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Dictyostelium morphogenesis Cornelis J Weijer 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. the soil leaf litter where they feed on bacteria and divide by binary fission. Under starvation conditions up to several hundreds of thousands cells aggregate chemotactically to form a multicellular structure (the slug) that, directed by light and temperature gradients, migrates to the soil surface to form a fruiting body. The fruiting body is composed of a stalk supporting a mass of spores. The spores disperse and under suitable conditions germinate to release amoeba, closing the life cycle. I review our understanding of the signalling mechanisms coordinating cellular behaviours responsible for pattern formation and morphogenesis. Addresses Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dundee DD1 5EH, UK e-mail: [email protected] The control of cell movement during development Current Opinion in Genetics & Development 2004, 14:392–398 This review comes from a themed issue on Pattern formation and developmental mechanisms Edited by Derek Stemple and Jean-Paul Vincent 0959-437X/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2004.06.006 Abbreviations ACA aggregation stage adenylylcyclase Dif differentiation inducing factor PIP3 Phosphatidyl-Inositol(3,4,5)-Phosphate PkA cAMP dependent protein kinase RGS regulator of G protein signalling Introduction One of the central aims of the study of development is to understand how distinct cellular behaviours (e.g. division, differentiation, apoptosis and movement) are coordinated in space and in time to result in reproducible pattern formation and morphogenesis. Coordination of these cellular behaviours requires extensive communication between cells of different types and cells and their environment. The social amoebae Dictyostelium discoideum, a simple genetically tractable organism situated at the threshold of single and multicellular organisms in the evolutionary tree of life, is well suited for the study of these interactions because its genome has been sequenced and it is amenable to experimental manipulation through targeted gene disruption and replacement [1]. Dictyostelium cells normally live as single cells in Current Opinion in Genetics & Development 2004, 14:392–398 As Dictyostelium development occurs under starvation conditions, only limited cell divisions occur during multicellular development. Morphogenesis primarily results from the arrangement of differentiating cells in a regulative spatial pattern. Key questions are: which signals guide the movement behaviour of thousands of cells during development, and how do movement and differentiation interact? Aggregation Starvation induces changes in the gene-expression programme that result in the cells acquiring the ability to produce, secrete and degrade cAMP [2,3]. Through the expression of the cAMP receptor, the cells also acquire the ability to respond chemotactically to cAMP gradients. It has emerged that chemotaxis results from the polarisation of the cytoskeletal dynamics persistently along the cAMP gradient. Unstimulated amoeboid cells are changing shape continuously by extension and retraction of pseudodpods in all directions resulting in a random walk [4]. In the presence of an external gradient of a chemoattractant such as cAMP, the cells persistently extend successive pseudopods in the direction of rising cAMP concentration while suppressing the extension of lateral pseudopods, which results in an efficient movement up the gradient. Much current research is directed towards understanding how cells detect cAMP gradients, polarise and move in response to it. Pseudopod extension is driven by actin polymerisation, which provides the driving force for extension and simultaneous disassembly of the myosin thick filaments in the cortex at the site of extension as well as localised delivery of membrane and or proteins to allow extension to occur [5,6,7]. Cells also need to pull up their back end and suppress the extension of lateral pseudopods which involves members of the myosin I www.sciencedirect.com Dictyostelium morphogenesis Weijer 393 family and is dependent on internal cAMP levels [8,9]. To move forward the cells must gain traction from the substrate on which they are moving, which involves the formation of multiple transient (10–20s) actin contact sites that have been shown to transduce traction forces to the substrate [10,11]. It appears that cells may undergo alternating phases of actin-driven