Multiscale models for movement in oriented environments and their application to hilltopping in butterflies

Hilltopping butterflies direct their movement in response to topography, facilitating mating encounters via accumulation at summits. In this paper, we take hilltopping as a case study to explore the impact of complex orienteering cues on population dynamics. The modelling employs a standard multiscale framework, in which an individual’s movement path is described as a stochastic ‘velocity-jump’ process and scaling applied to generate a macroscopic model capable of simulating large populations in landscapes. In this manner, the terms and parameters of the macroscopic model directly relate to statistical inputs of the individual-level model (mean speeds, turning rates and turning distributions). Applied to hilltopping in butterflies, we demonstrate how hilltopping acts to aggregate populations at summits, optimising mating for low-density species. However, for abundant populations, hilltopping is not only less effective but also possibly disadvantageous, with hilltopping males recording a lower mating rate than their non-hilltopping competitors.     

Oil droplets and yolk spheres during development of Medaka embryos

The sizes of oil droplets (globules) and the yolk sphere in the Medaka Oryzias latipes egg were measured in the developmental period from fertilization to hatching. Oil droplets coalesced with one another in the process of shifting toward the vegetal pole, and a single large oil droplet was finally located at the vegetal pole region in most eggs 2 days post-fertilization. The volume of the yolk sphere steeply decreased in the period from 2 to 8 days post-fertilization. The volume of oil droplets also declined linearly from 4 to 10 days post-fertilization. Lipid components exhibited no distinct change during embryogenesis. In order to verify whether oil droplets were required for development of Medaka embryos, oil droplets were artificially removed from the early developing embryos without the chorion (egg envelope). Naked embryos without the oil droplet developed normally to fry in the sterilized incubation medium and grew to the same mature fry as those grown from the control embryos.     

Profound Effects of Population Density on Fitness-Related Traits in an Invasive Freshwater Snail

Population density can profoundly influence fitness-related traits and population dynamics, and density dependence plays a key role in many prominent ecological and evolutionary hypotheses. Here, we evaluated how individual-level changes in population density affect growth rate and embryo production early in reproductive maturity in two different asexual lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail that is an important model system for ecotoxicology and the evolution of sexual reproduction as well as a potentially destructive worldwide invader. We showed that population density had a major influence on individual growth rate and early-maturity embryo production, effects that were often apparent even when comparing treatments that differed in population density by only one individual. While individual growth rate generally decreased as population density increased, we detected a hump-shaped relationship between embryo production and density, with females from intermediate-density treatments producing the most embryos and females from low- and high-density treatments producing the fewest embryos. The two lineages responded similarly to the treatments, indicating that these effects of population density might apply more broadly across P. antipodarum. These results indicate that there are profound and complex relationships between population density, growth rate, and early-maturity embryo production in at least two lineages of this important model system, with potential implications for the study of invasive populations, research on the maintenance of sex, and approaches used in ecotoxicology.     

Developmental competence of porcine oocytes selected by brilliant cresyl blue and matured individually in a chemically defined culture medium

The objective of this study was to investigate the effects of oocyte selection using brilliant cresyl blue (BCB) and culture density during individual in vitro maturation (IVM) on porcine oocyte maturity and subsequent embryo development using a chemically defined medium. Cumulus-oocyte complexes (COCs) were classified as BCB-positive or BCB-negative after exposure to a BCB solution for 90 min. The classified COCs were matured in a group (15 COCs per 100-μL droplet) or individually (1 COC per 1-, 2.5-, 5-, or 10-μL droplet). Meiotic competence, intraoocyte glutathione concentration, and developmental competence after intracytoplasmic sperm injection were monitored. The BCB selected oocytes competent for nuclear and cytoplasmic maturation. Furthermore, meiotic competence for oocytes matured individually in a 5-μL droplet was superior (P < 0.05) to that of oocytes matured in a 1-μL droplet. Also, the culture density in a 5-μL droplet during IVM resulted in a higher (P < 0.05) rate of cleaved embryos than that in a 1-μL droplet and produced a similar rate of blastocysts compared with that of a group culture system. Conversely, BCB selection did not improve cleavage and blastocyst formation. In conclusion, it was possible to predict porcine oocytes competent for maturation using oocyte selection with BCB. Moreover, a 5-μL droplet during the individual IVM culture was most suitable for oocyte maturation and subsequent embryo development, although every culture density used in this study supported development up to the blastocyst stage.     

