MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
*Author for correspondence (e-mail: rrk{at}mrc-lmb.cam.ac.uk)
Accepted September 26, 2001
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SUMMARY |
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We propose that prespore cells cross-induce the differentiation of prestalk-O cells by making DIF-1, and that this is one of the regulatory loops that sets the proportion of prespore-to-prestalk cells in the aggregate.
Key words: Dictyostelium, DIF-1, Proportioning mechanism, Cell differentiation
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INTRODUCTION |
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It is generally accepted that cell-type proportioning requires communication between prestalk and prespore cells (MacWilliams et al., 1985; Loomis, 1993; Gross, 1994; Schaap et al., 1996; Aubry and Firtel, 1999; Kessin, 2001), but understanding the nature of this communication is complicated because there are four subtypes of prestalk cell to consider, each of which presumably has its own induction conditions (Berks and Kay, 1990; Thompson and Kay, 2000). Prestalk-A, prestalk-O and prestalk-AB cells (pstA, pstO and pstAB cells) constitute the anterior prestalk zone (Jermyn et al., 1989; Jermyn and Williams, 1991; Jermyn et al., 1996; Early et al., 1993; Early, 1999), whereas anterior-like cells (ALCs) form a scattered population in the posterior prespore zone (Sternfeld and David, 1981; Devine and Loomis, 1985).
One signal molecule involved in stalk cell formation is a small chlorinated alkyl phenone called DIF-1 (Morris et al., 1987; Kay et al., 1999). It was discovered as an inducer of mature stalk cell differentiation in culture, but shown subsequently to induce differentiation of prestalk cells (Kopachik et al., 1983; Williams et al., 1987; Early et al., 1995) and repress differentiation of prespore cells (Kay and Jermyn, 1983; Early and Williams, 1988). Mutants that lack DIF-1 also lack pstO cells and have an increased proportion of prespore cells (Thompson and Kay, 2000); surprisingly, pstA cells seem unaffected, suggesting that they have a separate inducer from DIF-1. Our working hypothesis is therefore that DIF-1 induces the differentiation of pstO cells in normal development and that DIF-1 levels in the aggregate regulate the ratio of pstO to prespore cells.
In order to understand the logic of the DIF-1 regulatory system, it is essential to know which cell type produces DIF-1. Originally, DIF-1 was viewed as a candidate for the activator of prestalk cell differentiation in a Gierer-Meinhardt reaction-diffusion scheme (Geirer and Meinhardt, 1972), which would imply production by prestalk cells (Gross et al., 1981). Later schemes had DIF-1 as a consumed substrate, which was made by all cells, but converted into the true activator by prestalk cells (Meinhardt, 1983); as an inhibitor of anterior-like cell formation, possibly made by these cells (MacWilliams et al., 1985); or as being produced by prespore cells, resulting in the cross-induction of prestalk cells (Loomis, 1993; Kay et al., 1999); or they did not to specify which cells made DIF-1, because the location of its breakdown was considered to be more important than its source (Schaap et al., 1996).
Experimental attempts to determine which cells make DIF-1 have produced contradictory results. One approach has been to measure DIF-1 production by separated prestalk and prespore cells, using a bioassay. This suggested that DIF-1 is either made by pstAB cells (Kwong et al., 1990) or (using a different bioassay) by prespore cells (Inouye, 1989). Alternatively, micro-dissection experiments, where DIF-1 is extracted and assayed from prestalk and prespore fragments of migrating slugs, show that the highest level of DIF-1 is in the prespore zone, suggesting that it is made by prespore cells (Brookman et al., 1987). These experiments have been limited by the sensitivity of the bioassays and by lack of any knowledge of how DIF-1 is biosynthesized. However, recent progress in this area has made new tools available.
