Cell Sorting daring Pattern Formation in Dictyostelium

Formation of the prestalk-prespore pattern in Dictyostelium was investigated in slugs and submerged clumps of cells. Prestalk and prespore cells were identified by staining with vital dyes, which are shown to be stable cell markers. Dissociated slug cells reaggregate and form slugs that contain a prestalk-prespore pattern indistinguishable from the original pattern. The pattern forms by sorting out of stained prestalk cells from unstained prespore cells. Sorting also occurs in clumps of dissociated slug cells submerged in liquid or agar. A pattern arises in 2 h in which a central core of stained cells is surrounded by a periphery of unstained cells. Sorting appears to be due to differential chemotaxis of stained and unstained cells to cAMP since exogenous cAMP (>10⁻⁷ M) reverses the normal direction of sorting-out such that stained cells sort to the periphery of the clumps.
Isolated portions of slugs regenerate a new prestalk-prespore pattern. Posterior isolates regenerate a pattern within 2 h due to sorting of a population of vitally stained 'anterior-like' cells present in posteriors. Anterior-like cells do not sort in intact slugs due to the influence of a diffusible inhibitor secreted by the anterior region. During posterior regeneration this signal is absent and anterior-like cells rapidly acquire the ability to sort. Anterior isolates regenerate a staining pattern more slowly than posterior isolates by a process that requires conversion of stained prestalk cells to unstained prespore cells.
The results suggest that pattern formation in Dictyostelium consists of two processes: establishment of appropriate proportions of two cell types and establishment of the pattern itself by a mechanism of sorting-out.     

Regulation of the anterior-like cell state by ammonia in Dictyostelium discoideum

Ammonia appears to be an important regulatory signal for several aspects of the Dictyostelium life cycle. The postulated role of ammonia in the determination of the prespore pathway in cells of the slug stage has led us to examine the effect of ammonia on the prestalk/prespore ratio of migrating slugs. In the presence of 10(-3) M ammonium chloride, the volume of the prestalk region decreases by 40.8%. The kinetics of the process make it unlikely that this is due to a shift in the differentiation pathway. A test of the hypothesis that the decrease in volume of the prestalk region is due to the conversion of prestalk cells to anterior-like cells shows that the percent of anterior-like cells in the posterior region increases by the amount predicted by the hypothesis. This suggests that ammonia may be the molecular signal, produced by the tip, that prevents anterior-like cells from chemotactically migrating to the tip and thereby becoming anterior cells. The effect of enzymatic removal of ammonia from vitally stained migrating slugs is the appearance of a series of dark stripes beginning at the posterior end and progressing forward. We interpret this as a result of progressive removal of anterior-like cells from tip dominance and essentially as the formation of new potential tips. Indeed, in a few cases one or even two of the stripes separate from the posterior of the cell mass and form small fruiting bodies. We consider the phenomenon of stripe formation further evidence that the tip acts on anterior-like cells through ammonia.     

Differentiation, cell sorting and proportion regulation in the slug stage of Dictyostelium discoideum

Recent experimental work suggests that under normal conditions cell sorting plays an important part in maintaining and re-establishing the axial pattern of cell types in the slug stage of the cellular slime mold Dictyostelium discoideum. Following removal of the anterior zone of the slug, anterior-like cells that are normally distributed throughout the posterior of the slug rapidly migrate to the anterior end of the transected slug, and new anterior-like cells appear in the posterior portion. These results provide evidence that the direct linkage between spatial location and differentiation hypothesized in positional information models of spatial pattern formation is not universal. In this paper we develop and analyze a class of mathematical models of the slug in which cell determination can be less rigidly tied to spatial location, and which involve chemotactic cell sorting to re-establish and maintain the spatial pattern of cell types. We show that these models can reproduce the qualitative aspects of the experimental observations and that sorting takes place on the observed time scale when reasonable values of the parameters are used.     

