The Dictyostelium prestalk cell inducer DIF regulates nuclear accumulation of a STAT protein by controlling its rate of export from the nucleus

Dd-STATc becomes tyrosine phosphorylated, dimerises and accumulates in the nuclei of Dictyostelium cells exposed to DIF, the chlorinated hexaphenone that directs prestalk cell differentiation. By performing cytoplasmic photobleaching of living cells, we show that DIF inhibits the nuclear export of Dd-STATc. Within Dd-STATc there is a 50 amino acid region containing several consensus CRM1 (exportin 1)-dependent nuclear export signals (NESs). Deletion of this region causes Dd-STATc to accumulate in the nucleus constitutively and, when coupled to GFP, the same region directs nuclear export. We show that the N-terminal-proximal 46 amino acids are necessary for nuclear accumulation of Dd-STATc and sufficient to direct constitutive nuclear accumulation when fused to GFP. Combining the photobleaching and molecular analyses, we suggest that DIF-induced dimerisation of Dd-STATc functionally masks the NES-containing region and that this leads to nett nuclear accumulation, directed by the N-terminal-proximal import signals. These results show that the regulated nuclear accumulation of a STAT protein can be controlled at the level of nuclear export and they also provide a better understanding of the mechanism whereby DIF directs cell type divergence.     

A Dictyostelium morphogen that is essential for stalk cell formation is generated by a subpopulation of prestalk cells

The stalk cell differentiation inducing factor (DIF) has the properties required of a morphogen responsible for pattern regulation during the pseudoplasmodial stage of Dictyostelium development. It induces prestalk cell formation and inhibits prespore cell formation, but there is as yet no strong evidence for a morphogenetic gradient of DIF. We have measured DIF accumulation by monolayers of isolated prestalk and prespore cells in an attempt to provide evidence for such a gradient. DIF is accumulated in the largest quantities by a subpopulation of prestalk cells that specifically express the DIF-inducible genes pDd56 and pDd26. Since it has been shown recently that cells that express pDd56 are localized in the central core of the prestalk cell region of the pseudoplasmodia, our current results suggest a morphogenetic gradient generated by this region.     

Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium discoideum

We have isolated cDNA clones derived from three mRNA sequences which are inducible by DIF, the putative stalk-specific morphogen of Dictyostelium. The three mRNA sequences are selectively expressed in cells on the stalk cell pathway of differentiation and we have compared them with previously characterized prestalk-enriched mRNA sequences. We find these latter sequences are expressed without a dependence on DIF, are much less highly enriched in prestalk over prespore cells and are expressed earlier during development than the DIF-inducible mRNA sequences. We propose two distinct mechanisms whereby a mRNA may become enriched in prestalk cells. An apparently small number of genes, represented by those we have isolated, is inducible by DIF and accumulates only in prestalk cells. We suggest that a second class of prestalk-enriched mRNA sequences are induced by cAMP to accumulate in all cells during aggregation and then become enriched in prestalk cells by selective loss from prespore cells.     

Stalk cell formation in monolayers from isolated prestalk and prespore cells of Dictyostelium discoideum: evidence for two populations of prestalk cells

Cells from the pseudoplasmodial stage of Dictyostelium discoideum differentiation were dispersed and separated on Percoll gradients into prestalk and prespore cells. The requirements for stalk cell formation in low-density monolayers from the two cell types were determined. The isolated prespore cells required both the Differentiation Inducing Factor (DIF) and cyclic AMP for stalk cell formation. In contrast, only part of the isolated prestalk cell population required both cyclic AMP and DIF, the remainder requiring DIF alone, suggesting the possibility that there were two populations of prestalk cells, one independent of cyclic AMP and one dependent on cyclic AMP for stalk cell formation. The finding that part of the prestalk cell population required only a brief incubation in the presence of DIF to induce stalk cell formation, whilst the remainder required a considerably longer incubation in the presence of both DIF and cyclic AMP was consistent with this idea. In addition, stalk cell formation from cyclic-AMP-dependent prestalk cells was relatively more sensitive to caffeine inhibition than stalk cell formation from cyclic-AMP-independent prestalk cells. The latter cells were enriched in the most anterior portion of the migrating pseudoplasmodium, indicating that there is spatial segregation of the two prestalk cell populations. The conversion of prespore cells to stalk cells took longer and was more sensitive to caffeine when compared to stalk cell formation from cyclic-AMP-dependent prestalk cells.     

