Studies on the mechanisms of neurulation in the chick: morphometric analysis of force distribution within the neuroepithelium during neural tube formation

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197-205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V-shaped neuroepithelium), 2) the midlateral walls (during transformation of the V-shaped neuroepithelium into a C-shaped neuroepithelium), and 3) the upper walls (during the transformation of the C-shaped neuroepithelium into a closed neural tube).     

Studies on the mechanisms of neurulation in the chick: interrelationship of contractile proteins, microfilaments, and the shape of neuroepithelial cells

Electron microscopy and indirect immunofluorescence were employed to correlate the distribution patterns of major contractile proteins (actin and myosin) with 1) the organizational state of microfilaments, 2) the apical cell surface topography, 3) the shape of the neuroepithelial cells, and 4) the degree of bending of the neuroepithelium during neurulation in chick embryos at Hamburger and Hamilton stages 5-10 of development. Both actin and myosin are present at these developmental stages and colocalize in the neural plate as well as in later phases of neurulation. During elevation of neural folds, actin- and myosin-specific fluorescence is always most intense in regions where the greatest degree of bending of the neuroepithelium takes place [e.g., the midline of the V-shaped neuroepithelium (early neural fold stage) and the midlateral walls of the "C"-shaped neuroepithelium (mid-neural-fold stage)]. This intense fluorescence coincides with 1) a particularly dense packing of microfilaments and 2) highly constricted cell apices. After neural folds make contact, there is an overall reduction in both the intensity of apical fluorescence and the thickness of apical microfilament bundles, especially in the roof and floor of the neural tube. The remaining fluorescence in the contact area is apparently related to cellular movements during fusion of neural folds.     

Effects of shape variations on the energy metabolism of the sand cricket Gryllus firmus: a geometric morphometric analysis

Respiration and energy metabolism are key processes in animals, which are severely constrained by the design of physical structures, such as respiratory structures. Insects have very particular respiratory systems, based on gas diffusion across tracheae. Since the efficiency of the tracheal respiratory system is highly dependent on body shape, the pattern of morphological variation during ontogeny could have important metabolic consequences. We studied this problem combining through-flow respirometry and geometric morphometrics in 88 nymphs of the sand cricket, Gryllus firmus. After measuring production in each individual, we took digital photographs and defined eight landmarks for geometric morphometric analysis. The analysis suggested that ontogenic deformations were mostly related to enlargement of the abdomen, compared to thorax and head. We found that (controlling for body size) metabolic variables and especially resting metabolism are positively correlated with a shape-component associated to an elongation of the abdomen. Our results are in agreement with the mechanics of tracheal ventilation in orthopterans, as gas circulation occurs by changes in abdominal pressures due to abdominal contractions and expansions along the longitudinal axis.     

How generation intervals shape the relationship between growth rates and reproductive numbers

Mathematical models of transmission have become invaluable management tools in planning for the control of emerging infectious diseases. A key variable in such models is the reproductive number R. For new emerging infectious diseases, the value of the reproductive number can only be inferred indirectly from the observed exponential epidemic growth rate r. Such inference is ambiguous as several different equations exist that relate the reproductive number to the growth rate, and it is unclear which of these equations might apply to a new infection. Here, we show that these different equations differ only with respect to their assumed shape of the generation interval distribution. Therefore, the shape of the generation interval distribution determines which equation is appropriate for inferring the reproductive number from the observed growth rate. We show that by assuming all generation intervals to be equal to the mean, we obtain an upper bound to the range of possible values that the reproductive number may attain for a given growth rate. Furthermore, we show that by taking the generation interval distribution equal to the observed distribution, it is possible to obtain an empirical estimate of the reproductive number.     

Experimental analysis on the variations of acid-base balance in the chick embryo

1. The pH remains steady, 7.57. 2. The pCO2 increases gradually. We believe this is fundamental for hatching. 3. Bicarbonate and base excess increase from acidosis to alkalosis due to absorption of the egg shell. 4. The pO2 increases in stage 41 and 42 due to pulmonary respiration. 5. The pO2 drop prior to hatching due to involution of the chorioalantoid membrane and increasing requirements.     

