Epidermal growth factor stimulates type-V collagen synthesis in cultured murine palatal shelves

Abstract. The problem studied was whether treatments that reorient vascular differentiation have a similar effect on the polarity of auxin transport. Hypocotyls of Phaseolus vulgaris L. were cut so that a transverse bridge connected the shoot and root directions. Within three days these bridges of tissue regenerated both vessels and sieve tubes along the new orientation, at 90° to the original axis. Experiments involving organ removal, wounds, and hormone application confirm previous suggestions that this differentiation follows the expected flow of the hormone auxin in the direction of the roots. Transport of (³H) indoleacetic acid through sections in which vascular reorientation occurred was polar: it was at least twice as great in the new direction of the roots than in the opposite direction. This new polarity of transport, at right angles to the original axis of the plant, can be readily understood if there is a positive feed-back between the differentiation of tissue polarity and auxin transport.     

Size,shape and orientation of cells in budding hydra and regulation of regeneration in cell aggregates

Summary Changes in the number, shape, volume, orientation and vacuolization of cells involved in the budding of hydra were measured in histological sections. Before evagination, a group of about 800 epithelial cells are visibly recruited for the bud to be produced and this number increases to about 5,000 within a day. Thereafter, bud size increases mainly by proliferation of the cells within the bud. Upon recruitment for budding, the epithelial cells assume a columnar shape, with a smaller contact area facing the mesoglea, accompanied by a decrease in volume which is mostly accounted for by devacuolization. In later stages, cells progressively resume the form typical for non-budding areas of hydra. Evagination proceeds without reorientation of epithelio-muscular fibers, whereas elongation of the bud is accompanied by fiber reorientation.The process of sorting out and regeneration in aggregates of previously dissociated hydra cells was followed using various ratios of endodermal to ectodermal epithelial cells. From different initial compositions, the ratio in the regenerate rapidly approaches 11, the ratio found in normal hydra tissue.The experimental findings are discussed in the context of theoretical notions on pattern formation, evagination, elongation and stability of layered structures.     

Unipolar orientation stability of the sex univalent in the grasshopper (Melanoplus sanguinipes)

The behavior of the X chromosome in living Melanoplus sanguinipes primary spermatocytes was examined utilizing phase contrast microscopy and micromanipulation. During early prometaphase I, the X univalent establishes a unipolar orientation which is normally stable. In 28 cells, the X chromosome did not reorient once during a total of 122 h of observation. Though normally stable, the X chromosome can be induced to reorient by micromanipulation. An anomalous increase in X reorientation was observed in one testicular preparation. Stable unipolar orientation is an intrinsic characteristic of the X chromosome and was not displayed by two small autosomal univalents discovered in one cell. The two autosomal univalents, resulting from asynapsis or desynapsis, oscillated back and forth within the spindle, averaging 26.0 and 17.6 min respectively between reorientation events. The behavior of the M. sanguinipes X chromosome reveals an orientation stability mechanism that does not involve tension created by forces from opposing kinetochoric spindle fibers.     

Brachiopod orientation to water movement: functional morphology

Previous work by the author has shown that Laqueus californianus. Terebratulina unguicula , and. to a lesser extent, Hemithyris psittacea will actively reorient to water currents in the laboratory; Terebratalia transversa will not. This active reorientation is effected primarily by the dorsal adjustor muscles; the ventral adjustors function to depress and tilt the shell. The torque around the pedicle generated by drag on the shell is low at moderate current speeds. The dorsal adjustor muscles are competent to resist these torques up to current speeds of 35 cm/s for L. californianus and H. psittacea and 56 cm/s for T. unguicula . Shell shape, gross pedicle form, and form of the pedicle foramen are unreliable indicators of a brachiopod's ability to actively reorient. Shape of the pedicle bulb and position and size of the pedicle connectives are better indicators but. on functional grounds, the dorsal adjustor muscle morphology is the best indicator of active reorientation. An angle α (the maximum possible rotation of the shell that can be produced by contraction of one of the dorsal adjustor muscles) correlates well with both the maximum observed rotations and the exhibition of active reorientation to currents. For fossil material, the existence of dorsal adjustor muscle scars that touch medially should also be a good indicator of active reorientation ability.     

Reorientation of mispositioned spindles in short astral microtubule mutant spc72Delta is dependent on spindle pole body outer plaque and Kar3 motor protein

Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic gamma-tubulin complex, can only generate very short (<1 microm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However, SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.     

