PLK-1 asymmetry contributes to asynchronous cell division of C. elegans embryos

Acquisition of lineage-specific cell cycle duration is an important feature of metazoan development. In Caenorhabditis elegans, differences in cell cycle duration are already apparent in two-cell stage embryos, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. This time difference is under the control of anterior-posterior (A-P) polarity cues set by the PAR proteins. The mechanisms by which these cues regulate the cell cycle machinery differentially in AB and P1 are incompletely understood. Previous work established that retardation of P1 cell division is due in part to preferential activation of an ATL-1/CHK-1 dependent checkpoint in P1, but how the remaining time difference is controlled is not known. Here, we establish that differential timing relies also on a mechanism that promotes mitosis onset preferentially in AB. The polo-like kinase PLK-1, a positive regulator of mitotic entry, is distributed in an asymmetric manner in two-cell stage embryos, with more protein present in AB than in P1. We find that PLK-1 asymmetry is regulated by A-P polarity cues through preferential protein retention in the embryo anterior. Importantly, mild inactivation of plk-1 by RNAi delays entry into mitosis in P1, but not in AB, in a manner that is independent of ATL-1/CHK-1. Together, our findings support a model in which differential timing of mitotic entry in C. elegans embryos relies on two complementary mechanisms: ATL-1/CHK-1-dependent preferential retardation in P1 and PLK-1-dependent preferential promotion in AB, which together couple polarity cues and cell cycle progression during early development.     

Lysophosphatidic acid signaling controls cortical actin assembly and cytoarchitecture in Xenopus embryos

The mechanisms that control shape and rigidity of early embryos are not well understood, and yet are required for all embryonic processes to take place. In the Xenopus blastula, the cortical actin network in each blastomere is required for the maintenance of overall embryonic shape and rigidity. However, the mechanism whereby each cell assembles the appropriate pattern and number of actin filament bundles is not known. The existence of a similar network in each blastomere suggests two possibilities: cell-autonomous inheritance of instructions from the egg; or mutual intercellular signaling mediated by cell contact or diffusible signals. We show that intercellular signaling is required for the correct pattern of cortical actin assembly in Xenopus embryos, and that lysophosphatidic acid (LPA) and its receptors, corresponding to LPA1 and LPA2 in mammals, are both necessary and sufficient for this function.     

Gas exchange and blood gases of Puna teal (Anas versicolor puna) embryos in the Peruvian Andes

Oxygen consumption, air cell gases, hematology, blood gases and pH of Puna teal (Anas versicolor puna) embryos were measured at the altitude at which the eggs were laid (4150 m) in the Peruvian Andes. In contrast to the metabolic depression described by other studies on avian embryos incubated above 3700 m, O₂ consumption of Puna teal embryos was higher than even that of some lowland avian embryos at equivalent body masses. Air cell O₂ tensions dropped from about 80 toor in eggs with small embryos to about 45 toor in eggs containing a 14-g embryo; simultaneously air cell CO₂ tension rose from virtually negligible amounts to around 26 torr. Arterial and venous O₂ tensions (32–38 and 10–12 toor, respectively, in 12- to 14-g embryos) were lower than described previously in similarly-sized lowland wild avian embryos or chicken embryos incubated in shells with restricted gas exchange. The difference between air cell and arterial O₂ tensions dropped significantly during incubation to a minimum of 11 torr, the lowest value recorded in any avian egg. Blood pH (mean 7.49) did not vary significantly during incubation. Hemoglobin concentration and hematocrits rose steadily throughout incubation to 11.5 g · 100 ml^-1 and 39.9%, respectively, in 14-g embryos.Abbreviations PO₂ partial pressure gradient of O₂ - BM body mass - D diffusion coefficient - G gas conductance (cm³·s^-1·torr^-1) - conductance to water vapor - IP internal pipping of embryos - P _ACO₂ partial pressure of carbon dioxide in air cell - P _AO₂ partial pressure of oxygen in air cell - P _aCO₂ partial pressure of carbon dioxide in arterial blood - P _aCO₂ partial pressure of oxygen in arteries - P _H barometric pressure (torr) - PCO₂ partial pressure of carbon dioxide - P _IO₂ partial pressure in ambiant air - PO₂ partial pressure of oxygen - P _VCO₂ venous carbon dioxide partial pressure - P _VO₂ mixed venous oxygen partial pressure - SE standard error - VO ₂ oxygen consumption     

