Cell type regulation in response to expression of ricin A in Dictyostelium

Expression of ricin A in either prespore or prestalk cells of Dictyostelium discoideum results in cell-autonomous lethality. Strains expressing the toxic gene under the control of a prestalk-specific regulatory region fail to culminate or form stalks, but form spores normally. Strains expressing ricin A under the control of a prespore-specific regulatory region form neither spores nor stalks. Regulation of the cell types results in conversion of prestalk cells to prespore cells when the prespore cells are poisoned. The newly converted cells then express ricin A and die. In contrast, we could not detect any significant conversion of prespore cells to prestalk cells when the prestalk cells are poisoned under our experimental conditions. This regulation of cell types suggests that the tendency of prestalk cells to regulate and become prespore cells is inhibited by the already established prespore cells. It appears that prespore cells control prestalk cell regulation by producing an inhibitor of prespore differentiation to which they themselves are insensitive.     

Dual regulation of the glycogen phosphorylase 2 gene Dictyostelium discoideum: the effects of DIF-1, cAMP, NH3 and adenosine

Cell differentiation in Dictyostelium results in the formation of two cell types, stalk and spore cells. The stalk cells undergo programmed cell death, whereas spore cells retain viability. The current evidence suggests that stalk cell differentiation is induced by Differentiation Inducing Factor (DIF), while spore cell differentiation occurs in response to cAMP. We have discovered the first developmentally regulated Dictyostelium gene, the glycogen phosphorylase gene 2 (gp2) gene, that can be induced by both DIF-1 and cAMP, suggesting the possibility of a new group of developmentally regulated genes that have DIF-1 and cAMP dual responsiveness. The gp2 gene was found to be expressed in both prestalk/stalk cells and prespore/spore cells. The DIF-1 competence of the gp2 gene required uninterrupted development, whereas the cAMP-competence for the gene required only starvation. Both DIF-1 and cAMP induction of the gene could be inhibited by NH3, a factor that is thought to act as a developmental signal in Dictyostelium. Another developmental signal, adenosine, was found to repress the DIF-1 induction of the gp2 gene. Two introns in the gp2 gene were examined for their involvement in the regulation of the gene, but no regulatory function was detected. A model for the regulation of the gp2 gene during the development is proposed.     

A Serum Response Factor homolog is required for spore differentiation in Dictyostelium

A homolog of the Serum Response Factor (SRF) has been isolated from Dictyostelium discoideum and its function studied by analyzing the consequences of its gene disruption. The MADS-box region of Dictyostelium SRF (DdSRF) is highly conserved with those of the human, Drosophila and yeast homologs. srfA is a developmentally regulated gene expressed in prespore and spore cells. This gene plays an essential role in sporulation as its disruption leads to abnormal spore morphology and loss of viability. The mutant spores were round and cellulose deposition seemed to be partially affected. Initial prestalk and prespore cell differentiation did not seem to be compromised in the mutant since the expression of several cell-type-specific markers were found to be unaffected. However, the mRNA level of the spore marker spiA was greatly reduced. Activation of the cAMP-dependent protein kinase (PKA) by 8-Br-cAMP was not able to fully bypass the morphological defects of srfA- mutant spores, although this treatment induced spiA mRNA expression. Our results suggest that DdSRF is required for full maturation of spores and participates in the regulation of the expression of the spore-coat marker spiA and probably other maturation genes necessary for proper spore cell differentiation.     

