Analytical studies on migrating movement of the pseudo-plasmodium ofDictyostelium discoideum

Summary Migrating movement of a pseudoplasmodium (slug) of the cellular slime mouldDictyostelium discoideum was analyzed using a time-lapse video tape recorder. Since slugs usually migrated with repeated interruptions of advance, migrating velocities were measured only within a period of forward movement. On the basis of some known facts and assumptions, a dynamical model for slug movement was formulated, which consists of motive force generated by slug cells against their intrinsic resistance and resistance of slime sheath at the tip. The migrating velocity of a slug depended neither on its width nor its volume, but solely on its length. Under any experimental conditions tested, a linear relationship always held between reciprocals of the two variables. The results were in good agreement with predictions of the model. Quantitative analyses of experimental results by the use of the model lead to the conclusions that a decrease in velocity at a low temperature is due to an increase in resistance of slime sheath at the tip, but that a decrease in velocity during prolonged migration is due to a decrease in motive force of constituent cells. An anterior isolate dissected from a slug migrated at a velocity greater than that of an intact slug of the same length. This was interpreted by the model to be due to the fact that the anterior cells have greater motive forces and intrinsic resistances than the posterior cells. The heterogeneous distributions of the two variables in the cell mass is discussed in reference to the mechanism of sorting out of cells.     

Cell sorting by differential cell motility: a model for pattern formation in Dictyostelium

In the slug stage of the cellular slime mold Dictyostelium discoideum, prespore cells and four types of prestalk cells show a well-defined spatial distribution in a migrating slug. We have developed a continuous mathematical model for the distribution pattern of these cell types based on the balance of force in individual cells. In the model, cell types are assumed to have different properties in cell motility, i.e. different motive force, the rate of resistance against cell movement, and diffusion coefficient. Analysis of the stationary solution of the model shows that combination of these parameters and slug speed determines the three-dimensional shape of a slug and cell distribution pattern within it. Based on experimental data of slug motive force and velocity measurements, appropriate sets of parameters were chosen so that the cell-type distribution at stationary state matches the distribution in real slugs. With these parameters, we performed numerical calculation of the model in two-dimensional space using a moving particle method. The results reproduced many of the basic features of slug morphogenesis, i.e. cell sorting, translocation of the prestalk region, elongation of the slug, and its steady migration.     

Quantitative analysis of the spatial distribution of ultrastructural differentiation markers during development ofDictyostelium discoideum

Summary The appearance and spatial distrubution of ultrastructural markers ofDictyostelium discoideum differentiation were quantitatively analysed. Our results combined with data from the literature on the functions of cells at various stages of development lead to the following conclusions. When food is no longer available all amoebae initially develop an autophagic apparatus in order to sustain metabolism. After slugs have been formed, autophagy is suppressed in the prespore cells. During aggregation a number of cells gradually form prespore characteristics. These cells arise at random but later they become located in the basal part of the tip-forming aggregate. From the early slug stage onwards, cells of the posterior two third region gradually enter into the prespore pathway. During prolonged slug migration the optimal acquirement of prespore characteristics is blocked. Cells of the anterior region show no active differentiation but they maintain the morphology and most of the functions of aggregating cells. At the rear-guard of the slug and later on in the basal region of the maturing fruiting body, a second anteriorlike region appears. Actual stalk cell differentiation takes place only at the apex and at the base of the developing fruiting body.     

A 3-D model used to explore how cell adhesion and stiffness affect cell sorting and movement in multicellular systems

A three-dimensional mathematical model is used to determine the effects of adhesion and cell signalling on cell movements during the aggregation and slug stages of Dictyostelium discoideum (Dd) and to visualize cell sorting. The building blocks of the model are individual deformable ellipsoidal cells, where movement depends on internal parameter state (cell size and stiffness) and on external cues from the neighboring cells, extracellular matrix, and chemical signals. Cell movement and deformation are calculated from equations of motion using the total force acting on each cell, ensuring that forces are balanced. The simulations show that the sorting patterns of prestalk and prespore cells, emerging during the slug stage, depend critically on the type of cell adhesion and not just on chemotactic differences between cells. This occurs because cell size and stiffness can prevent the otherwise faster cells from passing the slower cells. The patterns are distinctively different when the prestalk cells are more or less adhesive than the prespore cells. These simulations suggest that sorting is not solely due to differential chemotaxis, and that differences in both adhesion strength and type between different cell types play a very significant role, both in Dictyostelium and other systems.     

