The control of chemotactic cell movement during Dictyostelium morphogenesis

Differential cell movement is an important mechanism in the development and morphogenesis of many organisms. In many cases there are indications that chemotaxis is a key mechanism controlling differential cell movement. This can be particularly well studied in the starvation-induced multicellular development of the social amoeba Dictyostelium discoideum. Upon starvation, up to 10(5) individual amoebae aggregate to form a fruiting body The cells aggregate by chemotaxis in response to propagating waves of cAMP, initiated by an aggregation centre. During their chemotactic aggregation the cells start to differentiate into prestalk and prespore cells, precursors to the stalk and spores that form the fruiting body. These cells enter the aggregate in a random order but then sort out to form a simple axial pattern in the slug. Our experiments strongly suggest that the multicellular aggregates (mounds) and slugs are also organized by propagating cAMP waves and, furthermore, that cell-type-specific differences in signalling and chemotaxis result in cell sorting, slug formation and movement.     

Becoming Multicellular by Aggregation; The Morphogenesis of the Social Amoebae Dicyostelium discoideum

The organisation and form of most organisms is generated during theirembryonic development and involves precise spatial and temporal controlof cell division, cell death, cell differentiation and cell movement.Differential cell movement is a particularly important mechanism in thegeneration of form. Arguably the best understood mechanism of directedmovement is chemotaxis. Chemotaxis plays a major role in the starvationinduced multicellular development of the social amoebae Dictyostelium.Upon starvation up to 10⁵ individual amoebae aggregate to form afruiting body. In this paper we review the evidence that the movement ofthe cells during all stages of Dictyostelium development is controlled bypropagating waves of cAMP which control the chemotactic movement ofthe cells. We analyse the complex interactions between cell-cell signallingresulting in cAMP waves of various geometries and cell movement whichresults in a redistribution of the signalling sources and therefore changes thegeometry of the waves. We proceed to show how the morphogenesis,including aggregation stream and mound formation, slug formation andmigration, of this relatively simple organism is beginning to be understoodat the level of rules for cell behaviour, which can be tested experimentallyand theoretically by model calculations.     

Dictyostelium morphogenesis

During starvation-induced Dictyostelium development, up to several hundred thousand amoeboid cells aggregate, differentiate and form a fruiting body. The chemotactic movement of the cells is guided by the rising phase of the outward propagating cAMP waves and results in directed periodic movement towards the aggregation centre. In the mound and slug stages of development, cAMP waves continue to play a major role in the coordination of cell movement, cell-type-specific gene expression and morphogenesis; however, in these stages where cells are tightly packed, cell-cell adhesion/contact-dependent signalling mechanisms also play important roles in these processes.     

Signal emission and signal propagation during early aggregation in Dictyostelium discoideum.

Waves of chemotactic movement during the early phase of aggregation in Dictyostelium discoideum are of 2 kinds, concentric waves produced by cells that emit cyclic AMP signals spontaneously, and spirals generated by excitations relayed continuously around loops of excitable cells. The period of a spiral wave is the time taken for the excitation to make one complete circuit of the pacemaker loop. We have compared signal emission from the 2 types of source in time-lapse films made at a variety of temperatures. Our results show that spiral waves have a characteristic period length throughout most if not all of the early phase of aggregation, and that the period of concentric waves is generally longer and more variable. Temperature has a pronounced effect on period length and a lesser effect on propagation velocity. We find that each individual wave is propagated at constant velocity over distances of 1-2 cm but that the velocity of successive waves declines. This decline probably reflects some cumulative effect of the chemotactic excitations on the excitable properties of the aggregating cells.     

Cyclic-AMP binding to the cell surface during development of Dictyostelium discoideum

Abstract. Cell aggregation in Dictyostelium discoideum is a chemotactic process mediated by cyclic adenosine monophosphate (CAMP), which is detected by cell surface receptors. The cAMP signal is degraded by cAMP phosphodiesterase. The possibility that cAMP signals are also used for cell communication in the multicellular stages was studied by determining whether the cAMP receptors, which are essential for signal transduction, continue to function in these stages. During slug migration, the number of binding sites per cell decreases to about 15% of the maximum level acquired during aggregation. At the onset of fruiting body formation, a three- to Four-Fold increase in cAMP binding activity occurs. This increase coincides with an increase in cAMP phosphodiesterase. Both phenomena suggest that cell-cell communication mediated by cAMP is used during culmination. During both slug migration and early culmination, the prestalk cells exhibit about twice as much binding activity as the prespore cells.     

