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Development 100, 745-755 (1987) Printed in Great Britain © The Company of Biologists Limited 1987 745 Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium discoideum K. A. JERMYN1, M. BERKS2, R. R. KAY2 and J. G. WILLIAMS1* 'Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Herts, EN6 3LD, UK MRC Laboratory' of Molecular Biology, Hills Road, Cambridge CB2 3QH, UK 2 * To whom reprint requests should be addressed Summary 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. Introduction difference in the carbohydrate modification of several lysosomal enzymes and this has been used as a marker of prestalk cell differentiation (Oohata, 1983; Knecht, Green, Loomis & Dimond, 1984; Loomis & Kuspa, 1984). There are also several cDNA clones derived from mRNA sequences enriched in prestalk over prespore cells which have been used to determine the timing of prestalk cell differentiation and to analyse the effect of cellular perturbations, such as disaggregation and the addition of cAMP, upon prestalk cell gene expression (Barklis & Lodish, 1983; Mehdy, Ratner & Firtel, 1983). Isolated cells of the strain V12M2, incubated at low cell density (Town, Gross & Kay, 1976), can be induced to form stalk cells by DIF. This is a low molecular weight hydrophobic compound, which has been purified and structurally identified (Kay, Dhokia & Jermyn, 1983; H. Morris, G. W. Taylor, M. Masento, K. Jermyn and R. Kay, unpublished results). The mutant strain HM44 is defective in the production of DIF (Kopachik et al. 1983) but remains responsive to exogenous DIF. Hence it can be used to determine whether the expression of a particular The migrating slug of Dictyostelium discoideum contains up to 105 cells which have aggregated together in response to pulsatile emissions of cAMP. Under normal circumstances, cells in the anterior, prestalk, region differentiate to form stalk cells and cells in the posterior region differentiate to spores. The two precursor cell types are readily purified, either by microdissection or by Percoll gradient centrifugation, and a number of biochemical markers which distinguish them have been identified (reviewed by Morrissey, 1982; Williams etal. 1986). Two-dimensional gel electrophoresis indicates a continuity of gene expression on the prespore-spore cell pathway of differentiation (Morrissey, Devine & Loomis, 1984; Ratner & Borth, 1983) and there are reliable enzymic, immunological, structural protein and cDNA markers of prespore cells. There is little apparent continuity on the stalk cell pathway of differentiation, with very few prestalk-specific proteins being expressed in prestalk and stalk cells (Ratner & Borth, 1983; Morrissey et al. 1984). However, there is a Key words: Dictyostelium discoideum, mRNA, DIF, prestalk cells. 746 K. A. Jermyn, M. Berks, R. R. Kay and]. G. Williams marker is dependent upon DIF. In the absence of DIF, prespore cell proteins accumulate but the prestalk-specific isozyme of acid phosphatase is not produced (Kopachik et al. 1983). A similar conclusion derives from the analysis of total cellular proteins by two-dimensional gel electrophoresis (Kopachik, Dhokia & Kay, 1985). We have employed this in vitro induction system to investigate the effect of DIF upon specific mRNA accumulation. We have used previously isolated prestalk-enriched cDNA clones and also cDNA clones which we isolated by differential screening for DIF-induced mRNA sequences. Analysis of the accumulation of these various mRNA sequences - in response to DIF, during normal development and in isolated prestalk and prespore cells - has led us to re-evaluate the usefulness of previously described cDNA clones as prestalk markers and to suggest the existence of two distinct mechanisms for establishing a differential mRNA concentration in the two cell types. in i h periods, depositing the cells in ice-cold KK2 to inhibit dedifferentiation and, after pelleting, snap freezing on dry ice. For terminally differentiated cells, freshly culminated Ax-2 fruiting bodies were harvested and stalks were separated as described by Devine, Morrissey & Loomis (1982) from freshly culminated Ax-2 cells. RNA isolation All RNA was isolated by a minor modification of the GTME method (Maniatis, Fritsch & Sambrook, 1982) except the spore RNA which was isolated according to Macleod, Firtel & Papkoff (1980). Following centrifugation, RNA was routinely subjected to 1 phenol and 1 chloroform extraction. Ribosomal RNA integrity was monitored on a 0-8% minigel (TBE pH8-3) after denaturing at 50°C for 15min. cDNA preparation A cDNA library was prepared by the method of Gubler & Hoffman (1983) using mRNA isolated from HM44 cells 12 h after DIF addition. The cDNA was cloned into the Pst\ site of the pUC8 vector, and transformed into DH5 cells. About 12000 colonies were generated, of