Ultrastructural and morphometric analysis of the separation of two thigh muscles in the chick

Limb muscles separate from one another in a complex but highly stereotyped sequence and spatial pattern. The process of separation is characterized by the progression of a region of increased extracellular space, the cleavage zone, along the proximodistal axis between the individual muscle anlagen. We analyzed ultrastructurally the muscles and cleavage zone during the separation of two representative muscles, the developing sartorius and iliotibialis in the chick thigh, to establish an accurate baseline for an analysis of the mechanisms of separation. Comparisons of the morphology and distribution of cells before and after separation show no evidence that muscles became separated by the massive influx of an exterior cell population; if populations invade the cleavage zone, they are small. We do find characteristic transitions within the cell population of the cleavage zone in situ that could accomplish cleavage without invoking massive cell movements. These progressive transitions within the cleavage zone include a loss of close cell-cell interactions, an increase in extracellular space, the assumption of a more stellate morphology by mesenchyme cells, and a gradual alteration in the composition of the extracellular matrix from one typical of early muscle to one typical of loose connective tissue. Myotubes do differentiate between the incipient muscles, ruling out the possibility that the location where muscles will separate is defined by sites where myotubes fail to differentiate. Instead, the myotubes in the cleavage zone gradually diminish in number and appear to be specifically recognized and removed from the cleavage zone by phagocytes. We suggest that the transitions within the cleavage zone, including the loss of muscle cells, are a result of the progressive differentiation of loose connective tissue. If so, then the spatial pattern and process of cleavage is a consequence of spatially programmed cell differentiation.     

Pinocytosis and locomotion of amoebae: XII. Dynamics and motive force generation during induced pinocytosis in A. proteus

Summary The mechanism of induced pinocytosis was investigated in Amoeba proteus by light and electron microscopy. The application of nine different inducing substances revealed that pinocytotic channel formation, elongation, vesiculation, shortening and disappearance are the result of the successive or simultaneous action of both traction and pressure forces, which are produced by the contractile activity of a plasma membrane-associated layer of filaments ranging from a few hundred nm to several in thickness. The initial phase of channel formation is caused by traction forces according to the membrane flow concept, whereas channel elongation and vesiculation mainly result from pressure forces in conjunction with the extrusion of small hyaline pseudopodia. Shortening and disappearance of the pinocytotic channels are brought about by local contractions of the cortical filament layer in the basal region of the hyaline pseudopodia. Experiments using latex beads as marker particles together with inducing substances show that a rapid membrane turnover during pinocytosis can be excluded, and that the plasma membrane slides as an entire structure over the underlying cytoplasm.The authors are most grateful to Mrs. J. Ruch for technical assistance     

Mitosis puts sisters in a strained relationship: force generation at the kinetochore

During mitosis, kinetochores couple chromosomes to the dynamic tips of spindle microtubules. These attachments convert chemical energy stored in the microtubule lattice into mechanical energy, generating force to move chromosomes. In addition to mediating robust microtubule attachments, kinetochores also integrate and respond to regulatory signals that ensure the accuracy of chromosome segregation during each cell division. Signals for corrective detachment act specifically on kinetochore-microtubule attachments that fail to generate normal levels of tension, although it is unclear how tension is sensed and how the attachments are released. In this review, we discuss the mechanisms by which kinetochore-microtubule attachments generate force during chromosome biorientation, and the pathways of maturation and regulation that lead to the formation of correct attachments.     

Shaping the shoot: the relative contribution of cell number and cell shape to variations in internode length between parent and hybrid apple trees

Genetic control of plant size and shape is a promising perspective,particularly in fruit trees, in order to select desirable genotypes.A recent study on architectural traits in an apple progeny showedthat internode length was a highly heritable character. However,few studies have been devoted to internode cellular patterningin dicotyledonous stems, and the interplay between the two elementarycell processes that contribute to their length, i.e. cell divisionand elongation, is not fully understood. The present study aimedat unravelling their contributions in the genetic variationof internode length in a selection of F₁ and parent genotypesof apple tree, by exploring the number of cells and cell shapewithin mature internodes belonging to the main axes. The resultshighlighted that both the variables were homogeneous in samplescollected either along a sagital line or along the pith width,and suggest that cell lengthening was homogeneous during internodedevelopment. They allowed the total number of cells to be estimatedon the internode scale and opened up new perspectives for simplifyingtissue sampling procedures for further investigations. Differencesin internode length were observed between the genotypes, inparticular between the parents, and partly resulted from a compensationbetween cell number and cell length. However, genetic variationsin internode length primarily involved the number of cells,while cell length was more secondary. These results argue foran interplay between cellular and organismal control of internodeshape that may involve the rib meristem. Key words: Elongation, growth, histogenesis, Malusxdomestica Borkh, pith Received 2 January 2008; Revised 22 January 2008 Accepted 29 January 2008     

Studies on the variations in generation times of rat hepatoma cells in culture

The proliferation kinetics of a cultured hepatoma cell line, HTC, was studied by time-lapse photography and autoradiography. Variability of G1 phase, S phase and intermitotic times of sister cells were compared with data on the variability of G1 phase, G2 phase, S phase and intermitotic times of unrelated cells. Evidence is presented that the variation in times spent in intermitosis by a pair of sisters is determined by the variation of G2 phase durations, while the variation in intermitotic times of unrelated cells is mainly determined by the variation of G1 phase times.     