Effect of ice nucleation by droplet of immobilized silver iodide on freezing of rabbit and bovine embryos

Silver iodide was immobilized by applying the insoluble reaction between sodium alginate and calcium chloride. The immobilized silver iodide was immersed into a freezing solution in order to trigger ice nucleation. Temperature change during cooling and postthaw in vitro development of embryos were examined in order to evaluate the effectiveness of the immobilized silver iodide (AgI alginate-gel droplet) on embryo development. Samples containing the AgI alginate-gel droplets released the latent heat of fusion at a higher subzero temperature than samples without the AgI alginate-gel droplets. When the AgI alginate-gel droplet was added to the freezing solution of rabbit and bovine embryos, they were successfully preserved in liquid nitrogen.     

Transport characteristics of expiratory droplets and droplet nuclei in indoor environments with different ventilation airflow patterns

Expiratory droplets and droplet nuclei can be pathogen carriers for airborne diseases. Their transport characteristics were studied in detail in two idealized floor-supply-type ventilation flow patterns: Unidirectional-upward and single-side-floor, using a multiphase numerical model. The model was validated by running interferometric Mie imaging experiments using test droplets with nonvolatile content, which formed droplet nuclei, ultimately, in a class-100 clean-room chamber. By comparing the droplet dispersion and removal characteristics with data of two other ceiling-supply ventilation systems collected from a previous work, deviations from the perfectly mixed ventilation condition were found to exist in various cases to different extent. The unidirectional-upward system was found to be more efficient in removing the smallest droplet nuclei (formed from 1.5 mum droplets) by air extraction, but it became less effective for larger droplets and droplet nuclei. Instead, the single-side-floor system was shown to be more favorable in removing these large droplets and droplet nuclei. In the single-side-floor system, the lateral overall dispersion coefficients for the small droplets and nuclei (initial size 相似文献   

The lipid-droplet proteome reveals that droplets are a protein-storage depot

BACKGROUND: Lipid droplets are ubiquitous organelles that are among the basic building blocks of eukaryotic cells. Despite central roles for cholesterol homeostasis and lipid metabolism, their function and protein composition are poorly understood. RESULTS: We purified lipid droplets from Drosophila embryos and analyzed the associated proteins by capillary LC-MS-MS. Important functional groups include enzymes involved in lipid metabolism, signaling molecules, and proteins related to membrane trafficking. Unexpectedly, histones H2A, H2Av, and H2B were present. Using biochemistry, genetics, real-time imaging, and cell biology, we confirm that roughly 50% of certain embryonic histones are physically attached to lipid droplets, a localization conserved in other fly species. Histone association with droplets starts during oogenesis and is prominent in early embryos, but it is undetectable in later stages or in cultured cells. Histones on droplets are not irreversibly trapped; quantitation of droplet histone levels and transplantation experiments suggest that histones are transferred from droplets to nuclei as development proceeds. When this maternal store of histones is unavailable because lipid droplets are mislocalized, zygotic histone production starts prematurely. CONCLUSIONS: Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.     

In vitro production of bovine embryos using individual oocytes

The development of a bovine in vitro embryo production system where individual oocytes could be followed through to the morula or blastocyst stage would be of interest to several fields of study and would allow us to characterise developmentally competent oocytes and their corresponding follicular environment. Several studies have, however, reported significantly reduced embryo development when oocytes or embryos were cultured individually compared to in groups. The aim of this study was to establish such an embryo production system, with embryo development rates similar to that observed under control (grouped) conditions. This study showed that conservation of the oocyte/embryo medium densities generally employed for grouped culture does not facilitate embryo development if oocytes/embryos are cultured individually. However, individual oocytes could effectively undergo IVM/IVF/IVC to the expanded blastocyst stage with some small modifications to the standard protocol. Individual IVF was effective if carried out in either 100 μl of medium in wells or in 50 μl droplets. Individual IVC, if carried out in 10 or 20 μl droplets of SOF with FCS added at either 0 or 24 hr, was effective in terms of blastocyst yields but 20 μl droplets did yield significantly fewer hatched blastocysts compared to grouped controls (P < 0.05). An entirely individual embryo production system was effective when it included individual IVM in 10 μl droplets of M199 + 10 ng/ml EGF resulting in day 8 blastocyst yields not significantly different from controls (38% vs. 35% respectively). The use of 10% FCS during individual IVM appeared, at least under our experimental conditions, to be detrimental to subsequent development. The uses of an individual system for embryo production are many and varied. The results of this study show clearly that a large proportion of bovine oocytes can develop to the blastocyst stage when matured, fertilized, and cultured individually. This opens the way for studies regarding the quality of specific oocytes in such a way as will greatly improve our understanding of the events of late folliculogenesis. © 1996 Wiley-Liss, Inc.     

