Developmental significance of a cortical cytoskeletal domain in Chaetopterus eggs

The cortex of Chaetopterus eggs contains a cytoskeletal domain (CD) which includes a specific class of dense granular organelles and a large proportion of the maternal mRNA. This CD, along with its constituent dense granular organelles and mRNA, can be displaced to atypical locations in the egg by centrifugation. To investigate the developmental significance of the CD, we have examined the early development of egg and zygote fragments, prepared by centrifugation, which contained the CD, the nucleus, or both. Specifically, we prepared nucleate egg and zygote fragments depleted in the CD, and two-cell embryos in which the CD was present in only one cell. Nucleate centripetal egg fragments were both unable to develop after fertilization and were depleted in the CD, as shown by electron microscopy, acridine orange staining of cortical organelles, and hybridization with poly(U) and cloned DNA probes. In contrast, about 20-35% of the nucleate centripetal fragments derived from one-cell zygotes developed into swimming larva. Correlated with this improved success of development, we found that these zygotic centripetal fragments contained significant levels of the CD, using the same methods listed above. More effective removal of the CD from zygotic centripetal fragments by stratification prior to fragmentation virtually eliminated their ability to develop. The CD and associated components could be displaced into only one of the first two blastomeres by centrifugation of zygotes immediately prior to the first cleavage. Embryos containing the CD in only one blastomere continued to cleave, but formed defective larva. The results suggest that the cortical CD is necessary for normal embryonic development.     

The location of maternal mRNA in eggs and embryos

Recent studies have shown that some maternal mRNAs are localized in specific cytoplasmic regions of eggs and embryos and are rearranged in concert with the cytoplasmic movements that fix the embryonic axes. The localization and ooplasmic segregation of mRNA molecules may be mediated by their association with specific egg cytoskeletal domains.     

Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs

A cortical granule-free domain (CGFD) overlies the metaphase chromatin in fully mature mouse eggs. Although a chromatin-induced localized release of cortical granules (CG) during maturation is thought to be a major contributing factor to its formation, there are indications that CG redistribution may also be involved in generating the CGFD. We performed experiments to determine the relative contributions of CG exocytosis and redistribution in generating the CGFD. We found that the CGFD-inducing activity was not specific to female germ cell chromatin and was heat stable but sensitive to DNase and protease treatment. Surprisingly, chelation of egg intracellular Ca(2+) levels did not prevent CGFD formation in response to microinjection of exogenous chromatin, suggesting that development of the CGFD was not a result of CG exocytosis. This finding was confirmed by the lack of CG exudate on the plasma membrane surface of the injected eggs and the absence of conversion of ZP2 to ZP2(f) during formation of the new CGFD. Moreover, clamping intracellular Ca(2+) did not prevent the formation of the CGFD during oocyte maturation, but did inhibit the maturation-associated release of CGs between metaphase I and II. Results of these experiments suggest that CG redistribution is the dominant factor in formation of the CGFD.     

The myoplasm of ascidian eggs: a localized cytoskeletal domain with multiple roles in embryonic development

The myoplasm of ascidian eggs is a localized cytoplasmic region containing a unique cytoskeletal domain. During ooplasmic segregation, the myoplasm moves first to the vegetal pole and then to the future posterior region of the fertilized egg, where it subsequently enters the muscle cell lineage during cleavage. In the vegetal pole region, the myoplasm defines a developmental center which later controls gastrulation and embryonic axis formation. In the posterior region, the myoplasm defines another developmental center, which specifies muscle cell development. Evidence is described suggesting that the integrity of the myoplasmic cytoskeletal domain is required for normal embryonic functions of the myoplasm.     

Nuclei of Arbacia and Chaetopterus eggs as photographed by infrared light

Photographs, taken with infrared light, of all stages of dividing Arbacia punctulata eggs show the configuration of mitotic figures and nuclei as brilliant white areas against the dark granular cytoplasmic background. The photographs are very different from those taken with visible light. The nucleus and cytoplasm of the unfertilized Chaetopterus pergamentaceus egg which are quite opaque with visible light are quite striking with infrared light.     