DIF-1 appears to be synthesized from a 12-carbon polyketide skeleton, which is decorated by chlorination and methylation (Fig. 1) (Kay, 1998). The polyketide synthase producing the DIF-1 precursor has not yet been identified, but the enzymes carrying out the last two steps of the biosynthetic pathway can be detected in cell lysates: the chlorinating enzyme is particulate and uses hydrogen peroxide as oxidant, whereas the methylating enzyme, des-methyl DIF-1 methyltransferase, is soluble and uses S-adenosyl methionine as methyl donor (Kay, 1998). The des-methyl DIF-1 methyltransferase gene, dmtA, has been identified and knocked out by homologous recombination (Thompson and Kay, 2000). Detectable des-methyl DIF-1 methyltransferase activity is abolished in the mutant and it has less than 1% of wild-type levels of DIF-1, proving that DmtA does catalyse the last step in DIF-1 biosynthesis. Using the tools made available by this work, all our evidence suggests that DIF-1 is made largely by prespore cells.
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MATERIALS AND METHODS |
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Cell growth and labelling
Cells were grown and developed at 22°C. Strain Ax2 was grown in axenic medium with shaking (Watts and Ashworth, 1970), strain V12M2 was grown on nutrient plates in association with Klebsiella aerogenes and washed free of bacteria in KK2 (20 mM K1K2PO4, 2 mM MgSO4, pH 6.2) before use (Kay, 1998). Slugs were produced by streaking 107 cells on to 1.8% L28 agar (Oxoid) containing 10% NS (100% is 20 mM KCl, 20 mM NaCl, 1 mM CaCl2) and allowed to develop in unilateral light. They were dissected using a sharpened insect needle and fragments were accumulated in ice-cold KK2 before freezing.
Prestalk and prespore cells were purified from 40 hours V12M2 slugs, after pronase/2,3-di-mercapto-propanol disaggregation, by Percoll gradient centrifugation (Ratner and Borth, 1983), with prespore cells re-purified on a second Percoll gradient. The purity of the fractions was monitored by staining with an antibody against prespore cells (Hayashi and Takeuchi, 1976). All steps were at 4°C, except for the disaggregation, which was for 5 minutes at 22°C. Separated cells were washed twice by centrifugation and resuspended in 10 mM MES pH 6.2/NS. After 10 minutes equilibration at 22°C, cell suspensions at 107 per ml were incubated at 22°C with [3H]THPH (1.9 nM and 110,000 cpm/ml) and 40 µM ancymidol (an inhibitor of DIF-3 breakdown) in a final volume of 0.75-1 ml. At the indicated times, the reaction was terminated by adding an equal volume of stop solution (90/2 ethyl acetate/acetic acid, containing 0.05 mg/ml butylated hydroxytoluene and 0.25 mg/ml tocopherol). The aqueous phase was extracted twice more with ethyl acetate and the combined organic phases dried down and analysed by HPLC using a 25 cm Sperisorb S5ODS 2 column (solvent A=2% acetic acid; B=2% acetic acid/methanol; gradient: 65-71% B in 36 minutes; 71-91% B in 30 minutes; 1 ml/minute). Internal standards were included to identify the radioactive peaks. The cpm in each fraction was determined by scintillation counting and that in the peaks corrected by subtraction of the background given by adjacent fractions of the HPLC. Fractional chlorination of THPH was calculated from the cpm in each peak fraction: (Cl-THPH + 2x(dM-DIF-1+DIF-1+DIF-3)) divided by the cpm in (THPH+Cl-THPH+dM-DIF-1+DIF-1+DIF-3). This allows for the double chlorination of dM-DIF-1 and DIF-1 and for the derivation of DIF-3 from DIF-1. Fractional methylation of THPH was calculated in a similar way.
V12M2 cells were labelled with 36Cl in submerged monolayers (Kay, 1998). At different times, the medium was taken off and non-polar compounds extracted using a C18 SepPak cartridge (Waters). Labelled DIF-1 was eluted with methanol, resolved by TLC and quantitated using a Phosphorimager. Plates were exposed for 2-6 days in a lead safe, to reduce background, and signal converted to cpm (after subtracting background of the TLC plate) by comparison with standards spotted onto the plate. Recovery of DIF-1 during work-up of the samples was monitored using [3H]-DIF-1 in some experiments and averaged 45%.
In situ hybridization and dmtA reporter construct
In situ hybridization was as described previously (Escalante and Loomis, 1995), except that a Riboprobe from dmtA cDNA was used. Specimens were bulk harvested at different stages of development and fixed with methanol. The green fluorescent protein reporter construct, plasmid pCT7, was constructed by inserting 2.5 kb of genomic sequence upstream of the dmtA-coding region (Thompson and Kay, 2000) into BglII/BamHI digested plasmid 63-GFP. The first six amino acids from dmtA are fused in-frame with green fluorescent protein GFP.