which about 400 were picked for secondary screening. The colonies were repliMaterials and methods cated onto Genescreen Plus or nitrocellulose filters and screened with 32P-labelled cDNA made with RNA isolated Dictyostelium discoideum growth and development from cells incubated in the absence or presence of DIF for Strains NC4 (DdB) (obtained from P. Newell), V12M2 12 h. The filters were prehybridized for at least 2h at 37°C (obtained from G. Gerisch) and the V12M2-derived mutant in 50% formamide, 5xSSC, 10mM-Na2HPO4/NaH2PO4 HM44 (Kopachik et al. 1983) were all grown in association pH6-72, lOxDenhardt's solution, 250 fig ml"1 salmon with Klebsiella aerogenes plated on SM agar (Sussman, 1966) testes DNA (heat denatured and sonicated), 1 % SDS. at 22°C. Ax-2 (from J. Ashworth) was grown axenically in Hybridization was carried put in the same buffer at 37°C suspension (Watts & Ashworth, 1970). Cells were normally overnight. The filters were washed for 5x15 min at 37°C in harvested and washed by centrifugation in 20 mM-KH2PO4/ prewarmed 50% formamide, 2xSSC, 1 % SDS and finally K2HPO4, pH60 (KK2). Ax-2 cells were developed on in lxSSC at room temperature, before being sealed in 1-8% KK2 agar at approximately 5xlO('cellscm~2. For polythene and exposed tofilmat —70°C using an intensifydevelopmental time courses, NC4 and V12M2 cells were ing screen. developed on 1-8% agar containing 40mM-Na2HPO4/ KH2PO4, 20mM-KCl and 2-5mM-MgCl2, pH6-2, at about Northern transfer 5xl0 6 cellscm~ 2 . Amoebae of the V12M2-derived strain, The RNA samples (5f(g) were electrophoresed through a HM44, were induced to form stalk cells in 14 cm Sterilin formaldehyde denaturing gel system and transferred onto tissue culture dishes as described by Kopachik et al. (1985). nitrocellulose as described by Maniatis et al. (1982) except Cells were incubated in the presence of cAMP for 10 h ('t0' that the filter was baked and prehybridized immediately of the induction) and then the medium was renewed and after transfer. Thefilterwas hybridized as described above DIF was added to half of the plates at 3000 units ml"'. except that 0 1 % SDS was used in all the solutions. Scanning of the auforadiograms was performed with an Purification of differentiated cell types LKB Ultrascan XL laser scanning densitometer. For migrating slugs, cells were developed as single streaks 9 1 of approximately 10 cells ml" on 2% agar containing 20mM-KCl, 20mM-NaCl and 1 mM-CaCl2 for cutting or 2 % Results water agar for Percoll gradient separation of prestalk and prespore cells (Ratner & Borth, 1983). These plates were (A) Isolation and characterization of cDNA clones kept moist and dark except for a single slit providing a derived from three mRNA sequences which unidirectional light source. Gradient-purified cells were accumulate in response to DIF separated by the method of Ratner & Borth (1983), except The HM44 mutant is defective in DIF production. In that 60% gradients were normally used. Slugs were the absence of DIF, only a Very low proportion manually dissected using a microknife (a mounted, flat(<1 %) of amoebae differentiate into stalk cells tened dissecting pin) to yield a front (10 %) or a rear (50 %) while, in its presence, a very high proportion (>95 %) portion. The very rearmost portion of the slug was cut away to minimize basal disc cell contamination and to sever the of stalk cells are formed. A cDNA clone bank was slime trail. Usually one or other type was rapidly collected prepared from HM44 cells incubated with cAMP for Prestalk-enriched mRNAs in Dictyostelium 10 h and then with DIF for a further 12 h. The bank was screened differentially with labelled cDNA prepared from the RNA used to construct the bank and with cDNA prepared from a control, non-DIF induced, RNA preparation. Nine cDNA clones which showed differential hybridization were isolated. Cross-hybridization with purified inserts from each of the nine cDNA clones and mapping with restriction enzymes showed them to derive from three different mRNA sequences. The longest cDNA clones derived from each of the three mRNA sequences were used in all subsequent experiments. These contained inserts of 0-65kb (pDd26), 2-4kb (pDd56) and 2-3 kb (pDd63) in length. Subsequent analysis, by Northern transfer, showed pDd26 to contain an almost full-length insert while pDd56 and pDd63, which derive from longer mRNA sequences, contain portions of the coding sequences proximal to the 3' end of their respective mRNA sequences. The fact that multiple cDNA clones for each mRNA were isolated (four clones derived from pDd56 mRNA, three clones from pDd26 mRNA and two clones from the pDd63 mRNA) suggests these to be the most abundant mRNA sequences induced by DIF. The mRNA hybridizing to pDd26 is 0-7 kb in length (Fig. 1). It is not detectably expressed before 6 h of induction and maximal expression is achieved