A study of PstB cells during Dictyostelium migration and culmination reveals a unidirectional cell type conversion process

Summary The prestalk region of the Dictyostelium slug has recently been shown by Williams and his collaborators to consist of two distinct cell types, pstA and pstB cells. Here the movement of these cells in both the slug and culmination stages has been examined with the use of vital dyes. In the slug some of the pstB cells are continually lost from the prestalk region as small clusters of cells. These cells move through the prespore region and temporarily lie in the rearguard region at the posterior end of the slug. They are finally left in the slug's slime track as single cells or groups of a few cells. When culmination is initiated the pstB cells move as a whole from the prestalk region to the base where they join the rearguard cells to form the basal disc of the fruiting body. Transplantation experiments reveal that the rearguard cells form an outer ring portion of the basal disc and the pstB cells form an inner portion to which the stalk attaches. The continuous loss of one cell type during the slug stage without any change in cell type proportions suggests that cell types are redifferentiating. Grafting and transplantation experiments reveal that there is a unidirectional flow of cells through successive steps of cell type conversion. Prespore cells redifferentiate as anterior-like cells which migrate to the prestalk region and become pstA cells. The pstA cells then replace the pstB cells that are lost from the slug.     

Patterns of glucose utilization and protein synthesis in the development of Dictyostelium discoideum

Abstract. Using autoradiography, we examined the distribution of glucose utilization with labelled deoxyglucose, and protein synthesis with labelled leucine. Glucose utilization showed an even distribution at the early stages of slug migration and then, after the appearance of distinct pre-stalk-prespore zones, it showed a uniformly high level in the posterior prepore cells and a uniformly low level in the anterior prestalk cells. Occasionally, an intermediate gradient, highest at the posterior end, was observed which indicated a possible intermediate stage of slug migration. In all stages of slug development, the protein synthesis showed a gradient which was highest at the posterior end. This suggests that the posterior spore-formation region involves more active metabolic activities than the anterior tip, which is responsible for the organization of the cell mass.     

Cell Patterning During Slug Migration and Early Culmination in Dictyostelium discoideum

Abstract. The effects of migration and culmination on patterning of presumptive (prespore and prestalk) cells and mature (spore and stalk) cells of D. discoideum were investigated. The ratio of prespore to total cells, as determined by staining with fluorescein-conjugated antispore globulin, was constant (77%) up until 8 h of slug migration, but then decreased to a level (64%) which thereafter remained unchanged during migration. Cells which lost prespore antigen during migration were located in the posterior (prespore) part next to the agar surface.
Upon induction of culmination, however, the ratio of prespore cells quickly increased to the normal level (77%) within 1–2 h. During the transition between migration and culmination prestalk and prespore cells were considerably intermixed within the cell mass, before the normal prestalk-prespore pattern was reestablished at the preculmination (Mexican hat) stage. Spore: stalk ratios within fruiting bodies were normal irrespective of the lengths of slug migration.     

A thermodynamical model of cell distributions in the slug of cellular slime mold

A theoretical model is proposed for the formation of cell distribution patterns in the slug stage of the cellular slime moldDictyostelium discoideum. The equilibrium distribution of two types of cells, prestalk and prespore, is obtained by minimizing the free energy, which is defined in terms of differential chemotaxis, differential cell adhesion and randomness of cell movement. Resulting distributions show various segregation patterns of cell types. The condition for cell sorting is obtained from stability analysis of the set of diffusion equations governing the evolution of cell type distribution and the concentration of chemoattractant. The intensities of differential chemotaxis and random cell movement are quantitatively evaluated from experimental data to show that two cell types can sort themselves completely by these forces.     

Kin Discrimination in Dictyostelium Social Amoebae

Evolved cooperation is stable only when the benefactor is compensated, either directly or through its relatives. Social amoebae cooperate by forming a mobile multicellular body in which, about 20% of participants ultimately die to form a stalk. This benefits the remaining individuals that become hardy spores at the top of the stalk, together making up the fruiting body. In studied species with stalked migration, P. violaceum, D. purpureum, and D. giganteum, sorting based on clone identity occurs in laboratory mixes, maintaining high relatedness within the fruiting bodies. D. discoideum has unstalked migration, where cell fate is not fixed until the slug forms a fruiting body. Laboratory mixes show some degree of both spatial and genotype‐based sorting, yet most laboratory fruiting bodies remain chimeric. However, wild fruiting bodies are made up mostly of clonemates. A genetic mechanism for sorting is likely to be cell adhesion genes tgrB1 and tgrC1, which bind to each other. They are highly variable, as expected for a kin discrimination gene. It is a puzzle that these genes do not cause stronger discrimination between mixed wild clones, but laboratory conditions or strong sorting early in the social stage diminished by later slug fusion could be explanations.     