The effects of cAMP on differentiation of cultured Schwann cells: progression from an early phenotype (04+) to a myelin phenotype (P0+, GFAP-, N-CAM-, NGF-receptor-) depends on growth inhibition

The present experiments were designed to clarify the relationship between cAMP elevation, proliferation and differentiation in Schwann cells. They were carried out on short-term cultures of cells obtained from neonatal rat sciatic nerves. It was found that the myelin-related phenotype was expressed in response to agents that elevate or mimic intracellular cAMP (forskolin, cholera toxin, cAMP analogues), provided cell division was absent. This phenotype included upregulation of the major myelin protein P0 and downregulation of GFAP, N-CAM, A5E3, and NGF receptor. In contrast, when cells were cultured in conditions where cell division occurred, elevation of intracellular cAMP produced an alternative response, characterized by DNA synthesis and absence of myelin-related differentiation. The cAMP mediated induction of an early Schwann cell antigen, 04, followed a different pattern since it was induced equally in dividing and nondividing cells. These observations are consistent with the proposal that during development of the rat sciatic nerve: (a) cAMP elevation, possibly induced by axon-associated factors, is a primary signal responsible for the induction of 04 expression in proliferating Schwann cells during the premyelination period; (b) subsequent withdrawal of cells associated with the larger axons from the cell cycle acts as a permissive secondary signal for induction of myelination, since in quiescent cells the ongoing cAMP elevation will trigger myelination.     

Spatiotemporal Patterning of discoidin I and II during Development of Dictyostelium discoideum

We have examined the distribution of Dictyostelium lectins (discoidin I and II) during development by means of a sample preparation method of a whole mount. Monoclonal antibodies which were bound to discoidins revealed unique patterns of discoidin distribution. Discoidin I was localized mainly at the periphery of the aggregates, while the base of the aggregates was devoid of discoidin I staining. Discoidin I was not prominent in the body of the aggregates but when a migrating slug culminated, discoidin I staining appeared in the prestalk region, this suggested that prestalk cells begin to express discoidin I at the onset of culmination. During fruit formation we observed discoidin I staining at the foremost anterior prestalk region of the culminant, which implies a heterogeneity of discoidin I expression among prestalk cells; such a heterogenous pattern has also been found in other prestalk-specific proteins. In addition, anterior-like cells (ALC), which were sorted at the apex and basal parts of a spore mass during culmination, were also strongly stained with anti-discoidin I mAb; interestingly, we observed the staining of ALC from the slug stage through fruit formation. No discoidin II was observed in a migrating slug that had already accumulated prespore antigen ligands for discoidin II; it appeared in prespore cells after the onset of culmination. The present results indicate that, in addition to the early expression of discoidin I, both discoidin I and II are expressed during culmination, and these lectins also seem to be involved in the late development of Dictyostelium .     

The Differentiation of a Cell Sorting Mutant of Dictyostelium discoideum

As a result of transfecting Dictyostelium discoideum with an actin 6/ lac Z fusion transgene, strain HW80 was created which expresses the β-galactosidase gene product uniformly throughout development. When mixed with an excess of unmarked wild-type cells, however, HW80 cells selectively migrate to the positions of anterior-like cells surrounding the prespore cell mass, and differentiate as if they were anterior-like cells. As the proportion of HW80 cells is increased, they also sort to positions adjacent to anterior-like cells and some differentiate as prespore cells. Thus sorting of HW80 cells toward the opposite ends of the prespore cell zone supersedes how they differentiate, suggesting that position influences whether cells differentiate as anterior-like or prespore cells.     

A model for the morphogenesis of strip reduction patterns in phototrophic euglenids: evidence for heterochrony in pellicle evolution

We propose a general developmental model that explains the evolutionary origin, diversification, and inheritance of pellicle strip patterns in phototrophic euglenids. Dividing cells of Euglena gracilis, E. viridis, and Phacus similis were observed with scanning electron microscopy in order to study the morphogenesis of posterior whorls of strip reduction. We found evidence that constant whorl numbers are maintained through cell division because of organized strip growth before and during cytokinesis. Alternating nascent strips form a new whorl of strip reduction at each of the anterior and posterior ends of daughter cells. Strips that terminated to form posterior whorls in the mother cell change in length during the development of daughter cells. In the mother cells of E. gracilis, the strips forming whorls I and II grow to become whorls II and III, respectively, in the daughter cells; the strips forming whorl III in the mother cell lengthen and meet with other strips already present at the posterior tip of daughter cells. This process of whorl morphogenesis during asexual reproduction is consistent with known variation in pellicle strip patterns and suggests that heterochrony played a major role in the ultrastructural evolution of phototrophic euglenids.     