extension at the front and myosin-driven contraction at the back [12]. Much work is directed towards the investigation of the molecular mechanism resulting in the molecular mechanism underlying signal detection and its translation in directed movement, and has been reviewed recently elsewhere [6,13,14]. Aggregation is caused by periodic cAMP synthesis and secretion by cells in an aggregation centre [15]. Detection and amplification of this signal by surrounding cells coupled with desensitisation of the cAMP-producing cells results in the propagation of waves of cAMP from the aggregation centre outward (Figure 1). These waves of cAMP guide the chemotactically moving cells towards the aggregation centre, where they accumulate into a threedimensional aggregate: the mound [16,17]. Initially, the cells move towards the aggregation centre as individuals, but after 10–20 waves have passed they form bifurcating aggregation streams, in which the cells make head-to-tail contacts via calcium-independent adhesion molecules, contact site A and side-to-side contacts via a calciumdependent contact molecule (Dd cadherin) [18,19]. Stream formation is dependent on the localisation of aggregation stage adenylylcyclase (ACA) in the rear of the aggregating cells, resulting in polarised cAMP secretion from the back of the cells [20]. cAMP wave propagation can be observed indirectly at the population level via the observation of propagating optical density waves that are associated with the periodic surges in cell movement of groups of cells in the direction of the cAMP signal during the rising phase of the cAMP wave, or at the individual cell level by following the localised translocation of PIP3 at the leading edge of the cell [21]. During aggregation the cAMP signals propagate as target patterns or spiral waves from the aggregation centre outward. Cells stay polarised as long as the cAMP signal is rising in time. However, upon passage of the wave, the chemotactic response adapts, which prevents the cells from turning Figure 1 (a) (b) (c) (d) Current Opinion in Genetics & Development Dictyostelium development is controlled by propagating cAMP waves. These waves possess distinct geometries typical for the different stages of development. The cAMP waves are detected indirectly as optical density waves associated with the chemotactic movement of the cells in response to the cAMP waves. Groups of cells moving directly during the rising phase of the waves scatter more light than non-moving, less polarised cells during the falling phase of the waves. (a) Spiral waves typical for the early aggregation phase, when the cells are still in a monolayer on agar. (b) Waves in a streaming aggregate. In the body of the aggregate, multi-armed spiral waves rotates counter clockwise, throwing off individual wavefronts that propagate from the centre outward to the periphery of the aggregate directing inward movement of the cells. (c) Multi-armed waves in a mound. In this image, the waves rotate counter clockwise, directing the clockwise movement of the cells in the mound. (d) A slug migrating from right to left, showing two optical density waves, indicated by arrows, that travel from left to right. These waves direct the migration of the prespore cells in the direction of the tip from right to left. www.sciencedirect.com Current Opinion in Genetics & Development 2004, 14:392–398 394 Pattern formation and developmental mechanisms around and chasing after the cAMP waves once they have passed. Adaptation of the chemotactic pathways, involving depolarisation of the cells may involve activation of a newly discovered RGS-domain containing kinase as well as the activation of PkA [9,22]. The number of cells in aggregation streams appears to be controlled by the local concentration of a secreted extracellular high molecular weight complex protein complex, counting factor, which through modulation of movement and adhesion may control the numbers of cells that stably migrate in an aggregation stream [23,24]. Mound and slug formation After the cells have aggregated, they form the hemispherical mound. Mounds are characterised by rotating waves of cAMP that direct the counter-rotational periodic movement of the cells. Cells start to differentiate into prespore and prestalk cells during aggregation, on the basis of physiological biases like nutritional state and cellcycle position at the time of starvation already present in the population before aggregation [25,26]. As a result, there is little correlation between