Extravascular fluid dynamics of the embryonic chick wing bud

While a number of models of positional information in the chick wing bud have involved the diffusion of morphogens to establish chemical gradients of morphogenetic activity, only recently have there been attempts to characterize the milieu in which such diffusion must take place. We report an analysis of the fluid dynamics of the extravascular (interstitial) spaces of stage 22-25 chick wing buds, into which aqueous aniline blue dye was microinjected as a visible, unreactive tracer. Six sites along the antero-posterior (A-P) and proximo-distal (P-D) axes were chosen for study. Injections of dye into the posterior half of the wing bud exhibited marked directionality of extravascular transport (mean of all posterior sites = 68%), while anterior injections showed little or no directionality (mean of all anterior sites = 13%). All instances of directed dye movement were disto-proximal, the same direction as the blood flow through the marginal veins. In embryos gassed in situ with CO2, which reversibly stopped the heartbeat and vascular flow, directionality was abolished, yet diffusion rates were unaffected. Posterior disto-proximal extravascular dye movement was correlated with the greater diameter, flow velocity, and volume flow rate of the posterior marginal vein, compared to the anterior marginal vein. Radial diffusion rates were measured, and posterior disto-proximal rates were corrected for measured disto-proximal directionality by the use of a simple diffusion-translation model. Three-way analysis of variance showed that directionality-uncorrected disto-proximal rates in posterior sites were not significantly different from anterior radial rates, but that directionality-corrected posterior rates did differ significantly (P less than 0.0001). A significant stage effect (P less than 0.005) and a significant interaction between the A-P axis and stage (P less than 0.05) were also found. We hypothesize that the spatial arrangement and flow patterns of the vasculature physically determine the fluid dynamics of the interstitium. Based on these observations, we also suggest that disto-proximal extravascular fluid movement in the posterior wing bud presents a barrier to the free diffusion of aqueous molecules, including morphogens originating in the "zone of polarizing activity."     

Effects of the actin-binding protein DNAase I on cytoplasmic streaming and ultrastructure of Amoeba proteus

Microinjection of DNAase I, which is known to form a specific complex with G-actin, induces characteristic changes in cytoplasmic streaming, locomotion and morphology of the contractile apparatus of A. proteus. Light microscopical studies show pronounced streaming originating from the uroid and/or the retracting pseudopods, which ceases 10--15 min after injection of DNAase I, at a time when ultrasctructural studies show that the actin filament system is very much reduced. These results suggest that a controlled reversible equilibrium between soluble and polymerized forms of actin is a necessary requirement for amoeboid movement. The topographic distribution of contractile filaments beneath the plasma membrane visualized by correlated light- and electron microscopy of DNAase I-injected cells establishes the importance of the membrane-bound filamentous layer for three major aspects of streaming: (1) Streaming originates by local contractions of a cell membrane-associated filament layer at the uroid and/or retracting pseudopods, creating a pressure flow. (2) This flow continues beneath the membrane, which is stabilized by filaments in the lateral regions between the posterior end, with a high hydrostatic pressure, and the anterior end, with a low hydrostatic pressure. (3) Pseudopods or extending areas are created by a local destabilization of the cell periphery caused by the separation of the filamentous layer from the plasma membrane.     

Time-lapse analysis reveals different modes of primordial germ cell migration in the medaka Oryzias latipes

Previous studies have shown that medaka primordial germ cells (PGC) are first distinguishable by olvas expression during late gastrulation, and that they migrate to the gonadal region through the lateral plate mesoderm. Here, we demonstrate that medaka nanos expression marks the germ line at early gastrulation stage. By marking the germ line with green fluorescent protein (GFP) fused to the nanos 3' untranslated region, we were able to visualize the behavior of PGC using time-lapse imaging. We show that there are three distinct modes of PGC migration that function at different stages of development. At early gastrulation stage, PGC actively migrate towards the marginal zone, a process that requires the function of a chemokine receptor, CXCR4. However, at late gastrulation stage, PGC change the mode and direction of their movement, as they are carried towards the midline along with somatic cells undergoing convergent movements. After aligning bilaterally, PGC actively migrate to the posterior end of the lateral plate mesoderm. This posterior movement depends on the activity of both HMGCoAR and a ligand of CXCR4, SDF-1a. These results demonstrate that PGC undergo different modes of migration to reach the prospective gonadal region of the embryo.     