Inductive and axial properties of prospective wing-bud mesoderm in the chick embryo

Prospective wing-bud mesoderm, stripped of ectoderm mechanically through the use of glass needles, or chemically by means of EDTA or trypsin, was obtained from donor embryos of stages 11 through 21. Grafts were made in both homopleural (aadd and apdv) and heteropleural (aadv and apdd) combinations to the right flank of host embryos of the same range of stages. Flank ectoderm from the host healed over the graft in a few hours and, in combinations between donors and hosts in the range of stages 12 through 17, the composite formed, with high frequency, a limb bud capped by an apical ectodermal ridge, and then developed into a supernumerary wing in which all proximodistal levels were represented. When either member of the combination was older than stage 17, only incomplete limbs, if any, were formed. Regardless of their orientation on the host, the supernumerary limbs always showed the axial characteristics appropriate to their side of origin.Supernumerary wings failed to form if the grafts were inserted into a space tunneled between flank ectoderm and its underlying mesoderm. If the covering ectoderm were deliberately torn and forced to heal over the graft, however, an ectodermal ridge formed and a supernumerary limb developed.It is concluded, therefore, that: (1) the wing-bud mesoderm, appropriately combined with flank ectoderm, has the property of morphological and axial self-differentiation by stage 12; (2) the apical ectodermal ridge is induced in flank ectoderm by prospective wing-bud mesoderm; (3) this inductive power is restricted to prospective wing-bud mesoderm from donors of stages 12 through 17; (4) the response competence is limited to flank ectoderm that has healed over the mesoderm; and (5) this competence is lost by the end of stage 17.     

Strain waveform dependence of stress fiber reorientation in cyclically stretched osteoblastic cells: effects of viscoelastic compression of stress fibers

Actin stress fibers (SFs) of cells cultured on cyclically stretched substrate tend to reorient in the direction in which a normal strain of substrate becomes zero. However, little is known about the mechanism of this reorientation. Here we investigated the effects of cyclic stretch waveform on SF reorientation in osteoblastic cells. Cells adhering to silicone membranes were subjected to cyclic uniaxial stretch, having one of the following waveforms with an amplitude of 8% for 24 h: triangular, trapezoid, bottom hold, or peak hold. SF reorientation of these cells was then analyzed. No preferential orientation was observed for the triangular and the peak-hold waveforms, whereas SFs aligned mostly in the direction with zero normal strain (~55°) with other waveforms, especially the trapezoid waveform, which had a hold time both at loaded and unloaded states. Viscoelastic properties of SFs were estimated in a quasi-in situ stress relaxation test using intact and SF-disrupted cells that maintained their shape on the substrate. The dynamics of tension F(SFs) acting on SFs during cyclic stretching were simulated using these properties. The simulation demonstrated that F(SFs) decreased gradually during cyclic stretching and exhibited a compressive value (F(SFs) < 0). The magnitude and duration time of the compressive forces were relatively larger in the group with a trapezoid waveform. The frequency of SF orientation had a significant negative correlation with the applied compressive forces integrated with time in a strain cycle, and the integrated value was largest with the trapezoid waveform. These results may indicate that the applied compressive forces on SFs have a significant effect on the stretch-induced reorientation of SFs, and that SFs realigned to avoid their compression. Stress relaxation of SFs might be facilitated during the holding period in the trapezoid waveform, and depolymerization and reorientation of SFs were significantly accelerated by their viscoelastic compression.     

The clustering of acetylcholine receptors and formation of neuromuscular junctions in regenerating mammalian muscle grafts

The present investigation was undertaken to study the relationship between acetylcholine receptor (AchR) clustering and endplate formation within regenerating skeletal muscle grafts. Silver staining of nerves was combined with rhodamine-alpha-bungarotoxin labeling of AchR clusters in heterotopic grafts of the rat soleus muscle. Two major graft procedures were used: whole muscle grafts and grafts which lacked the zone of original motor endplates (MEP-less grafts). These categories were subdivided into standard grafts, where subsequent innervation was allowed, and noninnervated grafts, which were experimentally deprived of innervation. Grafting brought about the death and removal of muscle fibers, followed by regeneration of myotubes within surviving basal lamina sheaths. A transient population of small extra-junctional AchR clusters spontaneously appears shortly after myotube formation in all four muscle graft types. Early myotubes of whole muscle grafts (both innervated and standard grafts, prior to the time of innervation) also develop presumptive secondary synaptic clefts and large, organized aggregations of AchRs at original synaptic sites. At later times, nerves regenerating into standard whole muscle and MEP-less grafts lead to the formation of numerous ectopic endplates. In whole muscle grafts, endplates may also form at original synaptic sites. Functional graft innervation is achieved in whole muscle and MEP-less grafts as early as 20 days postgrafting. The results of this study support the existence of still-unknown factors associated with the original synaptic site which can direct postsynaptic differentiation independent of innervation. They also demonstrate that functional endplates may form in mammalian muscle grafts at both original synaptic sites and ectopic locations, thus indicating that the zone of original synaptic sites is not necessary for the establishment of numerous functional and morphologically well-differentiated endplates.     