the time of arrival in the mound and differentiation fate and the emerging cell types initially form a ‘salt and pepper’ pattern in the mound. Some prestalk cells then sort out to form the tip and the slug tip guides the movement of all other cells thus acting as an organiser. The tip’s action as an organiser can be mimicked by the periodic injection of cAMP pulses of the right frequency and duration [17], suggesting that the tip is a source of periodic cAMP waves, in agreement with the fact that prestalk cells express ACA and the extracellular cAMP phosphodiesterase pdeA [27,28]. It is not known which signals control tip-cell fate but it is becoming clear that to proceed from the aggregate to the mound stage cell–cell adhesion and/or contact start to play an important role. Mutants defective in the putative single-pass transmembrane contact molecules lagC, lagD and cotC cannot proceed beyond the aggregation stage and are defective in tip formation [29]. Surprisingly, mutants in some of the newly discovered nine pass transmembrane receptors of the Phag family, shown to be essential for phagocytosis, display severe defects in slug formation and culmination, suggesting a possible role for these receptors in cell–cell adhesion [30]. Sorting of prestalk cells towards the tip requires the invasive movement of prestalk cells through a tightly packed mass of other cells and most likely involves differential cell adhesion. Sorting of prestalk cells is facilitated by secretion of a disintegrin family protein and deletion of this protein results in a delayed sorting and defective cell-type specification, demonstrating that sorting and cell-type specification are tightly interlinked [31]. In slugs, optical density waves can be seen to propagate from the middle of the prestalk zone to the back, reflectCurrent Opinion in Genetics & Development 2004, 14:392–398 ing the periodic movement of the cells forward (Figure 1). These optical waves are strictly dependent on the tip. Cells in the tip often rotate perpendicularly to the direction of slug migration, especially when it is lifted from the substrate. In the posterior part of the slug, the cells move forward periodically and all cells move on average with the speed of the whole slug. It has been shown that the assumptions of cAMP wave propagation and chemotaxis in response to these waves is, in principle, sufficient to explain morphogenesis from single-cell via aggregation, stream and mound formation to cell sorting and slug formation. The interactions between cell-signalling and cell movement can be described in a robust way by relatively simple mathematical models. In one of these models the cells are described as a viscous fluid that moves in response to the cAMP waves generated in an excitable manner by the cells. This model is able to explain the formation of aggregation streams, resulting in the formation of a hemispherical mound. With the additional assumptions that the prestalk and prespore cells that differentiate in the mound in a salt and pepper pattern differ in two properties, their ability to relay the cAMP signal and their ability to move chemotactically in response to the cAMP signal it is possible to obtain cell sorting, where the prestalk cells move to the top of the mound form a slug, which falls over and migrates away (Figure 2). It would appear that these processes are sufficient to explain Dictyostelium morphogenesis up to the culmination stage [32,33]. However, the situation is almost certainly more complex because strains lacking the ACA can still form slugs when they overexpress the catalytic subunit of protein kinase A. The suggestion is that either there exists an ACA-independent mechanism to produce periodic cAMP signals (e.g. involving cAMP generation by other adenylylcyclases ACB and/or ACG and the recently discovered cAMP-stimulated cAMP phosphodiesterase [34]) or that there exists altogether different mechanisms that can control cell movement, such as contact following [35]. The latter mechanism does not, however, explain what directs the movement of the cells in the tip. It is possible that the slime sheath secreted by prestalk cells [36], which surrounds the slug as a ‘stocking’, may keep the cells together and may also give some polarity to the slug but its role, although presumably important, has not yet been investigated in detail. Differentiation A major goal is to understand the relationship between cell movement and the signals that control differentiation. These signals must be able to