Concentration distribution around a growing gas bubble in tissue

This paper presents the concentration distribution around a growing nitrogen gas bubble in the blood and other tissues of divers who surface too quickly, when the ambient pressure through the decompression process is variable and constant. This effort is a modification of Sirinivasan et al. model (1999) [9]. The mathematical model is solved analytically to find the growth rate of a gas bubble in a tissue after decompression in the ambient pressure. Moreover, the concentration distribution around the growing bubble is introduced. The growth process is affected by ascent rate , tissue diffusivity D_T, initial concentration difference ΔC₀, surface tension σ and void fraction ?₀.     

Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos

BACKGROUND: Acquisition of lineage-specific cell cycle duration is a central feature of metazoan development. The mechanisms by which this is achieved during early embryogenesis are poorly understood. In the nematode Caenorhabditis elegans, differential cell cycle duration is apparent starting at the two-cell stage, when the larger anterior blastomere AB divides before the smaller posterior blastomere P(1). How anterior-posterior (A-P) polarity cues control this asynchrony remains to be elucidated.RESULTS: We establish that early C. elegans embryos possess a hitherto unrecognized DNA replication checkpoint that relies on the PI-3-like kinase atl-1 and the kinase chk-1. We demonstrate that preferential activation of this checkpoint in the P(1) blastomere contributes to asynchrony of cell division in two-cell-stage wild-type embryos. Furthermore, we show that preferential checkpoint activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Galpha signaling.CONCLUSION: Our findings establish that differential checkpoint activation contributes to acquisition of distinct cell cycle duration in two-cell-stage C. elegans embryos and suggest a novel mechanism coupling asymmetric division to acquisition of distinct cell cycle duration during development.     

Cellular interactions involved in the determination of the early C. elegans embryo

Classical work implied that early nematode embryogenesis is completely mosaic. This view was lately challenged by the demonstration that in C. elegans an early interaction has to occur to induce the production of muscle from a blastomere. Here, early embryonic blastomeres were inactivated by laser microsurgery. The cell lineages of irradiated embryos were compared to those of intact embryos. It is shown that one blastomere, MS, is required for the specification of mesodermal pharyngeal fates and another blastomere, P2, for the specification of hypodermal fates from the descendants of the AB blastomere, whereas the proper specification of the nervous system requires the presence of both. The irradiation of a third blastomere shows that interactions also occur within the ectoderm. I propose that the body plan of the C. elegans embryo may be established by two primary signals followed by secondary interactions. The suggested mechanisms are reminiscent of those involved in amphibian development.     

Muscle lineage cytoplasm can change the developmental expression in epidermal lineage cells of ascidian embryos

Cytoplasm from muscle lineage blastomeres of an ascidian embryo can cause cells of a nonmuscle lineage to produce larval tail muscle acetylcholinesterase. Muscle cytoplasm was partitioned microsurgically into epidermal lineage blastomeres at the eight-cell stage. Posterior half-embryos (the two B3 cells) of Ascidia nigra were obtained first by separating the anterior and posterior blastomere pairs at the four-cell stage. At third cleavage, the two B3 cells divide into an ectodermal cell pair that gives rise solely to epidermal tissues, and a mesodermal-endodermal blastomere pair from which the tail muscle cells are derived. When the ectodermal and mesendodermal blastomere pairs were isolated from one another by microsurgery and reared as partial embryos, only cells originating from the mesendodermal blastomeres produced a histochemical acetylcholinesterase reaction. Immediately after cleavage of the isolated B3 cells into ectodermal and mesendodermal cell pairs, the cleavage furrows could be made to disappear by pressing firmly on the mesendodermal cells with a microneedle. Repeated up and down pressure with the microneedle at a new position across the mesendodermal cells caused furrows to reestablish in the new position, thereby incorporating mesodermal cytoplasm and increasing the size of the ectodermal cells. The cytoplasmically altered ectodermal blastomere pairs, which became detached from the mesendodermal cells by this microsurgical procedure, continued to divide and were reared to “larval” stages. One-third of these epidermal partial larvae produced patches of cells containing acetylcholinesterase. These results lend further support to the theory that choice of particular differentiation pathways (embryonic determination) in ascidian embryos is mediated by segregation of specific egg cytoplasmic determinants.     