Category-specific semantic deficits in focal and widespread brain damage: a computational account

To examine the potential role of calcium in regulating Dictyostelium development, we reduced free cytosolic and total cell Ca2+ in Dictyostelium cells by expressing a constitutively active form of a human erythrocyte plasma membrane calcium pump. The pump-expressing cells lacked a thapsigargin-mediated increase in cytoplasmic calcium, consistent with a reduced level of total cellular Ca2+. During aggregation, the cells initially formed a large number of aggregation centers, many of which coalesced to form mounds that were smaller than those of wild-type cells, and the cells did not exhibit the normal formation of elongated aggregation streams. The majority of the mounds either arrested at this stage with the formation of small protrusions or formed very aberrant finger-like structures, indicating an essential role for cellular calcium in morphogenesis. We used pump and wild-type cells differentially labeled by expressing different wavelength (green and blue) forms of green fluorescent protein and three-dimensional (3-D) reconstruction of serial fluorescent imaging to visualize the movement of pump and wild-type cells within the aggregate. The results showed that the pump cells exhibited very aberrant cell movement and sorting within the forming mound, suggesting that the reduced cytosolic calcium affects movement required for tip formation. When allowed to form chimeric organisms with wild-type cells, pump cells preferentially localized to two bands, one at the prestalk/prespore boundary and the other in the very posterior of the organism, suggesting that pump cells are unable to properly sort. Expression of the calcium pump had little effect on the induction of prestalk- or prespore-specific genes, whereas extended treatment with EGTA blocked induction of both classes of cell-type-specific genes. Our results suggest a role for intracellular Ca2+ in controlling cell sorting and morphogenesis in Dictyostelium.     

The anterior-like cells in Dictyostelium are required for the elevation of the spores during culmination

Shortly after initiation of Dictyostelium fruiting body formation, prespore cells begin to differentiate into non-motile spores. Although these cells lose their ability to move, they are, nevertheless, elevated to the tip of the stalk. Removal of the amoeboid anterior-like cells, located above the differentiating spores in the developing fruiting body, prevents further spore elevation although the stalk continues to elongate. Furthermore, replacement of the anterior-like cells with anterior-like cells from another fruiting body largely restores the ability to lift the spores to the top of the stalk. However, if amoeboid prestalk cells are used to replace the anterior-like cells, there is no restoration of spore elevation. Finally, when a droplet of mineral oil replaces differentiating spores, it is treated as are the spores: the mineral oil is elevated in the presence of anterior-like cells and becomes arrested on the stalk in the absence of anterior-like cells. Because a similar droplet of mineral oil is totally ignored by slug tissue, it appears that there is a dramatic transformation in the treatment of non-motile matter at this point in Dictyostelium development.     

Null mutations of the Dictyostelium cyclic nucleotide phosphodiesterase gene block chemotactic cell movement in developing aggregates

Extracellular cAMP is a critical messenger in the multicellular development of the cellular slime mold Dictyostelium discoideum. The levels of cAMP are controlled by a cyclic nucleotide phosphodiesterase (PDE) that is secreted by the cells. The PDE gene (pdsA) is controlled by three promoters that permit expression during vegetative growth, during aggregation, and in prestalk cells of the older structures. Targeted disruption of the gene aborts development, and complementation with a modified pdsA restores development. Two distinct promoters must be used for full complementation, and an inhibitory domain of the PDE must be removed. We took advantage of newly isolated PDE-null cells and the natural chimerism of the organism to ask whether the absence of PDE affected individual cell behavior. PDE-null cells aggregated with isogenic wild-type cells in chimeric mixtures, but could not move in a coordinated manner in mounds. The wild-type cells move inward toward the center of the mound, leaving many of the PDE-null cells at the periphery of the aggregate. During the later stages of development, PDE-null cells in the chimera segregate to regions which correspond to the prestalk region and the rear of the slug. Participation in the prespore/spore population returns with the restoration of a modified pdsA to the null cells.     

RtoA links initial cell type choice to the cell cycle in Dictyostelium

In Dictyostelium, initial cell type choice is correlated with the cell-cycle phase of the cell at the time of starvation. We have isolated a mutant, ratioA (rtoA), with a defect in this mechanism that results in an abnormally high percentage of prestalk cells. The rtoA gene has been cloned and sequenced and codes for a novel protein. The cell cycle is normal in rtoA. In the wild type, prestalk cells differentiate from those cells in S or early G2 phase at starvation and prespore cells from cells in late G2 or M phase at starvation. In rtoA mutants, both prestalk and prespore cells originate randomly from cells in any phase of the cell cycle at starvation.     