maintain the correct proportion of the prespore and prestalk celltypes in an environment of extensive cell movement and changes in shape of the slug. In the slug, the different cell types are arranged in a simple axial pattern: pstA (prestalk A) cells in the tip, a band of pstO cells, prespore cells with intermingled anterior-like cells and rearguard cells that www.sciencedirect.com Dictyostelium morphogenesis Weijer 395 Figure 2 t=15min t=0min t=40min t=200min t=30min t=0min t=60min t=40min t=250min t=80min t=80min Current Opinion in Genetics & Development Model calculations of wave propagation and cell movement from aggregation to slug migration using a hydrodynamic model in which the cells are described as a viscous fluid that move in response to propagating cAMP waves generated in an excitable manner by the cells [32]. The top row depicts the aggregation up to the mound stage. The first image starts with the randomly distributed cells (yellow) that are organised by a spiral wave of cAMP (purple). They form aggregation streams and finally a hemispherical mound [45]. The middle row shows cell sorting and the formation of a slug. The mound consist of two cells types: 20% yellow prestalk cells and 80% green prespore cells. They are initially randomly mixed. The cAMP waves (purple) organise the movement of the cells. The prestalk cells are more excitable and move more strongly in response to a cAMP wave. They move towards the centre of the mound and up to form the tip. The separation of the cells feeds back on the signal propagation resulting in the formation of a twisted scroll wave. This leads to an intercalation of the cells and an upward extension of the slug [46]. The bottom row shows that a slug organised by a scroll wave can move [32]. are precursors of the basal disk in the back of the slug [37]. It seems evident that this requires adaptive signalling dynamics but the signals and the details of their regulation are not understood in detail. cAMP pulses control the expression of aggregation-stage genes necessary for cAMP relay and cell–cell contact and can control prespore cell specific gene expression in later development [2,3]. Prespore cells, in turn, produce DIF, which controls the differentiation of pstO cells [37,38,39]. DIF spreads by simple diffusion from the prespore zone in adjacent regions where it controls the differentiation of prestalk O cells and possibly rear guard cells, in agreement with the fact that Dif-dependent pstO translocation of StatC is seen in both zones [40]. Cells in the pstA zone express www.sciencedirect.com ACA and studies investigating the cAR1-dependent nuclear translocation of the transcription factor statA have shown that cAM P levels are high in the tip, whereas cAMP levels are lower elsewhere in the slug [28,41], compatible with the idea that all the prestalk cells in the tip and only the anterior-like cells in the posterior part of the slug relay the cAMP signal. It is not known what maintains the expression of ACA in the cells in the tip and in anterior-like cells, but it could involve signalling through newly discovered orphan serpentine receptors, as deletion of these receptors results in defective tip formation [42]. The mechanisms of cell-type proportioning and detailed signal-transduction pathways to cell-type specific gene expression still need to be resolved. Current Opinion in Genetics & Development 2004, 14:392–398 396 Pattern formation and developmental mechanisms The switch from migrating slugs to fruiting body formation (culmination) appears to be controlled by a fall in ammonia concentration. The identification of several ammonia transporters, some of which are expressed in the very tip and when deleted lead to a slugger (slugs that fail to culminate) phenotype, supports the importance of ammonia as a morphogen [43]. Ammonia signals most likely through the histidine kinase DhkC to the response regulator domain of the internal cAMP phosphodiesterase RegA, which is a major determinant in the control of intracellular cAMP levels [3,44]. High ammonia is expected to result in activation of regA and low internal cAMP levels. A drop in ammonia is expected to result in a rise of intracellular cAMP and stalk-cell differentiation. The authors describe the premier site for Dictyostelium resources that includes genome analysis and stock centre information. 