Settling of fixed erythrocyte suspension droplets

The fractionation of micron-size particles according to physical properties of size, density and surface characteristics by centrifugation and electrophoresis is hindered when the particles behave collectively rather than individually. The formation and sedimentation of droplets containing particles is an extreme example of collective behavior and a major problem for these separation methods when large quantities of particles need to be fractionated. In this paper, experiments that measured droplet sizes and settling rates for a variety of particles and droplets are described. Expressions are developed relating the particle concentration in a drop to measurable quantities of the fluids and particles. The number of particles in each droplet was then estimated along with the effective droplet density and certain trends are noted. Since a major application of this work is the purification of biological cells in the range of 10 microns, for which monodisperse inert particles are not available, red blood cells from different animals fixed in glutaraldehyde provided model particle groups with the necessary size range, visibility and stability for these fluid dynamical studies.     

Regulation of lipid-droplet transport by the perilipin homolog LSD2

BACKGROUND: Motor-driven transport along microtubules is a primary mechanism for moving and positioning organelles. How such transport is regulated remains poorly understood. For lipid droplets in Drosophila embryos, three distinct phases of transport can be distinguished. To identify factors regulating this transport, we biochemically purified droplets from individual phases and used 2D gel analysis to search for proteins whose amount on droplets changes as motion changes. RESULTS: By mass spectrometry, we identified one such protein as LSD2. Similar to its mammalian counterpart Perilipin, LSD2 is responsible for regulating lipid homeostasis. Using specific antibodies, we confirmed that LSD2 is present on embryonic lipid droplets. We find that lack of LSD2 causes a specific transport defect: Droplet distribution fails to undergo the dramatic changes characteristic of the wild-type. This defect is not due to a complete failure of the core transport machinery--individual droplets still move bidirectionally along microtubules with approximately normal velocities and kinetics. Rather, detailed biophysical analysis suggests that developmental control of droplet motion is lost. We show that LSD2 is multiply phosphorylated in a developmentally controlled manner. LSD2 phosphorylation depends on the transacting signal Halo, and LSD2 can physically interact with the lipid-droplet-associated coordinator Klar, identifying LSD2 as a central player in the mechanisms that control droplet motion. CONCLUSIONS: LSD2 appears to represent a new class of regulators, a protein that transduces regulatory signals to a separable core motor machinery. In addition, the demonstration that LSD2 regulates both transport and lipid metabolism suggests a link between lipid-droplet motion and lipid homeostasis.     

Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.     

A determinant for directionality of organelle transport in Drosophila embryos

BACKGROUND: Motor-driven transport along microtubules is a primary cellular mechanism for moving and positioning organelles. Many cargoes move bidirectionally by using both minus and plus end-directed motors. How such cargoes undergo controlled net transport is unresolved. RESULTS: Using a combination of genetics, molecular biology, and biophysics, we have identified Halo, a novel regulator of lipid droplet transport in early Drosophila embryos. In embryos lacking Halo, net transport of lipid droplets, but not that of other cargoes, is specifically altered; net transport is minus-end directed at developmental stages when it is normally plus-end directed. This reversal is due to an altered balance of motion at the level of individual organelles; without Halo, travel distances and stall forces are reduced for plus-end and increased for minus-end motion. During development, halo mRNA is highly upregulated just as net plus-end transport is initiated (phase II), and its levels drop precipitously shortly before transport becomes minus-end directed (phase III). Exogenously provided Halo prevents the switch to net minus-end transport in phase III in wild-type embryos and induces net plus-end transport during phase II in halo mutant embryos. This mechanism of regulation is likely to be of general importance because the Drosophila genome encodes a family of related proteins with similar sequences, each transiently expressed in distinct domains. CONCLUSIONS: We conclude that Halo acts as a directionality determinant for embryonic droplet transport and is the first member of a new class of transport regulators.     

