Slug Migration Research Articles

The MADS-box transcription factor SRFA regulates different aspects of Dictyostelium discoideum development

The MADS-box transcription factor SRFA is expressed at several stages of Dictyostelium discoideum development. In early developmental stages is expressed in the prestalk region while it is expressed in the prespore region at culmination. The complex pattern of expression is obtained through the use of four different promoter regions that are active in different cell types and at different developmental stages. The analyses of srfA deficient strains has shown that this transcription factor is necessary for slug migration, morphogenesis and spore differentiation. Differential screening between Wild-type and srfA(-) strains has allowed the isolation of five cDNA clones whose expression is dependent on srfA. These genes are expressed at culmination in Wild-type strains but not in srfA strains. The expression of only one or these genes can be induced in srfA- strains by treatment with the Protein Kinase A activator 8-Br-cAMP, indicating a complex interaction between Protein Kinase A and SRFA in the regulation of Dictyostelium discoideum development.

A Model forDictyosteliumSlug Movement

Dictyosteliumslug movement results from the coordinated movement of its 105constituent cells. We have shown experimentally that cells in the tip of the slug show a rotational cell movement while the cells in the back of the slug move periodically forward (Siegert & Weijer, 1992). We have put forward the hypothesis that cell movement in slugs is controlled by chemotaxis to scroll waves generated in the tip which convert to twisted scroll or planar waves in the back of the slug (Bretschneideret al., 1995). The coordinated movement of all individual cells in response to these waves could then result in forward movement of the slug. We now test this hypothesis by extending our model for mound formation (Bretschneideret al., 1997) to include two cell types with different signalling and movement properties. All cells are able to relay cAMP and move chemotactically in response to cAMP gradients. Cells interact by adhesion, pressure and friction with neighbouring cells and the extracellular matrix. The model can generate stable scroll waves propagating from the tip to the back of a slug which coordinate forward cell movement and result in slug migration. We use the model to investigate the influence of cell type specific differences in excitability, adhesion and cell interactions on slug motion.

A myb-related protein required for culmination in Dictyostelium.

The avian retroviral v-myb gene and its cellular homologues throughout the animal and plant kingdoms contain a conserved DNA binding domain. We have isolated an insertional mutant of Dictyostelium unable to switch from slug migration to fruiting body formation i.e. unable to culminate. The gene that is disrupted, mybC, codes for a protein with a myb-like domain that is recognized by an antibody against the v-myb repeat domain. During development of myb+ cells, mybC is expressed only in prestalk cells. When developed together with wild-type cells mybC- cells are able to form both spores and stalk cells very efficiently. Their developmental defect is also bypassed by overexpressing cAMP-dependent protein kinase. However even when their defect is bypassed, mybC null slugs and culminates produce little if any of the intercellular signalling peptides SDF-1 and SDF-2 that are believed to be released by prestalk cells at culmination. We propose that the mybC gene product is required for an intercellular signaling process controlling maturation of stalk cells and spores and that SDF-1 and/or SDF-2 may be implicated in this process.

Mound-Cell Movement and Morphogenesis inDictyostelium

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 theDictyosteliummound, 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 inDictyostelium.

A way of following individual cells in the migrating slugs of Dictyostelium discoideum.

In the development of the cellular slime mold Dictyostelium discoideum there is a stage in which the aggregated amoebae form a migrating slug that moves forward in a polar fashion, showing sensitive orientation to environmental cues, as well as early signs of differentiation into anterior prestalk and posterior prespore cells. Heretofore it has been difficult to follow the movement of the individual cells within the slug, but a new method is described in which small, flat (one cell thick) slugs are produced in a glass-mineral oil interface where one can follow the movement of all the cells. Observations of time-lapse videos reveal the following facts about slug migration: (i) While the posterior cells move straight forward, the anterior cells swirl about rapidly in a chaotic fashion. (ii) Turning involves shifting the high point of these hyperactive cells. (iii) Both the anterior and the posterior cells move forward on their own power as the slug moves forward. (iv) There are no visible regular oscillations within the slug. (v) The number of prestalk and prespore cells is proportional for a range of sizes of these mini-slugs. All of these observations on thin slugs are consistent with what one finds in normal, three-dimensional slugs.

