Molecular characterization of the gastrula in the turtle Emys orbicularis: an evolutionary perspective on gastrulation

Due to the presence of a blastopore as in amphibians, the turtle has been suggested to exemplify a transition form from an amphibian- to an avian-type gastrulation pattern. In order to test this hypothesis and gain insight into the emergence of the unique characteristics of amniotes during gastrulation, we have performed the first molecular characterization of the gastrula in a reptile, the turtle Emys orbicularis. The study of Brachyury, Lim1, Otx2 and Otx5 expression patterns points to a highly conserved dynamic of expression with amniote model organisms and makes it possible to identify the site of mesoderm internalization, which is a long-standing issue in reptiles. Analysis of Brachyury expression also highlights the presence of two distinct phases, less easily recognizable in model organisms and respectively characterized by an early ring-shaped and a later bilateral symmetrical territory. Systematic comparisons with tetrapod model organisms lead to new insights into the relationships of the blastopore/blastoporal plate system shared by all reptiles, with the blastopore of amphibians and the primitive streak of birds and mammals. The biphasic Brachyury expression pattern is also consistent with recent models of emergence of bilateral symmetry, which raises the question of its evolutionary significance.     

Geometry and mechanics of teleost gastrulation and the formation of primary embryonic axes

Examination of normal shaping dynamics and immediate and long-term responses to blastoderm cutting in zebrafish and loach embryos prior to the onset of gastrulation and during the course of epiboly revealed that anteroposterior (AP) and dorsoventral (DV) polarity formation is connected with shaping of the blastoderm circumferential region, which stretches along and shrinks across its movement axes and originates the non-isotropic fields of tensile stresses. Based on data from cutting experiments and quantitative morphology, we reconstructed the movement-shaping patterns of epiboly and embryonic shield formation. We revealed that AP and DV axes originate as a mass cell movement subject to the movement-shaping equivalence principle, which means the spatial series of differently shaped areas corresponding to the time succession of the same area shaping. Maintenance of the main body axes in orthogonal orientation depends on the mechanical equilibrium principle allowing for converting shape asymmetry into that of tensile stresses and vice versa. The causal relationship between the main movement-shaping axes and that of embryonic polarity was proved in cutting experiments in which the DV axis direction was subject to rearrangement so as to adjust to the new direction of mass cell movement axes induced by healing the wound in the blastoderm circumferential region.     

Changes to the geometry and fluid mechanics of the carotid siphon in the pediatric Moyamoya disease

Background: The Moyamoya disease is a cerebrovascular disease that causes occlusion of the distal end of the internal carotid artery, leading to the formation of multiple tiny collateral arteries. To date, the pathogenesis of Moyamoya is unknown. Improved understanding of the changes to vascular geometry and fluid mechanics of the carotid siphon during disease may improve understanding of the pathogenesis, prognosis techniques and disease management. Methods: A retrospective analysis of Magnetic Resonance Angiography (MRA) images was performed for Moyamoya pediatric patients (MMD) (n = 23) and control (Ctrl) pediatric patients (n = 20). The Ctrl group was composed of patients who complained of headache and had normal MRA. We performed segmentation of MRA images to quantify geometric parameters of the artery. Computational fluid dynamics (CFD) was performed to quantify the hemodynamic parameters. Results: MMD internal carotid and carotid siphons were smaller in cross-sectional areas, and shorter in curved vascular length. Vascular curvature remained constant over age and vascular size and did not change between Ctrl and MMD, but MMD carotid siphon had lower tortuosity in the posterior bend, and higher torsion in the anterior bend. Wall shear stress and secondary flows were significantly lower in MMD, but the ratio of secondary flow kinetic energy to primary flow kinetic energy were similar between MMD and Ctrl. Conclusion: There were alterations to both the geometry and the flow mechanics of the carotid siphons of Moyamoya patients but it is unclear whether hemodynamics is the cause or the effect of morphological changes observed.     