in the terminal stages of stalk cell differentiation. Accumulation of this mRNA is totally dependent upon the presence of DIF. The mRNA hybridizing to pDd56 is 3-2 kb in length and shows a somewhat more complex pattern of accumulation (Fig. 1). The major peak in mRNA abundance occurs at about 6-8 h after DIF induction, with a decline in concentration thereafter. However, at a higher level of exposure of this Northern transfer and in all other experiments using a number of different mRNA preparations, the pDd56 mRNA showed a low, but definite, level of expression within l h of DIF addition (data not shown). Thus expression of the pDd56 mRNA is much more rapidly induced by DIF than is the pDd26 mRNA but again accumulation is totally dependent upon DIF. The pDd63 cDNA clone hybridizes to a mRNA of 5-8kb in length. This mRNA accumulates very rapidly after DIF addition, reaching 25 to 30 % of its maximal level by 1 h of incubation with a broad peak of accumulation at 4-6 h after DIF addition. At early times of incubation, the mRNA is almost totally dependent upon DIF for its accumulation, with only a very low level of expression detectable in overexposed autoradiograms in the absence of DIF. After prolonged incubation without DIF, a low level of expression becomes apparent (Fig. 1). The pDd63 gene might be expected to be responsive to a very low level of DIF, as it is highly induced within 1 h of the 747 addition of exogenous DIF. Hence, we believe the expression observed in the control cells results from the low level of DIF production known to occur in the HM44 mutant (Kopachik et al. 1983). The data presented in Table 1 suggest that this is indeed the case. In this experiment, DIF was added to cells at levels which are suboptimal for stalk cell induction and the accumulation of pDd63 mRNA was analysed. There was a clear uncoupling of terminal stalk cell differentiation from gene induction with, for 1 2 4 6 8 12 o +- +- +- +- +- + - pDd63 pDd56 pDd26 Iff —5-8 kb •ft — 32kb — 0-75kb Fig. 1. The kinetics of induction of three mRNA sequences dependent upon DIF for their accumulation. A DIF induction was performed with HM44 under the standard conditions and cells were harvested at the indicated times. The symbol 0 indicates the time at which DIF was added (Fig. 1); the numbers indicate the number of hours of incubation in the absence (—) or presence (+) of DIF. Total cellular RNA was purified and 5jUg samples were electrophoresed through an agarose gel and transferred to nitrocellulose. The filters were hybridized with the indicated DNA which was labelled by nick translation. The size estimates derive from a separate experiment where the RNA samples were coelectrophoresed with RNA size markers. 748 K. A. Jermyn, M. Berks, R. R. Kay and J. G. Williams Table 1. The induction ofpDd63 mRNA accumulation by low levels of DIF Concentration of DIF (units ml" 1 ) Stalk cells induction (%)• 12 500 1250 125 12-5 100 15 5 0-7 Fraction of maximal pDd63 mRNA induction (%)t 100 30 20 3 * Normalized to the fraction of amoebae (75 %) forming stalk cells at the highest DIF concentration (12 500 units ml" 1 ). t Measured 4 h after the addition of DIF. example, a level of DIF sufficient to induce only 5 % stalk cell formation giving 20 % of the control level of pDd63 expression. The three cDNA clones hybridize to mRNA sequences which differ in their size, in their kinetics of accumulation and disappearance after DIF induction and in the totality of their dependence upon exogenous DIF. However, two of the sequences encode related proteins. A more heavily exposed autoradiogram of the hybridization with pDd56 reveals the presence of an RNA of higher molecular weight and reprobing of the same filter with pDd63 shows this to be coincident with the pDd63 mRNA (data not shown). The cross-hybridizing mRNA has precisely the same kinetics of accumulation as the pDd63 mRNA, both in the presence and absence of DIF. Washing at an elevated stringency abolishes the signal obtained using pDd56 (see Fig. 4B). Reciprocal cross-hybridization, i.e. of the pDd63 cDNA clone with the pDd56 mRNA, is not detected. This apparent anomaly is explained by recent partial sequence analysis of a genomic clone containing the pDd63 gene which shows the 5' proximal portion of the gene to have a higher degree of homology to the pDd56 mRNA than the 3' proximal portion (A. Ceccarelli, S. J. McRobbie, H. Mahbubani and J. G. Williams, unpublished results). Since the pDd63 cDNA clone contains only the 3' proximal half of the pDd63 mRNA it does not cross-hybridize to the pDd56 mRNA under these conditions. (B) The kinetics of accumulation of the three mRNA sequences during normal development is consistent with their being induced by DIF The three DIF-induced mRNA sequences display quite different kinetics of accumulation after DIF induction (Fig. 1). During