Geometric morphometric analysis of the effect of temperature on wing size and shape in Aedes albopictus

Wing geometry helps to identify mosquito species, even cryptic ones. On the other hand, temperature has a well‐known effect on insect metric properties. Can such effects blur the taxonomic signal embedded in the wing? Two strains of Aedes albopictus (laboratory and field strain) were examined under three different rearing temperatures (26, 30 and 33 °C) using landmark‐ and outline‐based morphometric approaches. The wings of each experimental line were compared with Aedes aegypti. Both approaches indicated similar associations between wing size and temperature. For the laboratory strain, the wing size significantly decreased as the temperature increased. For the field strain, the largest wings were observed at the intermediate temperature. The two morphometric approaches describing shape showed different sensibilities to temperature. For both strains and sexes, the landmark‐based approach disclosed significant wing shape changes with temperature changes. The outline‐based approach showed lesser effects, detecting significant changes only in laboratory females and in field males. Despite the size and shape changes induced by temperature, the two strains of Ae. albopictus were always distinguished from Ae. aegypti. The present study confirms the lability of size. However, it also suggests that, despite environmentally‐induced variation, the architecture of the wing still provides a strong taxonomic signal.     

Misexpression of BRE gene in the developing chick neural tube affects neurulation and somitogenesis

The brain and reproductive expression (BRE) gene is expressed in numerous adult tissues and especially in the nervous and reproductive systems. However, little is known about BRE expression in the developing embryo or about its role in embryonic development. In this study, we used in situ hybridization to reveal the spatiotemporal expression pattern for BRE in chick embryo during development. To determine the importance of BRE in neurogenesis, we overexpressed BRE and also silenced BRE expression specifically in the neural tube. We established that overexpressing BRE in the neural tube indirectly accelerated Pax7⁺ somite development and directly increased HNK-1⁺ neural crest cell (NCC) migration and TuJ-1⁺ neurite outgrowth. These altered morphogenetic processes were associated with changes in the cell cycle of NCCs and neural tube cells. The inverse effect was obtained when BRE expression was silenced in the neural tube. We also determined that BMP4 and Shh expression in the neural tube was affected by misexpression of BRE. This provides a possible mechanism for how altering BRE expression was able to affect somitogenesis, neurogenesis, and NCC migration. In summary, our results demonstrate that BRE plays an important role in regulating neurogenesis and indirectly somite differentiation during early chick embryo development.     

Cellular mechanisms of paired electrical stimulation in ferret ventricular myocardium: relationship between myocardial force and stimulus interval change

Summary The subcellular mechanisms of twitch-force potentiation with paired electrical stimulation was studied in ferret ventricular myocardium using the bioluminescent calcium indicator aequorin. It is demonstrated for the first time that interpolation of an extrasystole in a train of conditioned twitches results in a beat-to-beat change in [Ca²⁺]_i and force. Steady-state twitch force and Ca _i ²⁺ were increased with paired stimulation. Increased [Ca²⁺]₀ in the setting of paired stimulation resulted in an increase in the amplitude of the postextrasystole and associated Ca²⁺ transient. Verapamil, a Ca²⁺ channel antagonist, had the opposite effect of increased [Ca²⁺]₀. Postextrasystole potentiation was still present, but diminished in amplitude. These results indicate that postextrasystole potentiation is in part due to a verapamil-depletable store (Ca²⁺). Postextrasystole potentiation is therefore predominantly dependent on sarcoplasmic reticulum (SR) Ca²⁺ loading. Ryanodine, an alkaloid which induces Ca²⁺ leakage from the SR, abolished postextrasystole potentiation; however, in the presence of ryanodine the extrasystole was potentiated. Caffeine, a phosphodiesterase inhibitor which induces SR Ca²⁺ release and impairs uptake, also abolished postextrasystole potentiation. As with ryanodine there was resultant potentiation of the extrasystole. In the case of caffeine the calcium transient consisted of a second slow component associated with extrasystole twitch potentiation. The results are consistent with sarcolemmal Ca²⁺ influx playing a role in potentiation of the extrasystole in the presence of an impaired SR. These data indicate that transsarcolemmal Ca²⁺ influx in the presence of impaired intracellular Ca²⁺ buffering can directly activate the myofilaments in agreement with reports on human myocardium.Abbreviations C conditioned stimulus - ESI extrasystolic interval - L_max active tension - PES postextrasystole - PESI postextrasystolic interval - SR sarcoplasmic reticulum - T test stimulus