Nature and distribution of the morphogen DIF in the Dictyostelium slug

The Dictyostelium slug contains a simple anterior-posterior pattern of prestalk and prespore cells. It is likely that DIF, the morphogen which induces stalk cells, is involved in establishing this pattern. Previous work has shown that a number of distinct species of DIF are released by developing cells and that cell-associated DIF activity increases rapidly during the slug stage of development. In this paper we describe a comparison of the DIF extracted from slugs with the DIF released into the medium. Analysis by high-pressure liquid chromatography (HPLC) using different solvent systems shows that the major species of DIF activity extracted from slugs coelutes with DIF-1, the major species of released DIF and is similarly sensitive to sodium borohydride reduction. Since DIF specifically induces the differentiation of prestalk cells, the anterior cells of the slug, it could be anticipated that DIF is localized in the prestalk region. We have therefore determined the distribution of DIF within the slug. Migrating slugs from strain V12M2 were manually dissected into anterior one-third and posterior two-third fragments and the DIF activity extracted. Surprisingly, we found that DIF was not restricted to the prestalk fragment. Instead there appears to be a reverse gradient of DIF in the slug with at least twice the specific activity of total DIF in the prespore region than in the prestalk region.     

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.     

Developmental characterization of the wheat germ agglutinin binding proteins, wst31 and wst34, enriched in prestalk and stalk cells of Dictyostelium discoideum

Abstract. The onset of prestalk differentiation of Dictyostelium discoideum has been thought to be triggered by differentiation inducing factor (DIF), which is secreted by differentiating cells. We characterized the cell-type specific proteins, wst31 (prestalk and stalk specific) and wst34 (stalk specific), using the mutant HM44 which is defective in DIF-production, and examined the effects of DIF and cAMP on the formation of the proteins. In the mutant HM44, wst34 was formed only in the presence of exogenous DIF as reported for other prestalk/stalk markers (e.g. pDd63 and acid phosphatase-2), which indicates the DIF-requirement for this protein. By contrast, the accumulation of wst31 in this mutant occurred in the presence of cAMP regardless of the presence of exogenous DIF. Thus, we propose a new and distinct state (or stage) in prestalk differentiation, where the expression of wst31 occurs but not that of pDd63 or acid phosphatase-2.     

Signalling pathways that direct prestalk and stalk cell differentiation in Dictyostelium

Prestalk cell differentiation in Dictyostelium is induced by DIF and two DIF-induced genes, ecmA and ecmB, have revealed the existence of multiple prestalk and stalk cell sub-types. These different sub-types are defined by the pattern of expression of subfragments derived from the ecmA and ecmB promoters. These markers have been utilised in three ways; for fate mapping in vivo, to investigate the molecular mechanisms underlying DIF signalling and to explore the relative requirement for DIF and other signalling molecules for prestalk and stalk cell differentiation in vitro. The heterogeneity of the prestalk and stalk populations seems to be reflected in differences in the cell signalling pathways that they utilise.     

Developmental regulation of a stalk cell differentiation-inducing factor in Dictyostelium discoideum

Previous work has shown that cells developing at high density release a low-molecular-weight factor that can induce isolated Dictyostelium discoideum amoebae of strain V12M2 to differentiate into stalk cells in the presence of cyclic AMP. We now show that this differentiation-inducing factor, called DIF, can be extracted from cells during normal development and that its production is strongly developmentally regulated. DIF is not detectable in vegetative cells but rises dramatically after aggregation to reach a peak during slug migration. DIF levels are very low in two mutants defective in aggregation. The postaggregative synthesis of DIF is stimulated by the addition of extracellular cyclic AMP. We propose that DIF is a morphogen controlling prestalk cell differentiation.     

The requirement for DIF for prestalk and stalk cell formation in Dictyostelium discoideum: a comparison of in vivo and in vitro differentiation conditions

In Dictyostelium discoideum stalk cell formation is induced by cyclic AMP and differentiation-inducing factor (DIF) when cells are plated in in vitro monolayers (Kay et al., 1979, Differentiation 13: 7-14). The in vivo developmental stages at which cells became independent of these factors were determined. Independence was defined as the stage at which dispersed cells no longer required the factors for stalk cell formation in low density monolayers. Cyclic AMP independent cells were first detected at around 12 hr of development, a time that corresponds to the transition between the tipped aggregate and the first finger stages. In contrast cells did not become independent of DIF until late culmination. The prestalk cell-specific isozyme acid phosphatase II and a stalk cell-specific 41,000 Mr antigen (ST 41) were expressed during differentiation in low density monolayers in the presence of both cyclic AMP and DIF, but neither component was expressed in the presence of cyclic AMP alone. This result implies that DIF is essential for both prestalk and stalk cell formation. The two components were expressed within 2 hr of each other during differentiation in vitro, whereas during development in vivo acid phosphatase II was first detected at the first finger stage and ST 41 was first detected during late culmination, 8-12 hr later. These contrasting results suggest that the conversion of prestalk cells to stalk cells is unrestrained in monolayers, following directly after prestalk cell induction, but restrained in vivo until the culmination stage. This interpretation is consistent with the finding that cells become independent of DIF early during in vitro differentiation (A. Sobolewski, N. Neave, and G. Weeks, 1983, Differentiation 25, 93-100), but do not become independent of DIF until the culmination stage when differentiating in vivo.     