normal development the major rise in DIF levels starts at about the tipped mound stage of development (Brookman, Town, Jermyn & Kay, 1982). If DIF acts to induce the accumulation of these sequences, they would be expected to show the relative order of appearance and kinetics of accumulation seen in the in vitro system starting after this stage. The data presented in Fig. 2A,B are from the strain V12M2, the parental strain of HM44, and from strain NC4. We used both strains because the timing of development is somewhat different, with strain V12M2 showing a more rapid progression through the early stages of differentiation and because we wished to compare the V12 parental strain with the more commonly used NC4 isolate. When correlated with the morphological changes occurring during development, both strains display similar kinetics of accumulation of the three mRNA sequences but there are clear differences. In V12M2, the mRNA hybridizing to pDd63 appears at the tipped aggregate stage of development (Fig. 2A). There is a rapid rise in expression to reach a peak at the slug stage and the mRNA then decreases in concentration to a very low level at culmination. In NC4 cells (Fig. 2B), the pattern of expression is similar but the mRNA persists at a high concentration until much later into development. (This is not the result of asynchrony in development as almost perfect synchrony was observed for NC4. The result probably underestimates the actual sharpness of the peak in V12M2 as it was not possible to obtain perfect synchrony with this strain and significant numbers of migrating slugs were present in the plates at the time culmination occurred in the majority of the aggregates.) The pDd56 mRNA also becomes detectable at the tipped aggregate stage in both strains. In V12M2 cells, it displays a sigmoidal accumulation curve similar to that observed upon DIF induction, with a very- low level initially and a sharp peak at the 'mexican hat' stage of development. The lag in NC4 cells is less pronounced, suggesting a difference in the kinetics of induction in the two strains. Again, as with the pDd63 mRNA, the pDd56 mRNA persists at a higher level late into the development of NC4 with no clear peak of accumulation being apparent. The mRNA hybridizing to the pDd26 mRNA is not detectable until the mexican hat stage of culmination in either NC4 or V12M2. Thus, the time course of accumulation of all three mRNA sequences matches their kinetics of appearance upon DIF induction very well. The only notable strain differences in developmental expression are the somewhat higher level of early accumulation of the pDd56 mRNA in NC4 cells and the more rapid disappearance of both the pDd63 and pDd56 mRNA sequences in V12M2 cells. Several cDNA clones have previously been described which hybridize to mRNA sequences selectively enriched in prestalk over prespore cells (Barklis & Lodish, 1983; Mehdy et al. 1983). These were Prestalk-enriched mRNAs in Dictyostelium 4 6 8 1 0 1 2 14 1 6 1 8 2 0 2 2 . 5 0^2 •ft 749 pDd63 .pDd63 pDd56 .pDd26 •*»# P Dd26 B NC4 10^ 12 JL 14 16 \ •« 18 •«* 20 • • M, 2 2 . 5 . -D11 Prestalk -pDd63 Fig. 2. The time course of accumulation of the three DIF-induced mRNA sequences during normal development. Amoebae of the strain V12M2 (Panel A), or NC4 (Panel B,C) were grown and set up for development as described in the Methods section. Development of the NC4 cells was almost perfectly synchronous with no prolonged period of slug migration. However, it is not possible to achieve such a high degree of synchrony with the strain V12M2 and there were significant numbers of migrating slugs in all plates harvested after the 12th hour of development. The symbols above the time points represent the morphological stages. For NC4 these were lOh, tight aggregates; 12h, tipped aggregates; 14h, first fingers; 16h, mexican hats; 18h, preculminants; 20h, midculminants; 22-5 h, culminants. For V12M2 these were 6h, tight aggregates; 8h, tipped aggregates; 10h, first fingers; 12h, standing slugs (with some slugs); 14h, mexican hats (with some slugs); 16h preculminants (with some slugs); 18h midculminants (with some slugs); 20h culminants (with some slugs). In the experiment shown in Panel C, the filter was cut transversely, after transfer. The two portions of the filter were separately hybridized with a pDd63 or a Dll probe in order to eliminate loading artefacts. This exposure of the autoradiogram shows a signal for both pDd56 and pDd63 only at the first finger stage but a longer exposure of the same filter shows these to be expression of both genes in tipped aggregates (data not shown). 