Cilia, KIF3 molecular motor and nodal flow

The establishment of left-right asymmetry during development of vertebrate embryos depends on leftward flow in the nodal cavity. The flow is produced by the rotational movement of the posteriorly tilted nodal cilia. However, it remains poorly understood how the nodal cilia are tilted posteriorly, and how the directionality of the flow is translated into gene expression patterns in the embryo. Recent studies have identified signaling molecules involved in these processes. First, planar cell polarity signaling has been shown to be involved in the posterior positioning of the basal bodies of nodal cilia, which leads to the posterior tilting of their rotation axes. Second, identification of putative receptors and signaling molecules suggests a link between the signaling molecules delivered by the nodal flow, and downstream signaling in the cells surrounding the nodal cavity and the lateral plate mesoderm.     

Diaphragmatic activity and lung liquid flow in the unanesthetized fetal sheep.

For some time it has been suggested that breathing movements are made "in utero" and recently measurements of tracheal pressure and lung liquid flow in chronic fetal preparations have led to the hypothesis that rapid changes in these parameters are the result of respiratory muscle activity. To test this hypothesis diaphragmatic electrical activity was measured in seven chronic unanesthetized fetal sheep preparations and correlated with lung liquid flow and tracheal pressure. Diaphragmatic activity led to a fall of tracheal pressure and movement of a small volume of lung liquid into the lung. After the activity ceased, tracheal pressure returned to normal and flow diminished to zero or was directed out of the lung. The breathing pattern was unassociated with the net movement of lung liquid out of the lung. A histogram of the interval between breaths revealed a changing pattern of activity throughout gestation. The pattern was significantly altered after premature delivery of one animal with a respiratory problem. These observations provide evidence that respiratory muscles are active "in utero" and that the pattern of activity changes throughout gestation.     

Nodal flow and the generation of left-right asymmetry

The establishment of left-right asymmetry in mammals is a good example of how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left-right axis. Nodal flow is autonomously generated by the rotation of cilia that are tilted toward the posterior on cells of the ventral node. These cilia are built by transport via the KIF3 motor complex. How nodal flow is interpreted to create left-right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of membrane-sheathed particles, called nodal vesicular parcels (NVPs), may result in the activation of the non-canonical Hedgehog signaling pathway, an asymmetric elevation in intracellular Ca(2+) and changes in gene expression.     

Mass transfer enhancement in moving biofilm structures

Biofilms are layers of microbial cells growing on an interface and they can form highly complex structures adapted to a wide variety of environmental conditions. Biofilm streamers have a small immobile base attached to the support and a flexible tail elongated in the flow direction, which can vibrate in fast flows. Herein we report numerical results for the role of the periodical movement of biofilm streamers on the nutrient uptake and in general on the solute mass transfer enhancement due to flow-induced oscillations. We developed what to our knowledge is a novel two-dimensional fluid-structure interaction model coupled to unsteady solute mass transport and solved the model using the finite element method with a moving mesh. Results demonstrate that the oscillatory movement of the biofilm tail significantly increases the substrate uptake. The mass transfer coefficient is the highest in regions close to the streamer tip. The reason for substrate transfer enhancement is the increase in speed of tip movement relative to the surrounding liquid, thereby reducing the thickness of the mass transfer boundary layer. In addition, we show that the relative mass transfer enhancement in unsteady conditions compared with the rigid static structure is larger at higher flow velocities, and this relative increase favors a more flexible structure.     