2. Iranfar N, Fuller D, Loomis WF: Genome-wide expression analyses of gene regulation during early development of Dictyostelium discoideum. Eukaryot Cell 2003, 2:664-670. A comprehensive analysis of expression of 3000 unique ESTs (expressed sequence tags) during development, showing the specific developmental regulation of 172 genes. 125 genes were expressed in response to extracellular pulses of cAMP. Three genes, carA (cAMP receptor), gbaB (G protein Beta), and pdsA (secreted phosphodiesterase), were consistently expressed early in development in the absence of cAMP pulses, they are necessary to get the cAMP oscillations going. PKA was demonstrated to have a critical role in transducing the cAMP signal to early gene expression. In the absence of constitutive PKA activity, expression of later genes was strictly dependent on ACA in pulsed cells. 3. Saran S, Meima ME, Alvarez-Curto E, Weening KE, Rozen DE, Schaap P: cAMP signaling in Dictyostelium. Complexity of cAMP synthesis, degradation and detection. J Muscle Res Cell Motil 2002, 23:793-802. 4. Soll DR, Wessels D, Heid PJ, Voss E: Computer-assisted reconstruction and motion analysis of the three-dimensional cell. Scientificworldjournal 2003, 3:827-841. Conclusions Multicellular Dictyostelium morphogenesis results from the chemotactic movement of thousands of differentiating cells coordinated by propagating waves of the chemoattractant cAMP produced by these cells. The dynamical interactions between the cAMP waves and the resulting chemotactic movement of the cells is formally sufficient to explain aggregation, stream and mound formation. Slug formation and culmination require the emergence of prespore and several prestalk cell types that show distinct cAMP signalling and movement properties. Only prestalk and anterior-like cells relay the cAMP signals while both prestalk and prespore cells move chemotactically in response to these signals. However, prestalk cells move more effectively than prespore cells, resulting in cell sorting. Many of the molecular details underlying the polarisation response to cAMP gradients and the mechanisms of actual movement, cell–cell contact and differentiation are beginning to be uncovered. It would appear that direct cell–cell contact mediated signalling will play an important role in the modulation of these processes, as is the case in higher organisms. Dictyostelium will thus, besides being a system of choice to investigate the molecular mechanisms underlying cell polarity and chemotaxis, be an excellent model system to investigate the principles underlying multicellular tissue organisation and cell-type proportioning. Acknowledgements We apologise to those whose work was not cited due to space constraints. I want to thank Dirk Dormann and Bakhtier Vasiev for their discussions. This work was supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Kreppel L, Fey P, Gaudet P, Just E, Kibbe WA, Chisholm RL, Kimmel AR: dictyBase: a new Dictyostelium discoideum genome database. Nucleic Acids Res 2004, 32(Suppl):D332-D333. Current Opinion in Genetics & Development 2004, 14:392–398 5. Thompson CR, Bretscher MS: Cell polarity and locomotion, as well as endocytosis, depend on NSF. Development 2002, 129:4185-4192. A temperature-sensitive mutant in NSF (NEM sensitive factor), an essential component of membrane transport, shows that NSF is essential for phagocytosis, membrane internalisation and cell movement. The cAMPinduced actin polymerisation (cringe response) is intact in the mutant at the restrictive temperature, but the cells round up and stop translocation, possibly due a defect in generating cell polarity. 6. Merlot S, Firtel RA: Leading the way: directional sensing through phosphatidylinositol 3-kinase and other signaling pathways. J Cell Sci 2003, 116:3471-3478. 7. Blagg SL, Stewart M, Sambles C, Insall RH: PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium. Curr Biol 2003, 13:1480-1487. This is the first piece of genetic evidence showing that PIR121 is involved in the negative regulation of SCAR, which controls the activity of the Arp2/3 complex and actin polymerisation. Pir121 null mutants show excessive actin polymerisation and continuous splitting of pseudopds implying important roles for SCAR in the control of actin polymerisation during psuedopod formation. 8. Falk DL, Wessels D, Jenkins L, Pham T, Kuhl S, Titus MA, Soll DR: Shared, unique and redundant functions of three members of the class I myosins (MyoA, MyoB and MyoF) in motility and chemotaxis in Dictyostelium. J Cell Sci 2003, 116:3985-3999. This study shows that members of the myosin I class of motor proteins are necessary for suppression of lateral psuedopods and efficient chemotaxis during aggregation in natural waves of cAMP. MyoF in particular is necessary for attaining an elongated shape during chemotaxis. 