Enzyme assays
Lysates were made by freeze/thawing. Desmethyl DIF-1 methyltransferase and DIF-1 dechlorinase were assayed in the high-speed supernatant as before (Kay, 1998; Nayler et al., 1992). A modified assay for the chlorinating enzyme (Kay, 1998) was used employing as substrate [3H]THPH. Lysates were prepared in 50 mM K1K2PO4 pH 7.5, 5 mM KCl, 2 mM MgSO4, 10% glycerol, 1 mM dTT, 1x protease inhibitors (1000x is 5 mg/ml leupeptin, 2.5 mg/ml pepstatin, 150 mg/ml benzaminide). Each 25 µl assay contained 1 µM [3H]THPH (0.1 µCi per assay) and 10 mM H2O2. After incubation at 25°C for 20 minutes, the reaction was terminated by adding 50 µl of stop (90/10/2 ethyl acetate/hexane/acetic acid, containing 5 mg/ml butylated hydroxytoluene and 1 mg/ml tocopherol), and the labelled compounds resolved by TLC using Whatman LK6D plates, developed with CH2Cl2, di-isopropyl ether, acetic acid (85/15/2). The solvent front was run for 15 cm, and the plate dried and developed a second time using the same solvent. Labelled bands were visualized by autoradiography after spraying with 3H-Enhance (NEN), scraped into scintillation vials and quantitated by scintillation counting.
General details
Protein was assayed using the BioRad dye-binding assay with bovine serum albumin as the standard. Extraction of RNA and northern blots were performed as previously described (Kay et al., 1993) using 32P-labelled probes and a Phosphorimager for quantitation.
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RESULTS |
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Although these experiments show that DmtA mRNA is preferentially expressed in prespore cells, they do not show whether it is also expressed in anterior-like cells, which are a minor population of prestalk cells scattered in the prespore zone. To investigate this possibility, we made a dmtA promoter fusion to green fluorescent protein (pCT7, carrying 2.5 kb of dmtA upstream sequence). As expected, transformants specifically express GFP in the prespore zone, from the tipped mound stage onwards (not shown). However, a non-staining population of cells could also be discerned within the prespore zone from migrating slugs. After manual dissection and disaggregation of the prespore zone, 87% of cells expressed GFP strongly and were clearly distinguishable from 13% that did not (using a strongly expressing clone, HM2121). This proportion of non-expressing cells in the prespore zone corresponds to the expected proportion of anterior-like cells (Sternfeld and David, 1982). As the GFP marker is relatively stable, it can be followed into the mature fruiting bodies. Squashes show expression in the spores, but not in the upper or lower cups [which derive from the ALCs (Sternfeld and David, 1982)] or in the mature stalk cells (Fig. 3). These results show that dmtA is much more strongly expressed in prespore cells than in anterior prestalk or ALC cells.
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Regulation of the expression of the DIF-1 biosynthetic enzymes
Expression of all tested prespore products can be induced by cAMP and this induction is invariably inhibited by DIF-1. The behaviour of prestalk products is more varied: many are induced by DIF-1, but there are some exceptions, and cAMP can be either stimulatory or inhibitory, depending on the gene in question (Kay et al., 1999). To test the response of the DIF-1 biosynthetic enzymes, cells were first pulsed with low levels of cAMP to bring them to a responsive state and then the effects of high, constant cAMP levels and of DIF-1 were determined. Fig. 4 shows that dmtA mRNA, methyltransferase enzyme activity and the chlorinating activity are all stimulated by cAMP addition but are little affected by DIF-1 alone. When both compounds are added together, DIF-1 strongly represses the induction of dmtA expression by cAMP and to a lesser extent, the induction of chlorinating activity.