Contribution of the submentalis muscle to feeding mechanics in the leopard frog, Rana pipiens

This study investigated the functional contributions of the submentalis muscle to the coordination of feeding behavior in the leopard frog, Rana pipiens. Additionally, the anatomical origins of the motor neurons innervating this muscle are identified and described. The m. submentalis is a small muscle connecting the distal mandibular tips. Depending upon the anuran species studied, this muscle contributes to mandibular bending and the degree to which the tongue is protracted, or has little or no role in feeding biomechanics. High-speed videography was used to quantify feeding attempts before versus after bilateral denervation of the m. submentalis. Additionally, the terminal branch of the trigeminal nerve prior to innervating the m. submentalis was retrogradely labeled to identify the origins of motor neurons innervating the muscle. For the kinematic analyses, denervation of the submentalis resulted in significant increases in the time to maximum tongue protrusion, and the duration of tongue protrusion. Neither mandibular bending, nor tongue length variables differed significantly between normal conditions and deafferented conditions. However, when unsuccessful feeding attempts were quantified following the denervation, failed attempts were nearly always due to the tongue not reaching the prey. None of the unsuccessful feedings prior to denervation were due to inadequate tongue protrusion. Anatomical data show a much larger rostral-caudal distribution of the trigeminal motor neurons than previously described for anurans. These data suggest a larger role for the submentalis muscle in Rana than in previously studied anurans with long protrusible tongues, and suggests a feedback mechanism from the trigeminal nerve to the nerves coordinating tongue protraction and retraction.     

Expression of Notch genes and their ligands during gastrulation in the chicken embryo

We examined the expression patterns of the two homologous genes, spinal cord-derived growth factor (SCDGF)/platelet-derived growth factor (PDGF)-C/fallotein and SCDGF-B/PDGF-D in the rat central nervous system. In the spinal cord, SCDGF/PDGF-C/fallotein was expressed in the floor plate at embryonic day (E) 11 and also in the ventricular zone at E16 but not in adult. However, SCDGF-B/PDGF-D was prominently expressed in the adult motoneurons, although faint expression was observed in the ventral ventricular zone at E16. Also in the brain, the expression of SCDGF/PDGF-C/fallotein was more remarkable at E16 than at adult. It was highly expressed in the cortex, pontine area and choroid plexus at E16. Contrary to SCDGF/PDGF-C/fallotein, SCDGF-B/PDGF-D expression was notable in several nuclei at adult.     

Variation and robustness of the mechanics of gastrulation: the role of tissue mechanical properties during morphogenesis

Diverse mechanisms of morphogenesis generate a wide variety of animal forms. In this work, we discuss two ways that the mechanical properties of embryonic tissues could guide one of the earliest morphogenetic movements in animals, gastrulation. First, morphogenetic movements are a function of both the forces generated by cells and the mechanical properties of the tissues. Second, cells could change their behavior in response to their mechanical environment. Theoretical studies of gastrulation indicate that different morphogenetic mechanisms differ in their inherent sensitivity to tissue mechanical properties. Those few empirical studies that have investigated the mechanical properties of amphibian and echinoderm gastrula-stage embryos indicate that there could be high embryo-to-embryo variability in tissue stiffness. Such high embryo-to-embryo variability would imply that gastrulation is fairly robust to variation in tissue stiffness. Cell culture studies demonstrate a wide variety of cellular responses to the mechanical properties of their microenvironment. These responses are likely to be developmentally regulated, and could either increase or decrease the robustness of gastrulation movements depending on which cells express which responses. Hence both passive physical and mechanoregulatory processes will determine how sensitive gastrulation is to tissue mechanics. Addressing these questions is important for understanding the significance of diverse programs of early development, and how genetic or environmental perturbations influence development. We discuss methods for measuring embryo-to-embryo variability in tissue mechanics, and for experimentally perturbing those mechanical properties to determine the sensitivity of gastrulation to tissue mechanics.     