Control of cell type proportions by a secreted factor in Dictyostelium discoideum

It has been shown that, in Dictyostelium discoideum, conversion of prestalk cells to prespore cells in suspension cultures is inhibited by coexisting prespore cells. To examine whether the inhibition of conversion requires direct cell contact or is mediated by substances secreted by the cells, prestalk cells and prespore cells were incubated in shaken suspension, separated from each other by a dialysis membrane, and conversion of the prestalk cells to prespore cells scored after 24 h. Prestalk-to-prespore conversion was significantly inhibited if the density of the prespore cells was sufficiently high. In contrast, prestalk cells had little influence on prestalk-to-prespore conversion. Media conditioned by prespore cells, but not by prestalk cells, also inhibited the conversion of prestalk cells. Adenosine, propionate, diethylstilboestrol and differentiation inducing factor (DIF), all of which are known to influence the prestalk/prespore differentiation, were examined for their effects on prestalk-to-prespore conversion. Among these, all except adenosine significantly inhibited the conversion. Based on these results, possible mechanisms for maintenance of the constant cell-type ratio in D. discoideum slugs were discussed.     

A slow sustained increase in cytosolic Ca2+ levels mediates stalk gene induction by differentiation inducing factor in Dictyostelium.

During Dictyostelium stalk cell differentiation, cells vacuolate, synthesize a cellulose cell wall and die. This process of programmed cell death is accompanied by expression of the prestalk gene ecmB and induced by the differentiation inducing factor DIF. Using cell lines expressing the recombinant Ca2+-sensitive photoprotein apoaequorin, we found that 100 nM DIF increases cytosolic Ca2+ ([Ca2+]i) levels from approximately 50 to 150 nM over a period of 8 h. The Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ) induced a similar increase in [Ca2+]i levels and induced expression of the prestalk gene ecmB to the same level as DIF. The [Ca2+]i increases induced by DIF and BHQ showed similar kinetics and preceded ecmB gene expression by approximately 1-2 h. The Ca2+ chelator 1,2-bis(o-aminophenoxy)-ethane-N,N,N'N'-tetra-acetic acid (BAPTA) efficiently inhibited the BHQ-induced [Ca2+]i increase and blocked DIF-induced expression of the ecmB gene. These data indicate that the effects of DIF on stalk gene expression are mediated by a sustained increase in [Ca2-]i. Sustained [Ca2+]i elevation mediates many forms of programmed cell death in vertebrates. The Dictyostelium system may be the earliest example of how this mechanism developed during early eukaryote evolution.     

Stalk cell formation in monolayers of Dictyostelium discoideum V12-M2

Stalk cell formation in low-cell-density monolayers of Dictyostelium discoideum, strain V12-M2, occurs following the sequential addition of cyclic AMP and the differentiation-inducing factor (DIF). Both cyclic AMP and DIF are essential for the appearance of the prestalk-specific isozyme alkaline phosphatase-II, which suggests that both factors are necessary for prestalk cell formation. The available evidence suggests that the cyclic AMP requirement for stalk cell formation is mediated through the cell surface cyclic AMP receptor. However, stalk cell formation is inhibited by caffeine and this inhibition is reversed by the cell-permeable analogue 8-Br-cyclic AMP, which suggests in addition a possible involvement for elevated intracellular cyclic AMP concentrations in stalk cell formation. During in vivo development cells first become independent of cyclic AMP at the tipped aggregate stage, but the acquisition of cyclic AMP independence is advanced by several hours when cells are incubated in the presence of cyclic AMP for 2 hours. Cells do not become independent of DIF until the culmination stage of development, which suggests the possibility that DIF is required for the conversion of prestalk cells to stalk cells. There is an absolute requirement for DIF for stalk cell formation in low-density monolayers of prestalk cells but only part of population exhibits a requirement for cyclic AMP, which suggests that the prestalk cell population consists of two distinct cell types. Stalk cell formation from prespore cells is totally dependent on both cyclic AMP and DIF.(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible role for DIF-2 in the formation of stalk cells during Dictyostelium development.

The differentiation inducing factor (DIF) is essential for stalk cell formation in monolayers of Dictyostelium discoideum and is necessary for the expression of several prestalk cell-specific genes. DIF activity has been fractionated into a major species, designated DIF-1, and several minor species, including DIF-2. Although DIF-1 is an excellent inducer of stalk cell formation from vegetative cells, it is a poor inducer of stalk cell formation from prestalk cells. In contrast, DIF-2 is more active for the conversion of prestalk cells into stalk cells, than for the conversion of vegetative cells to stalk cells. The same results were obtained regardless of whether chemically synthesized or naturally occurring components were utilized. In addition, stalk cell formation was three- to fourfold higher when vegetative cells were incubated with DIF-1 for a suboptimal period and then subsequently incubated with DIF-2, than when cells were incubated with DIF-2 first and then subsequently with DIF-1. These results indicate a distinct role for DIF-2 during stalk cell formation and suggest the possibility that DIF-1 and DIF-2 act sequentially.