5-trisphosphate (InsP(3)) receptor. However, InsP(3) microinjection does not mimic sperm-induced Ca(2+) oscillations, leading to the suggestion that the spermatozoon causes Ca(2+) release by sensitizing the InsP(3) receptor to basal levels of InsP(3). This contradicts recent evidence that the spermatozoon triggers Ca(2+) oscillations by introducing a phospholipase C or else an activator of phospholipase C. Here we show for the first time that sperm-induced Ca(2+) oscillations may be mimicked by the photolysis of caged InsP(3) in both mouse metaphase II eggs and germinal vesicle stage oocytes. Eggs, and also oocytes that had displayed spontaneous Ca(2+) oscillations, gave long-lasting Ca(2+) oscillations when fertilized or when caged InsP(3) was photolyzed. In contrast, oocytes that had shown no spontaneous Ca(2+) oscillations did not generate many oscillations when fertilized or following photolysis of caged InsP(3). Fertilization in eggs was most closely mimicked when InsP(3) was uncaged at relatively low amounts for extended periods. Here we observed an initial Ca(2+) transient with superimposed spikes, followed by a series of single transients with a low frequency; all characteristics of the Ca(2+) changes at fertilization. We therefore show that InsP(3) can mimic the distinctive pattern of Ca(2+) release in mammalian eggs at fertilization. It is proposed that a sperm Ca(2+)-releasing factor operates by generating a continuous small amount of InsP(3) over an extended period of time, consistent with the evidence for the involvement of a phospholipase C.     

Effect of substrate stiffness on early mouse embryo development

It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68±15% vs. 59±18%), blastocyst (64±9.1% vs. 50±18%) and hatching blastocyst stages (54±25% vs. 21±16%) and an increase in the number of trophectodermal cell (TE,65±13 vs. 49±12 cells) compared to control embryos cultured in PD (mean±S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.     

Synthesis of small molecular weight RNA components during the early stages of sea urchin embryo development

Sea urchin embryos of Psammechinus miliaris contain three major and two minor small molecular weight RNA components called S₁, S₂, S₃ and S₄, S₅, respectively. The synthesis of S₁ and S₂ is initiated in the time interval from 4 to 8 h after fertilization. Hatching and formation of nucleoli take place about 9 – 9.5 h after fertilization and around this time period the synthesis of S₃ is initiated, simultaneously with a qualitative shift in RNA synthesis from predominantly of the heterodisperse type to a synthesis of ribosomal RNA. Shortly after the appearance of nucleoli the synthesis of S₃ is more than twice as fast as S₂. S₁ and S₂ are methylated to about the same extent; S₃ is unmethylated. 60–70% of S₁ and S₂ and 10% of S₃ are localized in the nuclear fraction. The gel electrophoretic mobility of “5” S RNA changes as a result of heat treatment.     

Membrane formation and membrane contacts during the development of the sea urchin embryo

Summary Sea urchin embryos, 8-cell stage to pluteus stage, fixed in osmium tetroxide and embedded in Epon 812 were observed by electron microscopy. At no point in the development were syncytial junctions between the embryonic cells found. During the cleavage stages the membrane contact was closer than in later stages. In early blastula stages intercellular clefts appeared which in the gastrula stage demarcate every cell. At the same time a ringshaped desmosome structure develops at the outer cell surface. In the pluteus stage a closer cell contact is re-established. With proceeding embryogenesis endoplasmic membranes will attach to the cell membrane. These membrane structures may even be of nuclear origin. Gradually, long protrusions, vesicles and lamellae begin to be formed from the nuclear membrane. The commencement of this nuclear activity coincides in time with the formation of nucleoli. At cell division the new cell membrane seemed to arise partly independently of the cleavage furrow from a system of cytoplasmic vesicles.The investigation was facilitated by grants from the Nordic Insulin Foundation.I am indebted to Dr. Torsten Olsson and Miss Brita Nilsson for procuring the material and to Mrs. Mariann Carleson for technical aid.     