Cellular pattern formation, establishment of polarity and segregation of colored cytoplasm in embryos of the nematode Romanomermis culicivorax

We have begun to analyze the early embryogenesis of Romanomermis culicivorax, an insect-parasitic nematode phylogenetically distant to Caenorhabditis elegans. Development of R. culicivorax differs from C. elegans in many aspects including establishment of polarity, formation of embryonic axes and the pattern of asymmetric cleavages. Here, a polarity reversal in the germline takes place already in P₁ rather than P₂, the dorsal-ventral axis appears to be inverted and gut fate is derived from the AB rather than from the EMS blastomere. So far unique for nematodes is the presence of colored cytoplasm and its segregation into one specific founder cell. Normal development observed after experimentally induced abnormal partitioning of pigment indicates that it is not involved in cell specification. Another typical feature is prominent midbodies (MB). We investigated the role of the MB region in the establishment of asymmetry. After its irradiation the potential for unequal cleavage in somatic and germline cells as well as differential distribution of pigment are lost. This indicates a crucial involvement of this region for spindle orientation, positioning, and cytoplasmic segregation. A scenario is sketched suggesting why and how during evolution the observed differences between R. culicivorax and C. elegans may have evolved.     

A test of the air-seeding hypothesis using sphagnum hyalocysts

“Air-seeding” is a proposed mechanism for the initiation of water stress embolism in dead plant cells. During air-seeding, external air is drawn into the lumen of a dead plant cell through a pore or crack in the cell wall. The resulting bubble may expand to fill the lumen, thus embolizing the cell. The data presented confirm that Sphagnum hyalocysts can embolize by air-seeding when the pressure difference across the air-water meniscus is given by ΔP = 0.3/D (derived from the capillary equation), where ΔP is the pressure difference across the meniscus (megapascal), and D is the diameter (micrometer) of the pore through which the air bubble enters.     

Time-lapse cinematography study of the germinal vesicle behaviour in mouse primary oocytes treated with activators of protein kinases A and C

Previous studies have shown that early embryos contain information that can alter the developmental fate of adjacent cells and transferred nuclei. In this report we show that a specific combination of cells from early murine embryos, a single blastomere from an eight-cell embryo placed under the zona pellucida with a two-cell embryo, results in a difference in incorporation of ³H-uridine and expression of two protein bands between the chimeric treatment group and the nonchimeric controls, a single blastomere from an eight-cell embryo in a separate zona pellucida and a two-cell embryo. The incorporation of ³H-uridine in the chimeric group and nonchimeric control group was significantly different at 45 hours after chimerization (P < .02). A stage-specific protein band (52k) on a polyacrylamide gel detected with fluorography was found to be qualitatively different (present more often; P < .01) and another stage-specific protein band (48k) was found to be quantitatively different (more protein; P equals; .07) in the chimeric treatment vs. the nonchimeric controls at 45 hours after chimerization. These results suggest communication between the cells resulting in a change in their incorporation of uridine and protein synthetic profiles.     

Intracellular air bubbles and interfacial tension inNicotiana miersii

Summary Air bubbles were introduced into living hair cells ofNicotiana miersii. The air entered through wounds inflicted on slightly flaccid trichomes from the base of a fruiting stem. Protoplasmic streaming often continued normally in the threads that were near or apparently touched the air bubble. When air bubbles were included within a plasmolyzing protoplast, the protoplasm nearest the air bubble appeared and behaved like that further away.The volume of an included air bubble is affected by many factors, but as the bubble gets smaller, the overriding factor determining the rate of decrease in volume is the surface tension. The effect of the surface tension on the pressure within the bubble is such that the slope, in a graph of the radius of the bubble to the third power against time, is a constant. The value of this slope constant varies directly with the surface tension, although the surface tension is not the only factor determining its magnitude. The rate of volume decrease of bubbles both in living and in dead cells tended to be constant for small bubbles, and the value of the slope for radius cubed vs. time ranged from – 5 ³/sec. to –14 ³/sec, with most values near –10 ³/sec. A theoretical value for the slope of a nitrogen bubble in water at 25 C. is calculated to be –94 ³/sec. A minimum estimate of the surface tension of the cell content surrounding the air bubble is therefore 1/10th of the value of water.The relatively high value of the surface tension is interpreted to indicate that the organization of the cell content at the surface of the air bubble is not of the structural complexity assumed for the plasmalemma.A portion of this paper was presented at the annual meeting of the Botanical Society of America, Physiology Section, Lafayette, Indiana, 1961.This investigation was partly supported by a grant (G 8716) from the National Science Foundation.     