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Prestalk and prespore cells of strain V12M2 were separated by density-gradient centrifugation, with all steps at 4°C (except disaggregation for 5 minutes at 22°C), to minimize re-differentiation of the cells. For maximum sensitivity, DIF-1 synthesis was measured by the use of the labelled polyketide precursor [3H]THPH, with the labelled products resolved by HPLC (Fig. 5). THPH was successively monochlorinated, then dichlorinated and methylated by the cells to make DIF-1. DIF-1 was then metabolized, to give DIF-3, though further metabolism was inhibited by using the cytochrome P450 inhibitor, ancymidol (Morandini et al., 1995).
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A final test
It is predicted that, if DIF-1 is a prespore product, then it should inhibit its own synthesis in longer-term experiments, where there is time for it to repress expression of its biosynthetic machinery. For these experiments cells were developed as monolayers with cAMP and labelled with 36Cl, so that DIF-1 production would depend on endogenous production of the polyketide precursor, as it does in normal development. Labelled DIF-1 becomes detectable in the medium at 8 hours and reaches a peak at around 12 hours of 16.8±13.6 pmole/108 cells, n=5, which corresponds to a concentration in the medium of about 0.7 nM (corrected for losses during work-up, determined using [3H]DIF-1). Fig. 7 shows that DIF-1, added at the start of development, strongly inhibits the production of labelled DIF-1 (and its metabolites DIF-3 and DM3) at 12 hours. Half-maximal inhibition requires 4.7 nM DIF-1 (mean of three experiments; range 1-10 nM), which may be an underestimate, because as much as 50% of 10 nM DIF-1 is broken down during the incubation (monitored with [3H]DIF-1). DIF-2, a homologue of DIF-1 with one fewer carbon atom in the alkyl side chain, and DIF-3 both inhibited DIF-1 synthesis in these experiments, whereas DM3, the third metabolite produced from DIF-1 (Traynor and Kay, 1991) is without effect, or slightly stimulatory.
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DISCUSSION |
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We showed first that the final enzyme of DIF-1 biosynthesis, encoded by the dmtA gene, is as strongly enriched in prespore cells as a standard prespore marker and that its expression is induced by cAMP and repressed by DIF-1, as expected of a prespore product. The chlorinating enzyme is less highly enriched in prespore cells than DmtA, suggesting that it must also be expressed in prestalk cells, though at a lower level than in prespores.
Using a sensitive labelling technique, we found that separated prespore cells make DIF-1 at more than 20 times the rate of prestalk cells, over incubation times of 5-20 minutes. Prestalk cells chlorinated the polyketide precursor with reasonable efficiency, but are almost completely deficient in the final methylation step that produces DIF-1, as expected from the distribution of chlorinating and methylating enzyme activity between the two cell types.
Finally, we used metabolic labelling with 36Cl (where DIF-1 production depends on endogenous polyketide synthesis) to show that DIF-1 inhibits its own synthesis, after a delay of about 1 hour. This is as expected if DIF-1 is a prespore product and could largely be explained by repression of dmtA gene expression by DIF-1.
In previous work, gradient-separated prestalk cells were found to make two to three times more DIF-1 than prespores (Kwong et al., 1990). Only the relatively insensitive bioassay was available to measure DIF-1 production, and so incubations of 4-24 hours were required to produce detectable DIF-1. However, by 2 hours of incubation, the prestalk cell population had re-differentiated to express prespore markers at levels comparable with the original prespore population; thus the cell-type actually making remains DIF uncertain in these experiments. By contrast, cell re-differentiation has been avoided in our experiments by using incubations of as little as 5 minutes, which is too short a time to allow any major changes in gene expression.
The different approaches that we have used all concur and we therefore conclude that DIF-1 is predominantly made by prespore cells, in accordance with its enrichment in the prespore zone of the slug (Brookman et al., 1987).
A minor production of DIF-1 by non-prespore cells cannot be precluded from our results. Indeed this is likely in early development, when low levels of DIF-1 and dmtA mRNA are made before prespore cells are thought to have differentiated (Brookman et al., 1982; Sobolewski et al., 1983; Kay, 1998; Thompson and Kay, 2000). Thus dmtA may be expressed at a low level in all cells early in development, before later switching to the observed prespore specificity.