Effect of antimediator preparations on the intercellular relations in early embryos of the sea urchin]

The dated treatment of the early embryos of an irregular (flat) sea urchin Scaphechinus mirabilis by neuropharmacological drugs (anti-neurotransmitters) during one of the first four cleavage divisions results in the impairment of intercellular connections and leads to the formation of twin embryos, dwarf embryos, embryos of the dumb-bell shape etc. In the experiments with some of the drugs under study such developmental abnormalities were not seen or were expressed much more weakly when serotonin or bufotenin (N,N-dimethylserotonin) were added to the medium. A suggestion is put forward that the early embryos possess an intracellular mechanism participating in the interaction between the cells and operating via endogenous monoamines, primarily serotonin.     

Plasma membrane proteins of embryo cells of various sea urchin species and hybrid embryo]

Differences are observed in plasma membrane proteins of S. intermedius and S. droebachiensis sea urchin embryo cells isolated at middle blastula stage by means of acrylamide-gel electrophoresis in presence of SDS, urea or non-ionic detergents--Triton X-100 or Brij 35. Electrophoretic mobilities of plasma membrane proteins of sea urchin hybrid embryo malemale S. intermedius X femalefemale S. droebachiensis were identical with electrophoretic mobilities of plasma membrane proteins of maternal species S. droebachiensis. Three sea urchin embryo species under study had just the same biosynthesis of plasma membrane proteins at middle blastula stage detected by 14C-aminoacids pulse-labeling followed by membrane isolation, electrophoresis and gel-autoradiography.     

Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo

The transition from early (E) to late (L) histone gene expression in developing sea urchin (Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L/E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.     

Fibronectin in the developing sea urchin embryo

The presence of fibronectin in developing sea urchin embryos was studied uing immunofluorescence staining. The fluorescence pattern indicates that fibronectin is found on the cell surfaces and between cells in the blastula and gastrula stages, indicating that it plays a role in cell adhesion. Its presence on invaginating cells also suggests its involvement in morphogenesis during early development.     

Determination and morphogenesis in the sea urchin embryo

The study of the sea urchin embryo has contributed importantly to our ideas about embryogenesis. This essay re-examines some issues where the concerns of classical experimental embryology and cell and molecular biology converge. The sea urchin egg has an inherent animal-vegetal polarity. An egg fragment that contains both animal and vegetal material will produce a fairly normal larva. However, it is not clear to what extent the oral-aboral axis is specified in embryos developing from meridional fragments. Newly available markers of the oral-aboral axis allow this issue to be settled. When equatorial halves, in which animal and vegetal hemispheres are separated, are allowed to develop, the animal half forms a ciliated hollow ball. The vegetal half, however, often forms a complete embryo. This result is not in accord with the double gradient model of animal and vegetal characteristics that has been used to interpret almost all defect, isolation and transplantation experiments using sea urchin embryos. The effects of agents used to animalize and vegetalize embryos are also due for re-examination. The classical animalizing agent, Zn2+, causes developmental arrest, not expression of animal characters. On the other hand, Li+, a vegetalizing agent, probably changes the determination of animal cells. The stability of these early determinative steps may be examined in dissociation-reaggregation experiments, but this technique has not been exploited extensively. The morphogenetic movements of primary mesenchyme are complex and involve a number of interactions. It is curious that primary mesenchyme is dispensable in skeleton formation since in embryos devoid of primary mesenchyme, the secondary mesenchyme cells will form skeletal elements. It is likely that during its differentiation the primary mesenchyme provides some of its own extracellular microenvironment in the form of collagen and proteoglycans. The detailed form of spicules made by primary mesenchyme is determined by cooperation between the epithelial body wall, the extracellular material and the inherent properties of primary mesenchyme cells. Gastrulation in sea urchins is a two-step process. The first invagination is a buckling, the mechanism of which is not understood. The secondary phase in which the archenteron elongates across the blastocoel is probably driven primarily by active cell repacking. The extracellular matrix is important for this repacking to occur, but the basis of the cellular-environmental interaction is not understood.(ABSTRACT TRUNCATED AT 400 WORDS)