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.

Twisted scroll waves organize Dictyostelium mucoroides slugs.

Cellular slime moulds (Dictyosteloids) are characterised by at least two different modes of slug migration. Most species, e.g. Dictyostelium mucoroides, produce a stalk continuously during slug migration, while a few species, e.g. Dictyostelium discoideum are characterised by stalk-less slug migration and only produce a stalk upon culmination. Experiments on D. discoideum and theoretical model calculations have shown that D. discoideum slugs are organized by a cAMP scroll wave in the tip which produces planar waves in the back. These waves guide cell movement in slugs: spiralling in the tip and forward movement parallel to the slug axis in the back. Simple changes in model parameters can lead to the formation of a twisted scroll wave which extends throughout the slug. In order to investigate whether such twisted scroll waves occur naturally we have analysed the movement of fluorescently labelled single cells in migrating D. mucoroides slugs. The results show that cells in the prespore zone of D. mucoroides slugs move in a spiral path. Although the velocity of single cells in D. mucoroides is faster than in D. discoideum, the net forward component of their movement is less due to their spiral trajectories. As a result D. mucoroides slugs move more slowly than D. discoideum slugs. The entire D. mucoroides slug also describes a spiralling path leaving corkscrew shaped stalks behind. Based on these observations we propose that cell movement in D. mucoroides slugs is controlled by a propagating twisted scroll wave of cAMP which extends throughout the length of the slug.

A marker of terminal stalk cell terminal differentiation in Dictyostelium

We describe the isolation of staB, a Dictyostelium gene that is selectively expressed in stalk cells. If an aggregate enters culmination immediately staB is expressed in the bottom half of the stalk and in the basal disc but if a migratory slug is formed it is also expressed in the top of the stalk and in a disc of cells that surmounts the spore head. The latter two populations of cells derive from the rear part of the prestalk region, the pstO cells, which would therefore seem to change their transcriptional competency during slug migration. The staB promoter contains a distal region that is required for expression within both the stalk and the basal disc and a proximal region that is required only for expression within the basal disc. This uncoupling of transcriptional specificities suggests that the signalling mechanisms that direct terminal differentiation in these two tissues differ in some way.

RZIP, a RING-leucine zipper protein that regulates cell fate determination during Dictyostelium development.

rZIP is an approx. 32 kDa, multi-domain protein of Dictyostelium discoideum whose structural motifs include a RING (zinc-binding) domain, a leucine zipper, a glutamine repeat, an SH3-binding region and a consensus phosphorylation site for MAP kinase. In vitro, rZIP forms homodimers and interacts specifically with the SH3 domain(s) of the Nck adaptor protein. rZIP is expressed maximally during cell differentiation at approximately equivalent levels in all cells. Disruption of the rZIP gene rzpA results in altered cellular aggregation, impaired slug migration, and aberrant patterning of prespore and prestalk cells, the major progenitor classes. In rzpA- strains, prespore-specific genes are overexpressed and prestalk expression zones are reduced. Conversely, constitutive overexpression of rzpA markedly decreases prespore-specific gene expression and significantly increases the expression of prestalk-specific genes. Further, induced transdifferentiation of prespore cells into prestalk cells is inhibited in rzpA-slugs. In light of these patterning defects, we suggest that the RING/zipper protein rZIP plays an important role in early cell fate decisions in Dictyostelium, acting as a positive regulator of prestalk differentiation and an inhibitor of prespore differentiation.

Modelling Morphogenesis: From Single Cells to Crawling Slugs

We present a three-dimensional hybrid cellular automata (CA)/partial differential equation (PDE) model that allows for the study of morphogenesis in simple cellular systems. We apply the model to the cellular slime mold Dictyostelium discoideum“from single cells to crawling slug”. Using simple local interactions we can achieve the basic morphogenesis with only three processes: production of and chemotaxis to cAMP and cellular adhesion. The interplay of these processes causes the amoebae to spatially self-organize leading to the complex behaviour of stream and mound formation, cell sorting and slug migration all without any change of parameters during the complete morphogenetic process.