Pre-and post-branchial blood respiratory status during acute hypercapnia or hypoxia in rainbow trout,Oncorhynchus mykiss

Simultaneous venous (pre-branchial) and arterial (post-branchial) extracorporeal blood circulations were utilized to monitor continuously the rapid and progressive effects of acute environmental hypercapnia (water partial pressure of CO₂ 4.8±0.2 torr) or hypoxia (water partial pressure of O₂ 25±2 torr) on oxygen and carbon dioxide tensions and pH in the blood of rainbow trout (Oncorhynchus mykiss). During hypercapnia, the CO₂ tension in the arterial blood increased from 1.7±0.1 to 6.2±0.2 torr within 20 min and this was associated with a decrease of arterial extracellular pH from 7.95±0.03 to 7.38±0.03; the acid-base status of the mixed venous blood changed in a similar fashion. The decrease in blood pH in vivo was greater than in blood equilibrated in vitro with a similar CO₂ tension indicating a significant metabolic component to the acidosis in vivo. Under normocapnic conditions, venous blood CO₂ tension was slightly higher than arterial blood CO₂ tension difference was abolished or reversed during the initial 25 min of hypercapnia indicating that CO₂ was absorbed from the water during this period. Arterial O₂ tension remained constant during hypercapnia; however, venous blood O₂ tension decreased significantly (from 22.0±2.6 to 9.0±1.0 torr) during the initial 10 min. Hypercapnia elicited the release of catecholamines (adrenaline and noradrenaline) into the blood. The adrenaline concentration increased from 6±3 to 418±141 nmol · l^-1 within 25 min; noradrenaline concentration increased from 3±0.5 to 50±21 nmol · l^-1 within 15 min. During hypoxia arterial blood O₂ tension declined progressively from 108.4±9.9 to 12.8±1.7 torr within 30 min. Venous blood O₂ tension initially was stable but then decreased abruptly as catecholamines were released into the circulation. The release of catecholamines occurred concomitantly with a sudden metabolic acidosis in both blood compartments and a rise in CO₂ tension in the mixed venous blood only.Abbreviations CCO₂ plasmatotal carbondioxide - CtO₂ blood oxygen content - PO₂ partial pressure of oxygen - PCO₂ partial pressure of carbon dioxide - PaO₂ arterial bloodPO₂ - PaCO₂ arterial bloodPCO₂ - PvCO₂ venous bloodPCO₂ - PwO₂ waterPO₂ - PwCO₂ waterPCO₂ - Hb haemoglobin - SHbO₂ haemoglobin oxygen saturation - HPLC high-performance liquid chromatography - rbc red blood cell(s) - Hct haematocrit     

DNA synthesis in developing two-cell mouse embryos

Mated CF1 (Carworth) female mice were sacrificed at 2 hr intervals between 29 and 43 hr after human chorionic gonadotrophin (HCG) administration. One- and two-cell eggs were incubated in [³H]thymidine for 1 hr. Labeled two-cell embryos were first observed at 31 hr and reached a maximum number at 35 hr. The S period is approximately 6 hr in duration. Although both blastomeres were labeled in most cases, embryos with only one labeled blastomere were more numerous at later times. In vitro labeling was corroborated by injecting [³H]thymidine directly into the isthmic portion of the oviduct. Embryos usually complete the second cleavage division 18–20 hr after onset of DNA synthesis. The cell cycle at the two-cell stage is thus characterized by a G₁ of close to 1 hr, a 6 hr S, and a G₂ of about 12 hr.Embryos developing in vitro frequently fail to progress beyond the two-cell stage. The block is not due to absence of DNA synthesis since these embryos were found to incorporate [³H]thymidine.     