9. Zhang H, Heid PJ, Wessels D, Daniels KJ, Pham T, Loomis WF, Soll DR: Constitutively active protein kinase A disrupts motility and chemotaxis in Dictyostelium discoideum. Eukaryot Cell 2003, 2:62-75. Cells expressing a constitutively active PkA are incapable of suppressing lateral pseudopods and maintain an ovoid shape, resulting in inefficient movement. They are incapable of moving in response to natural waves of cAMP implying PkA activation in dismantling cell polaritity at the peak of the wave. 10. Uchida KS, Yumura S: Dynamics of novel feet of Dictyostelium cells during migration. J Cell Sci 2004, 117:1443-1455. The authors show that cells periodically form small, localised and close contacts with the substratum and that these contact points are rich in filamentous actin. By correlating these actin-rich contact points with the local deformations of an elastic substrate on which these cells move, it is shown that these contact points are the places where the cell transmits traction forces necessary for movement to the substrate. 11. Bretschneider T, Diez S, Anderson K, Heuser J, Clarke M, Muller-Taubenberger A, Kohler J, Gerisch G: Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells. Curr Biol 2004, 14:1-10. Using TIRF (total internal reflection fluorescence) microscopy the authors show that cells make short lived (7–10sec) Arp2/3-rich foci in a myosin-IIindependent fashion. These high-resolution experiments furthermore www.sciencedirect.com Dictyostelium morphogenesis Weijer 397 show that the Arp2/3/foci often induce waves of actin polymerisation propagating through the cell in a wave-like fashion, showing that actin polymerisation machinery operates far from equilibrium. The Arp2/3-rich foci are observed not only in the leading edge but also in the middle of the cell, implying that the actin cytoskeleton is remodelled continuously everywhere in the cell. It appears likely that these foci are the same as the actin-rich contact sites shown in [10] to coincide with the points of transmission of traction forces to the substrate. 12. Uchida KS, Kitanishi-Yumura T, Yumura S: Myosin II contributes to the posterior contraction and the anterior extension during the retraction phase in migrating Dictyostelium cells. J Cell Sci 2003, 116:51-60. This paper shows conclusively that cells undergo alternating cycles of extension and contraction and it correlates these behaviours with the generation of traction forces showing that both extension and contraction at the rear contribute to forward movement. 13. Devreotes P, Janetopoulos C: Eukaryotic chemotaxis: distinctions between directional sensing and polarization. J Biol Chem 2003, 278:20445-20448. A clear review describing rivalling hypotheses for cell polarisation. 14. Postma M, Bosgraaf L, Loovers HM, Van Haastert PJ: Chemotaxis: signalling modules join hands at front and tail. EMBO Rep 2004, 5:35-40. A review of current molecular insight in the mechanisms underlying coordination of events at the leading and trailing edges of the cell during chemotactic movement. 15. Kimmel AR, Parent CA: The signal to move: D. discoideum go orienteering. Science 2003, 300:1525-1527. 16. Dormann D, Weijer CJ: Chemotactic cell movement during development. Curr Opin Genet Dev 2003, 13:358-364. 17. Dormann D, Weijer CJ: Propagating chemoattractant waves coordinate periodic cell movement in Dictyostelium slugs. Development 2001, 128:4535-4543. 18. Harris TJ, Ravandi A, Awrey DE, Siu CH: Cytoskeleton interactions involved in the assembly and function of glycoprotein-80 adhesion complexes in dictyostelium. J Biol Chem 2003, 278:2614-2623. 19. Wong E, Yang C, Wang J, Fuller D, Loomis WF, Siu CH: Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to aberrant cell sorting and cell-type proportioning during Dictyostelium development. Development 2002, 129:3839-3850. The cadA gene encodes the Ca2þ-dependent cell adhesion molecule DdCAD-1, one of the major calcium-dependent adhesion molecules. It is expressed soon after the initiation of development in Dictyostelium. Cells that lack DdCAD-1 are able to complete development and form fruiting bodies, but they make abnormal slugs, culmination is delayed by 6 hours, and the yield of spores is reduced by 50%. The proportion of prestalk cells in cadA slugs showed a 2.5-fold increase over the parental strain. This indicates that, in addition to cell–cell adhesion, DdCAD-1 plays a role in cell-type proportioning and pattern formation. 