Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate

There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.     

Overexpression of an activated rasG gene during growth blocks the initiation of Dictyostelium development.

Transformants that expressed either the wild-type rasG gene, an activated rasG-G12T gene, or a dominant negative rasG-S17N gene, all under the control of the folate-repressible discoidin (dis1gamma) promoter, were isolated. All three transformants expressed high levels of Ras protein which were reduced by growth in the presence of folate. All three transformants grew slowly, and the reduction in growth rate correlated with the amount of RasG protein produced, suggesting that RasG is important in regulating cell growth. The pVEII-rasG transformant containing the wild-type rasG gene developed normally despite the presence of high levels of RasG throughout development. This result indicates that the down regulation of rasG that normally occurs during aggregation of wild-type strains is not essential for the differentiation process. Dictyostelium transformants expressing the dominant negative rasG-S17N gene also differentiated normally. Dictyostelium transformants that overexpressed the activated rasG-G12T gene did not aggregate. The defect occurred very early in development, since the expression of car1 and pde, genes that are normally induced soon after the initiation of development, was repressed. However, when the transformant cells were pulsed with cyclic AMP, expression of both genes returned to wild-type levels. The transformants exhibited chemotaxis to cyclic AMP, and development was synergized by mixing with wild-type cells. Furthermore, cells that were pulsed with cyclic AMP for 4 h before being induced to differentiate by plating on filters produced small, but otherwise normal, fruiting bodies. These results suggest that the rasG-G12T transformants are defective in cyclic AMP production and that RasG - GTP blocks development by interfering with the initial generation of cyclic AMP pulses.     

Regulation of DdrasG gene expression during Dictyostelium development.

DdrasG gene expression during the early development of Dictyostelium discoideum has been examined in detail. The amount of DdrasG-specific mRNA increased approximately twofold during the first 2 to 3 h of development and then declined rapidly, reaching negligible levels by the aggregation stage. The increase in mRNA levels that occurred during the first 2 to 3 h of development also occurred during differentiation in cell suspensions and was enhanced when cells were shaken rapidly. This initial increase was unaffected by cell density. When cells were set up to differentiate on filters, the addition of a glucose-amino acid mixture slightly delayed differentiation and had a similar effect on the expression of the gene. The decline in DdrasG expression during development did not occur when cells were treated with cycloheximide, suggesting that the expression of a developmentally regulated gene product is essential for the reduction of DdrasG gene mRNA. There was no decrease in DdrasG mRNA level during differentiation in shake suspension, but the decrease did occur upon application of pulses of cyclic AMP to shaking cultures. The application of a continuously high level of cyclic AMP delayed the increase in expression of the gene and did not result in the subsequent decline. These results suggest that the induction of a functional cyclic AMP relay system is important in reducing DdrasG gene mRNA levels.     

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.     

Membrane traffic during embryonic development: epithelial formation, cell fate decisions and differentiation

The analysis of membrane trafficking has in the past mainly dealt with single cells in culture. Recent studies of membrane trafficking in Drosophila focus on how cells are organized in tissues and form epithelia during embryogenesis. During these processes, the specific involvement of distinct biosynthetic and endocytic routes is starting to be understood. Once organized in epithelia, cells communicate with each other to make cell fate decisions through morphogen gradients and lateral inhibition. Endocytosis seems to play unexpected roles in shaping morphogen gradients and in biasing lateral inhibition events. Once committed to a developmental program, cells differentiate. In the case of neurons, trafficking through the biosynthetic and endocytic pathways may give the necessary speed of response and versatility to axons that navigate through a changing environment during pathfinding.     

Ribosomal protein gene expression is cell type specific during development in Dictyostelium discoideum

Starvation for amino acids initiates the developmental cycle in the cellular slime mold, Dictyostelium discoideum. Upon starvation one of the earliest developmental events is the selective loss of the ribosomal protein mRNAs from polysomes. This loss depends upon sequences in the 5' non-translated leader of the ribosomal protein (r-protein) mRNAs. Here evidence is presented which indicates that those cells which will become prestalk cells express the ribosomal protein genes during development under starvation conditions. Cells which enter the prespore pathway shut off r-protein synthesis. The promoter and 5' non-translated leader sequences from two ribosomal protein genes, the rp-L11 and the rp-S9 genes, are fused to the Escherichia coli beta-galactosidase reporter gene. While beta-galactosidase enzyme activity is detected in situ in most growing cells, by 15 h of development beta-galactosidase enzyme activity is largely lost from the prespore cells although strong beta-galactosidase enzyme activity is present in the prestalk cells. These observations suggest the possibility that the ribosomal protein mRNAs are excluded from polysomes in a cell-type-specific manner.     