The spd-2 gene is required for polarization of the anteroposterior axis and formation of the sperm asters in the Caenorhabditis elegans zygote

In the Caenorhabditis elegans zygote, polarization of the anteroposterior (AP) axis occurs during a brief period of reorganization that follows fertilization and results in the establishment of discrete cytoplasmic and cortical domains. In the cytoplasm, germ-line or P granules are circulated by an actomyosin-driven fountain flow of cytoplasm and localize to the posterior, while in the cortex, two proteins required for AP polarity, PAR-2 and PAR-3, localize to the posterior and the anterior, respectively. The identity of the positional cue that determines AP axis orientation is not known, although it has been postulated to be a component of the sperm pronucleus/centrosome complex (SPCC) as the position of the SPCC correlates with the orientation of the AP axis and the direction of the fountain flows. Here, we show that mutations in the spd-2 gene disrupt polarization of the AP axis. In mutant zygotes, the fountain flow of cytoplasm and associated asymmetric cortical contractions are absent, P granules do not localize, and cortical PAR-3 does not become asymmetrically distributed. Interestingly, cortical PAR-2 localizes randomly to either or both poles. The random positioning of PAR-2 requires PAR-3 and indicates that a spd-2-dependent mechanism normally modulates PAR-2/PAR-3 interactions to correctly position PAR-2 at the posterior. spd-2 mutations also disrupt formation of the SPCC by delaying and attenuating the formation of sperm asters until after the period of reorganization, suggesting that spd-2 mutations disrupt formation of the positional cue. Our results also indicate that sperm asters are not essential for pronuclear migration but are required for rapid female pronuclear movement and premitotic positioning of the pronuclei.     

Dynamic topology of the cephalochordate to amniote morphological transition: a self-organized system of Russian dolls

This note presents a mechanistic explanation of the transition between the morphology of cephalochordates to that of amniotes. By a careful study of the morphogenetic movements which occur during the early stages of development of a typical amniote (a chicken embryo), we are able to show that the formation of a vertebrate body follows a sequence: first, formation of dorsal folds, then head and heart as dorsal and ventral folds, and finally another dorsal fold, which eventually builds up the chorion. This order has a physical origin linked to the velocity field of the tissue flow. These folds form at right angles to the flow direction, and the topology of the chordates flow is hyperbolic. This mechanism explains the differences between the successive bauplans, by the cumulate forward and backward movement of the flow. Eventually, the entire phenomenon can be described as a self-organized system of Russian dolls, by which the heart finds itself inside the embryo, and the embryo itself inside the chorion. In addition, the phenomenon has a mirror symmetry in the anterior and in the posterior part, thereby explaining naturally the existence of animals having a caudal heart.     

Coordinated Flagellar and Ciliary Beating in the Protozoon Tritrichomonas foetus1

ABSTRACT. Tritrichomonas foetus is a flagellated protozoon found in urogenital tract of cattle. Its free movement in liquid medium is powered by the coordinated movement of three flagella projecting towards the anterior region of the cell, and one recurrent flagellum that forms a junction with the cell body and ends as a free projection in the posterior region of the cell. We have used video microscopy and digital image processing to analyze the relationships between the movements of these flagella. The anterior flagella beat in a ciliary type pattern displaying effective and recovery strokes, while the recurrent flagellum beats in a typical flagellar wave form. One of the three anterior flagella has a distinctive pattern of beating. It beats straight in its forward direction as opposed to the ample beats performed by the others. Frequency measurements obtained from cells swimming in a viscous medium shows that the beating frequency of the recurrent flagelium is approximate twice the frequency for the three anterior flagella. We also observed that the costa and the axostyle do not show any active motion. On the contrary, they form a cytoskeletal base for the anchoring and orientation of the flagella.     

Perception of sound-source motion by the human brain

We assessed the human brain network for sound-motion processing using the same virtual stimulus in three independent functional imaging experiments. All experiments show a bilateral posterior network of activation, including planum temporale (PT) and parieto-temporal operculum (PTO). This was demonstrated in contrasts between sound movement and two control conditions: externalized stationary stimuli (in the midline or to the side of the head) and midline sounds within the head with similar spectro-temporal structure. We suggest specific computational mechanisms in PT for disambiguation of the intrinsic spectro-temporal features of a sound and the spectro-temporal effect of sound movement. The results support the existence of a posteriorly directed temporo-parietal pathway for obligatory perceptual processing of sound-source motion.     