Reduction of nucleolar size in reaggregated Dictyostelium cells

The ultra-structure of the nucleolus in Dictyostelium discoideum cells was studied by electron microscopy. Large nucleoli on the periphery of the nucleus in cells of the multi-cellular pseudoplasmodium (slug) were maintained during long migration. Disaggregation of the slug cells induced a reduction in the size of the large nucleoli. The size of the reduced nucleoli in the reaggregated cells were maintained during the long migration and culmination of reconstructed slug. The electron density of the cytoplasm clearly distinguishes the prespore from the prestalk region, and it takes about 6 h for the complete recovery of cell-to-cell contact after reaggregation.     

Gender Specific Brood Cells in the Solitary Bee Colletes halophilus (Hymenoptera; Colletidae)

We studied the reproductive behaviour of the solitary bee Colletes halophilus based on the variation in cell size, larval food amount and larval sex in relation to the sexual size dimorphism in this bee. Brood cells with female larvae are larger and contain more larval food than cells with males. Occasionally males are reared in female-sized cells. We conclude that a female C. halophilus in principal anticipates the sex of her offspring at the moment brood cell construction is started. Additionally a female is able to ‘change her mind’ about the sex of her offspring during a single brood cell cycle. We present a model that can predict the sex of the larvae in an early stage of development.     

ON THE PROGENITOR CELL MIGRATION VELOCITY

An attempt is presented to extract cell kinetic information from histomorphological features. It is applicable to rapidly proliferating tissues like the intestinal epithelium. Each replicating tissue has an origin where cells are formed and a periphery toward which cells migrate. The migration path along which they move is denominated as tissue radius on which all cell positions are mapped. Cell migration on the radius is associated with cell proliferation at tissue origin. Each mitosis there is associated with the displacement of all cells distal to it by one cell position. The more mitoses positioned between a cell and tissue origin, the greater its migration velocity. It is possible therefore to derive the cell migration velocity v(x) from the cumulative mitotic distribution on the radius, N(x). v(x) = N(x)/t_m (t_m= mitotic time). In this form v(x) represents also cell production at any point on the radius and may serve for the computation of other cell kinetic parameters like generation time. These arguments are illustrated on the rat incisor tooth inner enamel epithelium which has been studied in the normal and rapidly erupting tooth.     

A generalized transport model for biased cell migration in an anisotropic environment

 A generalized transport model is derived for cell migration in an anisotropic environment and is applied to the specific cases of biased cell migration in a gradient of a stimulus (taxis; e.g., chemotaxis or haptotaxis) or along an axis of anisotropy (e.g., contact guidance). The model accounts for spatial or directional dependence of cell speed and cell turning behavior to predict a constitutive cell flux equation with drift velocity and diffusivity tensor (termed random motility tensor) that are explicit functions of the parameters of the underlying random walk model. This model provides the connection between cell locomotion and the resulting persistent random walk behavior to the observed cell migration on longer time scales, thus it provides a framework for interpreting cell migration data in terms of underlying motility mechanisms. Received: 8 April 1999     

Length regulation in the Dictyostelium discoideum slug is a late event.

Time-lapse video light microscopy was used to study the emergence and maturation of the migratory slug from a D. discoideum aggregate. The anterior part, the tip of this simple multicellular organism, establishes migration prior to the definition of the rear, and hence the length of the slug. It was found that newly formed slugs of wild-type strain WS380B can reach lengths greater than 1 cm, yet mature slugs of this strain are rarely longer than 2-3 mumm. Often the tip extended out of the aggregation mound upon an arching pillar of cells. After the tip first touched the substratum, it commenced migration with a rapid succession of movement steps. Here we show that at the initiation of migration, a differential rate of cell movement along the developing slug axis results in a series of complicated changes, before the stable and mature shape of the slug is formed. Our results lead to new conclusions about D. discoideum slug formation and shape maintenance. Evidence is presented for regulation of slug length.     