Eye development: stable cell fate decisions in insect colour vision

The retina provides an example of a fundamental property of developing cells: cell fate decisions are stable. A recent paper reports a double negative feedback loop that leads to bistable fate decisions in the colour-detecting photoreceptors of Drosophila.     

Molecular requirements for cell fate determination during T-lymphocyte development.

Antigen-specific T lymphocytes recognize peptide antigens in conjunction with the products of the self major histocompatibility complex (MHC). In addition, they are immunologically self-tolerant. To acquire these characteristics, thymocytes undergo a stringent cellular selection process during development. The study of thymocyte development at the molecular level is impeded in mammalian systems by the heterogeneity of the thymocyte population in each individual. However, the use of mice transgenic for the T-cell receptor successfully circumvented this problem and made it possible to elucidate some of the requirements for positive selection, which leads to thymocyte differentiation, survival, and MHC restriction, and negative selection, which leads to programmed cell death, clonal deletion, and self-tolerance. T-cell fate is determined primarily by the nature of the interaction between a complex composed of the T-cell receptor and CD4 or CD8 molecules on the T-cell surface and the peptide antigens that are bound to MHC products and are displayed by other nonlymphoid cells present in the thymus. The molecular analysis of the receptor-ligand interactions involved in this process in transgenic mice provides opportunities to dissect cell fate determination in an intact mammalian system and to understand the molecular basis for immunological self-tolerance and MHC-restriction.     

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.     

Cyclic GMP and cyclic AMP changes in response to folic acid pulses during cell development of Dictyostelium discoideum.

Folic acid pulses induced developmental processes in agip 71, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. Cells that had received folic acid pulses were able to form EDTA-stable cell aggregates and to complete full differentiation to fruiting bodies. In these cells no autonomous periodic activities were observed by light scattering. Folic acid pulses elicited increases in the concentrations of cyclic GMP and cyclic AMP. In undifferentiated cells, folic acid caused a rapid increase in the level of cyclic GMP without a significant change in the level of cyclic AMP. In an advanced developmental state folic acid caused an increase in cyclic AMP in addition to two successsive peaks of cyclic GMP. Experiments performed with the parent strain, Ax-2, also showed that during the development towards aggregation competence, cells acquired the ability to produce a cyclic AMP peak in response to folic acid.     

Developmental decisions in Dictyostelium discoideum.

A few hours after the onset of starvation, amoebae of Dictyostelium discoideum start to form multicellular aggregates by chemotaxis to centers that emit periodic cyclic AMP signals. There are two major developmental decisions: first, the aggregates either construct fruiting bodies directly, in a process known as culmination, or they migrate for a period as "slugs." Second, the amoebae differentiate into either prestalk or prespore cells. These are at first randomly distributed within aggregates and then sort out from each other to form polarized structures with the prestalk cells at the apex, before eventually maturing into the stalk cells and spores of fruiting bodies. Developmental gene expression seems to be driven primarily by cyclic AMP signaling between cells, and this review summarizes what is known of the cyclic AMP-based signaling mechanism and of the signal transduction pathways leading from cell surface cyclic AMP receptors to gene expression. Current understanding of the factors controlling the two major developmental choices is emphasized. The weak base ammonia appears to play a key role in preventing culmination by inhibiting activation of cyclic AMP-dependent protein kinase, whereas the prestalk cell-inducing factor DIF-1 is central to the choice of cell differentiation pathway. The mode of action of DIF-1 and of ammonia in the developmental choices is discussed.     

Asymmetric cell division during T cell development controls downstream fate

During mammalian T cell development, the requirement for expansion of many individual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell division (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs specifically during the β-selection stage of T cell development, and subsequent divisions are predominantly symmetric. ACD is controlled by interaction with stromal cells and chemokine receptor signaling and uses a conserved network of polarity regulators. The disruption of polarity by deletion of the polarity regulator, Scribble, or the altered inheritance of fate determinants impacts subsequent fate decisions to influence the numbers of DN4 cells arising after the β-selection checkpoint. These findings indicate that ACD enables the thymic microenvironment to orchestrate fate decisions related to differentiation and self-renewal.