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.     

alphaSU2, an epithelial integrin that binds laminin in the sea urchin embryo

Receptor-mediated activation of adenylyl cyclase (ACA) in Dictyostelium requires CRAC protein. Upon translocation to the membrane, this pleckstrin homology (PH) domain protein stimulates ACA and thereby mediates developmental aggregation. CRAC may also have roles later in development since CRAC-null cells can respond to chemotactic signals and participate in developmental aggregation when admixed with wild-type cells, but they do not complete development within such chimeras. To test whether the role of CRAC in postaggregative development is related to the activation of ACA, chemotactic aggregation was bypassed in CRAC-null cells by activating the cAMP-dependent protein kinase (PKA). While such strains formed mounds, they did not complete fruiting body morphogenesis or form spores. Expression of CRAC in the prespore cells of these strains rescued sporulation and fruiting body formation. This later function of CRAC does not appear to require its PH domain since the C-terminal portion of the protein (CRAC-DeltaPH) can substitute for full-length CRAC in promoting spore cell formation and morphogenesis. No detectable ACA activation was observed in any of the CRAC-null strains rescued by PKA activation and expression of CRAC-DeltaPH. Finally, we found that the development of CRAC-null ACA-null double mutants could be rescued by the activation of PKA together with the expression of CRAC-DeltaPH. Thus, there appears to be a required function for CRAC in postaggregative development that is independent of its previously described function in the ACA activation pathway.     

ROLE OF CYCLIC AMP IN THE POLARIZED MOVEMENT OF THE MIGRATING PSEUDOPLASMODIUM OF DICTYOSTELIUM DISCOIDEUM

Cyclic AMP is known to act as a chemotactic agent that directs the movement of aggregating Dictyostelium discoideum cells. Its role in the multicellular organization of this organism was studied with special reference to the polarized movement of the migrating pseudoplasmodium (slug). The results showed that the tip of the slug has the ability to function as an aggregation center, and that slug cells are chemotactically sensitive to cyclic AMP. The addition of calcium or magnesium appeared to enhance formation of cell streams, thus facilitating detection of chemotactic response of slug cells, but this addition was not required for the response itself. These indicate that the polar movement of the slug may be principally controlled by cyclic AMP.     

Origins of the prestalk-prespore pattern in Dictyostelium development

Using cell-autonomous markers we have traced the origins of prespore cells and two types of prestalk cells (pstA and pstB cells) during slug formation. We show that cell sorting and positional information both contribute to Dictyostelium morphogenesis. The initial pattern established at the mound stage is topologically quite different from that of the slug. Confirming previous studies, we find that prespore cells occupy most of the aggregate but are absent from a thin layer at the base and from the emerging tip. PstB cells are almost entirely localized to the basal region during the early stages of tip formation. Thus prespore and pstB cell differentiation appear to occur in response to localized morphogenetic signals. In the case of pstB cells, these signals presumably emanate from the base and not, as might be expected, from the tip. When first detectable, pstA cells are scattered throughout the aggregate. They then appear to migrate to the apex, where the tip forms.     

Mechanics as a Means of Information Propagation in Development

New research demonstrates that mechanics can serve as a means of information propagation in developing embryos. Historically, the study of embryonic development has had a dichotomy between morphogens and pattern formation on the one hand and morphogenesis and mechanics on the other. Secreted signals are the preeminent means of information propagation between cells and used to control cell fate, while physical forces act downstream or in parallel to shape tissue morphogenesis. However, recent work has blurred this division of function by demonstrating that mechanics can serve as a means of information propagation. Adhesive or repulsive interactions can propagate through a tissue as a wave. These waves are rapid and directional and can be used to control the flux of cells through a developmental trajectory. Here, two examples are reviewed in which mechanics both guides and mediates morphogenesis and two examples in which mechanics intertwines with morphogens to regulate cell fate.     