20. Kriebel PW, Barr VA, Parent CA: Adenylyl cyclase localization regulates streaming during chemotaxis. Cell 2003, 112:549-560. Kriebel et al. show that Dictyostelium cells, like neutrophils, polarise in response to uniform stimulation of cAMP, which is normally produced by ACA. Furthermore, it shows that the posterior localisation of ACA in aggregating cells is necessary for efficient stream formation most likely because it leads to localised end-to-end signalling between cells in aggregation streams. 21. Dormann D, Weijer G, Parent CA, Devreotes PN, Weijer CJ: Visualizing PI3 kinase-mediated cell-cell signaling during Dictyostelium development. Curr Biol 2002, 12:1178-1188. In this paper, we show that cells in aggregation streams and mounds show periodic PIP3 production at the leading edge and that this correlates with a surge in forward movement. Cells in slugs, however, appear to be polarised permanently in the direction of slug migration and this polarisation depends on close range cell–cell signalling or direct cell– cell contact 22. Sun B, Firtel RA: A regulator of G protein signaling-containing kinase is important for chemotaxis and multicellular development in dictyostelium. Mol Biol Cell 2003, 14:1727-1743. 23. Tang L, Gao T, McCollum C, Jang W, Vicker MG, Ammann RR, Gomer RH: A cell number-counting factor regulates the www.sciencedirect.com cytoskeleton and cell motility in Dictyostelium. Proc Natl Acad Sci USA 2002, 99:1371-1376. 24. Brock DA, Ehrenman K, Ammann R, Tang Y, Gomer RH: Two components of a secreted cell number-counting factor bind to cells and have opposing effects on cAMP signal transduction in Dictyostelium. J Biol Chem 2003, 278:52262-52272. 25. Huang HJ, Pears C: Cell cycle-dependent regulation of early developmental genes. Biochim Biophys Acta 1999, 1452:296-302. 26. Araki T, Abe T, Williams JG, Maeda Y: Symmetry breaking in Dictyostelium morphogenesis: Evidence that a combination of cell cycle stage and positional information dictates cell fate. Dev Biol 1997, 192:645-648. 27. Weening KE, Wijk IV, Thompson CR, Kessin RH, Podgorski GJ, Schaap P: Contrasting activities of the aggregative and late PDSA promoters in Dictyostelium development. Dev Biol 2003, 255:373-382. 28. Verkerke-van Wijk I, Fukuzawa M, Devreotes PN, Schaap P: Adenylyl cyclase A expression is tip-specific in Dictyostelium slugs and directs StatA nuclear translocation and CudA gene expression. Dev Biol 2001, 234:151-160. 29. Kibler K, Svetz J, Nguyen TL, Shaw C, Shaulsky G: A cell-adhesion pathway regulates intercellular communication during Dictyostelium development. Dev Biol 2003, 264:506-521. The authors show that three single-pass transmembrane putative calcium-independent adhesion molecules comC, LagC and lagD are essential for the transition of development from the late aggregate to the mound stage. Evidence is presented that these genes fall into a regulatory network in which comC inhibits lagC and activates lagD expression, lagC and lagD are mutually inductive, and the cell adhesion gene lagC is the terminal node in this signaling network. 30. Benghezal M, Cornillon S, Gebbie L, Alibaud L, Bruckert F, Letourneur F, Cosson P: Synergistic control of cellular adhesion by transmembrane 9 proteins. Mol Biol Cell 2003, 14:2890-2899. 31. Varney TR, Ho H, Petty C, Blumberg DD: A novel disintegrin domain protein affects early cell type specification and pattern formation in Dictyostelium. Development 2002, 129:2381-2389. 32. Vasiev B, Weijer CJ: Modelling of Dictyostelium discoideum slug migration. J Theor Biol 2003, 223:347-359. We have shown through the development of a physico-chemical model that cAMP wave propagation by prestalk and anterior-like cells and chemotactic movement of both prestalk and prespore cells are sufficient to explain slug formation and migration. In this model, the prestalk and prespore tissues are described as two distinct incompressible viscous fluids, that move in response to cAMP waves, produced and propagated by the prestalk cells. By varying the dynamics of cAMP signalling, the chemotactic responsiveness of the prestalk and prespore cells and the cell–cell interactions as described by changes in tissue viscosity, it is possible to analyse their influence on slug migration and shape. 33. Umeda T, Inouye K: Cell sorting by differential cell motility: a model for pattern formation in Dictyostelium. J Theor Biol 2004, 226:215-224. See annotation [32]. 