The Drosophila Actin Regulator ENABLED Regulates Cell Shape and Orientation during Gonad Morphogenesis

Organs develop distinctive morphologies to fulfill their unique functions. We used Drosophila embryonic gonads as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. We developed a membrane GFP marker to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). We show that Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. We propose that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction.     

A 'chemotactic dipole' mechanism for large-scale vortex motion during primitive streak formation in the chick embryo

Primitive streak formation in the chick embryo involves significant coordinated cell movement lateral to the streak, in addition to the posterior-anterior movement of cells in the streak proper. Cells lateral to the streak are observed to undergo 'polonaise movements', i.e. two large counter-rotating vortices, reminiscent of eddies in a fluid. In this paper, we propose a mechanism for these movement patterns which relies on chemotactic signals emitted by a dipolar configuration of cells in the posterior region of the epiblast. The 'chemotactic dipole' consists of adjacent regions of cells emitting chemo-attractants and chemo-repellents. We motivate this idea using a mathematical analogy between chemotaxis and electrostatics, and test this idea using large-scale computer simulations. We implement active cell response to both neighboring mechanical interactions and chemotactic gradients using the Subcellular Element Model. Simulations show the emergence of large-scale vortices of cell movement. The length and time scales of vortex formation are in reasonable agreement with experimental data. We also provide quantitative estimates for the robustness of the chemotaxis dipole mechanism, which indicate that the mechanism has an error tolerance of about 10% to variation in chemotactic parameters, assuming that only 1% of the cell population is involved in emitting signals. This tolerance increases for larger populations of cells emitting signals.     

The active and passive ciliary motion in the embryo node: A computational fluid dynamics model

The breaking of left–right symmetry in the mammalian embryo is believed to occur in a transient embryonic structure, the node, when cilia create a leftward flow of liquid. The two-cilia hypothesis proposes that the node contains two kinds of primary cilia: motile cilia that rotate autonomously to generate the leftward fluid flow and passive cilia that act as mechano-sensors, responding to flow. While studies support this hypothesis, the mechanism by which the sensory cilia respond to the fluid flow is still unclear. In this paper, we present a computational model of two cilia, one active and one passive. By employing computational fluid dynamics, deformable mesh computational techniques and fluid–structure interaction analysis, and solving the three-dimensional unsteady transport equations, we study the flow pattern produced by the movement of the active cilium and the response of the passive cilium to this flow. Our results reveal that clockwise rotation of the active cilium can generate a counter-clockwise elliptical rotation and overall lateral displacement for its neighboring passive one, of measurable magnitude and consistent pattern. This supports the plausibility of the two-cilia hypothesis and helps quantify the motion pattern for the passive cilium induced by this regional flow.     

Making the connection: expanding the role of restoration genetics in restoring and evaluating connectivity

The success of restoration activities is affected by connectivity with the surrounding landscape. From a genetic perspective, landscape connectivity can influence gene flow, effective size, and genetic diversity of populations, which in turn have impacts on the fitness and adaptive potential of species in restored areas. Researchers and practitioners are increasingly using genetic data to incorporate elements of connectivity into restoration planning and evaluation. We show that genetic studies of connectivity can improve restoration planning in three main ways. First, by comparing genetic estimates of contemporary and historical gene flow and population size, practitioners can establish historical baselines that may provide targets for restoration of connectivity. Second, empirical estimates of dispersal, landscape resistance to movement, and adaptive genetic variance can be derived from genetic data and used to parameterize existing restoration planning tools. Finally, restoration actions can also be targeted to remove barriers to gene flow or mitigate pinch‐points in corridors. We also discuss appropriate methods for evaluating the restoration of gene flow over timescales required by practitioners. Collaboration between restoration geneticists, ecologists, and practitioners is needed to develop practical and innovative ways to further incorporate connectivity into restoration practice.     