These results suggest a simple and robust mechanism for cell-type proportioning: prespore cells make DIF-1, and so cross-induce the differentiation of pstO cells. If there are too many prespore cells, DIF-1 levels will rise, favouring recruitment of pstO cells; if there are too few, DIF-1 levels will fall, favouring recruitment of prespore cells. An additional feedback mechanism, working in the same direction, is the inactivation of DIF-1 by prestalk cells, owing to their possession of the DIF-1 dechlorinase enzyme (Insall et al., 1992; Kay et al., 1993).
Finally, two unexpected observations deserve comment. First, we noticed that expression of dmtA is often graded in the prespore zone of the slug, with the highest level at the front. Such a graded expression pattern has only been described previously for certain prespore promoter constructs, and not for a natural mRNA (Haberstroh and Firtel, 1990; Balint-Kurti et al., 1998). As there is some evidence that DmtA is rate-limiting in converting the polyketide THPH to DIF-1 (Kay, 1998), this observation hints that DIF-1 production itself may be graded in the prespore zone. If such a gradient exists in synthetic capacity, it might help to explain why the front of the prespore zone has the greatest propensity to regenerate a prestalk zone in regulation experiments (Durston, 1976; MacWilliams, 1982; Lokeshwar and Nanjundiah, 1983). Second, we found that DIF-3 inhibits DIF-1 synthesis in living cells, apparently by inhibiting the chlorination of THPH. The enzyme making DIF-3 is found at high levels at the front of the slug (Kay et al., 1993) and it is therefore possible that the DIF-3 produced here helps to repress any residual DIF-1 synthesis in the prestalk zone.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Aubry, L. and Firtel, R. (1999). Integration of signaling networks that regulate Dictyostelium differentiation. Annu. Rev. Cell Dev. Biol. 15, 469-517.[Medline]
Balint-Kurti, P., Ginsburg, G. T., Liu, J. and Kimmel, A. R. (1998). Non-autonomous regulation of a graded, PKA-mediated transcriptional activation signal for cell patterning. Development 125, 3947-3954.
Barklis, E. and Lodish, H. F. (1983). Regulation of Dictyostelium discoideum mRNAs specific for prespore or prestalk cells. Cell 32, 1139-1148.[Medline]
Berks, M. and Kay, R. R. (1990). Combinatorial control of cell differentiation by cAMP and DIF-1 during development of Dictyostelium discoideum. Development 110, 977-984.[Abstract]
Bonner, J. T. and Slifkin, M. K. (1949). A study of the control of differentiation: the proportions of stalk and spore cells in the slime mold Dictyostelium discoideum. Am. J. Bot. 36, 727-734.
Brookman, J. J., Jermyn, K. A. and Kay, R. R. (1987). Nature and distribution of the morphogen DIF in the Dictyostelium slug. Development 100, 119-124.[Abstract]
Brookman, J. J., Town, C. D., Jermyn, K. A. and Kay, R. R. (1982). Developmental regulation of stalk cell differentiation-inducing factor in Dictyostelium discoideum. Dev. Biol. 91, 191-196.[Medline]
Devine, K. M. and Loomis, W. F. (1985). Molecular characterization of anterior-like cells in Dictyostelium discoideum. Dev. Biol. 107, 364-372.[Medline]
Durston, A. J. (1976). Tip formation is regulated by an inhibitory gradient in the Dictyostelium discoideum slug. Nature 263, 126-129.[Medline]
Early, A. (1999). Signalling pathways that direct prestalk and stalk cell differentiation in Dictyostelium. Semin. Cell Dev. Biol. 10, 587-595.[Medline]
Early, A., Abe, T. and Williams, J. (1995). Evidence for positional differentiation of prestalk cells and for a morphogenetic gradient in Dictyostelium. Cell 83, 91-99.[Medline]
Early, A. E. and Williams, J. G. (1988). A Dictyostelium prespore-specific gene is transcriptionally repressed by DIF in vitro. Development 103, 519-524.[Abstract]
Early, A. E., Gaskell, M. J., Traynor, D. and Williams, J. G. (1993). Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination. Development 118, 353-362.