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells¹.Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.Open in a separate windowClick here to view.^{(58M, flv)}     

Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro

Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.     

Simulation of Airborne Microbial Droplet Transport

The framework for a simulation model which describes the dispersion of individual droplets of water containing viable microbes is presented. The model accounts for physical, chemical, biological, and measured meteorological parameters of each droplet at each of many short time steps. Repeating the modeling process for many droplets will simulate a cloud of droplets. The model is compared with the Tulelake, Calif., release in 1988 and found to show very similar patterns of deposition within 30 m (the maximum observation distance of the source. A hypothesis for the survival sequence in the microbe-containing droplets is discussed.     

Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo culture

Due to the internal nature of mammalian development, much of the research performed is of a static nature and depends on interpolation between stages of development. This approach cannot explore the dynamic interactions that are essential for normal development. While roller culture overcomes the problem of inaccessibility of the embryo, the constant motion of the medium and embryos makes it impossible to observe and record development. We have developed a static mammalian culture system for imaging development of the mouse embryo. Using this technique, it is possible to sustain normal development for periods of 18-24 h. The success of the culture was evaluated based on the rate of embryo turning, heart rate, somite addition, and several gross morphological features. When this technique is combined with fluorescent markers, it is possible to follow the development of specific tissues or the movement of cells. To highlight some of the strengths of this approach, we present time-lapse movies of embryonic turning, somite addition, closure of the neural tube, and fluorescent imaging of blood circulation in the yolk sac and embryo.     

Influence of in vitro systems on embryo survival and fetal development in cattle

In vitro systems are commonly used for the production of bovine embryos. Comparisons between in vivo and in vitro produced embryos illustrate that the morphology of preimplantation-stage embryos differ significantly, the survival of embryos and fetuses is decreased, the size distributions of the populations of conceptuses and fetuses are altered throughout gestation, and placental development is significantly changed. Taken together these findings indicate that exposure to some in vitro environments during the first 7 days of life can profoundly influence fetal and placental development in cattle. An understanding of how in vitro oocyte maturation, in vitro fertilization, and embryo culture systems influence both fetal and placental development should result in systems that consistently produce normal embryos, fetuses, and calves.     

Effect of retinoids and growth factor on in vitro bovine embryos produced under chemically defined conditions

Experiments were conducted to investigate the beneficial effects of adding retinol (RT) and retinoic acid (RA) to bovine oocyte maturation media and insulin-like growth factor-I (IGF-I) to embryo culture under chemically-defined conditions. In Experiment 1.1, in vitro maturation (IVM) was performed in basic maturation media (bMM) and supplemented with 0.3microM RT or 0.5microM RA. For embryo development presumptive zygotes and embryos were placed in droplets of potassium simplex optimized medium (KSOM). Addition of RT and RA to bMM improved (p<0.05) blastocyst formation as compared with control treatments. In Experiment 1.2, using embryos originating from oocytes previously treated with RT and RA, the presumptive zygotes were placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The number of 2-4-cell stage embryos developing to the blastocyst and expanded blastocyst stages were greater (p<0.05) when embryo culture media was supplemented with IGF-I. In Experiment 2.1, IVM was conducted with bMM+FSH containing 0.3microM RT or 0.5microM RA. For embryo development, presumptive zygotes were placed in droplets of KSOM. Addition of RT or RA to IVM medium also enhanced (p<0.05) blastocyst formation. The supplementation of embryo culture media with IGF-I resulted in a greater number (p<0.05) of 2-4-cell stage embryos developing into blastocysts, expanded blastocysts and hatched blastocysts. In Experiment 2.2, using embryos originating from oocytes previously treated with RT and RA, presumptive zygotes were also placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The supplementation of embryo culture media with IGF-I resulted in a greater (p<0.05) number of 2-4-cell stage embryos developing to the blastocyst, expanded blastocyst and hatched blastocyst stages.