Regeneration of the active zone at the frog neuromuscular junction

The active zone is a unique specialization of the presynaptic membrane and is believed to be the site of transmitter release. The formation of the active zone and the relationship of this process to transmitter release were studied at reinnervated neuromuscular junctions in the frog. At different times after a nerve crush, the cutaneous pectoris muscles were examined with intracellular recording recording and freeze-fracture electron microscopy. The P face of a normal active zone typically consists of two double rows of particles lined up in a continuous segment located opposite a junctional fold. In the initial stage of reinnervation, clusters of large intramembrane particles surrounding membrane elevations appeared on the P face of nerve terminals. Like normal active zones, these clusters were aligned with junctional folds. Vesicle openings, which indicate transmitter release, were seen at these primitive active zones, even though intramembrane particles were not yet organized into the normal pattern of two double rows. The length of active zones at this stage was only approximately 15% of normal. During the secondary stage, every junction was reinnervated and most active zones had begun to organize into the normal pattern with normal orientation. Unlike normal, there were often two or more discontinuous short segments of active zone aligned with the same junctional fold. The total length of active zone per junctional fold increased to one-third of normal, mainly because of the greater number of segments. In the third stage, the number of active zone segments per junctional fold showed almost no change when compared with the secondary stage. However, individual segments elongated and increased the total length of all active zone segments per junctional fold to about two-thirds of the normal length. The dynamic process culminated in the final stage, during which elongating active zones appeared to join together and the number of active zone segments per junctional fold decreased to normal. Thus, in most regions, regeneration of the active zones was complete. These results suggest that the normal organization of two double rows is not necessary for the active zone to be functional. Furthermore, localization of regenerating active zones is related to junctional folds and/or their associated structures.     

Cell electrophoresis of amphibian blastula and gastrula cells; the relationship of surface charge and morphogenetic movement

By use of a novel modification in cell electrophoresis technique, an “electrophoretic map” has been made of selected embryonic regions at four different stages of embryogenesis in Rana pipiens. Hitherto, it had been impossible to make surface charge determinations for cells from small, selected regions of early amphibian embryos.Embryonic regions were dissected, and the tissue was disaggregated in EDTA. For cell electrophoresis, cells were suspended in low ionic strength medium with 15% Ficoll, and timed over 14.6 micrometers in a voltage gradient of 20 V/cm.The data reveal a correlation between three parameters: that of surface charge, morphogenetic movement, and adhesiveness. Embryonic regions that undergo a rapid increase in surface charge with the onset of gastrulation, are the same regions that initiate morphogenetic activity. Regions in which surface charge remains constant tend to be morphogenetically less active. Adhesive estimates were based on observations of degree of rounding of cells in situ. There is an apparent correlation, in ectoderm, mesoderm, and endoderm, of increased surface charge with decreased adhesiveness (and vice versa).     

On the geometry and mechanics of tooth position in the white shark,Carcharodon carcharias

The teeth of captured specimens, of prepared museum specimens, and of high-speed videotape images of the white shark, Carcharodon carcharias, were compared with respect to (1) deviation of each tooth from the animal's midline and (2) the crown angle of the functional teeth along the jaw margin. Tooth position was measured either directly using a meter stick apparatus or derived from tracings of the video footage. Tooth positions were not statistically unique in any region of the upper or lower jaw but demonstrated less variability in crown angle within 30° of the midline (71.48° ± 10°). Videotape analysis of feeding sharks indicated an 8.7° increase in crown angle of the centermost teeth during bites where the jaws were closed through an angle of 20–35° and a 15.7° reduction in this same parameter during jaw adduction through 35° or more. Such changes in tooth orientation (relative to the rear of the buccal cavity) are ascribed to flexure of the cartilaginous jaws and cranium by the cranial musculature and possibly also to sliding of the tooth bed over the jaw. Outward rotation of the teeth and jaw rami describes a plucking action during feeding or prey sampling, while larger bites rotate the frontmost teeth inward towards the gullet. Functionally, this may make the teeth more effective at grasping small prey items or gouging chunks from larger prey. However, testing of the load required to remove teeth showed no significant increase in tensile resistance with reduced crown angle. © 1995 Wiley-Liss, Inc.     