Development of a bovine luteal cell in vitro culture system suitable for co-culture with early embryos

The cross talk between the corpus luteum (CL) and the early embryo, potentially relevant to pregnancy establishment, is difficult to evaluate in the in vivo bovine model. In vitro co-culture of bovine luteal cells and early embryos (days?2?C8 post in vitro fertilization) may allow the deciphering of this poorly understood cross talk. However, early embryos and somatic cells require different in vitro culture conditions. The objective of this study was to develop a bovine luteal cell in vitro culture system suitable for co-culture with early embryos in order to evaluate their putative steroidogenic and prostanoid interactions. The corpora lutea of the different stages of the estrous cycle (early, mid, and late) were recovered postmortem and enriched luteal cell populations were obtained. In experiments 1 and 2, the effects of CL stage, culture medium (TCM, DMEM-F12, or SOF), serum concentration (5 or 10%), atmosphere oxygen tension (5 or 20%), and refreshment of the medium on the ability of luteal cells to produce progesterone (P₄) were evaluated. The production of P₄ was significantly increased in early CL cultures, and luteal cells adapted well to simple media (SOF), low serum concentrations (5%), and oxygen tensions (5%). In experiment 3, previous luteal cell cryopreservation did not affect the production of P₄, PGF_2??, and PGE₂ compared to fresh cell cultures. This enables the use of pools of frozen?Cthawed cells to decrease the variation in cell function associated with primary cell cultures. In experiment 4, mineral oil overlaying culture wells resulted in a 50-fold decrease of the P₄ quantified in the medium, but had no effect on PGF_2?? and PGE₂ quantification. In conclusion, a luteal cell in vitro culture system suitable for the 5-d-long co-culture with early embryos was developed.     

Hydraulic propagation of pressure along immature and mature xylem vessels of roots of Zea mays measured by pressure-probe techniques

Turgor pressure was measured in cortical cells and in xylem elements of excised roots and roots of intact plants of Zea mays L. by means of a cell pressure probe. Turgor of living and hence not fully differentiated late metaxylem (range 0.6–0.8 MPa) was consistently higher than turgor of cortical cells (range 0.4–0.6 MPa) at positions between 40 and 180 mm behind the root tip. Closer to the tip, no turgor difference between the cortex and the stele was measured. The turgor difference indicated that late-metaxylem elements may function as nutrient-storage compartments within the stele. Excised roots were attached to the root pressure probe to precisely manipulate the xylem water potential. Root excision did not affect turgor of cortical cells for at least 8 h. Using the cell pressure probe, the propagation of a hydrostatic pressure change effected by the root pressure probe was recorded in mature and immature xylem elements at various positions along the root. Within seconds, the pressure change propagated along both early and late metaxylems. The half-times of the kinetics, however, were about five times smaller for the early metaxylem, indicating they are likely the major pathway of longitudinal water flow. The hydraulic signal dissipated from the source of the pressure application (cut end of the root) to the tip of the root, presumably because of radial water movement along the root axis. The results demonstrate that the water status of the growth zone and other positions apical to 20 mm is mainly uncoupled from changes of the xylem water potential in the rest of the plant.Abbreviations and Symbols CPP cell pressure probe - EMX early metaxylem - LMX Late metaxylem - P_c cell turgor - P_r root pressure - RPP root pressure probe - t_1/2,c half-time of water exchange across a single cell - t_1/2 half-time of water exchange across multiple cells We thank Antony Matista for his expert assistance in the construction and modification of instruments. The work was supported by grant DCB8802033 from the National Science Foundation and grant 91-37100-6671 from USDA, and by the award of a Feodor Lynen-Fellowship from the Alexander von Humboldt-Foundation (Germany) to J.F.     