Differentiation, cell sorting and proportion regulation in the slug stage of Dictyostelium discoideum

Recent experimental work suggests that under normal conditions cell sorting plays an important part in maintaining and re-establishing the axial pattern of cell types in the slug stage of the cellular slime mold Dictyostelium discoideum. Following removal of the anterior zone of the slug, anterior-like cells that are normally distributed throughout the posterior of the slug rapidly migrate to the anterior end of the transected slug, and new anterior-like cells appear in the posterior portion. These results provide evidence that the direct linkage between spatial location and differentiation hypothesized in positional information models of spatial pattern formation is not universal. In this paper we develop and analyze a class of mathematical models of the slug in which cell determination can be less rigidly tied to spatial location, and which involve chemotactic cell sorting to re-establish and maintain the spatial pattern of cell types. We show that these models can reproduce the qualitative aspects of the experimental observations and that sorting takes place on the observed time scale when reasonable values of the parameters are used.     

Distribution of cAMP-dependent protein kinase during development in Dictyostelium discoideum

During the developmental cycle of Dictyostelium discoideum cyclic AMP functions as both a chemotactic signal for aggregation and a regulatory molecule during later events of differentiation. Morphological and biochemical data suggest that cAMP may direct cells during morphogenesis and differentiation. We utilized microtechniques to determine the stage- and cell-specific levels of the cAMP-dependent protein kinase, the probable intracellular cAMP receptor. Kinase activity was low and non-cAMP-dependent in amoebae and early aggregates but increased and became cAMP-dependent in aggregates after the formation of tight cell contacts. Maximum kinase activity and cAMP dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single stage occurred during culmination when the activity in the stalks was approximately one-fourth of that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. In all other stages tested cAMP-dependent protein kinase activity was equal in prespore and prestalk cells.     

Membrane potentials of Dictyostelium discoideum cells at the pseudoplasmodial stage

Abstract The difference in membrane potentials between prestalk cells and prespore cells has been examined with reference to the formation of cellular pattern in the pseudoplasmodium (slug) of D. discoideum . Each cell at a different concentration of cAMP had a characteristic membrane potential. In addition, differences in and reversal of membrane potentials occurred between the two types of cell. The results indicate that the changes in membrane potential in both types of cell are closely correlated with the changes in chemotactic movement in response to cAMP.     

A model for cell type localization in the migrating slug of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> based on differential chemotactic sensitivity to cAMP and differential sensitivity to suppression of chemotaxis by ammonia

The three basic cell types in the migrating slug of Dictyostelium discoideum show differential chemotactic response to cyclic AMP (cAMP) and differential sensitivity to suppression of the chemotaxis by ammonia. The values of these parameters indicate a progressive maturation of chemotactic properties during the transdifferentiation of slug cell types. We present a model that explains the localization of the three cell types within the slug based on these chemotactic differences and on the maturation of their chemotactic properties.     

Chemotaxis to cAMP and slug migration in Dictyostelium both depend on migA, a BTB protein.

Chemotaxis in natural aggregation territories and in a chamber with an imposed gradient of cyclic AMP (cAMP) was found to be defective in a mutant strain of Dictyostelium discoideum that forms slugs unable to migrate. This strain was selected from a population of cells mutagenized by random insertion of plasmids facilitated by introduction of restriction enzyme (a method termed restriction enzyme-mediated integration). We picked this strain because it formed small misshapen fruiting bodies. After isolation of portions of the gene as regions flanking the inserted plasmid, we were able to regenerate the original genetic defect in a fresh host and show that it is responsible for the developmental defects. Transformation of this recapitulated mutant strain with a construct carrying the full-length migA gene and its upstream regulatory region rescued the defects. The sequence of the full-length gene revealed that it encodes a novel protein with a BTB domain near the N terminus that may be involved in protein-protein interactions. The migA gene is expressed at low levels in all cells during aggregation and then appears to be restricted to prestalk cells as a consequence of rapid turnover in prespore cells. Although migA- cells have a dramatically reduced chemotactic index to cAMP and an abnormal pattern of aggregation in natural waves of cAMP, they are completely normal in size, shape, and ability to translocate in the absence of any chemotactic signal. They respond behaviorally to the rapid addition of high levels of cAMP in a manner indicative of intact circuitry connecting receptor occupancy to restructuring of the cytoskeleton. Actin polymerization in response to cAMP is also normal in the mutant cells. The defects at both the aggregation and slug stage are cell autonomous. The MigA protein therefore is necessary for efficiently assessing chemical gradients, and its absence results in defective chemotaxis and slug migration.     