The expression profile and function of Satb2 in zebrafish embryonic development

The present study shows the expression profile and function of the homeobox gene, satb2 during zebrafish embryonic development. Satb2 was ubiquitously expressed from the 1 cell stage to the 10-somite stage in zebrafish embryos. Satb2 showed stage-specific expression profiles such as in the pronephric duct at 24 hpf, the branchial arches at 36 hpf, and the ganglion cell layer of the retina and fins at 48 hpf. Additionally, satb2 knockdown embryos were arrested at 50–60% epiboly, and transplantation experiments with satb2 knockdown cells showed migration defects. Interestingly, satb2 knockdown cells also exhibited down-regulation of dynamin II and VAMP4, which are involved in exocytosis and endocytosis, respectively. Furthermore, satb2 knockdown cells have a disorganized actin distribution and an underdeveloped external yolk syncytial layer, both of which are involved in epiboly. These results suggest that satb2 has a functional role in epiboly. This role may potentially be the regulation of endo-exocytic vesicle transport-dependent cell migration and/or the regulation of the development of the yolk syncytial layer.     

A flow fluorimetric analysis of the cell cycle during growth and differentiation inDictyostelium discoideum

Summary We report a flow fluorimetric analysis of the DNA content of cells and nuclei from vegetative populations and various developmental stages of the cellular slime mouldDictyostelium discoideum using the dyes Hoechst 33258 and mithramycin. Nuclei from all of these populations showed an identical single DNA-content peak, indicating that most vegetative cells and most cells in all developmental stages are in one phase of the cell cycle. Our own data and findings in the literature indicate that this phase is G₂. On the other hand, we also found that various stages, subpopulations of cells at early stages and the different differentiated cell types in the slug stage differ in DNA content per cell. Any particular population typically has one major peak of DNA content, with a modal value that is characteristic for the cell type and for the developmental stage. These differences presumably reflect differences in mitochondrial DNA content per cell.     

Quantitative studies on cell differentiation during morphogenesis of the cellular slime mold Dictyostelium discoideum

Taking advantage of the fact that differentiation of the prespore cell of Dictyostelium discoideum is characterized by synthesis of a prespore specific antigen, the process of its differentiation during the course of morphogenesis was quantitatively studied by determining the proportion of prespore cells and their cellular contents of the antigen, using the method of microfluorometry in combination with immunocytochemistry with antispore serum. The cells synthesizing the antigen became first detectable in the early aggregation center which was about to form a papilla. As the papilla elongated, the number of prespore cells rapidly increased up to the stationary level (70–80% of total cells) before completion of slug formation. During the process antigenic contents of prespore cells were gradually increased and leveled off in the early migration stage. When culmination was induced, antigenic contents were markedly increased to the maximum, which was followed by a sudden decrease immediately before spore formation. On the other hand, the proportions of prespore to total cells were kept constant at the stationary level all through the migration and culmination stages, in spite of a persistent decrease during culmination in the total number of cells due to continuous differentiation of the prestalk into the mature stalk cells. These results were discussed in relation to possible mechanisms of differentiation in this organism.     

Changes in β-Galactosidase activity during differentiation and dedifferentiation inDictyostelium discoideum

β-Galactosidase (EC 3.2.1.23) ofDictyostelium discoideum was investigated for its properties and activity during differentiation and dedifferentiation. β-Galactosidase of this organism had a pH optimum at 3.5. The specific activity of this enzyme was increased gradually from the time of initiation of differentiation and reached a peak at the aggregation stage. Then the activity of the enzyme showed a slight decrease followed by a further increase and reached a maximum at early culmination. During dedifferentiation of cells disaggregated from a slug, the activity of the enzyme was increased, reached a maximum after 3 hr of incubation and then decreased nearly to the original level of activity after completion of dedifferentiation. This increase in the enzyme activity coincided with decomposition of acid mucopolysaccharide contained in the prespore specific vacuoles, and both processes were sensitive to cycloheximide. No increase in the activity of acetylglucosaminidase (EC 3.2.1.30), another lysosomal enzyme, was observed during the process. Possible roles of β-galactosidase in cell type conversion as well as in dedifferentiation of the prespore cell were discussed.     

The scale-invariance of spatial patterning in a developing system

Summary Regulating systems, that is, those which exhibit scale-invariant patterns in the adult, are supposed, to do so on account of interactions between cells during development. The nature of these interactions has to be such that the system of positional information (map) in the embryo also regulates. To our knowledge, this supposition regarding a regulating map has not been subjected to a direct test in any embryonic system. Here we do so by means of a simple and novel criterion and use it to examine tip regeneration in the mulicellular stage (slug) ofDictyostelium discoideum. When anterior, tip-containing fragments of slugs are amputated, a new tip spontaneously regenerates at the cut surface of the (remaining) posterior fragment. The time needed for regeneration to occur depends on the relative size of the amputated fragment but is independent of the total size of the slug. We conclude from this finding that there is at least one system underlying positional information in the slug which regulates.