tip genes act in parallel pathways of early Dictyostelium development

Analysis of Dictyostelium strains carrying null mutations in tipA showed a primary defect in cell sorting and the formation of tips on the developing mound. To study the process affected in tipA^– mutants further, other mutants with a similar phenotype were isolated and characterized. These studies showed three new Dictyostelium genes: tipB, tipC, and tipD. All the tip mutants aggregate into larger than average mounds, which split up and form many tips on their surfaces. Furthermore, each mutant exhibits reduced or aberrant cell‐sorting behavior, never makes migrating slugs, and has severely reduced fruiting body and spore production. The mRNA of each tip gene is present in vegetative cells and does not vary significantly with development. Prespore and prestalk gene expression is reduced or delayed in the tip mutants indicating cell type differentiation is dependent on the function of these genes. Developing mutant cells in chimeric mixtures with wild‐type cells demonstrated that the defects in each tip mutant behave cell autonomously. The overexpression of TipA in a tipB^– background and the overexpression of TipB in a tipA^– background significantly improved the morphogenesis of these mutants. These were the only situations in which the expression of one tip gene could compensate for the lack of a different tip gene. Except for the tipA^–/tipB^– strain, double mutations in the tip genes have additive effects, causing a more severe mutant phenotype with defects earlier in development than single mutants. The tipA^–/tipB^– double mutant does not show additive effects and is very similar to the tipA^– single mutant. Analysis of the effects of double mutations and overexpression indicates that members of this class of genes appear to act through parallel pathways of differentiation and tip formation in early Dictyostelium development. Furthermore, TipA and TipB appear to have some overlapping functions or are involved in the same pathway. The multitipped phenotype observed in all the mutants may be a general result of perturbing early developmental events such as cell type differentiation and cell type proportioning. Dev. Genet. 25:64–77, 1999. © 1999 Wiley‐Liss, Inc.     

Dictyostelium mucoroides cells exhibit cell cycle-dependent sorting behavior as observed in D. discoideum development

Evidence has been obtained indicating that the cell's position in the cell cycle at the onset of starvation is a naturally occurring variable closely involved in the subsequent sorting and pattern formation during the development of Dictyostelium discoideum Ax2. It is of interest to know whether a similar phenomenon is also noticed in species other than D. discoideum and also without any treatment of cells for cell synchronization. For this, the sorting behavior of D. mucoroides-7 ( Dm7 ) cells and its relation to the cell-cycle phase at the onset of starvation were analyzed, using non-synchronized Dm7 cells pulse-labeled with 5'-bromo-2-deoxyuridine (BrdU). The results demonstrate that Dm7 cells starved at the early G2 phase aggregate most rapidly, but are eventually sorted out to the posterior prespore zone of migrating slugs. In contrast, cells starved at the mid late G2 phase exhibited slower aggregation, but were sorted out to the anterior zone (tip), this being basically similar to the sorting behavior of D. discoideum cells. Measurements of cell numbers and nuclearity provided evidence that approximately 80% of cells progressed their cell-cycle after the formation of multicellular structures (mounds), probably coupling with prespore differentiation as in the case of D. discoideum . Thus, cell cycle-dependent sorting during Dictyostelium development is most likely to be a common phenomenon in different species.     

Zinc ions promote prestalk-to-stalk and prespore-to-stalk conversions in Dictyostelium discoideum

Abstract To clarify the mechanism of stalk cell differentiation in Dictyostelium discoideum (strain NC4), we have examined the effects of Zn²⁺ on in vitro cell differentiation of prestalk and prespore cells isolated from normally formed slugs. Prestalk cells did not differentiate into stalk cells under submerged conditions, but in the presence of the stalk-inducing factor-1 (DIF-1) at 100 nM or Zn²⁺ at 5 mM, a small number of the cells (< 15%) differentiated into stalk cells. Interestingly, Zn²⁺ in combination with DIF-1 induced the prestalk-to-stalk conversion at high efficiencies (approx. 60%). Furthermore, isolated prespore cells were also converted to stalk cells at high efficiencies (approx. 50%) in the presence of both DIF-1 and Zn²⁺, while the conversion poorly occurred in the absence of Zn²⁺. These results indicate that Zn²⁺ may mimic some cellular interaction(s) which are required for stalk cell formation in this strain.     