The histidine kinase homologue DhkK/Sombrero controls morphogenesis in Dictyostelium

A key event in Dictyostelium development is the formation of the Mexican hat. This corresponds to a commitment step in morphogenesis that irreversibly signals progression from the slug stage to the fruiting body. We describe the characterization of the dhkK gene that controls this morphogenetic step. Null mutants of dhkK repeatedly attempt, and fail, to undergo morphogenesis from the slug to the Mexican hat, causing them to exhibit a "slugger" phenotype, which cannot be corrected by co-development with wild-type cells. The dhkK gene encodes a putative receptor histidine kinase whose expression is enriched in prestalk cells in the slug. Uniquely for a histidine kinase, DhkK is located in the nuclear envelope. Entry into culmination requires the DhkK response regulator domain, which appears to directly regulate cyclic AMP signaling.     

A temperature-sensitive adenylyl cyclase mutant of Dictyostelium

Dictyostelium development starts with the chemotactic aggregation of up to 10(6) amoebae in response to propagating cAMP waves. cAMP is produced by the aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Temperature-sensitive mutants of ACA were selected from a population of aca null cells transformed with a library of ACA genes, a major segment of which had been amplified by error-prone PCR. One mutant (tsaca2) that can complement the aggregation null phenotype of aca null cells at 22 degrees C but not at 28 degrees C was characterized in detail. The basal catalytic activity of the enzyme in this mutant was rapidly and reversibly inactivated at 28 degrees C. Using this mutant strain we show that cell movement in aggregates and mounds is organized by propagating waves of cAMP. Synergy experiments between wild-type and tsaca2 cells, shifted to the restrictive temperature at various stages of development, showed that ACA plays an important role in the control of cell sorting and tip formation.     

Chemotactic cell movement during Dictyostelium development and gastrulation

Many developmental processes involve chemotactic cell movement up or down dynamic chemical gradients. Studies of the molecular mechanisms of chemotactic movement of Dictyostelium amoebae up cAMP gradients highlight the importance of PIP3 signaling in the control of cAMP-dependent actin polymerization, which drives the protrusion of lamellipodia and filopodia at the leading edge of the cell, but also emphasize the need for myosin thick filament assembly and motor activation for the contraction of the back of the cell. These process become even more important during the multicellular stages of development, when propagating waves of cAMP coordinate the chemotactic movement of tens of thousands of cells, resulting in multicellular morphogenesis. Recent experiments show that chemotaxis, especially in response to members of the FGF, PDGF and VEGF families of growth factors, plays a key role in the guidance of mesoderm cells during gastrulation in chick, mouse and frog embryos. The molecular mechanisms of signal detection and signaling to the actin-myosin cytoskeleton remain to be elucidated.     

Guanylate Cyclase Activity in Dictyostelium discoideum and Its Increase during Cell Development

Following consumption of the food supply, cells of the cellular slime mould Dictyostelium discoideum aggregate and form a multicellular organism. The mechanism for cell aggregation is chemotaxis. The chemotactic signal in D. discoideum is released periodically from aggregation centers and propagated from cell to cell. cAMP mediates cell aggregation by acting as chemotactic attractant and as propagator of the signal. cAMP signals are measured by cell-surface receptors. Recent evidence indicates a role for cGMP during cAMP-mediated cell aggregation in D. discoideum .
During cell differentiation to aggregation competence, cAMP binding sites appear at the cell surface, and the activity of the enzymes adenylate cyclase and phosphodiesterase increases several-fold. In the present work we investigate the synthesis of cGMP in D. discoideum . Conditions for the assay of guanylate cyclase in cell homogenates are described. Guanylate cyclase activity was followed during cell differentiation to aggregation competence and found to increase fourfold. These results indicate that cGMP is involved in cell differentiation of D. discoideum . In contrast to adenylate cyclase, which is activated by cAMP, guanylate cyclase was under our conditions activated neither by cAMP, nor by folic acid.