Twisted scroll waves organize Dictyostelium mucoroides slugs.

Cellular slime moulds (Dictyosteloids) are characterised by at least two different modes of slug migration. Most species, e.g. Dictyostelium mucoroides, produce a stalk continuously during slug migration, while a few species, e.g. Dictyostelium discoideum are characterised by stalk-less slug migration and only produce a stalk upon culmination. Experiments on D. discoideum and theoretical model calculations have shown that D. discoideum slugs are organized by a cAMP scroll wave in the tip which produces planar waves in the back. These waves guide cell movement in slugs: spiralling in the tip and forward movement parallel to the slug axis in the back. Simple changes in model parameters can lead to the formation of a twisted scroll wave which extends throughout the slug. In order to investigate whether such twisted scroll waves occur naturally we have analysed the movement of fluorescently labelled single cells in migrating D. mucoroides slugs. The results show that cells in the prespore zone of D. mucoroides slugs move in a spiral path. Although the velocity of single cells in D. mucoroides is faster than in D. discoideum, the net forward component of their movement is less due to their spiral trajectories. As a result D. mucoroides slugs move more slowly than D. discoideum slugs. The entire D. mucoroides slug also describes a spiralling path leaving corkscrew shaped stalks behind. Based on these observations we propose that cell movement in D. mucoroides slugs is controlled by a propagating twisted scroll wave of cAMP which extends throughout the length of the slug.

The initiation of basal disc formation in Dictyostelium discoideum is an early event in culmination.

We have analysed expression of the ecmA and ecmB genes of Dictyostelium by enzymatic double staining using beta-galactosidase and beta-glucuronidase reporter gene constructs. Cells expressing the ecmA gene first appear as scattered cells at the mound stage of development and we show that this is also true for cells expressing the ecmB gene. During tip formation the ecmA-expressing cells move to the apex of the mound, while the ecmB-expressing cells accumulate in the base. The ecmB-expressing cells constitute part of the basal disc if the culminant is formed in situ but are discarded if a migratory slug is formed. During slug migration they are replaced by a band of ecmB-expressing cells, situated in the front half of the prespore zone and tightly apposed to the substratum. When culmination is triggered these cells rapidly move to the back half of the prestalk zone, possibly acting as a point of attachment to the substratum. Ultimately, they are joined by cells at the back of the slug, the rearguard cells, to form the basal disc. Thus, contrary to previous belief, basal disc formation is initiated very early during culmination and occurs by the forward movement of cells located in the anterior of the prespore zone.

Transport of a tracer slug in a fractured permeable formation

This paper concerns the migration of a tracer slug injected into a fractured permeable formation. Basic transport processes are formulated. Dimensionless parameters governing transport phenomena in the domain are identified, and their effects are determined. A simplified conceptual model is employed for the quantitative evaluation of the physical phenomena associated with tracer migration in the domain. The analytical approach applied in this study provides some basic characteristics concerning the build-up and decay of tracer breakthrough curves. Characteristics of fracture networks incorporating a large number of fracture sectors are evaluated using a numerical finite difference scheme. It is shown that various time and length scales characterize the phenomena involved in tracer slug migration in a fractured permeable formation. It is found that, in cross-sections located at a small distance from the tracer injection point, two maximum values of tracer concentration are expected in the breakthrough curves. In cross-sections located at comparatively large distances from the tracer injection point, the breakthrough curves are similar to those characterizing an ideal porous media.

The extracellular matrix of the Dictyostelium discoideum slug.

In this review, we detail the current understanding of the extracellular matrix (ECM) of the migratory slug phase of the cellular slime mould, Dictyostelium discoideum. We describe some structural and non-structural molecules which comprise the ECM, and how these molecules reflect both plant and animal ECM systems. We also describe zones of the multicellular slug that are known to make ECM components, including the role of the prestalk cells and the slug epithelium-like layer. Finally, we review the contributions of studies on mutants to our understanding of the ECM of D. discoideum, and relate this to differentiation and development in more complex eukaryotic systems.

