Dictyostelium: a model for regulated cell movement during morphogenesis

Dictyostelium has played an important role in unraveling the pathways that control cell movement and chemotaxis. Recent studies have started to elucidate the pathways that control cell sorting, morphogenesis, and the establishment of spatial patterning in this system. In doing so, they provide new insights into how cell movements within a multicellular organism are regulated and the importance of pathways that are similar to those that regulate chemotaxis of cells on two-dimensional surfaces during aggregation.     

Mound-cell movement and morphogenesis in Dictyostelium

To examine the mechanisms of cell locomotion within a three-dimensional (3-D) cell mass, we have undertaken a systematic 3-D analysis of individual cell movements in the Dictyostelium mound, the first 3-D structure to form during development of the fruiting body. We used time-lapse deconvolution microscopy to examine two strains whose motion represents endpoints on the spectrum of motile behaviors that we have observed in mounds. In AX-2 mounds, cell motion is slow and trajectories are a combination of random and radial, compared to KAX-3, in which motion is fivefold faster and most trajectories are rotational. Although radial or rotational motion was correlated with the optical-density wave patterns present in each strain, we also found small but significant subpopulations of cells that moved differently from the majority, demonstrating that optical-density waves are at best insufficient to explain all motile behavior in mounds. In examining morphogenesis in these strains, we noted that AX-2 mounds tended to culminate directly to a fruiting body, whereas KAX-3 mounds first formed a migratory slug. By altering buffering conditions we could interchange these behaviors and then found that mound-cell motions also changed accordingly. This demonstrates a correlation between mound-cell motion and subsequent development, but it is not obligatory. Chimeric mounds composed of only 10% KAX-3 cells and 90% AX-2 cells exhibited rotational motion, suggesting that a diffusible molecule induces rotation, but many of these mounds still culminated directly, demonstrating that rotational motion does not always lead to slug migration. Our observations provide a detailed analysis of cell motion for two distinct modes of mound and slug formation in Dictyostelium.     

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.     

Morphogenetic cell movement in Dictyostelium

Dictyostelium morphogenesis starts with the chemotactic aggregation of starving individual cells. The cells move in response to propagating waves of the chemoattractant cyclic AMP initiated by cells in the aggregation centre. During aggregation the cells begin to differentiate into several types with different signalling and chemotactic properties. These cell types sort out from each other to form an axial pattern in the slug. There is now good evidence that periodic chemotactic signals not only control aggregation, but also later stages of morphogenesis. These signals take the form of target patterns, spirals, multi-armed spirals and scroll waves. I will discuss their role in the control of cell movement during mound and slug formation and in the formation of the fruiting body.     

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.     

Regulation of cellular differentiation during Dictyostelium morphogenesis.

In Dictyostelium there are multiple prestalk cell types that have a complex pattern of directed cell movement during slug formation and culmination. Three extracellular signals, cyclic AMP, DIF and ammonia, control cell type differentiation. Recently there has been considerable progress in understanding their modes of action and interaction.     

Signalling molecules involved in cellular differentiation during Dictyostelium morphogenesis

GSK-3, Dd-STATa, PKA, rZIP and Ras all play important roles in cell type determination of Dictyostelium discoideum. The fact that homologs of these proteins also function in metazoan development emphasizes the importance of Dictyostelium as a model microbial organism for studying the molecular mechanisms that regulate development. The recent elaboration of the central role for GSK-3 in cell type determination has been of particular importance. The stimulatory effect of extracellular cAMP on GSK-3 activity has been shown to act through the cell surface receptor cAR3 and a tyrosine protein kinase ZAK1, which directly activates and phosphorylates GSK-3. Several proteins, including Dd-STATa, have been identified as substrates for GSK-3, and are therefore potential transducers of the signals involved in cell type determination.     