Survival of maternal mRNA in anucleate and unfertilized mouse eggs

Unfertilized mouse eggs were parthenogenetically activated in vitro and then bisected. Anucleate fragments were aged in vitro, and their protein synthesis was analyzed by two-dimensional polyacrylamide gel electrophoresis. Proteins were compared with those which were synthesized by aging unfertilized eggs and those which were translated in vitro from mRNA extracted from the unfertilized eggs. Normally cleaving parthenogenetic eggs served as controls. Cytoplasts and unfertilized eggs synthesized considerable quantities of protein after 2 days in culture. The protein patterns of cytoplasts and unfertilized eggs shifted in this time mainly within a group of proteins with a molecular mass of about 35 kDa. This shift was also seen in controls between day 1 and 2 but was delayed in unfertilized eggs. There was no clear appearance of new proteins in aging cytoplasts, which might have indicated a selective activation of maternal mRNA at a certain time after the activation stimulus, nor was such a change apparent in unfertilized eggs. The survival of maternal allozymes of glucose phosphate isomerase was tested in cytoplasts derived from fertilized eggs. The allozymes remained active during 4 days of aging and did not change their quantitative correlation.     

Body handedness is directed by genetically determined cytoskeletal dynamics in the early embryo

Although substantial progress has been made recently in understanding the establishment of left-right asymmetry in several organisms, little is known about the initial step for any embryo. In gastropods, left-right body handedness is determined by an unknown maternally inherited single gene or genes at closely linked loci and is associated with the sense of spiral cleavage in early embryos. Contrary to what has been believed, we show that temporal and spatial cytoskeletal dynamics for the left- and right-handed snails within a species are not mirror images of each other. Thus, during the third cleavage of Lymnaea stagnalis, helical spindle inclination (SI) and spiral blastomere deformation (SD) are observed only in the dominant dextral embryos at metaphase-anaphase, whereas in the recessive sinistral embryos, helicity emerges during the furrow ingression. Actin depolymerization agents altered both cleavages to neutral. Further, we found a strong genetic linkage between the handedness-specific cytoskeletal organization and the organismal handedness, using backcrossed F4 congenic animals that inherit only 1/16 of dextral strain-derived genome either with or without the dextrality-determining gene(s). Physa acuta, a sinistral-only gastropod, exhibits substantial SD and SI levotropically. Thus, cytoskeletal dynamics have a crucial role in determination of body handedness with further molecular, cellular, and evolutionary implications.     

Spatial distribution of messenger RNA in the cytoskeletal framework of ascidian eggs

Maternal poly(A)⁺RNA, histone mRNA, and actin mRNA exhibit unique spatial distributions in the different ooplasmic regions of ascidian eggs. These RNAs also appear to migrate with their respective ooplasms during the episode of extensive cytoplasmic rearrangement that occurs after fertilization, suggesting they are associated with a structural framework. The role of the cytoskeletal framework (CF) in determining the spatial distribution of maternal mRNA was tested by subjecting Triton X-100 extracted (Styela plicata) eggs and early embryos to in situ hybridization with poly(U) and cloned DNA probes. Grain counts indicated that substantial proportions of the egg poly(A)⁺RNA, histone mRNA, and actin mRNA were present in the CF and that there was no alteration in the extent of mRNA-CF interactions during the period between fertilization and the two-cell stage. Analysis of grain distributions indicated that poly(A)⁺RNA, histone mRNA, and actin mRNA were concentrated in the same regions of detergent-extracted eggs as they are in intact eggs. The proportions and spatial distribution of these RNAs in the CF were not affected when the actin cytoskeleton was destabilized by cytochalasin B or DNAse I. The data suggest that maternal mRNA is associated with the CF, that this association is responsible for mRNA rearrangement during ooplasmic segregation, and that mRNA-CF interactions are not dependent on the integrity of the actin cytoskeleton.     

Cyclin: A protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division

Cleavage in embryos of the sea urchin Arbacia punctulata consists of eight very rapid divisions that require continual protein synthesis to sustain them. This synthesis is programmed by stored maternal mRNAs, which code for three or four particularly abundant proteins whose synthesis is barely if at all detectable in the unfertilized egg. One of these proteins is destroyed every time the cells divide. Eggs of the sea urchin Lytechinus pictus and oocytes of the surf clam Spisula solidissima also contain proteins that only start to be made after fertilization and are destroyed at certain points in the cell division cycle. We propose to call these proteins the cyclins.     

Cellular distribution of mammalian DNA topoisomerase II is determined by its catalytically dispensable C-terminal domain.

