The effects of altering the position of cleavage planes on the process of localization of developmental potential in ctenophores.

During the transition from the four- to the eight-cell stage in ctenophore embryos, each blastomere produces one daughter cell with the potential to form comb plate cilia and one daughter cell that does not have this potential. If the second cleavage in a two-cell embryo is blocked, at the next cleavage these embryos frequently form four blastomeres which have the configuration of the blastomeres in a normal eight-cell embryo. At this division there is also a segregation of comb plate-forming potential. By compressing a two-cell embryo in a plane perpendicular to the first plane of cleavage it is possible to produce a four-cell blastomere configuration that is identical to that produced following the inhibition of the second cleavage. However, under these circumstances the segregation of comb plate potential does not occur. These results suggest that the appropriate plane of cleavage must take place for a given cleavage cycle, in order for localizations of developmental potential to be properly positioned within blastomeres.     

Simulation of the mechanism of determining the position of the cleavage furrow in cytokinesis of sea urchin eggs

In cytokinesis of sea urchin eggs, the numerical density of astral microtubules extending close to the cell surface has been thought to determine the position of the cleavage furrow. In the present study, a new model was constructed to simulate the relationship between the microtubule density and the furrow formation. In the model, gradients of the microtubule density drive fluid membrane proteins whose accumulation triggers the formation of contractile-ring microfilaments. The model could explain the behavior of the cleavage furrow under various experimental conditions. These simulations revealed two aspects of furrow formation. One is that in some cases, the cleavage furrow appears in a surface region where the microtubule density has neither a minimum nor a maximum. In all furrow regions, however, the second derivative of the microtubule-density function has large positive values. Membrane proteins greatly slow down to accumulate in such a region. The other is that the cleavage furrow is mobile, not fixed in one position, because of the fluidity of membrane proteins. These results strongly suggested that the mitotic apparatus determines the position of the cleavage furrow by redistributing membrane proteins through gradients of the microtubule density at the cell surface.     

Spiral cleavage and cell position contribute to the specification of the dorsoventral axis in the embryo of the archaeogastropod Haliotis tuberculata

In the embryo of Haliotis tuberculata spiral cleavage induces size differences between the quadrants in the 4-cell embryo. These size differences, together with the formation of compact cell configurations, induce asymmetrical positions of equivalent cells in the 8- and 16-cell embryo. The asymmetries in size and position influence the final specification of the dorsoventral asymmetry in the 32-cell embryo, as well as formation of the mesentoblast. © 1994 Wiley-Liss, Inc.     

Mutations altering the cleavage specificity of a homing endonuclease

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein–DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences.     

Cytological effects of urethan on cleavage induction in parthenogenetically activated sea urchin eggs

The effect of urethan on artificial induction of cleavage in eggs of the sea urchin, Hemicentrotus pulcherrimus, was studied. When the eggs were exposed for 20 minutes to seawater containing urethan (final concentration, 0.08 M) after butyric acid-activation and then treated with hypertonic seawater, the cleavage rate was enchanced by about 1.5 times as compared with the nontreated eggs. In the eggs exposed to urethan–seawater for over 70 minutes many clear spots appeared throughout the cytoplasm. Simultaneously, the pigment granules, which had been embedded within the cortex, migrated to the inner cytoplasm and encircled a monastral centrosphere and clear spots. The clear spots were composed of microtubules much like cytasters, and in the central region of them centrioles were not yet found. The eggs in which the pigment granules disappeared from the cortex may be more susceptible to cleavage induction by succeeding hypertonic treatment.     

Determination of first cleavage plane: the relationships between the orientation of the mitotic apparatus for first cleavage and the position of meiotic division-related structures in starfish eggs

In order to understand when the orientation of the first cleavage plane is fixed along the animal-vegetal axis in starfish eggs, the behavior of the sperm aster was examined by indirect immunofluorescence staining. After duplication, the sperm aster organizes the mitotic apparatus for first cleavage perpendicular to the cleavage plane. The sperm aster located in the egg periphery just after fertilization and moved to the site close to the animal pole rather than the egg center by meiosis II. At early metaphase II, duplication of the sperm aster was detected but the axis of the resultant sperm diaster randomly pointed. Subsequently, its axis had already turned perpendicular to the animal-vegetal axis before pronucleus fusion. These results indicate that the orientation processes of the sperm diaster consist of positioning before its duplication and successive determining its azimuth. Furthermore, the azimuth and position of the mitotic apparatus for first cleavage did not change by shifting or eliminating the meiotic division-related structures such as the germinal vesicle, meiotic spindle, and female pronucleus by micromanipulation. These results show that none of them determines the first cleavage plane. Therefore, we discuss the pointing mechanism of the first cleavage plane without the influence of these meiotic division-related structures.     