A gene encoding, prespore-cell-inducing factor in Dictyostelium discoideum

Two factors that exist in conditioned medium (CM) of Dictyostelium discoideum induce amoebae to differentiate into prespore cells when they are incubated at a very low cell density in submerged monolayer culture. Previously, we purified one of them, a glycoprotein factor with an apparent molecular mass of 106 kDa, and we named it psi factor (psi, prespore-inducing factor). Based on the partial amino acid sequence of the purified psi factor, we have isolated the corresponding cDNA clone, which is expressed maximally at the loose mound stage. The cDNA encodes a novel protein and the predicted molecular mass of the mature secreted protein is 60 kDa. Knockout mutant strains of the psi factor gene, psiA(-), were created by targeted integration. Although these mutant strains appear to develop normally, CM from these mutants showed reduced prespore-cell-inducing activity. Rescuing the mutant strains by expression of psi factor under control of a constitutive promoter causes overproduction of psi factor protein and CM from such cells showed a 20-fold higher level of prespore-cell-inducing activity than that from wild-type cells. Further, CM from parental cells induced prespore cell division, while that from psiA null strains showed no cell division inducing activity. Our results indicate that psi factor protein is a novel type of growth factor that does not belong to any of the families of growth factor so far identified in animals.     

Calcium levels during cell cycle correlate with cell fate of Dictyostelium discoideum

Levels of intracellular calcium, (Ca(2+))(i), from different stages of cell cycle of Dictyostelium discoideum were monitored using the fluorescent Ca(2+)-sensitive dye, Indo 1. Combinations of Ca(2+)-ionophore (A23187) and Ca(2+)-chelator (EGTA) resulted in the inhibition of progression of cell cycle. This delay was caused due to block in G(2)/M-->S phase transition of the cell cycle. Rescue of the cell cycle progression was made with 0.5 m m of exogenous Ca(2+). High (Ca(2+))(i)levels overlapped with the S-phase, of the cell cycle.Results indicate that a high (Ca(2+))(i)level during S-phase is not required for cell cycle progression but for cell-type choice mechanism at the onset of starvation, and these cells tend to follow the prestalk pathway.     

Copine A is expressed in prestalk cells and regulates slug phototaxis and thermotaxis in developing Dictyostelium

Copines are calcium-dependent membrane-binding proteins found in many eukaryotic organisms. We are studying the function of copines using the model organism, Dictyostelium discoideum. When under starvation conditions, Dictyostelium cells aggregate into mounds that become migrating slugs, which can move toward light and heat before culminating into a fruiting body. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to form fruiting bodies and instead arrest at the slug stage. In this study, we compared the slug behavior of cells lacking the cpnA gene to the slug behavior of wild-type cells. The slugs formed by cpnA- cells were much larger than wild-type slugs and exhibited no phototaxis and negative thermotaxis in the same conditions that wild-type slugs exhibited positive phototaxis and thermotaxis. Mixing as little as 5% wild-type cells with cpnA- cells rescued the phototaxis and thermotaxis defects, suggesting that CpnA plays a specific role in the regulation of the production and/or release of a signaling molecule. Reducing extracellular levels of ammonia also partially rescued the phototaxis and thermotaxis defects of cpnA- slugs, suggesting that CpnA may have a specific role in regulating ammonia signaling. Expressing the lacZ gene under the cpnA promoter in wild-type cells indicated cpnA is preferentially expressed in the prestalk cells found in the anterior part of the slug, which include the cells at the tip of the slug that regulate phototaxis, thermotaxis, and the initiation of culmination into fruiting bodies. Our results suggest that CpnA plays a role in the regulation of the signaling pathways, including ammonia signaling, necessary for sensing and/or orienting toward light and heat in the prestalk cells of the Dictyostelium slug.     

Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.     

Induction of Cell Differentiation of Isolated Cells in Dictyostelium discoideum by Low Extracellular pH

In Dictyostelium discoideum , the formation of multicellular masses is necessary for cell differentiation. However, the present study shows that amoebae of strain V12M2 efficiently differentiate to prespore or stalk cells under submerged incubation in a simple medium containing cAMP and salts without cell contact, only if the pH of the medium is maintained at acidic values; differentiation scarcely occurs in the neutral pH range. The optimum pH values for prespore and stalk cell differentiation are 5.1 and 4.5, respectively. In addition to the extracellular pH, Mg ions and the concentration of cAMP also affect the choice of the differentiation pathway. The time courses of differentiation of both cell types under optimum conditions are also presented.     

Dictyopyrones, novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell differentiation in Dictyostelium discoideum

Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 microM promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 microM suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 microM dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 microM slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.     

盘基网柄菌细胞分化及细胞比例的调控

本文综述了盘基网柄菌(Dictyosteliumdiscoideum)发育过程中调控细胞分化及细胞比例的一些信号分子,包括分化诱导因子(DIF-1、SDF-2)、糖原合成酶激酶(GSK-3)、环状亮氨酸拉链蛋白(rZIP)等,介绍了这些信号分子的功能及其作用机制。     

Cell-cycle progression during the development of Dictyostelium discoideum and its relation to the subsequent cell-sorting in the multicellular structures

Previous studies have shown that the cell-cycle phase at the onset of starvation is a naturally occurring variable that is closely involved in the subsequent sorting and differentiation of cells during Dictyostelium development. Here the cell-cycle progression during the development of D. discoideum Ax-2 cells and its relation to the subsequent cell-sorting were analyzed in detail using synchronized cells and their pulse-labeling by 5'-bromodeoxyuridine (BrdU). Measurements of cell number and nuclearity provided evidence that about 80% of cells progressed their cell-cycle after formation of multicellular structures (mounds). Many cells (T7 cells) starved at mid–late G2-phase (just before the PS-point from which cells initiate development when starved) progressed to the cell-cycle after mound formation. In contrast, a less amount of cells (T1 cells) starved at late G2-phase (just after the PS-point) progressed through the cell-cycle after mound formation. The significance of cell-cycle progression presented here is discussed, with reference to cell differentiation and pattern formation.     

盘基网柄菌中分化诱导因子信号与细胞命运决定

对盘基网柄菌发育过程中分化诱导因子(DIF)的作用及其机制进行了综述,包括DIF对盘基网柄菌前柄细胞、柄细胞分化的作用以及DIF的生物合成、DIF的诱导、降解失活、DIF对细胞命运和细胞比例的调节及其作用机制等。     

Membrane sorting in the endocytic and phagocytic pathway of Dictyostelium discoideum

To study sorting in the endocytic pathway of a phagocytic and macropinocytic cell, monoclonal antibodies to membrane proteins of Dictyostelium discoideum were generated. Whereas the p25 protein was localized to the cell surface, p80 was mostly present in intracellular endocytic compartments as observed by immunofluorescence as well as immunoelectron microscopy analysis. The p80 gene was identified and encodes a membrane protein presumably involved in copper transport. Expression of chimeric proteins revealed that the cytoplasmic domain of p80 was sufficient to cause constitutive endocytosis and localization of the protein to endocytic compartments. Dileucine- and tyrosine-based endocytic signals described previously in mammalian systems were also capable of targeting chimera to endocytic compartments. In phagocytosing cells no membrane sorting was observed during formation of the phagosome. Both p25 and p80 were incorporated non-selectively in nascent phagosomes, and then retrieved shortly after phagosome closure. Our results emphasize the fact that very active membrane traffic takes place in phagocytic and macropinocytic cells. This is coupled with precise membrane sorting to maintain the specific composition of endocytic compartments.     

Expression pattern of 5''-nucleotidase in Dictyostelium.