TOR complex 2 integrates cell movement during chemotaxis and signal relay in Dictyostelium

Dictyostelium cells form a multicellular organism through the aggregation of independent cells. This process requires both chemotaxis and signal relay in which the chemoattractant cAMP activates adenylyl cyclase through the G protein-coupled cAMP receptor cAR1. cAMP is produced and secreted and it activates receptors on neighboring cells, thereby relaying the chemoattractant signal to distant cells. Using coimmunoprecipitation and mass spectrometric analyses, we have identified a TOR-containing complex in Dictyostelium that is related to the TORC2 complex of Saccharomyces cerevisiae and regulates both chemotaxis and signal relay. We demonstrate that mutations in Dictyostelium LST8, RIP3, and Pia, orthologues of the yeast TORC2 components LST8, AVO1, and AVO3, exhibit a common set of phenotypes including reduced cell polarity, chemotaxis speed and directionality, phosphorylation of Akt/PKB and the related PKBR1, and activation of adenylyl cyclase. Further, we provide evidence for a role of Ras in the regulation of TORC2. We propose that, through the regulation of chemotaxis and signal relay, TORC2 plays an essential role in controlling aggregation by coordinating the two essential arms of the developmental pathway that leads to multicellularity in Dictyostelium.     

DWnt4 regulates cell movement and focal adhesion kinase during Drosophila ovarian morphogenesis

Cell motility is regulated by extracellular cues and by intracellular factors that accumulate at sites of contact between cells and the extracellular matrix. One of these factors, focal adhesion kinase (FAK), regulates the cycle of focal adhesion formation and disassembly that is required for cell movement to occur. Recently, Wnt signaling has also been implicated in the control of cell movement in vertebrates, but the mechanism through which Wnt proteins influence motility is unclear. We demonstrate that Drosphila Wnt4 is required for cell movement and FAK regulation during ovarian morphogenesis. Dfrizzled2, Disheveled, and protein kinase C are also required. The DWnt4 cell motility pathway is distinct from both the canonical Wnt pathway and the planar polarity pathway. Our data suggest that DWnt4 facilitates motility through regulation of focal adhesions.     

Dictyostelium discoideum cell membranes contain masked chemotactic receptors for cyclic AMP

Aggregating Dictyostelium discoideum cells possess highly specific receptors for the chemoattractant cAMP on their cell surface. Isolated membranes as well as intact cells are shown to contain a large number of latent cAMP receptors. These are reversibly unmasked in the presence of a high salt concentration (0.1–2 M) or in the presence of millimolar concentrations of Ca²⁺.     

Conserved microtubule-actin interactions in cell movement and morphogenesis

Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions--regulatory and structural. These interactions comprise at least three conserved 'mechanochemical activity modules' that perform similar roles in these diverse cell functions.     

Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation

Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. We analyze here the formation of the Drosophila melanogaster cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. Our study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, we identify a genetic pathway in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data we propose a model for D. melanogaster cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. We suggest that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis.     

Transmethylation inhibitors decrease chemotactic sensitivity and delay cell aggregation in Dictyostelium discoideum

In Dictyostelium discoideum, extracellular cyclic AMP (cAMP) induces chemotaxis and cell aggregation. Suspensions of cAMP-sensitive cells respond to a cAMP pulse with a rapid, transient increase of protein carboxyl methylation. The transmethylation inhibitors cycloleucine, L-homocysteine thiolactone, and coformycin decrease chemotactic sensitivity and delay cell aggregation when administered in concentrations which do not influence cAMP binding to cell surface receptors or the activity of total phosphodiesterase. The ability of the drugs to inhibit chemotaxis could be correlated with their capacity to convert the initial transient positive response of carboxyl methylation to cAMP into a negative one. This suggests that both protein O-methyltransferase and protein methylesterase are activated after stimulation of aggregative cells with cAMP, the net effect being a transient, positive response of methylation. In the presence of a sufficiently large dose of inhibitor, methyltransferase is inhibited, whereas methylesterase activity is much less affected, so that a transient negative response of methylation to cAMP is observed. The slow, positive response of carboxyl methylation to cAMP which occurs ca. 2.5 to 5 min after stimulus administration is not affected by inhibitors of transmethylation. These results suggest that methylation reactions are involved in the chemotactic response of D. discoideum cells to cAMP.     

