Jeffrey G. Williams

Optimization and in situ detection of Escherichia coli β-galactosidase gene expression in Dictyostelium discoideum

We show that a fusion gene, containing the promoter and 5-noncoding region of a Dictyostelium discoideum actin 6 gene linked to the Escherichia coli beta-galactosidase (beta Gal) gene (lacZ), directs the production of functionally active beta Gal in D. discoideum and that the enzyme can be detected by staining in situ; a procedure which will be of great value in analyzing cell-type-specific gene expression. We illustrate this by fusing lacZ to the promoter of the prespore-specific gene, D19, and localizing expressing cells in migrating slugs. Optimal expression requires the inclusion of termination and polyadenylylation signals and we describe pDDlac, a vector containing a multiple cloning site upstream from a lacZ-Dictyostelium terminator fusion, which can be used to analyze regulated promoters.

Direct induction of dictyostelium prestalk gene expression by DIF provides evidence that DIF is a morphogen

We have isolated a gene that is very rapidly induced at the transcriptional level by DIF--a low molecular weight, diffusible factor necessary for stalk cell differentiation in Dictyostelium cells developing in vitro. The gene encodes a protein containing an N-terminal signal peptide preceding approximately 70 tandem repeats of a highly conserved 24 amino acid sequence with a high cysteine content. These features suggest it is an extracellular structural protein. During normal development, the gene is maximally expressed in the slug, in which the mRNA is very highly enriched in prestalk over prespore cells. The gene is not detectably expressed until the tipped aggregate stage, several hours later than prespore genes, suggesting that prespore cell differentiation precedes prestalk cell differentiation. The demonstration that DIF induces a gene normally only expressed in the prestalk zone of the slug provides strong evidence that DIF is a Dictyostelium morphogen.

Origins of the prestalk-prespore pattern in Dictyostelium development

Using cell-autonomous markers we have traced the origins of prespore cells and two types of prestalk cells (pstA and pstB cells) during slug formation. We show that cell sorting and positional information both contribute to Dictyostelium morphogenesis. The initial pattern established at the mound stage is topologically quite different from that of the slug. Confirming previous studies, we find that prespore cells occupy most of the aggregate but are absent from a thin layer at the base and from the emerging tip. PstB cells are almost entirely localized to the basal region during the early stages of tip formation. Thus prespore and pstB cell differentiation appear to occur in response to localized morphogenetic signals. In the case of pstB cells, these signals presumably emanate from the base and not, as might be expected, from the tip. When first detectable, pstA cells are scattered throughout the aggregate. They then appear to migrate to the apex, where the tip forms.

Culmination in dictyostelium is regulated by the cAMP-dependent protein kinase

We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.

Positively and negatively acting signals regulating stalk cell and anterior-like cell differentiation in dictyostelium

The Dictyostelium ecmB gene encodes an extracellular matrix protein and is inducible by the stalk cell morphogen DIF. It is expressed in a subset of prestalk (pstB) cells in the slug and surrounding pstA cells first express it at culmination. A region of the ecmB promoter can direct transcription in all anterior prestalk cells, but a separate, downstream region acts to prevent its expression in pstA cells prior to culmination. This may be the site of interaction of a repressor, regulated by an extracellular antagonist to DIF. At culmination, expression of the ecmB gene also becomes greatly elevated in anterior-like cells as they move to surround the spore mass. A distal region of the ecmB promoter directs increased expression in those anterior-like cells that surmount the spore head. This divergence in gene expression suggests that anterior-like cells and anterior prestalk cells experience different inductive conditions at culmination.