Comparison of the Dictyostelium rasD and ecmA genes reveals two distinct mechanisms whereby an mRNA may become enriched in prestalk cells

The Dictyostelium ras gene, rasD, encodes an mRNA that is more abundant in prestalk than prespore cells in the migratory slug. Its expression is inducible by extracellular cAMP but is not inducible by the prestalk and stalk cell morphogen differentiation inducing factor (DIF). We show that a rasD-lacZ fusion gene is first expressed in approximately one half of the cells in the aggregate, including some cells that also express a prespore-specific marker. The amount of rasD-lacZ fusion protein in prespore cells then diminishes as the slug is formed. Analysis of a rasD-lacZ fusion protein with an N terminal substitution that reduces protein stability within the cell provides strong confirmatory evidence that the ras gene product becomes enriched in prestalk cells by selective repression of gene expression in prespore cells. In contrast, the DIF-inducible ecmA gene is expressed only in those cells that will become prestalk cells in the migratory slug. These results show that there are two different ways in which an mRNA may become enriched in prestalk cells and support the view that DIF is the inducer of prestalk cell differentiation.

Glycoprotein Complexes Interacting with Cellulose in the "Cell Print" Zones of the Dictyostelium discoideum Extracellular Matrix

Cellulose is one of the commonest structural biopolymers. How cellulose is organized in extracellular matrices is a mystery. Here we investigate a model system, the extracellular matrix (ECM) of Dictyostelium discoideum which is composed of proteins and cellulose. A group of glycoproteins, the sheathins, which colocalize with cellulose in the ECM of D. discoideum are characterized. Sheathins are dimeric or trimeric forms of molecular mass 53-68 kDa, where the monomers are 12-35 kDa. The sheathin subunits are similar but not identical proteins. The sheathin family comprises sheathin 68, (68-kDa trimer); sheathin 62, (62-kDa dimer); sheathin 55, (55-kDa dimer), and sheathin 53, (53-kDa dimer). The subunits which assemble into the four sheathins represent at least three gene products: ShC, ShD, and ShE which are linked by disulphide bonds, Protein sequence analysis shows two of the sheathin genes encode products ShC and ShD with very similar amino terminal sequences. This group of D. discoideum ECM glycoproteins has homology with two other much larger ECM proteins of D. discoideum, ST430 and ST310, which are located in a more dispersed fashion in the ECM. Sheathins are tightly but non-covalently associated with the ECM, and this association requires strong denaturing conditions for disruption, e.g., SDS or 8 M urea. Sheathins form a component of the "cell prints" which are believed to have a role in cell-ECM interactions and slug cell migration.

Optical flow analysis of the ventral cellular layer of the migrating Dictyostelium discoideum slug

A digital image analysis system for extracting motion information from time-varying digital light microscopy images is presented. This system is then used to map out the movement profile of the surface layer of cells in contact with the substratum through the extracellular matrix (ECM) of the migrating Dictyostelium discoideum slug. From digital high magnification light microscopy images, the morphology of moving cells within the tail region of a young migrating wild-type WS380B slug is described, and compared with the morphology of streaming D. discoideum cells. It is shown that: (i) when the migrating tip of the slug touches the agar substrate, cells in the anterior ventral surface layer of the tip region slow dramatically; (ii) overall cell movement in the ventral surface layer of the migrating D. discoideum slug is slower than the movement of the slug as a whole; and (iii) in less than 10% of cases a wave of movement (groups of cells synchronously slowing down and then accelerating forward) propagates down the slug axis at approx. 1.2 microns s-1. The time interval between waves may be related to the time interval between tip-to-substratum contact that is periodically re-established during normal WS380B slug migration after each aerial projection of the tip.

Asense is a Drosophila neural precursor gene and is capable of initiating sense organ formation.