Influence of patellofemoral articular geometry and material on mechanics of the unresurfaced patella

Patellar resurfacing during knee replacement is still under debate, with several studies reporting higher incidence of anterior knee pain in unresurfaced patellae. Congruency between patella and femur impacts the mechanics of the patellar cartilage and strain in the underlying bone, with higher stresses and strains potentially contributing to cartilage wear and anterior knee pain. The material properties of the articulating surfaces will also affect load transfer between femur and patella. The purpose of this study was to evaluate the mechanics of the unresurfaced patella and compare with natural and resurfaced conditions in a series of finite element models of the patellofemoral joint. In the unresurfaced analyses, three commercially available implants were compared, in addition to an 'ideal' femoral component which replicated the geometry, but not the material properties, of the natural femur. Hence, the contribution of femoral component material properties could be assessed independently from geometry changes. The ideal component tracked the kinematics and patellar bone strain of the natural knee, but had consistently inferior contact mechanics. In later flexion, compressive patellar bone strain in unresurfaced conditions was substantially higher than in resurfaced conditions. Understanding how femoral component geometry and material properties in unresurfaced knee replacement alters cartilage contact mechanics and bone strain may aid in explaining why the incidence of anterior knee pain is higher in the unresurfaced population, and ultimately contribute to identifying criteria to pre-operatively predict which patients are suited to an unresurfaced procedure and reducing the incidence of anterior knee pain in the unresurfaced patient population.     

The effect of three-dimensional glottal geometry on intraglottal quasi-steady flow distributions and their relationship with phonation

Vocal fold geometry plays an important role in human phonation. The intraglottal quasi-steady pressure and velocity distributions depend upon the shape, size, and diameter of the glottis. This study reports the effects of the variation of glottal shapes on intraglottal pressures and velocities using a Plexiglas model with a glottis having nine symmetric glottal angles (uniform, as well as convergent and divergent 5°, 10°, 20° and 40°), while the minimal glottal diameter was held constant at 0.06 cm. The empirical data were supported by penalty finite element computational results. The results suggest that larger convergent glottal angles correspond to increased pressures and decreased velocities in the glottis upstream of the minimum glottal location, with a reversal of this pattern at the minimal glottal diameter location. The pressure dip near the glottal entrance for divergent glottal angles was greatest for the 10° divergence angle condition, and was sequentially less for 5°, 20°, and 40°. Flow resistance was greater for a convergent angle than a divergent angle of the same value, and least for the 10° divergent condition. Pressure recovery in the glottis suggested that the optimal glottal diffuser angle was near 10°. Results suggest that the glottal geometry has a critical relationship with phonation (especially for vocal efficiency), and therefore important significance to understanding artistic voice and clinical voice management.     

The natural variability of morphogenesis: a tool for exploring the mechanics of gastrulation movements in amphibian embryos

The investigation of natural variability of metric morphological characters in frog gastrulation revealed that in genetically and environmentally homogeneous samples of embryos their variability is of a higher order of magnitude than that known for quantitative metric characters in adult organisms. Matching the coefficients of variation of characters under consideration to the specific rates of their changes in normal development revealed a strong positive correlation between the rates of morphological change and the amount of morphological variance. The increase in the variance is mainly in characters concerned with shaping of moving embryonic areas and arises as a result of a positive feedback between the movement of a given area and recruitment of cells from surrounding areas into the movement. The account of natural variation suggests a new model of amphibian gastrulation whose essential feature is the intimate connection between the movement and shaping of the dorsal blastopore lip of the gastrula.     

Sputum mediator profiling and relationship to airway wall geometry imaging in severe asthma

Background

Severe asthma is a heterogeneous disease and the relationship between airway inflammation and airway remodelling is poorly understood. We sought to define sputum mediator profiles in severe asthmatics categorised by CT-determined airway geometry and sputum differential cell counts.

Methods

In a single centre cross-sectional observational study we recruited 59 subjects with severe asthma that underwent sputum induction and thoracic CT. Quantitative CT analysis of the apical segment of the right upper lobe (RB1) was performed. Forty-one mediators in sputum samples were measured of which 21 mediators that were assessable in >50% of samples were included in the analyses.