Temperature-sensitive Gbeta mutants discriminate between G protein-dependent and -independent signaling mediated by serpentine receptors

Deletion of the single gene for the Dictyostelium G protein beta-subunit blocks development at an early stage. We have now isolated temperature-sensitive alleles of Gbeta to investigate its role in later development. We show that Gbeta is directly required for adenylyl cyclase A activation and for morphogenetic signaling during the entire developmental program. Gbeta was also essential for induction of aggregative gene expression by cAMP pulses, a process that is mediated by serpentine cAMP receptors (cARs). However, Gbeta was not required for cAR-mediated induction of prespore genes and repression of stalk genes, and neither was Gbeta needed for induction of prestalk genes by the differentiation inducing factor (DIF). cAMP induction of prespore genes and repression of stalk genes is mediated by the protein kinase GSK-3. GSK-3 also determines cell-type specification in insects and vertebrates and is regulated by the wingless/wnt morphogens that are detected by serpentine fz receptors. The G protein-dependent and -independent modes of cAR-mediated signaling reported here may also exist for the wingless/wnt signaling pathways in higher organisms.     

Spalten, a protein containing Galpha-protein-like and PP2C domains, is essential for cell-type differentiation in Dictyostelium

We have identified a novel gene, Spalten (Spn) that is essential for Dictyostelium multicellular development. Spn encodes a protein with an amino-terminal domain that shows very high homology to Galpha-protein subunits, a highly charged inter-region, and a carboxy-terminal domain that encodes a functional PP2C. Spn is essential for development past the mound stage, being required cell autonomously for prestalk gene expression and nonautonomously for prespore cell differentiation. Mutational analysis demonstrates that the PP2C domain is the Spn effector domain and is essential for Spn function, whereas the Galpha-like domain is required for membrane targeting and regulation of Spn function. Moreover, Spn carrying mutations in the Galpha-like domain that do not affect membrane targeting but affect specificity of guanine nucleotide binding in known GTP-binding proteins are unable to fully complement the spn- phenotype, suggesting that the Galpha-like domain regulates Spn function either directly or indirectly by mediating its interactions with other proteins. Our results suggest that Spn encodes a signaling molecule with a novel Galpha-like regulatory domain.     

Evidence that a combined activator-repressor protein regulates Dictyostelium stalk cell differentiation

The ecmA gene is expressed in Dictyostelium prestalk cells and is inducible by differentiation-inducing factor (DIF), a low-molecular-weight lipophilic substance. The ecmB gene is expressed in stalk cells and is under negative control by two repressor elements. Each repressor element contains two copies of the sequence TTGA in an inverted relative orientation. There are activator elements in the ecmA promoter that also contain two TTGA sequences, but in the same relative orientation. Gel retardation assays suggest that the same protein binds to the ecmB repressor and the ecmA activator. We propose that DIF induces prestalk cell differentiation by activating this protein and that the protein also binds to the promoters of stalk-specific genes, acting as a repressor that holds cells in the prestalk state until culmination is triggered.     

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.     

Adenylyl cyclase G, an osmosensor controlling germination of Dictyostelium spores

Dictyostelium cells express a G-protein-coupled adenylyl cyclase, ACA, during aggregation and an atypical adenylyl cyclase, ACG, in mature spores. The ACG gene was disrupted by homologous recombination. acg- cells developed into normal fruiting bodies with viable spores, but spore germination was no longer inhibited by high osmolarity, a fairly universal constraint for spore and seed germination. ACG activity, measured in aca-/ACG cells, was strongly stimulated by high osmolarity with optimal stimulation occurring at 200 milliosmolar. RdeC mutants, which display unrestrained protein kinase A (PKA) activity and a cell line, which overexpresses PKA under a prespore specific promoter, germinate very poorly, both at high and low osmolarity. These data indicate that ACG is an osmosensor controlling spore germination through activation of protein kinase A.     