Mammalian cells express two genetically distinct isoforms of DNA topoisomerase II, designated topoisomerase IIalphaand topoisomerase IIbeta. We have recently shown that mouse topoisomerase IIalpha can substitute for the yeast topoisomerase II enzyme and complement yeast top2 mutations. This functional complementation allowed functional analysis of the C-terminal domain (CTD) of mammalian topoisomerase II, where the amino acid sequences are divergent and species-specific, in contrast to the highly conserved N-terminal and central domains. Several C-terminal deletion mutants of mouse topoisomerase IIalpha were constructed and expressed in yeast top2 cells. We found that the CTD of topoisomerase IIalphais dispensable for enzymatic activity in vitro but is required for nuclear localization in vivo. Interestingly, the CTD of topoisomerase IIbetawas also able to function as a signal for nuclear targeting. We therefore examined whether the CTD alone is sufficient for nuclear localization in vivo . The C-terminal region was fused to GFP (green fluorescent protein) and expressed under the GAL1 promoter in yeast cells. As expected, GFP signal was exclusively detected in the nucleus, irrespective of the CTD derived from either topoisomerase IIalphaor IIbeta. Surprisingly, when the upstream sequence of each CTD was added nuclear localization of the GFP signal was found to be cell cycle dependent: topoisomerase IIalpha-GFP was seen in the mitotic nucleus but was absent from the interphase nucleus, while topoisomerase IIbeta-GFP was detected predominantly in the interphase nucleus and less in the mitotic nucleus. Our results suggest that the catalytically dispensable CTD of topoisomerase II is sufficient as a signal for nuclear localization and that yeast cells can distinguish between the two isoforms of mammalian topoisomerase II, localizing each protein properly.     

Possible involvement of calmodulin in maturation and activation of Chaetopterus eggs

We report the isolation of calmodulin from oocytes of Chaetopterus pergamentaceus. The identification of this protein is based on (1) activation of beef heart cAMP phosphodiesterase, (2) heat stability, (3) sensitivity to chlorpromazine, and (4) electrophoretic mobility identical to that of porcine brain calmodulin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of either Ca2+ or EGTA. We treated oocytes with chlorpromazine and W-7 to investigate the involvement of calmodulin in meiosis initiation and egg activation. Very low concentrations of chlorpromazine inhibited germinal vesicle breakdown (GVBD). This effect was shown to be dependent upon bright indirect light, since the drug was much less effective at GVBD inhibition under conditions of very low illumination. Higher concentrations of chlorpromazine and W-7 (100 microM) inhibited GVBD and activated eggs with intact germinal vesicles as determined by fertilization envelope formation and the onset of ameboid activity. Neither egg activation nor inhibition of calmodulin stimulation of phosphodiesterase activity in vitro was affected by light. These results are consistent with a role for calmodulin in egg activation and GVBD, but suggest that chlorpromazine in bright light may prevent GVBD by some mechanism other than calmodulin inhibition.     

A yellow crescent cytoskeletal domain in ascidian eggs and its role in early development

In this investigation, Triton X-100 extraction was utilized to examine the cytoskeleton of ascidian eggs and embryos. The cytoskeleton contained little carbohydrate or lipid and only about 20–25% of the total cellular protein and RNA. It was enriched in polypeptides of molecular weight (M_r) 54, 48, and 43 × 10³. The 43 × 10³M_r polypeptide was identified as actin based on its M_r, isoeletric point, and affinity for DNase I. Electron microscopy of the detergent-extracted eggs showed that they contained cytoskeletal domains corresponding to colored cytoplasmic regions of specific morphogenetic fate in the living egg. A yellow crescent cytoskeletal domain in the myoplasm was examined and shown to consist of a plasma membrane lamina (PML) and a deeper lattice of filaments which appeared to connect the yellow crescent pigment granules to the PML. The PML probably consists of integral membrane proteins stabilized by an underlying network of actin filaments since NBD-phallacidin stained this area of the egg cortex and the PML was extracted from the cytoskeleton by DNase I treatment. The yellow crescent cytoskeletal domain was found throughout the cortex of the unfertilized egg. During ooplasmic segregation it progressively receded into the vegetal hemisphere and was subsequently partitioned to the presumptive muscle and mesenchyme cells of the 32-cell embryo. It is suggested that contraction of the actin network in the yellow crescent cytoskeletal domain is the motive force for ooplasmic segregation. This structure may also serve as a framework for the positioning of morphogenetic determinants involved in muscle cell development.     