Evolutionary implications of the mode of D quadrant specification in coelomates with spiral cleavage

In annelids, molluscs, echiurans and sipunculids the establishment of the dorsal-ventral axis of the embryo is associated with D quadrant specification during embryogenesis. This specification occurs in two ways in these phyla. One mechanism specifies the D quadrant via the shunting of a set of cytoplasmic determinants located at the vegetal pole of the egg to one blastomere of the four cell stage embryo. In this case, at the first two cleavages of embryogenesis there is an unequal distribution of cytoplasm, generating one macromere which is larger than the others at the four cell stage. The D quadrant can also be specified by a contact mediated inductive interaction between one of the macromeres at the vegetal pole with micromeres at the animal pole of the embryo. This mechanism operates at a later stage of development than the cytoplasmic localization mechanism and is associated with a pattern of cleavage in which the first two cleavages are equal. An analysis of the phylogenetic relationships within these phyla indicates that the taxa which determine the D quadrant at an early cleavage stage by cytoplasmic localization tend to be derived and lack a larval stage or have larvae with adult characters. Those taxa where the D quadrant is specified by induction include the ancestral groups although some derived groups also use this mechanism. The pulmonate mollusc Lymnaea uses an inductive mechanism for specifying the D quadrant. In these embryos each of the four vegetal macromeres has the potential of becoming the D macromere; however under normal circumstances one of the two vegetal crossfurrow macromeres almost invariably becomes the D quadrant. Experiments are described here in which the size of one of the blastomeres of the four cell stage Lymnaea embryo is increased; this macromere invariably becomes the D quadrant. These experiments suggest that developmental change in relative blastomere size during the first two cleavages in spiralian embryos that normally cleave equally may have provided a route that has led to the establishment of the cytoplasmic localization mechanism of D quadrant formation.     

Some aspects of cleavage in Pomatoceros triqueter eggs

Summary The first cleavage division of Pomatoceros triqueter eggs is described. Time-lapse microcinematographic and electron microscopic studies revealed that, prior to division, the plasma membrane was folded into pleats. These were not present after division. This fact pointed to an unfolding of the plasma membrane which enabled it to cover the increased surface area resulting from cleavage. It is suggested that the pre-cleavage folds are derived from the membranes of the cortical granules, which continue releasing their contents into the perivitelline region following fertilization and first division. Filamentous material in the form of a band was present subjacent to the plasma membrane in the region of the furrow. The individual filaments of this band measure 5 to 7 nm in cross section, the dimensions being similar to those described for other kinds of dividing cells.The authors wish to thank Messrs. P. C. Lloyd, D. Williams and J. Dingley for technical assistance, and Professor B. M. Jones and Dr. G. G. Selman for their helpful criticisms.     

Exogenous peroxidase in the eggs of rats during cleavage]

The presence of exogenous peroxidase of the rat's eggs during cleavage at the 1-, 2-, 8-cellular stages and the blastocysts were studied after 30 minute incubation with horseradish peroxidase (molecular weight 40.000, approximate size of the molecule 40 A). The activity of exogenous peroxidase in the area of zona pellucida was established in the perivitelline space, in the cytoplasm - in the pinocytosis vesicles, in certain greater vacuoles and in certain secondary lysosomes. At the stage of blastocyst the penetration of exogenous protein was greater than at earlier stages. The reaction product was found here in the intercellular space and in the blastocyst cavity. The obtained data suggest possible penetration of the protein from the surrounding area into the egg during its passing the uterine tube. The substances influencing the differentiation of the egg or teratogens may penetrate the egg in the same way.     

Destabilization of the neuromuscular junction by proteolytic cleavage of agrin results in precocious sarcopenia

Etiology and pathogenesis of sarcopenia, the progressive decline in skeletal muscle mass and strength that occurs with aging, are still poorly understood. We recently found that overexpression of the neural serine protease neurotrypsin in motoneurons resulted in the degeneration of their neuromuscular junctions (NMJ) within days. Therefore, we wondered whether neurotrypsin-dependent NMJ degeneration also affected the structure and function of the skeletal muscles. Using histological and functional analyses of neurotrypsin-overexpressing and neurotrypsin-deficient mice, we found that overexpression of neurotrypsin in motoneurons installed the full sarcopenia phenotype in young adult mice. Characteristic muscular alterations included a reduced number of muscle fibers, increased heterogeneity of fiber thickness, more centralized nuclei, fiber-type grouping, and an increased proportion of type I fibers. As in age-dependent sarcopenia, excessive fragmentation of the NMJ accompanied the muscular alterations. These results suggested the destabilization of the NMJ through proteolytic cleavage of agrin at the onset of a pathogenic pathway ending in sarcopenia. Studies of neurotrypsin-deficient and agrin-overexpressing mice revealed that old-age sarcopenia also develops without neurotrypsin and is not prevented by elevated levels of agrin. Our results define neurotrypsin- and age-dependent sarcopenia as the common final outcome of 2 etiologically distinct entities.     

Histones in the first cleavage cycle of fertilised sea urchin eggs

Nuclei were isolated from Echinus eggs through the first cleavage cycle by modification of existing techniques. When these nuclei were extracted with 2 M NaCl and the supernatant diluted to 0.15 M, large amounts of non-histone proteins remained in solution. The precipitated nucleoprotein contained expected amounts of DNA and a protein analogous to mammalian histone H1. Extrachromosomal histone H1 was eliminated by the modified isolation procedure. Amounts of nuclear proteins soluble in 0.15 M NaCl reached a peak in G2. Histones and non-histone proteins were phosphorylated postfertilization, in early prophase and in telophase.