Establishment and maintenance of stable spatial patterns in lacZ fusion transformants of Polysphondylium pallidum.

Polysphondylium pallidum cells were transformed with a construct containing the Dictyostelium discoideum ecmA promoter fused to a lacZ reporter gene. Two stably transformed lines, one in which beta-galactosidase (beta-gal) is expressed in apical cells of the fruiting body (p63/2.1), and one in which it is expressed in basal cells (p63/D), have enabled us to infer how cells move during aggregation and culmination. Several types of cell movement proposed to occur during slime mold culmination, such as random cell mixing and global cell circulation, can be ruled out on the basis of our observations. Cells of the two transformant lines express beta-gal very early in development. In both cases, stained cells are randomly scattered in a starving population. By mid to late aggregation, characteristic spatial patterns emerge. Marked cells of p63/2.1 are found predominantly at tips of tight aggregates; those of p63/D accumulate at the periphery. These patterns are conserved throughout culmination, showing that marked cells maintain their relative positions within the multicellular mass following aggregation. Neither the apical nor the basal pattern appears to be regulated within the primary sorogen by de novo gene expression or by cell sorting as whorls are formed. However, marked cells within a whorl re-establish the original pattern in secondary sorogens. This must be achieved by cell migration, since beta-gal is not re-expressed.     

Requirement of Cyclic Adenosine 3′,5′-Monophosphate for the Thermosensitive Effects of Rts1 in a Cyclic Adenosine 3′,5′-Monophosphate-Less Mutant of Escherichia coli

Previous publications showed that a covalently closed circular (CCC) Rts1 plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 42 degrees C but not at 32 degrees C. At 42 degrees C, the CCC Rts1 DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rts1 on host bacteria, Rts1 was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rts1 did not exert the thermosensitive effect on these cells, and CCC Rts1 DNA was formed even at 42 degrees C. Upon addition of cAMP to E. coli CA7902(Rts1), cell growth and formation of CCC Rts1 DNA were inhibited at 42 degrees C. The addition of cAMP to E. coli PP47(Rts1) did not cause inhibitory effects on either cell growth or CCC Rts1 DNA formation at 42 degrees C. The inhibitory effect of cAMP on E. coli CA7902(Rts1) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rts1 DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [(3)H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rts1 DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rts1 DNA formation and cell growth at 42 degrees C.     

Orientation of cells during slug formation inDictyostelium

Summary Scanning electron microscopic observations ofDictyostelium discoideum cell masses during slug formation revealed two populations around the anterior tip; one group of cells resembled elongated aggregation stream cells and their orientation suggested that they move to the tip, whereas the other group of cells were isodiametric and showed no obvious orientation. In seeking further evidence for a role of differential cAMP chemotaxis in the orientation and movement of slug cells the anterior prestalk cells were compared to the posterior prespore cells in two chemotaxis tests. When a cell mass is placed on cAMP agar the prestalk cells exhibited better movement to cAMP sources but when the gradient was generated in a diffusion chamber the prestalk cells did not. This evidence suggested that the cells which are better able to generate a cAMP gradient might form part of the anterior zone of the slug.     