750 K. A. Jermyn, M. Berks, R. R. Kay and J. G. Williams selected by differential screening with mRNA isolated from cells fractionated on density gradients (Tsang & Bradbury, 1981; Ratner & Borth, 1983). In addition to correlating expression of the DIF-inducible mRNA sequences with morphological stages, we have also determined their time of expression relative to D l l , a prestalk gene typical of previously isolated sequences. The pDd63 and pDd56 mRNA sequences are first detectably expressed approximately 2h later than the major rise in concentration of the Dll mRNA (Fig. 2C). (C) A class of transcripts, which become enriched in prestalk cells, does not require DIF for their expression We have determined the effect of DIF on a number of the mRNA sequences previously described as prestalk-enriched and find a variable pattern of response. The cysteine proteinase 2 mRNA encodes a Dictyostelium protein homologous to the sulphydryl proteinase of higher eukaryotes (Pears, Mahbubani & Williams, 1985). It is totally absent from vegetative cells, begins to accumulate late during cellular aggregation and is prematurely expressed in response to exogenous cAMP. In previous work from this laboratory, it has been reported to be approximately threefold enriched in prestalk over prespore cells (Pears et al. 1985), but we have more recently obtained a somewhat lower estimate of a twofold enrichment using a different gradient fractionation procedure (Pears & Williams, 1987). This gene has been independently isolated and characterized elsewhere (Mehdy et al. 1983; Datta, Gomer & Firtel, 1986) and the protein has been shown to be specifically localized in prestalk cells (Gomer, Datta & Firtel, 1986). Since the mRNA is expressed at a high relative level in prespore cells it may be that it is selectively translated or the protein may be differentially stable in the two cell types. The mRNA is present at t0 in the standard induction (i.e. the time of DIF addition) and, in the absence of DIF (Fig. 3), it displays approximately the same kineticsof disappearance as in normal development (Pears et al. 1985). The effect of DIF is to accelerate disappearance of the mRNA from the cells, presumably as a result of their differentiation into stalk cells. The prestalk-enriched mRNA sequences isolated by Barklis and Lodish have been shown to fall into two classes termed Prestalk 1 (Pstl) and Prestalk 2 (Pst2) (Barklis & Lodish, 1983; Chisholm, Barklis & Lodish, 1984). These two classes differ in their time course of accumulation and in their response to cellular disaggregation and incubation with exogenous cAMP. The Dll (Pstl) gene encodes a hydrophilic, cysteine-rich protein of unknown function (Barklis, Pontius & Lodish, 1985). The mRNA is expressed at 0 1 8 12 „ Time after -DIF addition Cysteine proteinase 2 -D11 • I »^» ••••V •PL1 2H6 -Ras Fig. 3. The effect of DIF upon previously characterized prestalk-enriched mRNA sequences. A Northern transfer was performed with the same RNA samples used for the experiment described in Fig. 1. The filter was hybridized with the indicated DNA probes and washed under the standard conditions (see Methods section) before autoradiography. The probe for the ras mRNA was a synthetic oligonucleotide of 23 residues in length with the sequence 5' CGATAACTATCTTCAATTGTTGG 3'. This is the complement of a sequence located in the 5' proximal coding portion of the ras mRNA (Reymond et al. 1984). The oligonucleotide was 5' end labelled using T4 polynucleotide kinase and y-32P-ATP. a low level during vegetative growth in axenic medium, accumulates to a 10- to 20-fold higher level during aggregation (Barklis & Lodish, 1983; Chisholm et al. 1984) and is induced in response to exogenous cAMP (D. Driscoll, personal communication; Oyama & Blumberg, 1986). The mRNA is present at t0 in a DIF induction but it differs from the cysteine proteinase 2 mRNA in that the concentration remains constant in the absence of DIF (Fig. 3). In contrast to cysteine proteinase 2, DIF has absolutely no effect on the concentration of the Dll mRNA. Since the cells undergo terminal differentiation into stalk cells during this time period, these results would suggest that the D l l mRNA is Prestalk-enriched mRNAs in Dictyostelium expressed in mature stalk cells and this is confirmed below. The PL1 mRNA is a Pst2 transcript, encoding a protein of unknown function. It is reported to be enriched in prestalk over prespore cells (Barklis & Lodish, 1983) but we do not find this to be the case (see below). In contrast to the D l l mRNA sequence, the PL1 mRNA is expressed only after the loose aggregate stage of development and the mRNA is not inducible with cAMP (Oyama & Blumberg, 1986). The PL1 mRNA is not expressed at t0 in a DIF induction (Fig. 3), a result consistent with its late stage of expression during normal development and its nonresponsiveness to cAMP. However, after 4-6 h of incubation in the presence of DIF-1, the PL1 mRNA is expressed. This observation suggests that the PL1 mRNA may play a role in the terminal stages of stalk cell differentiation and its presence