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.     

Multicellular stalk-like structures in Saccharomyces cerevisiae

Stalk formation is a novel pattern of multicellular organization. Yeast cells which survive UV irradiation form colonies that grow vertically to form very long (0.5 to 3.0 cm) and thin (0.5 to 4 mm in diameter) multicellular structures. We describe the conditions required to obtain these stalk-like structures reproducibly in large numbers. Yeast mutants, mutated for control of cell polarity, developmental processes, UV response, and signal transduction cascades were tested and found capable of forming stalk-like structures. We suggest a model that explains the mechanism of stalk formation by mechanical environmental forces. We show that other microorganisms (Candida albicans, Schizosaccharomyces pombe, and Escherichia coli) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms. Diploid yeast stalks sporulate at an elevated frequency, raising the possibility that the physiological role of stalks might be disseminating spores.     

A functional analysis of imprinting in parthenogenetic embryonic stem cells

A detailed analysis of the developmental potential of parthenogenetic embryonic stem cells (PGES) was made in vivo and in vitro, and a comparison was made with the development of cells from parthenogenetic embryos (PG). In vivo, in chimeras with normal host cells (N), PGES cells showed a restricted tissue distribution consistent with that of PG cells, suggesting faithful imprinting in PGES cells with respect to genes involved in lineage allocation and differentiation. Restricted developmental potential was also observed in teratomas formed by ectopic transfer under the kidney capsule. In contrast, the classic phenotype of growth retardation normally observed in PG<==>N chimeras was not seen, suggesting aberrant regulation in PGES cells of genes involved in growth regulation. We also analysed the expression of known imprinted genes after ES cell differentiation. Igf2, H19 and Igf2r were all appropriately expressed in the PGES derived cells following induction of differentiation in vitro with all-trans retinoic acid or DMSO, when compared with control (D3) and androgenetic ES cells (AGES). Interestingly, H19 was found to be expressed at high levels following differentiation of the AGES cells. Due to the unexpected normal growth regulation of PGES<==>N chimeras we also examined Igf2 expression in PGES derived cells differentiated in vivo and found that this gene was still repressed. Our studies show that PGES cells provide a valuable in vitro model system to study the effects of imprinting on cell differentiation and they also provide invaluable material for extensive molecular studies on imprinted genes. In addition, the aberrant growth phenotype observed in chimeras has implications for mechanisms that regulate the somatic establishment and maintenance of some imprints. This is of particular interest as aberrant imprinting has recently been invoked in the etiology of some human diseases.     

Production and activity of spore differentiation factors (SDFs) in Dictyostelium

SDF-1 and SDF-2 are peptides that promote terminal spore differentiation under submerged conditions. The present study shows that they accumulate differentially and are released during the development of wild-type cells and can promote spore formation in cells disaggregated from wild-type culminants. SDF-1 accumulates during the slug stage and is released in a single burst at the onset of culmination while SDF-2 accumulates during early culmination and is released in a single burst from mid-culminants. The effects of SDF-1 and SDF-2 on stalk cell formation in cell monolayers were investigated. SDF-1 by itself induces stalk cell formation in some strains and also synergizes with the stalk-cell-inducing factor, DIF-1. cAMP has an inhibitory effect on stalk cell formation when either DIF-1 or SDF-1 are present on their own but is almost not inhibitory when both are present. SDF-2 alone does not induce stalk cell formation and appears to inhibit the response to DIF-1. At the same time, it increases the extent of vacuolization of the stalk cells that are produced. We propose that the release of SDF-1 and then of SDF-2 may mark irreversible steps in the developmental programme associated, respectively, with culmination and spore maturation.