Ammonia differentially suppresses the cAMP chemotaxis of anterior-like cells and prestalk cells indictyostelium discoideum

A drop assay for chemotaxis to cAMP confirms that both anterior-like cells (ALC) and prestalk cells (pst cells) respond to cAMP gradients. We present evidence that the chemotactic response of both ALC and pst cells is suppressed by ammonia, but a higher concentration of ammonia is required to suppress the response in pst cells. ALC show a chemotactic response to cAMP when moving on a substratum of prespore cells in isolated slug posteriors incubated under oxygen. ALC chemotaxis on a prespore cell substratum is suppressed by the same concentration of ammonia that suppresses ALC chemotaxis on the agar substratum in drop assays. Chemotaxis suppression is mediated by the unprotonated (NH₃) species of ammonia. The observed suppression, by ammonia, of ALC chemotaxis to cAMP supports our earlier hypothesis that ammonia is the tip-produced suppressor of such chemotaxis. We discuss implications of ammonia sensitivity of pst cells and ALC with regard to the movement and localization of ALC and pst cells in the slug and to the roles played by ALC in fruiting body formation. In addition, we suggest that a progressive decrease in sensitivity to ammonia is an important part of the maturation of ALC into pst cells.     

Lignin isolated from primary walls of hybrid aspen cell cultures indicates significant differences in lignin structure between primary and secondary cell wall.

Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.     

MOM-5 frizzled regulates the distribution of DSH-2 to control C. elegans asymmetric neuroblast divisions

Asymmetric cell divisions produce all 302 neurons of the C. elegans hermaphrodite. Here, we describe a role for a C. elegans Dishevelled homolog, DSH-2, in an asymmetric neuroblast division. In dsh-2 mutants, neurons normally descended from the anterior neuroblast daughter of the ABpl/rpppa blast cell were frequently duplicated, while non-neuronal cells produced by the posterior daughter cell were often missing. These observations indicate that in the absence of dsh-2 function, the posterior daughter cell was transformed into a second anterior-like cell. Loss of mom-5, a C. elegans frizzled homolog, produced a similar phenotype. We also show that the DSH-2 protein localized to the cell cortex in most cells of the embryo. In the absence of MOM-5/Fz, DSH-2 was localized to the cytoplasm, suggesting that MOM-5 regulates asymmetric cell division by controlling the localization of DSH-2. Although all neurons in C. elegans are produced by an invariant pattern of cell divisions, our results indicate that cell signaling may contribute to asymmetric neuroblast division during embryogenesis.     

Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several gram-negative bacteria

The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria.     

Studies of cell-surface glorin receptors, glorin degradation, and glorin-induced cellular responses during development of Polysphondylium violaceum

The chemoattractant mediating cell aggregation in the slime mold Polysphondylium violaceum is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethylester (glorin). Here we examine the binding properties of tritiated glorin to intact P. violaceum cells. Scatchard analysis of binding data yielded slightly curvilinear plots with Kd values in the range of 20 and 100 nM. The number of glorin receptors increased from 35,000 in the vegetative stage to 45,000 per cell during aggregation. Later, during culmination receptor numbers decreased to undetectable levels (less than 1000). The receptor binding kinetics show binding equilibrium within 30 s at 0 degrees C, and ligand dissociation occurs from two kinetically distinct receptors whose half-times were 2 s for 72% of the bound glorin and 28 s for the remainder. The enzymatic degradation of glorin did not affect binding data during incubations of up to 1 min at 0 degrees C. Two glorinase activities were observed. An ornithine delta-lactam cleaving activity with a Km of ca. 10(-4) M and a propionic acid removing activity (Km 10(-5) M), both of which were detected mainly on the cell surface. Cleavage of the lactam occurred at a higher rate than removal of propionic acid. Lactam-cleaved glorin showed no chemotactic activity nor did it bind to cell-surface glorin receptors. Cell-surface-bound glorinase activity and glorin-induced cGMP synthesis were developmentally regulated, peaking at aggregation. In the most sensitive stage half-maximal responses (cGMP synthesis, chemotaxis, light-scattering) were elicited in the 10-100 nM range. Neither cAMP synthesis nor glorin-induced glorin synthesis was observed. Guanine nucleotides specifically modulated glorin receptor binding on isolated membranes, and, conversely, glorin modulated GTP gamma S binding to membrane preparations. Our results support the notion that glorin mediates chemotactic cell aggregation in P. violaceum acting via cell-surface receptors, G-proteins, and cGMP accumulation.