On the genetic control of cell cycles during morphogenesis in Ulva mutabilis

Development of the wild type and two temperature-sensitive mutants of the multicellular green alga Ulva mutabilis is compared. The mutants develop normal phenotypes at 22°C and abnormal phenotypes at 15°C. Normal development starts by formation of a filament consisting of a row of cells. The growth rate, the generation times, and the cell length at division change in a coordinated manner according to the positions of the cells within the filament. In the mutant cs₂ transfer to 15°C inhibits all cytoplasmic divisions during early development. In the mutant cs₆ the first three divisions proceed normally. Then cytoplasmic division is blocked in the most distal cells, while the proximal cells continue to divide according to a branched pattern. In the cs₂ mutant cell determination seems to occur at 15°C, while the differentiation of the determined cells can only occur at 22°C. In the mutant cs₆ the cells are not determined at 15°C. The cs₆⁺ gene, as well as the previously described Slender-like genes, presumably has a short period of activity and is concerned with more fundamental epigenetic processes than the cs₂⁺-gene and the previously described precocious-like genes, which seem to have more prolonged periods of activity.     

The trishanku gene and terminal morphogenesis in Dictyostelium discoideum

SUMMARY Multicellular development in the social amoeba Dictyostelium discoideum is triggered by starvation. It involves a series of morphogenetic movements, among them being the rising of the spore mass to the tip of the stalk. The process requires precise coordination between two distinct cell types—presumptive (pre-) spore cells and presumptive (pre-) stalk cells. Trishanku ( triA ) is a gene expressed in prespore cells that is required for normal morphogenesis. The triA⁻ mutant shows pleiotropic effects that include an inability of the spore mass to go all the way to the top. We have examined the cellular behavior required for the normal ascent of the spore mass. Grafting and mixing experiments carried out with tissue fragments and cells show that the upper cup, a tissue that derives from prestalk cells and anterior-like cells (ALCs), does not develop properly in a triA⁻ background. A mutant upper cup is unable to lift the spore mass to the top of the fruiting body, likely due to defective intercellular adhesion. If wild-type upper cup function is provided by prestalk and ALCs, trishanku spores ascend all the way. Conversely, Ax2 spores fail to do so in chimeras in which the upper cup is largely made up of mutant cells. Besides proving that under these conditions the wild-type phenotype of the upper cup is necessary and sufficient for terminal morphogenesis in D. discoideum , this study provides novel insights into developmental and evolutionary aspects of morphogenesis in general. Genes that are active exclusively in one cell type can elicit behavior in a second cell type that enhances the reproductive fitness of the first cell type, thereby showing that morphogenesis is a cooperative process.     

The cell adhesion molecule DdCAD-1 regulates morphogenesis through differential spatiotemporal expression in Dictyostelium discoideum

During development of Dictyostelium, multiple cell types are formed and undergo a coordinated series of morphogenetic movements guided by their adhesive properties and other cellular factors. DdCAD-1 is a unique homophilic cell adhesion molecule encoded by the cadA gene. It is synthesized in the cytoplasm and transported to the plasma membrane by contractile vacuoles. In chimeras developed on soil plates, DdCAD-1-expressing cells showed greater propensity to develop into spores than did cadA-null cells. When development was performed on non-nutrient agar, wild-type cells sorted from the cadA-null cells and moved to the anterior zone. They differentiated mostly into stalk cells and eventually died, whereas the cadA-null cells survived as spores. To assess the role of DdCAD-1 in this novel behavior of wild-type and mutant cells, cadA-null cells were rescued by the ectopic expression of DdCAD-1-GFP. Morphological studies have revealed major spatiotemporal changes in the subcellular distribution of DdCAD-1 during development. Whereas DdCAD-1 became internalized in most cells in the post-aggregation stages, it was prominent in the contact regions of anterior cells. Cell sorting was also restored in cadA(-) slugs by exogenous recombinant DdCAD-1. Remarkably, DdCAD-1 remained on the surface of anterior cells, whereas it was internalized in the posterior cells. Additionally, DdCAD-1-expressing cells migrated slower than cadA(-) cells and sorted to the anterior region of chimeric slugs. These results show that DdCAD-1 influences the sorting behavior of cells in slugs by its differential distribution on the prestalk and prespore cells.