Escalante, R. and Loomis, W. F. (1995). Whole-mount in situ hybridization of cell-type-specific mRNAs in Dictyostelium. Dev. Biol. 171, 262-266.[Medline]
Geirer, A. and Meinhardt, H. (1972). A theory of biological pattern formation. Kybernetik 12, 30-39.[Medline]
Gross, J. D. (1994). Developmental decisions in Dictyostelium discoideum. Microbiol. Rev. 58, 330-351.[Abstract]
Gross, J. D., Town, C. D., Brookman, J. J., Jermyn, K. A., Peacey, M. J. and Kay, R. R. (1981). Cell patterning in Dictyostelium. Philos. Trans. R. Soc. London Ser. B 295, 497-598.[Medline]
Haberstroh, L. and Firtel, R. A. (1990). A spatial gradient of expresssion of a cAMP-regulated prespore cell type-specific-gene in Dictyostelium. Genes Dev. 4, 596-612.[Abstract]
Hayashi, M. and Takeuchi, I. (1976). Quantitative analysis on cell differentiation during morphogenesis of the cellular slime mold Dictyostelium discoideum. Dev. Biol. 50, 302-309.[Medline]
Inouye, K. (1989). Control of cell type proportions by a secreted factor in Dictyostelium discoideum. Development 107, 605-610.[Abstract]
Insall, R., Nayler, O. and Kay, R. R. (1992). DIF-1 induces its own breakdown in Dictyostelium. EMBO J. 11, 2849-2854.[Abstract]
Jermyn, K., Traynor, D. and Williams, J. (1996). The initiation of basal disc formation in Dictyostelium discoideum is an early event in culmination. Development 122, 753-760.
Jermyn, K. A., Duffy, K. T. I. and Williams, J. G. (1989). A new anatomy of the prestalk zone in Dictyostelium. Nature 340, 144-146.[Medline]
Jermyn, K. A. and Williams, J. G. (1991). An analysis of culmination in Dictyostelium using prestalk and stalk-specific cell autonomous markers. Development 111, 779-787.[Abstract]
Kay, R. R. (1998). The biosynthesis of differentiation-inducing factor, a chlorinated signal molecule regulating Dictyostelium development. J. Biol. Chem. 273, 2669-2675.
Kay, R. R. and Jermyn, K. A. (1983). A possible morphogen controlling differentiation in Dictyostelium. Nature 303, 242-244.[Medline]
Kay, R. R., Large, S., Traynor, D. and Nayler, O. (1993). A localized differentiation-inducing-factor sink in the front of the Dictyostelium slug. Proc. Natl. Acad. Sci. USA 90, 487-491.[Abstract]
Kay, R. R., Flatman, P. and Thompson, C. R. L. (1999). DIF signalling and cell fate. Semin. Cell Dev. Biol. 10, 577-585.[Medline]
Kessin, R. H. (2001). Dictyostelium. Cambridge: Cambridge University Press.
Kopachik, W., Oohata, W., Dhokia, B., Brookman, J. J. and Kay, R. R. (1983). Dictyostelium mutants lacking DIF, a putative morphogen. Cell 33, 397-403.[Medline]
Kwong, L., Xie, Y., Daniel, J., Robbins, S. M. and Weeks, G. (1990). A Dictyostelium morphogen that is essential for stalk cell formation is generated by a subpopulation of prestalk cells. Development 110, 303-310.[Abstract]
Lokeshwar, B. L. and Nanjundiah, V. (1983). Tip regeneration and positional information in the slug of Dictyostelium discoideum. J. Embryol. Exp. Morphol. 73, 151-162.[Medline]
Loomis, W. F. (1993). Lateral inhibition and pattern formation in Dictyostelium. Curr. Topics Dev. Biol. 28, 1-46.[Medline]
MacWilliams, H. K. (1982). Transplantation experiments and pattern mutants in cellular slime mold slugs. In Developmental Order: Its Origin and Regulation (40th Symposium of the Society for Developmental Biology) (ed. P. B. Green), pp. 463-483. New York: A. R. Liss.
MacWilliams, H., Blaschke, A. and Prause, I. (1985). Two feedback loops may regulate cell-type proportions in Dictyostelium. Cold Sprimg Harbor Symp. Quant. Biol. 50, 779-785.