in mature stalk cells (see below) is consistent with this hypothesis. The 2H6 cDNA clone hybridizes to a mRNA of unknown coding potential, which is enriched in prestalk cells (Mehdy et al. 1983) and which seems to show the properties of a Pstl mRNA. This mRNA is expressed at a finite level at t0 in the DIF induction and increases in abundance at 4-6 h after DIF addition (Fig. 3). This is the time of overt stalk cell differentiation and we find that this mRNA sequence is expressed at culmination in stalk cells; although in contrast to PL1, which shows similar kinetics of induction, the 2H6 sequence is also expressed in mature spore cells. The Dictyostelium ras gene is expressed during vegetative growth; the mRNA declines in abundance during early development and reaccumulates late during aggregation when it is reported to be enriched in prestalk over prespore cells (Mehdy et al. 1983; Reymond, Gomer, Medhy & Firtel, 1984). The ras mRNA is expressed in HM44 cells at t0 in the induction and DIF has no effect on accumulation of the mRNA (Fig. 3). Thus, the cysteine proteinase 2, D l l , ras and 2H6 mRNA sequences, which are enriched in prestalk over prespore cells, share one important common feature with respect to DIF induction. They are all expressed at high levels in HM44 cells incubated in submerged conditions in the presence of cAMP for 10h (i.e. t0 in the standard induction conditions). Hence, in this in vitro system at least, they do not require DIF for their expression. (D) The DIF-induced mRNA sequences are more highly enriched in the prestalk-stalk cell pathway of differentiation than previously described sequences We have determined the relative degree of enrichment of these different mRNA sequences in purified 751 Table 2. The relative degree of enrichment of prestalk and prespore mRNA sequences in purified cells mRNA sequence pDd63 pDd56 pDd26 Dll PL1 ras 2H6 cysteine proteinase 2* D19t Approximate prestalk/prespore ratio Approximate stalk/spore ratio S10:l 3=10:1 Not expressed 4:1 1:1 3:1 5:1 1-5:1 50:1 50:1 50:1 50:1 50:1 6:1 1:1 1:1-5 s=l:10 N.D. *Data derived from Pears & Williams (1987). tD19 is a prespore 2 clone (Barklis & Lodish, 1983) which was used to monitor the purity of the prestalk fraction. prestalk and prespore cells. Migrating slugs were fractionated by manual microdissection or, after disaggregation, by Percoll gradient centrifugation (Ratner & Borth, 1983). Microdissection yields an anterior, prestalk fraction, which should in principle be totally devoid of prespore cells. The posterior portion of slugs is predominantly composed of prespore cells but about 10-15 % of the cells display the morphological and biochemical properties of prestalk cells and hence are termed 'anterior-like cells' (Sternfeld & David, 1981, 1982; Devine & Loomis, 1985). Based upon staining with a prespore-specific antiserum, the prespore cells purified by density centrifugation were estimated to be between 5 and 10 % contaminated with prestalk cells. The degree of enrichment observed for the various mRNA sequences was very similar using RNA isolated from cells purified by either of the two methods. The Dll, the PL1 and the ras mRNA sequences all show a lower prestalk enrichment than previously reported. The most extreme example is PL1, which we find to be present at approximately equal levels in prespore and prestalk cells (Fig. 4A, Table 2). We do not understand the reason for this contradiction but it appears unlikely to be due to a strain difference, since M. Oyama and D. Blumberg (personal communication) have obtained similar results to ours using slugs of the strain NC4. Hence, it may reflect some difference in conditions of development or gradient fractionation. The cysteine proteinase 2, D l l , 2H6 and ras mRNA sequences are somewhat prestalkenriched. We have previously estimated the cysteine proteinase 2 mRNA to be approximately threefold to fourfold enriched using gradient fractions prepared by the method of Tsang & Bradbury (1981), but we obtain a slightly lower estimate of a twofold enrichment using cells fractionated by the gradient pro- 752 K. A. Jennvn. M. Berks, R. R. Kav and]. G. Williams A Dissection Gradient Pst PspPstPspSt Sp I I I I I I _pDd63 — D11 -PL1 §!••• — Ras pDd26 D19 B PstPspStSp Low w stringency 49 High * stringency w • IB pDd63 pDd56 pDd63 V pDd56 Fig. 4. A comparison of specific mRNA abundance in prestalk and prespore cells. Prestalk. prespore. stalk and spore cells were purified as described in the Methods section and a 5,ug sample of the RNA from each cell population was analysed by Northern transfer using the indicated probes (Fig. 4A). The D19 cDNA clone hybridizes to a prespore-enriched mRNA sequence (Barklis & Lodish. 1983) and is included as a measure of the purity of the prestalk fraction. The filter hybridized with pDd56 (Fig. 4B) was washed firstly at low (3xSSC. 50% formamide. 