Multiple roles for cAMP-dependent protein kinase during Dictyostelium development☆

The cAMP-dependent protein kinase (PKA) holoenzyme of Dictyostelium comprises a single regulatory (R) and catalytic (C) subunit, and both proteins increase in concentration during cellular aggregation. In order to determine the role of the kinase, we have constructed mutants of the R subunit that are defective in cAMP binding, in inhibition of the C subunit, or in both functions. Analysis of these mutants suggests that overexpression of the unmutated R subunit, which is known to block development, occurs by direct inactivation of the C subunit rather than by an effect on intracellular cAMP levels. Cells with an inactive C subunit (PKA- cells) are defective in cAMP relay, the production of cAMP in response to extracellular cAMP stimulation. This presumably accounts for their inability to undertake aggregation. When mixed with wild-type cells, PKA- cells migrate toward the signalling centre but remain confined to the periphery of the tight aggregate and are lost from the back of the migratory slug. This suggests that PKA may be required during the late, multicellular stages of development. Consistent with this, we find that a number of postaggregative genes are not expressed in PKA- cells, even when they are allowed to synergise with normal cells.

Patterns of cell movement within the Dictyostelium slug revealed by cell type-specific, surface labeling of living cells

There are cells scattered in the rear, prespore region of the Dictyostelium slug that share many of the properties of the prestalk cells and that are therefore called anterior-like cells (ALCs). By placing the gene encoding a cell surface protein under the control of an ALC-specific promoter and immunologically labeling the living cells, we analyze the movement of ALCs within the slug. There is a posterior to anterior cellular flow, and the ALCs change their movement pattern as they enter the prestalk zone. Prestalk cells are periodically shed from the migrating slug. They must be replaced if the correct ratio of prestalk to prespore cells is to be maintained, and we present evidence for the transdifferentiation of prespore into prestalk cells, with ALCs functioning as intermediates in the transition. The slug has, therefore, a surprisingly dynamic structure, both with respect to cellular differentiation and cell movement.

The extensive homology between mRNA sequences of normal and SV40-transformed human fibroblasts

The poly(A)-containing messenger RNA of normal diploid fibroblast and SV40-transformed progeny cells are compared by cross-hybridizing cDNA. We find a high degree of homology between the mRNA from normal and transformed cells. Despite imperfections in the procedure, the technique permits the conclusion that, at most, 3% of the mRNA in the transformed cell has sequences not present in the normal parental cell. Furthermore, much of the difference appears to occur in low and intermediate complexity classes of mRNA molecules. Extension homology in the mRNA sequences of disparate cell lines may be a general phenomenon, and even HeLa cell mRNA is nearly identical to that of diploid human fibroblasts.

Cytoplasmic acidification facilitates but does not mediate DIF-induced prestalk gene expression in Dictyostelium discoideum.

Stalk cell differentiation in Dictyostelium can be induced by the differentiation-inducing factor, DIF, or by conditions that decrease intracellular pH (pHi). We have investigated whether cytoplasmic acidification acts directly to induce expression of pDd56 and pDd63, two DIF-regulated genes, specifically expressed in prestalk cells. The weak base methylamine, which increases pHi, inhibits DIF-induced transcription. The weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO), which decreases pHi, stimulates DIF-induction of the two prestalk genes. After relatively long incubation periods, DMO also induces a low level of prestalk gene expression in the absence of added DIF. However, unlike DIF-mediated induction, the apparent DMO-mediated induction decreases to undetectable levels when the cell density is reduced from 10(7) to 10(5) cells/ml. This indicates that DMO does not itself induce gene expression, but acts to enhance the effects of an autonomously secreted stalk-inducing factor, presumably DIF. These results suggest that the effects of DIF on gene expression are regulated by intracellular pH, but do not support a role for protons as direct intermediates in the DIF signal transduction pathway.

Identification and localization of proteins encoded by two DIF-inducible genes of Dictyostelium

We show that pDd56 and pDd63, two related DIF-inducible genes of Dictyostelium, respectively encode the ST310 and ST430 polypeptides identified by Morrissey, Devine, and Loomis (1984, Dev. Biol. 103, 414-424). We localize the two proteins by immunoelectron microscopy to the extracellular matrix surrounding the stalk cells and the stalk tube. Coupled with their predicted amino acid sequence and biochemical properties, this suggests that they are structural proteins of the stalk.