Bubble coalescence behaviour in biological media

Summary Volumetric mass transfer coefficients (k_La) were measured by a steady state method in a twin bubble column to characterize the coalescence behaviour of the medium. Employing Hansenula polymorpha cultivation broths, k_La values were compared with those measured in model media in the presence and absence of antifoam agents. The ratio of the volumetric mass transfer coefficient in the system investigated to that in water, , was employed to characterize the cultivation medium.Symbols a Specific gas/liquid interfacial area with regard to the liquid volume in reactor - d_e Dynamical equilibrium bubble diameter - d_H Perforated plate hole diameter - d_p Primary bubble diameter - d_S Sauter bubble diameter - F_v Liquid feed rate - H Bubbling layer height - k_L Gas/liquid mass transfer coefficient - k_La Volumetric mass transfer coefficient - m k_La/(k_La)_r coalescence index - m_corr Corrected coalescence index [Eq. (3)] - OTR Oxygen transfer rate - P_O Dissolved O₂-partial pressure in BS2 - P₁ Dissolved O₂-partial pressure in BS1 - P_O P_O/P_S relative oxygen saturation in BS2 - P₁ P₁/P_S relative oxygen saturation in BS1 - P_S Saturation dissolved oxygen partial pressure - R_c dn_B/dt coalescence rate - S Substrate concentration - t_F Time since the beginning of the cultivation - X Biomass concentration - V₁ Liquid volume in BS1 - w_SG Superficial gas velocity in BS1 - G Gas holdup in BS1 - ₁ V₁/F_v mean liquid residence time in BS1 - BS1 O₂ absorber column - BS2 O₂ desorber column - D Desmophen (antifoam agent) - NS Nutrient salt solution (Table 1)     

Membrane tension regulates water transport in yeast

Evidence that membrane surface tension regulates water fluxes in intact cells of a Saccharomyces cerevisiae strain overexpressing aquaporin AQY1 was obtained by assessing the osmotic water transport parameters in cells equilibrated in different osmolarities. The osmotic water permeability coefficients (P_f) obtained for yeast cells overexpressing AQY1 incubated in low osmolarity buffers were similar to those obtained for a double mutant aqy1aqy2 and approximately three times lower (with higher activation energy, E_a) than values obtained for cells incubated in higher osmolarities (with lower E_a). Moreover, the initial inner volumes attained a maximum value for cells equilibrated in lower osmolarities (below 0.75 M) suggesting a pre-swollen state with the membrane under tension, independent of aquaporin expression. In this situation, the impairment of water channel activity suggested by lower P_f and higher E_a could probably be the first available volume regulatory tool that, in cooperation with other osmosensitive solute transporters, aims to maintain cell volume. The results presented point to the regulation of yeast water channels by membrane tension, as previously described in other cell systems.     

Electron Microscopic Study of Development in a Sea Cucumber,Stichopus tremulus (Holothuroidea), from Unfertilized Egg through Hatched Blastula

In this, the first fine structural study of sea cucumber embryology, eggs and embryos of Stichopus tremulus developing at 7.5°C are described from spawning through hatched blastulae. Spawned eggs are at about first meiotic metaphase and are surrounded by a jelly layer that remains around the embryos until hatching. No vitelline coat can be demonstrated, but whether it is truly absent or removed by electron microscopic processing is not known. Insemination initiates a rapid cortical reaction, completed within 2 min., which involves a wave of cortical granule exocytosis and fertilization envelope formation. The compactly fibrous fertilization envelope is about 50 nm thick and appears to consist entirely of ejected cortical granule material (if one assumes that there is no vitelline coat). As the fertilization envelope elevates, no hyaline layer appears in the perivitelline space. The first and second polar bodies are emitted, respectively, at about 9 and 15 min. after insemination. The first seven or so cleavages are equal, radial, and occur approximately every 4 hr. The blastocoel opens up at the four-cell stage and, during the earlier cleavages, remains connected with the perivitelline space via numerous gaps between the roughly spherical blastomeres. At the 64-cell stage, these gaps begin to close as the blastomeres start to become cuboidal; in addition, an embryonic cuticle is produced on the apical surface of each blastomere. In embryos of several hundred cells, the blastomeres become associated apicolaterally by junctional complexes, each consisting of a zonula adherens and a septate junction. Several hours before hatching, a single cilium is produced at the apical surface of most blastomeres. At hatching (about 50 hr after insemination), the ciliated blastula leaves behind the fertilization envelope and jelly layer. Swimming blastulae soon begin to elongate in the animal-vegetal axis, and a basal lamina develops on blastomere surfaces facing the blastocoel. The discussion includes a fine structural comparison of egg coats among the five classes of the phylum Echinodermata.