Results

Independent of airway geometry, sputum MMP9 and IL-1β were elevated in those groups with a high sputum neutrophil count while sputum ICAM was elevated in those subjects with a low sputum neutrophil count. In contrast, sputum CCL11, IL-1α and fibrinogen were different in groups stratified by both sputum neutrophil count and airway geometry. Sputum CCL11 concentration was elevated in subjects with a low sputum neutrophil count and high luminal and total RB1 area, whereas sputum IL1α was increased in subjects with a high sputum neutrophil count and low total RB1 area. Sputum fibrinogen was elevated in those subjects with RB1 luminal narrowing and in those subjects with neutrophilic inflammation without luminal narrowing.

Conclusions

We have demonstrated that sputum mediator profiling reveals a number of associations with airway geometry. Whether these findings reflect important biological phenotypes that might inform stratified medicine approaches requires further investigation.     

Na activation delays and their relation to inactivation in frog skeletal muscle

Summary Delays in the development of activation of Na currents were studied using voltage-clamped frog skeletal muscle fibers. Na currents elicited by a depolarizing voltage step from a hyperpolarized membrane potential were delayed in their activation when compared to Na currents elicited from the resting potential. The magnitude of the delay increased with larger hyperpolarizing potentials and decreased with larger depolarizing test potentials. Delays in activation observed following chloramine-T treatment that partially removes inactivation did not differ from delays observed before treatment. Longer exposures of the muscle fiber to chloramine-T led to a complete loss of inactivation, coincident with an elimination of the hyperpolarization-induced delays in activation. Steady-state slow inactivation was virtually unaffected by prolonged exposures of the fibers to chloramine-T that eliminated fast inactivation. The results show that chloramine-T acts at a number of sites to alter both activation and inactivation. Markov model simulations of the results show that chloramine-T alters fundamental time constants of the system by altering both activation and inactivation rate constants.     

Preferential polar pathways in the cuticle and their relationship to ectodesmata

Summary Ectodesmata-like structures, referred to here as mercurous or mercury precipitates (MP) and considered to be identical to precipitates observed after treatment of leaf tissue with Gilson solution for demonstration of ectodesmata, were demonstrated with cuticle enzymatically isolated from Allium bulb scales and leaves mounted on ascorbic acid-enriched agar or gelatin. The MP distribution patterns obtained with isolated cuticle, in the absence of a cell wall, were identical to those observed with living tissue. Since the distribution in either the presence or absence of the cell wall was similar, the distribution pattern must be determined by the cuticle and not by the cell wall. Disruption of the physical arrangement of epicuticular wax by brushing or removal with chloroform altered the distribution pattern and increased the frequency of MP. This was interpreted to mean that epicuticular wax plays an important role and also that the necessary reductant was not localized in specific structures in the cell wall. Based on this evidence, it appears that ectodesmata, as demonstrated with Gilson solution, are not specific cell-wall structures, whether plasmic or not plasmic. More likely, the MP observed in the cell wall reflect areas in the cuticle permeable to mercuric chloride and undoubtedly to other polar compounds. The presence of such pathways in the cuticle, long established as the prime barrier to penetration of polar compounds, has marked implications in foliar uptake and excretion.Michigan Agricultural Experiment Station Journal Article No. 4971. This study was supported-in-part by Public Health Service Grant CC 00246 from the National Communicable Disease Center, Atlanta, Georgia, and Food and Drug Administration Grant FD 00223.     

Q-bands in Lilium and their relationship to C-banded heterochromatin

In L. pardalinum, narrow bands of quinacrine fluorescence are distributed throughout the chromosomes. These vary in intensity from dull to bright, and their constant pattern allows all chromosomes to be recognized. Bright bands occur at some centromeres, and near all three nucleolar constrictions. In L. longiflorum, similar Q-bands occur along chromosomes, but they are less distinctive and their pattern does not closely match that of L. pardalinum. Also, L. longiflorum does not have bright regions at or near primary and secondary constrictions. Most Q-bands do not coincide with dark Giemsa C-bands, except for the bright nucleolar and centromeric regions in L. pardalinum. All C-banded heterochromatin stains identically after SSC pretreatment, dark with Giemsa and bright with quinacrine.— The many Q-bands of varying intensity, wide distribution and constant pattern, unrelated to C-bands, may be analogous to mammalian Q-bands. Such universality is expected if Q-bands area fundamental component of chromosome architecture.