In order to analyze the expression pattern of the 5′-nucleotidase (5nt) gene in Dictyostelium, we made a fusion construct in which the 5nt promoter directed the expression of β-galactosidase gene. The reporter gene was not active in vegetative amoebae but was expressed during the aggregation stage. At the slug stage, 5nt was highly expressed in pstAB cells. As the slug moved along the substratum, high activity of β-galactosidase was detected in cells that were left behind in the slime trail. In the completed fruiting body, 5nt was expressed in the lower cup, the anterior like cells (ALC) and the basal disc.     

Control of spatial patterning and cell-type proportioning in Dictyostelium

The spatial patterning of prestalk and prespore cells in the slug arises from the differential sorting of newly differentiated cell types as the mound forms. This pattern is highly organized along an anterior-posterior axis and is constant irrespective of the size of the organism. Cell-type differentiation is plastic until late in development. A change in the ratio of cell types resulting from removal of part of the slug leads to a rapid restoration of the original ratio of the cell types through a pathway involving dedifferentiation, redifferentiation, and sorting of the existing cells. This review provides insight into various molecules, morphogens, and pathways regulating spatial patterning and cell-type proportioning.     

The Regulation of Dictyostelium Development by Transmembrane Signalling

ABSTRACT. Dictyostelium discoideum has a well characterized life cycle where unicellular growth and multicellular development are separated events. Development is dependent upon signal transduction mediated by cell surface, cAMP receptor/G protein linkages. Secreted cAMP acts extracellularly as a primary signal and chemoattractant. There are 4 genes for the distinct cAMP receptor subtypes, CAR1, CAR2, CAR3 and CAR4. These subtypes are expressed with temporally and spatially specific patterns and cells carrying null mutations for each gene have distinct developmental phenotypes. These results indicate an essential role for cAMP signalling throughout Dictyostelium development to regulate such diverse pathways as cell motility, aggregation (multicellularity), cytodifferentiation, pattern formation and cell type-specific gene expression.     

The role of the cell cycle in differentiation of the cellular slime mould Dictyostelium discoideum

Summary During development and differentiation of the cellular slime mould Dictyostelium discoideum there appears to be a relationship between the cell cycle and cell fate: amoebae halted in G2 phase during early development differentiate into spores whereas stalk cells are formed from amoebae halted in GI phase. It is proposed that this is because a major effect of the cell cycle is to generate heterogeneity in the cell surface properties of the developing amoebae.     

Coh A,a mutation affecting the post aggregative related (par) cohesive system of Dictyostelium discoideum

Three stage-specific cohesive systems operate in D. discoideum: VEG, elaborated by vegetative cells: AR, by aggregation competent cells; and PAR, by post aggregation stage cells. Previous study of a mutant strain JC-5 had shown the stability of its PAR system (but not the AR) to be temperature sensitive. However, the phenotypic expression of this mutation termed Coh A is complicated by the presence in that strain of a preexisting mutant gene Rde A, which accelerates developmental events generally and alters the pattern of morphogenesis. Genetic evidence presented here indicates that the two mutations have been separated by parasexual recombination yielding a Coh A, Rde A⁺ segregant class of which strain JC-36 is a prototype. At the permissive temperature, JC-36 follows a morphogenetic sequence like that of the wild type in respect to timing, morphogenetic pattern, and spore appearance. At the restrictive temperature, it forms normal aggregates at the usual time but exhibits two morphogenetic aberrancies during post aggregative development. First, fruit construction is arrested at a stage approximating the 16 hr “Bottle” stage of the wild type, though more squat and blunt tipped, and then the aggregate regresses. Cytodifferentiation into spores and stalk cells is also blocked. Second, a shift of slugs migrating normally at the permissive temperature to the restrictive causes the latter to disintegrate progressively as they leave clumps of cells behind them within the flattened sheath. JC-36 cells developing at the restrictive temperature also exhibited a decrease in EDTA resistant cohesivity attributable on two grounds to the sensitivity of the PAR system. In addition, the disappearance of the AR system completed in the wild type by the Mexicanhat (18–19 hr) stage is indefinitely arrested at an intermediate level in JC-36.