34. Meima ME, Weening KE, Schaap P: Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum. J Biol Chem 2003, 278:14356-14362. 35. Umeda T, Inouye K: Possible role of contact following in the generation of coherent motion of Dictyostelium cells. J Theor Biol 2002, 219:301-308. 36. Morrison A, Blanton RL, Grimson M, Fuchs M, Williams K, Williams J: Disruption of the gene encoding the EcmA, extracellular matrix protein of Dictyostelium alters slug morphology. Dev Biol 1994, 163:457-466. 37. Maeda M, Sakamoto H, Iranfar N, Fuller D, Maruo T, Ogihara S, Morio T, Urushihara H, Tanaka Y, Loomis WF: Changing patterns of gene expression in dictyostelium prestalk cell subtypes recognized by in situ hybridization with genes from microarray analyses. Eukaryot Cell 2003, 2:627-637. 38. Thompson CR, Fu Q, Buhay C, Kay RR, Shaulsky G: A bZIP/bRLZ transcription factor required for DIF signaling in Dictyostelium. Development 2004, 131:513-523. Current Opinion in Genetics & Development 2004, 14:392–398 398 Pattern formation and developmental mechanisms A bZIP/bRLZ transcription factor is here identified as a central regulator of all known Dif responses and the DimA null mutant phenocopies the DmtA, Dif null mutant, in that it does not make PstO cells. 39. Kay RR, Thompson CR: Cross-induction of cell types in Dictyostelium: evidence that DIF-1 is made by prespore cells. Development 2001, 128:4959-4966. 40. Fukuzawa M, Abe T, Williams JG: The Dictyostelium prestalk cell inducer DIF regulates nuclear accumulation of a STAT protein by controlling its rate of export from the nucleus. Development 2003, 130:797-804. Shows that Dif directs tyrosine phosphorylation and nuclear accumulation of StatC (signal transducer and activator of transcription). The nuclear accumulation of statC is controlled by the inhibition of StatC nuclear export, as shown via cytoplasmic photobleaching of StatC–GFP in living cells. The paper furthermore shows that StatC functions as a transcriptional activator in a stress-response pathway. 41. Dormann D, Abe T, Weijer CJ, Williams J: Inducible nuclear translocation of a STAT protein in Dictyostelium prespore cells: implications for morphogenesis and cell-type regulation. Development 2001, 128:1081-1088. We show by monitoring the nuclear translocation of statA, which is dependent on the level of extracellular cAMP, that slug tips are regions of high extracellular cAMP. The absence of nuclear translocation of statA in prespore cells is the result of ambient levels of extracellular cAMP in the prespore zone being too low to cause nuclear translocation of StatA. An increase in extracellular cAMP concentration by injection of cAMP in the extracellular space in the prespore region of the slug results in rapid translocation of StatA in prespore cells. These findings are in agreement with findings described in [28], where extracellular cAMP was manipulated by ectopic expression of the aggregation stage Current Opinion in Genetics & Development 2004, 14:392–398 adenylylcyclase (ACA) in prespore cells, resulting in nuclear translocation of statA in prespore cells. These results are compatible with the idea that prestalk and anterior-like cells relay the cAMP signal and prespore cells do not. 42. Raisley B, Zhang M, Hereld D, Hadwiger JA: A cAMP receptor-like G protein-coupled receptor with roles in growth regulation and development. Dev Biol 2004, 265:433-445. 43. Follstaedt SC, Kirsten JH, Singleton CK: Temporal and spatial expression of ammonium transporter genes during growth and development of Dictyostelium discoideum. Differentiation 2003, 71:557-566. Three ammonium transporters (amtA–C) are identified in Dictyostelium, which may play a role in ammonia signaling, in addition to physically altering the levels of ammonia within cells. AmtC is expressed in the most anterior tip of fingers and slugs, corresponding to cells that mediate ammonia’s effect on the choice between slug migration and culmination. Indeed, amtC null cells have a slugger phenotype suggesting that it may play a role in this choice. 44. Singleton CK, Zinda MJ, Mykytka B, Yang P: The histidine kinase dhkC regulates the choice between migrating slugs and terminal differentiation in Dictyostelium discoideum. Dev Biol 1998, 203:345-357. 45. Vasiev B, Siegert F, Weijer CJ: A hydrodynamic model for Dictyostelium discoideum mound formation. J Theor Biol 1997, 184:441. 46. Vasiev B, Weijer CJ: Modeling chemotactic cell sorting during Dictyostelium discoideum mound formation. Biophys J 1999, 76:595-605. www.sciencedirect.com