37°C) and then at high stringency (0-6XSSC. 50% formamide. 37°C). cedure of Ratner & Borth (1983) and by slug-cutting (Pears & Williams, 1987). We find the ras mRNA sequence to be only approximately threefold enriched in prestalk over prespore cells (Fig. 4A. Table 2). The D l l mRNA sequence is fourfold to fivefold enriched in prestalk over prespore cells but this is again a lower enrichment than previous results suggest (Barklis & Lodish, 1983). A proportion of the apparent expression of these mRNA sequences in prespore cells could result from contamination with prestalk cells. There must, however, be a finite level of expression of these genes in prespore cells because we can show, using these same fractions, that the pDd63 mRNA sequence is much more highly enriched in prestalk over prespore cells (Fig. 4A. Table 2). In a large series of experiments, using many different preparations of purified cells, we have established that the low level of apparent prespore expression we observe for the pDd63 mRNA can be accounted for entirely by contamination with prestalk or anterior-like cells (Williams et al. 1987). The pDd56 mRNA is expressed in slugs but at submaximal levels. We have not determined the extent of enrichment to the same degree of precision but, using gradient-purified fractions, we find the pDd56 mRNA is enriched in prestalk over prespore cells. In the experiment shown in Fig. 4B. we simultaneously assayed the relative degree of enrichment of both the pDd56 and pDd63 by hybridizing with the pDd56 probe at low stringency. As discussed above, the two sequences share sufficient sequence homology to cross-hybridize under these conditons. This result clearly shows the pDd56 mRNA is as highly prestalk enriched as the pDd63 mRNA. The pDd26 mRNA sequence is not expressed at a detectable level in the slug. This sequence is detectable in stalk cells isolated from fruits in the terminal stages of culmination, but it is not detectable in purified spore cells (Fig. 4A). The pDd63 and pDd56 mRNA sequences are also expressed in stalk, but not in spore cells (Fig. 4A.B). The D l l and PL1 mRNA sequences are both highly enriched in stalk over spore cells, but the 2H6 and ras mRNA sequences are expressed at a significant level in both cell types (Fig. 4A) as is the cysteine proteinase 2 mRNA (Pears & Williams, 1987). Discussion During normal development on agar. the mutant strain HM44 becomes arrested after aggregation at a stage prior to slug formation (Kopachik et al. 1983). Cells within the aggregate have been shown to express morphological and biochemical markers characteristic of prespore cells (Kopachik et al. 1983. Prestalk-enriched mRNAs in Dictyostelium 1985). The prestalk-specific isozyme of acid phosphatase does not accumulate in HM44 cells in the absence of DIF but is produced in the presence of DIF (Kopachik et al. 1983). This result provides strong support for a major role of DIF in directing prestalk cell differentiation. In apparent contrast to this result, we observe a high level of expression of several prestalk-enriched mRNA sequences in HM44 cells developing in the absence of DIF. We believe the explanation for this apparent paradox lies in the two different mechanisms whereby a product may come to be enriched within a specific cell type; differential accumulation or differential loss. The most compelling evidence for selective loss of a gene product in prespore cells derives from several monoclonal antibodies which stain a major fraction of the cell population early during aggregation but which stain only prestalk cells in the migrating slug (Tasaka, Noce & Takeuchi, 1983; Krefft, Voet, Gregg & Williams, 1985). The D l l , cysteine proteinase 2, 2H6 and ras genes are expressed prior to tip formation and they are rapidly induced by cAMP which may, therefore, act as the initial trigger for their accumulation during normal development (Barklis & Lodish, 1983; Mehdy et al. 1983; Pears et al. 1985). Only the most extreme cell-sorting models for pattern formation in Dictyostelium involve cellular differentiation concomittant with the initiation of cAMP signalling. Hence, it seems reasonable to assume that transcripts of these genes are present at equal concentrations in the precursors of both prestalk and prespore cells. It is then only necessary to assume a differential loss in prespore cells, due to an altered rate of transcription or post-transcriptional processing, to account for the different mRNA concentrations observed in the two cell types. Since cAMP is present in the in vitro assay with HM44, we would expect these genes to be expressed and, with the above interpretation of their prestalk-enrichment, their nonresponsiveness to DIF would be explained. There is, of course, no direct evidence that these genes are expressed in all cells during aggregation. It