Neural precursor cells in Drosophila arise from the ectoderm in the embryo and from imaginal disc epithelia in the larva. In both cases, this process requires daughterless and the proneural genes achaete, scute and lethal-of-scute of the achaete-scute complex. These genes encode basic helix-loop-helix proteins, which are nuclear transcription factors, as does the asense gene of the achaete-scute complex. Our studies suggest that asense is a neural precursor gene, rather than a proneural gene. Unlike the proneural achaete-scute gene products, the asense RNA and protein are found in the neural precursor during its formation, but not in the proneural cluster of cells that gives rise to the neural precursor cell. Also, asense expression persists longer during neural precursor development than the proneural gene products; it is still expressed after the first division of the neural precursor. Moreover, asense is likely to be downstream of the proneural genes, because (1) asense expression is affected in proneural and neurogenic mutant backgrounds, (2) ectopic expression of asense protein with an intact DNA-binding domain bypasses the requirement for achaete and scute in the formation of imaginal sense organs. We further note that asense ectopic expression is capable of initiating the sense organ fate in cells that do not normally require the action of asense. Our studies therefore serve as a cautionary note for the inference of normal gene function based on the gain-of-function phenotype after ectopic expression.

Pattern Formation in a Cell-Cycle Dependent Manner during the Development of Dictyostelium discoideum: (pattern formation/cell sorting/cell cycle/PS-point/Dictyostelium discoideum).

strain Ax-2, cells grow axenically and multiply by binary fission with a doubling time of about 7.2 hr provided sufficient nutrients are supplied. Upon exhaustion of the nutrient supply, starving cell populations start differentiating with the acquisition of aggregation competence and aggregate to form multicellular structures by che- motaxis to 3’,5’-cyclic adenosine monophosphate (CAMP) (2, 15). During aggregation, cells also acquire EDTA-resistant cohesiveness (9). The cell aggregate (mound) thus formed exerts orga- nized movement and zonal differentiation as a migrating pseudoplasmodium (slug), and even- tually develops into a sorocarp consisting of a mass of spores (sorus), a supporting cellular stalk, and a basal disc. Differentiation of the two cell types, anterior prestalk and posterior prespore cells, is evident at the slug stage. The growth and differentiation in Ax-2 cells are temporarily sepa- rated from each other and easily controlled by nutritional conditions. In addition, determinations of the fate map of cell types are simpler than in other organisms. Accordingly, this organism has been particularly useful for elucidating the molecu- lar mechanism of growthidifferentiation transition and also the regulatory mechanisms controlling eukaryotic cell-type determination and pattern formation. There have been numerous studies on the following developmental stages: 1 ) growth and preaggregation, 2) aggregation, 3) tip formation and prestalk/prespore differentiation, 4) slug formation and migration, and 5) culmination and terminal stalk/spore differentlation. The studies

Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination : Development 118, 353–362

The ecmA gene of Dictyostelium encodes an extracellular matrix protein and is selectively expressed in prestalk cells. We show that its promoter contains discrete elements that direct expression in different subpopulations of prestalk cells. Prestalk(pst)A cells occupy the front half of the prestalk region. Expression in pstA cells requires DNA sequences close to the cap site of the gene and a separate, upstream region that acts in combination with the gene proximal sequences. PstO cells are situated in the rear half of the prestalk region and at least two separate and redundant promoter regions direct expression within them. All constructs that are expressed in pstO cells are also expressed in anterior-like cells (ALCs); cells that resemble prestalk cells but which are scattered throughout the prespore region. This observation suggests that pstO cells and ALCs may be very similar in their properties. If development occurs under conditions in which a migratory slug is not formed, there is an ordered movement of cells into the stalk tube. PstA cells enter the stalk tube first, followed by a proportion of the pstO cells. The remainder of the pstO cells contribute to the upper cup, an ALC-derived subpopulation of prestalk cells which is located at the apex of the spore head. After prolonged slug migration, a discrete pstO zone appears not to be maintained and, at culmination, pstO cells are found scattered throughout the stalk.