CHROMOSOME MICROMANIPULATION : III. Spindle Fiber Tension and the Reorientation of Mal-Oriented Chromosomes

Kinetochore reorientation is the critical process ensuring normal chromosome distribution. Reorientation has been studied in living grasshopper spermatocytes, in which bivalents with both chromosomes oriented to the same pole (unipolar orientation) occur but are unstable: sooner or later one chromosome reorients, the stable, bipolar orientation results, and normal anaphase segregation to opposite poles follows. One possible source of stability in bipolar orientations is the normal spindle forces toward opposite poles, which slightly stretch the bivalent. This tension is lacking in unipolar orientations because all the chromosomal spindle fibers and spindle forces are directed toward one pole. The possible role of tension has been tested directly by micromanipulation of bivalents in unipolar orientation to artificially create the missing tension. Without exception, such bivalents never reorient before the tension is released; a total time "under tension" of over 5 hr has been accumulated in experiments on eight bivalents in eight cells. In control experiments these same bivalents reoriented from a unipolar orientation within 16 min, on the average, in the absence of tension. Controlled reorientation and chromosome segregation can be explained from the results of these and related experiments.     

Vessel differentiation along different tissue polarities

Organized vessel differentiation in an isolated system was studied on a quantitative basis. An auxin source was oriented on isolated pieces of turnip storage root ( Brassica campestris cv. Rapifera) to allow diffusion a) in the direction of, b) at right angle to, and c) opposite to the original polarity. New vessel members differentiated within 44 h, and a minimum average auxin (IAA) concentration of 3.10⁻⁶ M was required to induce initial vessel differentiation. The differentiation rates in three experimental orientations were 167, 60 and 43 cells h⁻¹ at 10⁻³ M IAA, and 1445, 1346 and 838 cells (log IAA concentration)⁻¹ after 96 h, respectively. The difference between the differentiation rates in the original polarity orientation (a) and in the orientation at right angle (b) is interpreted as reflecting reorientation itself, which requires a minimum time.     

Developmental reorientation of transverse cortical microtubules to longitudinal directions: a role for actomyosin-based streaming and partial microtubule-membrane detachment

Transversely oriented cortical microtubules in elongating cells typically reorient themselves towards longitudinal directions at the end of cell elongation. We have investigated the reorientation mechanism along the outer epidermal wall in maturing leek (Allium porrum L.) leaves using a GFP-MBD microtubule reporter gene and fluorescence microscopy. Incubating leaf segments for 14-18 h with the anti-actin or anti-actomyosin agents, 20 microm cytochalasin D or 20 mM 2,3-butanedione monoxime, inhibited the normal developmental reorientation of microtubules to the longitudinal direction. Observation of living cells revealed a small subpopulation of microtubules with their free ends swinging into oblique or longitudinal directions, before continuing to assemble in the new direction. Electron microscopy confirmed that longitudinal microtubules are partly detached from the plasma membrane. Incubating leaf segments with 0.2% 1 degree-butanol, an activator of phospholipase D, which has been implicated in plasma membrane-microtubule anchoring, promoted the reorientation, presumably by promoting microtubule detachment from the membrane. Stabilizing microtubules with 10 microm taxol also promoted longitudinal orientation, even in the absence of cytoplasmic streaming. These results were consistent with confocal microscopy of live cells before and after drug treatments, which also revealed that the slow (days) global microtubule reorientation is superimposed over short-term (hours) regional cycling in a clockwise and an anti-clockwise direction. We propose that partial detachment of transverse microtubules from the plasma membrane in maturing cells exposes them to hydrodynamic forces of actomyosin-driven cytoplasmic streaming, which bends or shifts pivoting microtubules into longitudinal directions, and thus provides an impetus to push microtubule dynamics in the new direction.     