may be that there is a subset of aggregating cells, fated to become stalk cells, which expresses the genes at a higher level than presumptive prespore cells. However, such a model requires that the genes later be turned on in 'definitive' prespore cells, where we observe their expression, or alternatively that the subset of expressing cells sediments with prespore cells on a gradient. Given the existence of other 'prestalk' markers initially expressed in most or all cells (Tasaka et al. 1983; Krefft et al. 1985), we feel it reasonable to suggest differential loss from prespore cells for these mRNA sequences. Support for this suggestion derives from our identification of pDd63 and pDd56, two mRNA sequences which are more 753 highly enriched in prestalk over prespore cells than these other sequences and which differ radically in their mode of induction and in their time of appearance during development. The level of expression in prespore fractions of pDd63 and pDd56 is low enough to be accounted for by prestalk cell contamination and thus they are the first potentially presta\k-speciftc mRNA sequences to be identified. As such, their time of appearance during normal development can be used as a definitive marker for the appearance of prestalk cells. The pDd63 and pDd56 mRNAs are first detectably expressed at the tipped aggregate stage of development, several hours later than the cysteine proteinase 2 (Pears et al. 1985) Dll, ras and 2H6 mRNA sequences (Fig. 4C). Again, this is consistent with our suggestion that these latter mRNA sequences accumulate in all cells, becoming selectively enriched in prestalk cells. The early appearance of such sequences has led to the suggestion that prestalk cell differentiation occurs very early during development. If we are correct in our interpretation of the mechanism whereby these mRNA sequences become enriched, then this conclusion is rendered invalid. Similarly, they cannot safely be used to assess the response of prestalk cells to inducing agents such as cAMP or DIF, or to determine the effect of other potential control signals such as cell contact (Chisholm etal. 1984). Of course, we cannot exclude the existence of prestalk-specific markers expressed earlier in development than pDd56 and pDd63. There are cells at the tight aggregate stage of development which do not express prespore-specific antigens (Takeuchi, Hayashi & Tasaka, 1977; Krefft et al. 1984) but there is no direct proof from fate mapping that these are the precursors of prestalk cells. There is also no indirect evidence from protein markers, because analysis of proteins by two-dimensional gel electrophoresis has shown that, while there is continuity in the genes expressed on the prespore-spore cell pathway of differentiation, this is not the case on the prestalkstalk pathway (Ratner & Borth, 1983; Morrissey etal. 1984). This has led to the suggestion that most prestalk-enriched proteins are involved in processes such as cAMP signal transduction and other processes peculiar to the slug stage (Morrissey, 1982). There is now good evidence that the ras mRNA sequence, which we confirm to be marginally enriched in prestalk over prespore cells, encodes a protein which is involved in signal relay (Reymond et al. 1986). The pDd63 and pDd56 mRNA sequences appear, therefore, to be exceptional in that they are expressed in both prestalk and stalk cells. The pDd56 and pDd63 genes encode closely related, cysteine-rich proteins composed of a tandem array of a simple peptide 754 K. A. Jermyn, M. Berks, R. R. Kay and J. G. Williams repeat (A. Ceccarelli, S. J. McRobbie, H. Mahbubani & J. G. Williams; unpublished results). These are characteristic features of extracellular structural proteins of lower eukaryotes and we have preliminary evidence that these two proteins form part of the stalk wall of Dictyostelium (S. J. McRobbie & J. G. Williams, unpublished results). The pDd56 and pDd63 mRNA sequences are rapidly induced in response to DIF but they differ in their precise kinetics of accumulation and in their level of expression in the absence of DIF. The pDd56 mRNA displays a sigmoidal accumulation curve and is totally dependent upon DIF for its accumulation. The pDd63 mRNA accumulates rapidly with linear kinetics and there is a low level of expression in the absence of DIF which we believe to be the result of slight leakiness in the DIF-deficient mutant. The difference in response to DIF may not reflect a fundamental difference in mechanism of induction. Both mRNA sequences increase in concentration within 1 h of DIF addition and both sequences display more similar patterns of accumulation during normal development of the strain NC4. The lag observed for pDd56, in the in vitro system with HM44, may, therefore, reflect its derivation from the strain V12M2. 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