Linking movement behaviour to dispersal and divergence in plethodontid salamanders

To better understand the evolutionary and ecological effects of dispersal, there is growing emphasis on the need to integrate direct data on movement behaviour into landscape-scale analyses. However, little is known about the general link between movement behaviour and large-scale patterns of dispersal and gene flow. Likewise, although recent studies suggest that nonrandom, directionally biased movement and dispersal can promote evolutionary divergence, the generality of this mechanism is unknown. We test the hypothesis that directionally biased movement and dispersal by plethodontid salamanders interact with the topography of headwater areas to affect genetic and phenotypic divergence. Movements by Gyrinophilus porphyriticus and Eurycea bislineata show contrasting directional biases: upstream bias in G. porphyriticus and downstream bias in E. bislineata. Consistent with predictions of how these biases interact with slope to affect dispersal and gene flow, genetic distance increased with slope in G. porphyriticus and decreased with slope in E. bislineata over a standardized distance of 1 km along six headwater streams. In both species, phenotypic divergence in relative trunk length was positively related to genetic divergence. These results indicate that landscape-scale patterns of dispersal and gene flow are closely related to movement behaviour in G. porphyriticus and E. bislineata, and underscore the value of information on movement behaviour for predicting and interpreting patterns of dispersal and gene flow in complex landscapes. This study also provides new evidence that directionally biased movement and dispersal can be important sources of intra- and interspecific variation in population divergence, and highlights the value of explicit, a priori predictions in landscape genetic studies.     

Directionality of wingless protein transport influences epidermal patterning in the Drosophila embryo.

Active endocytotic processes are required for the normal distribution of Wingless (Wg) protein across the epidermal cells of each embryonic segment. To assess the functional consequences of this broad Wg distribution, we have devised a means of perturbing endocytosis in spatially restricted domains within the embryo. We have constructed a transgene expressing a dominant negative form of shibire (shi), the fly dynamin homologue. When this transgene is expressed using the GAL4-UAS system, we find that Wg protein distribution within the domain of transgene expression is limited and that Wg-dependent epidermal patterning events surrounding the domain of expression are disrupted in a directional fashion. Our results indicate that Wg transport in an anterior direction generates the normal expanse of naked cuticle within the segment and that movement of Wg in a posterior direction specifies diverse denticle cell fates in the anterior portion of the adjacent segment. Furthermore, we have discovered that interfering with posterior movement of Wg rescues the excessive naked cuticle specification observed in naked (nkd) mutant embryos. We propose that the nkd segment polarity phenotype results from unregulated posterior transport of Wg protein and therefore that wild-type Nkd function may contribute to the control of Wg movement within the epidermal cells of the segment.     

Crossveinless d is a vitellogenin-like lipoprotein that binds BMPs and HSPGs, and is required for normal BMP signaling in the Drosophila wing

The sensitivity of the posterior crossvein in the pupal wing of Drosophila to reductions in the levels and range of BMP signaling has been used to isolate and characterize novel regulators of this pathway. We show here that crossveinless d (cv-d) mutations, which disrupt BMP signaling during the development of the posterior crossvein, mutate a lipoprotein that is similar to the vitellogenins that comprise the major constituents of yolk in animal embryos. Cv-d is made in the liver-like fat body and other tissues, and can diffuse into the pupal wing via the hemolymph. Cv-d binds to the BMPs Dpp and Gbb through its Vg domain, and to heparan sulfate proteoglycans, which are well-known for their role in BMP movement and accumulation in the wing. Cv-d acts over a long range in vivo, and does not have BMP co-receptor-like activity in vitro. We suggest that, instead, it affects the range of BMP movement in the pupal wing, probably as part of a lipid-BMP-lipoprotein complex, similar to the role proposed for the apolipophorin lipid transport proteins in Hedgehog and Wnt movement.     

Effect of Temperature on the Dispersion of 1,2-dibromo-3-chloropropane in Soil

A study was conducted to evaluate the effect of temperature on the dispersion of 1,2-dibromo-3-chloropropane (DBCP) in soil. The facility of solution of DBCP in water was also investigated. Results of these studies show that the movement of DBCP from an injection site is temperature dependent. These data also indicate that DBCP dissolves readily in water and that dissolved chemical serves to limit the rate of outward dispersion once the liquid DBCP which was added has changed state. The mechanics of DBCP dispersion following soil injection are discussed.