Maeda, M., Kuwayama, H., Yokoyama, M., Nishio, K., Morio, T., Urushihara, H., Katoh, M., Tanaka, Y., Saito, T., Ochiai, H. et al. (2000). Developmental Changes in the Spatial Expression of Genes Involved in Myosin Function in Dictyostelium. Dev. Biol. 223, 114-119.[Medline]
Masento, M. S., Morris, H. R., Taylor, G. W., Johnson, S. J., Skapski, A. C. and Kay, R. R. (1988). Differentiation-inducing factor from the slime mould Dictyostelium discoideum and its analogues. Biochem. J. 256, 23-28.[Medline]
Meinhardt, H. (1983). A model for the prestalk/prespore patterning in the slug of the slime mold Dictyostelium discoideum. Differentiation 24, 191-202.[Medline]
Morandini, P., Offer, J., Traynor, D., Nayler, O., Neuhaus, D., Taylor, G. W. and Kay, R. R. (1995). The proximal pathway of metabolism of the chlorinated signal molecule differentiation-inducing factor-1 (DIF-1) in the cellular slime mould Dictyostelium. Biochem. J. 306, 735-743.[Medline]
Morris, H. R., Taylor, G. W., Masento, M. S., Jermyn, K. A. and Kay, R. R. (1987). Chemical structure of the morphogen differentiation inducing factor from Dictyostelium discoideum. Nature 328, 811-814.[Medline]
Nadin, B. M., Mah, C. S., Scharff, J. R. and Ratner, D. I. (2000). The regulative capacity of prespore amoebae as demonstrated by fluorescence-activated cell sorting and green fluorescent protein. Dev. Biol. 217, 173-178.[Medline]
Nayler, O., Insall, R. and Kay, R. R. (1992). Differentiation-inducing-factor dechlorinase, a novel cytosolic dechlorinating enzyme from Dictyostelium discoideum. Eur. J. Biochem. 208, 531-536.[Abstract]
Rafols, I., Amagai, A., Maeda, Y., MacWilliams, H. K. and Sawada, Y. (2000). Cell type proportioning in Dictyostelium slugs: lack of regulation within a 2.5-fold tolerance range. Differentiation 67, 107-116.
Raper, K. B. (1940). Pseudoplasmodium formation and organization in Dictyostelium discoideum. J. Elisha Mitchell Sci. Soc. 56, 241-282.
Ratner, D. and Borth, W. (1983). Comparison of diffentiating Dictyostelium discoideum cell types separated by an improved method of density gradient centrifugation. Exp. Cell Res. 143, 1-13.[Medline]
Sakai, Y. (1973). Cell type conversion in isolated prestalk and prespore fragments of the cellular slime mold Dictyostelium discoideum. Dev. Growth Differ. 15, 11-19.
Schaap, P., Tang, Y. H. and Othmer, H. G. (1996). A model for pattern formation in Dictyostelium discoideum. Differentiation 60, 1-16.
Sobolewski, A., Neave, N. and Weeks, G. (1983). The induction of stalk cell differentiation in submerged monolayers of Dictyostelium discoideum. Characterization of the temporal sequence for the molecular requirement. Differentiation 25, 93-100.
Sternfeld, J. and David, C. N. (1981). Cell sorting during pattern formation in Dictyostelium. Differentiation 20, 10-21.
Sternfeld, J. and David, C. N. (1982). Fate and regulation of anterior-like cells in Dictyostelium slugs. Dev. Biol. 93, 111-118.[Medline]
Thompson, C. R. L. and Kay, R. R. (2000). The role of DIF-1 signaling in Dictyostelium development. Mol. Cell 6, 1509-1514.[Medline]
Traynor, D. and Kay, R. R. (1991). The DIF-1 signaling system in Dictyostelium metabolism of the signal. J. Biol. Chem. 266, 5291-5297.
Watts, D. J. and Ashworth, J. M. (1970). Growth of myxamoebae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem. J. 119, 171-174.[Medline]
Williams, J. G., Ceccarelli, A., McRobbie, S., Mahbubani, H., Kay, R. R., Early, A., Berks, M. and Jermyn, K. A. (1987). Direct induction of Dictyostelium prestalk gene expression by DIF provides evidence that DIF is a morphogen. Cell 49, 185-192.[Medline]
Williams, K. L., Fisher, P. R., MacWilliams, H. K. and Bonner, J. T. (1981). Cell patterning in Dictyostelium discoideum. Differentiation 18, 61-63.[Medline]