Spatial reorientation by geometry with freestanding objects and extended surfaces: a unifying view

The macroscopic, three-dimensional surface layout geometry of an enclosure apparently provides a different contribution for spatial reorientation than the geometric cues associated with freestanding objects arranged in arrays with similar geometric shape. Here, we showed that a unitary spatial representation can account for the capability of animals to reorient both by extended surfaces and discrete objects in a small-scale spatial task. We trained domestic chicks to locate a food-reward from an opening on isolated cylinders arranged either in a geometrically uninformative (square-shaped) or informative (rectangular-shaped) arrays. The arrays were located centrally within a rectangular-shaped enclosure. Chicks trained to access the reward from a fixed position of openings proved able to reorient according to the geometric cues specified by the shape of the enclosure in all conditions. Chicks trained in a fixed position of opening with geometric cues provided both by the arena and the array proved able to reorient according to each shape separately. However, chicks trained to access the reward from a variable position of openings failed to reorient. The results suggest that the physical constrains associated with the presence of obstacles in a scene, rather than their apparent visual extension, are crucial for spatial reorientation.     

Limb outgrowth in the chick embryo induced by dissociated and reaggregated cells of the apical ectodermal ridge.

Mesodermal cores of the stage 19 chick leg bud were capped with an intact apical ectodermal ridge (AER) or with strips cut from centrifugal pellets formed from Pronase-dissociated AERs. They were then covered with embryonic back-skin ectoderm and grown as grafts to the somite region of a host embryo. Control mesoderms were capped with centrifugal aggregates of nonridge limb ectoderm or similarly treated back-skin ectoderm, with ethanol-killed AERs or with no ectodermal cells other than the enveloping back-skin ectoderm.Controls were vascularized slowly and atypically and showed little outgrowth, forming only proximal skeletal structures. Recombinants equipped with AER cells were vascularized more fully and promptly and began vigorous growth after brief delay, forming legs with all skeletal segments represented, including claw-tipped toes. The latter were arranged in anteroposterior order corresponding to the original polarity of the mesoderm.Histological sections of recombinants made with cytologically distinctive quail AERs reveal that the cap of ridge cells, whether initially intact or reaggregated beneath the back-skin envelope, undergo a period of reorganization, forming a typical AER at the apex of the chimeric appendage after 48 hr. Meanwhile vigorous growth of the recombinant continues.These results show that the AER can cooperate with nonlimb ectoderm in promoting the morphogenesis of successively more distal levels of the limb skeleton. They also show that dissociated ridge cells can reorganize a typical AER at the apex of the limb mesoblast, meanwhile exercising their inductive effect on it.     

Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant

Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity.     

Axons of Xenopus neural tube respond to reversals of neural tube orientation

Axonal trajectories of the Kolmer-Agduhr (KA) neurons of Xenopus embryos, were observed after anterior-posterior (A-P) inversions of neural tube grafts to determine whether KA axons follow cell-inherent directional cues, cues from their immediate environment, or rostrocaudal signals from the embryo. KA axons form one of the earliest ascending spinal pathways in Xenopus and are visible in the lateral marginal zone of whole mounts processed for GABA immunoreactivity. Grafts were made at trunk levels at stages 22–24, 3–5 h before the first KA neurons were detectable and prior to axonal outgrowth. Embryos were fixed and immunostained 6–36 h later. KA trajectories within and adjacent to reversed grafts were compared to those of nonrotated control grafts and to neural tube lengths comparable in position and in length in unoperated embryos. Most KA axons within rotated grafts followed the graft's orientation. However, others changed direction, taking novel routes including turning to conform to the orientation of the host embryo. Reorientations were most common near the posterior host/graft interface. Some host KA cells also reoriented, always within a few hundred microns of the graft interface. Taken together, these growth patterns show that most KA axons within the grafts grow normally with respect to the original polarity of the graft neural tube and maintain that direction even into tissue of opposite polarity, suggesting that their routes are mainly determined by cell-intrinsic and/or local tissue factors. However, the reorientation of many other axons, particularly near graft seams, implies that KA axons can respond to local fluctuations in directional or segment identity signals generated in both host and graft after this perturbation. © 1996 John Wiley & Sons, Inc.     