甘肃省苹果蠹蛾卵的空间分布格局

为准确掌握苹果蠹蛾Cydia pomonella(L.)的田间发生动态,本文对苹果园中苹果蠹蛾卵的空间分布格局和抽样技术进行了研究。结果表明:在第2代卵发生高峰期,苹果叶片上的苹果蠹蛾卵量显著高于果实上的卵量(叶片上着卵量占总卵量的58.2%,P<0.05),叶片上的卵主要分布于叶片正面(正面着卵量占叶片上总卵量的69.6%,P<0.05);在树冠不同方位上,东、南两面的着卵量最大,但在不同空间层次上分布的卵量没有差异。卵在叶片和果实上均呈聚集分布,且聚集强度随种群密度的升高而增加,其聚集是由环境因素造成的。文中还根据Taylor幂法则参数建立了苹果蠹蛾卵的理论抽样数模型。     

An ultraviolet-sensitive maternal mRNA encoding a cytoskeletal protein may be involved in axis formation in the ascidian embryo

Ultraviolet (uv) irradiation of the vegetal hemisphere of fertilized eggs during ooplasmic segregation inhibits subsequent gastrulation and axis formation in ascidian embryos. The molecular basis of this phenomenon was investigated in by comparing in vivo protein synthesis and in vitro mRNA translation in normal and uv-irradiated embryos of the ascidian Styela clava. Analysis of protein synthesis by [35S]methionine incorporation, two-dimensional (2D) gel electrophoresis, and autoradiography showed that only 21 (or about 5%) of 433 labeled polypeptides were missing or decreased in labeling intensity in uv-irradiated embryos. The most prominent of these was a 30,000 molecular weight (pI 6.0) polypeptide (p30). Extraction of gastrulae with the nonionic detergent Triton X-100 showed that p30 is retained in the detergent insoluble residue, suggesting that it is associated with the cytoskeleton. Several lines of evidence suggest that p30 may be involved in axis formation. First, p30 labeling peaks during gastrulation, when the embryonic axis is being established. Second, axis formation and p30 labeling are abolished by the same threshold uv dose, which is distinct from that required to inactivate muscle cell development. Third, the uv sensitivity period for abolishing p30 labeling and axis formation are both restricted to ooplasmic segregation. In vitro translation of egg RNA followed by 2D gel electrophoresis and autoradiography of the protein products showed that p30 is encoded by a maternal mRNA. The translation of p30 mRNA was abolished by uv irradiation of fertilized eggs during ooplasmic segregation suggesting that this message is a uv-sensitive target. The results are consistent with the hypothesis that uv irradiation blocks gastrulation and axis formation by inhibiting the translation of maternal mRNA localized in the vegetal hemisphere of the fertilized egg.     

Switching of membrane organelles between cytoskeletal transport systems is determined by regulation of the microtubule-based transport

Intracellular transport of membrane organelles occurs along microtubules (MTs) and actin filaments (AFs). Although transport along each type of the cytoskeletal tracks is well characterized, the switching between the two types of transport is poorly understood because it cannot be observed directly in living cells. To gain insight into the regulation of the switching of membrane organelles between the two major transport systems, we developed a novel approach that combines live cell imaging with computational modeling. Using this approach, we measured the parameters that determine how fast membrane organelles switch back and forth between MTs and AFs (the switching rate constants) and compared these parameters during different signaling states. We show that regulation involves a major change in a single parameter: the transferring rate from AFs onto MTs. This result suggests that MT transport is the defining factor whose regulation determines the choice of the cytoskeletal tracks during the transport of membrane organelles.     

Asymmetric distribution of actin mRNA and cytoskeletal pattern generation in polarized epithelial cells

We analysed the distribution of actin mRNA in intestinal epithelial cells using in situ hybridization of 35S-labelled cytoplasmic beta-actin RNA. We found that the distribution of actin mRNA generally parallels that of polymerized actin, i.e. there is an accumulation of actin mRNA in the apical end of villous epithelial cells. Furthermore, the development of this asymmetric localization of actin mRNA appears to parallel the elaboration of the cytoskeleton during cellular differentiation. We discuss the possibility that the interaction between actin and its mRNA may be important for the establishment and maintenance of cytoskeletal pattern in polarized epithelial cells.