Orientation of Smooth Muscle-Derived A10 Cells in Culture by Cyclic Stretching: Relationship between Stress Fiber Rearrangement and Cell Reorientation

Mechanical stress causes various responses in cells both in vivo and in vitro. Realignment of cells and stress fibers is one of the remarkable phenomena that are induced by the stress. However, the mechanism by which their realignment is controlled is largely unknown. In this study, effects of mechanical stretch on the morphology of cultured cells were examined using a cyclic and reciprocal cell stretching apparatus. A10 cells, a cell line derived from rat aortic smooth muscle, were used as a model, since they are spindle-shaped and have remarkable stress fibers aligned along the longitudinal cell axis. Therefore, the orientation of the cell and stress fibers could be easily identified. When the cells were cultured on elastic silicone membranes and subjected to cyclic and reciprocal stretch with an amplitude of 20% at a frequency of 60 cycles per minute, actin stress fibers were aligned obliquely to the direction of stretching with angles of 50 to 70 degrees within about 15 min after the onset of stretching. Then, after 1-3 hr of cyclic stretching, the long axes of a majority of the cells were also reoriented to similar directions to the stress fibers. The stretch-induced cell reorientation was blocked by 1 muM cytochalasin B, but not by colcemid. These results indicate that the orientation of cells and actin filaments are closely related and actin filaments play a critical role in the early step of the cell reorientation.     

Auxin Redistribution during First Positive Phototropism in Corn Coleoptiles : Microtubule Reorientation and the Cholodny-Went Theory

In red-light grown corn (Zea mays L. cv Brio42.HT) coleoptiles, cortical microtubules adjacent to the outer cell wall of the outer epidermis reorient from transverse to longitudinal in response to auxin depletion and after phototropic stimulation in the lighted side of the coleoptile. This was used as an in situ assay of cellular auxin concentration. The fluence-response relation for the blue light-induced reorientation is compared with that for first positive phototropism and the dose-response relationship for the auxin-dependent reorientation. The result supports the theory by Cholodny and Went, claiming that phototropic stimulation results in auxin displacement across the coleoptile. In terms of microtubule orientation, this displacement becomes even more pronounced after preirradiation with a weak blue light pulse from above.     

Ectodermal Wnt-6 promotes Myf5-dependent avian limb myogenesis

Limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Although several signaling molecules have been reported to be involved in the process of limb myogenesis, none of their activities has led to a consolidate idea about the limb myogenic pathway. Particularly, the role of ectodermal signals in limb myogenesis is still obscure. Here, we investigated the role of the ectoderm and ectodermal Wnt-6 during limb muscle development. We found that ectopic expression of Wnt-6 in the limb bud specifically extends the expression domains of Pax3, Paraxis, Myf5, Myogenin, Desmin and Myosin heavy chain (MyHC) but inhibits MyoD expression. Ectoderm removal results in a loss of expression of all of these myogenic markers. We show that Wnt-6 can compensate the absence of the ectoderm by rescuing the expression of Pax3, Paraxis, Myf5, Myogenin, Desmin and MyHC but not MyoD. These results show that, in chick, at least two signals from the limb ectoderm are necessary for muscle development. One of the signals is Wnt-6, which plays a unique role in promoting limb myogenesis via Pax3/Paraxis-Myf5, whereas the other putative signaling pathway involving MyoD expression is negatively regulated by Wnt-6 signaling.     

A mechanistic motor-clutch model that explains cell shape dynamics to cyclic stretch

Many cells in the body experience cyclic mechanical loading, which can impact cellular processes and morphology. In vitro studies often report that cells reorient in response to cyclic stretch of their substrate. To explore cellular mechanisms involved in this reorientation, a computational model was developed by adapting previous computational models of the actin–myosin–integrin motor-clutch system developed by others. The computational model predicts that under most conditions, actin bundles align perpendicular to the direction of applied cyclic stretch, but under specific conditions, such as low substrate stiffness, actin bundles align parallel to the direction of stretch. The model also predicts that stretch frequency impacts the rate of reorientation and that proper myosin function is critical in the reorientation response. These computational predictions are consistent with reports from the literature and new experimental results presented here. The model suggests that the impact of different stretching conditions (stretch type, amplitude, frequency, substrate stiffness, etc.) on the direction of cell alignment can largely be understood by considering their impact on cell–substrate detachment events, specifically whether detachments preferentially occur during stretching or relaxing of the substrate.