A marker of terminal stalk cell terminal differentiation in Dictyostelium

Differentiation (1997) 61:223–228

© Springer-Verlag 1997

O R I G I NA L A RT I C L E

&roles:Vicky Robinson · Jeffrey Williams

A marker of terminal stalk cell terminal differentiation in Dictyostelium

&misc:Accepted in revised form: 27 November 1996

&p.1:Abstract We describe the isolation of staB, a Dictyostelium gene that is selectively expressed in stalk cells. If an aggregate enters culmination immediately staB is expressed in the bottom half of the stalk and in the basal disc but if a migratory slug is formed it is also expressed in the top of the stalk and in a disc of cells that surmounts the spore head. The latter two populations of cells derive from the rear part of the prestalk region, the pstO cells, which would therefore seem to change their transcriptional competency during slug migration. The staB promoter contains a distal region that is required for expression within both the stalk and the basal disc and a proximal region that is required only for expression within the basal disc. This uncoupling of transcriptional specificities suggests that the signalling mechanisms that direct terminal differentiation in these two tissues differ in some way.&bdy:

Introduction The Dictyostelium culminant consists of a mass of spores atop a stalk that is embedded into a supporting structure called the basal disc. Both the stalk and the basal disc are composed of dead, highly vacuolated cells but the cells within the two structures have different origins. The stalk derives from the pstA and pstO cells, the two sub-types of prestalk cell that respectively comprise the front and back halves of the prestalk region and which are defined by their ability to utilise different parts of the promoter of the ecmA gene [4]. The basal disc derives from the pstB cells, a band of prestalk cells within the body of the V. Robinson1 Imperial Cancer Research Fund, Clare Hall Laboratory, S. Mimms, Herts, EN6 3LD, UK J. Williams (✉) MRC Laboratory of Molecular Cell Biology and Department of Biology, University College London, Gower St, London WC1E 6BT, UK 1 Present

address: The National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AR, UK&/fn-block:

slug that express the ecmA gene at a very low level but which express the closely related ecmB gene at a much higher relative level [3, 14]. At culmination, the slug sits on end and the prestalk cells move up to the apex and then down through the spore mass. Because movement into the stalk tube occurs in an ordered fashion, and because of their relative starting positions within the prestalk region, the pstA cells form the bottom of the stalk while the pstO cells form its top [4]. As they start their downward movement into the stalk tube the pstA cells activate expression of the ecmB gene [1, 13]. In approximately one half of the pstO cells activation of the ecmB gene also occurs at the stalk tube entrance but in the other half the ecmB gene is activated in situ within the papilla [13]. The latter population are known as the upper cup cells. Expression within the stalk tube and within the upper cup cells are directed by different parts of the ecmB promoter [1]. Simultaneously with the onset of these events in the papilla, the band of pstB cells moves underneath the tip to initiate formation of the basal disc [3, 14]. The DNA sequence elements within the ecmB promoter that direct expression within the basal disc have yet to be identified. Both the ecmA and the ecmB genes are inducible by the stalk-cell-specific morphogen DIF and were isolated by virtue of this property [11]. The differential screening procedure which yielded the ecmA and ecmB cDNA clones produced a third differentiation-inducing factor (DIF)-inducible cDNA, termed pDd26. It differs from ecmA and ecmB in a number of respects. In monolayer assays the pDd26 gene is much slower to respond to DIF and its transcripts also accumulate much later during normal development [11]. The pDd26 mRNA is absent from prestalk cells but is expressed in stalk cells and has been used as a marker of stalk cell differentiation in a number of studies [16, 20]. Because of its value as a marker of terminal stalk cell differentiation, we set out to isolate a genomic clone for pDd26. In the event we cloned a gene, staB, that is closely related to pDd26 and which is also selectively expressed late during stalk cell differentiation. (N.B. Because there

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is now a general agreement to apply the Demerec nomenclature to Dictyostelium genes we propose renaming pDd26 as staA (stalkA), hence the name staB for the new gene. For the sake of clarity we sometimes use the term pDd26 in this paper). We have used staB:lacZ promoter fusions to investigate the process of culmination, and the results shed new light on the nature of differentiation within the stalk and within the basal disc.

Methods Cell culture and development AX-2 cells [21] were cultured and transformed as described previously [5]. For development cells were washed three times in KK2 (16.5 mM KH2PO4, 3.8 mM K2HPO4, pH 6.2) and spread onto 0.45-µm nitrocellulose filters (Millipore Co., Bedford, U.K.) or on to 2% non-nutrient Bacto agar (Difco Laboratories, Detroit, USA.) plates. Incubations were at 22°C in a humid box. When slugs were to be formed incubation was again on water agar and in a humid box with a single slit that created a unidirectional light source. Gene isolation, sequence analysis and construction of promoter fusions to lacZ A library of Dictyostelium genomic DNA, prepared by partial digestion with Sau3A and cloning into a plasmid vector, was

Fig. 1 Nucleotide sequence of the minimal promoter region and coding region of the staB gene. The gene was isolated as a partial Sau3A fragment that contains 1.6 kb sequence upstream of the translational initiation codon. Normal activity is retained when only 527 nucleotides of upstream sequence is present and therefore only this part of the promoter sequence is presented. The coding region of staB contains only five substitutions with respect to the pDd26 cDNA [17] and these are underlined. However, staB is clearly a different gene because the sequences of the 3’ noncoding regions differ greatly (we have not mapped the 5’ end of the staB mRNA, so we do not know the exact extent of the 5’ noncoding region)&ig.c:/f

screened with the entire insert from the pDd26 cDNA clone [17]. The genomic clone that was isolated, staB, was used as a template for dideoxy sequencing and polymerase chain reaction (PCR). Restriction enzyme recognition sites were incorporated at the ends of primers used for PCR, which was performed over 20 cycles in a 100-µl reaction volume containing 10 ng template, 500 ng each primer, 200 nM each dNTP, 1 X PCR buffer (10 mM Tris-HCl, 4.5 mM MgCl2, 50 mM KCl, 0.1 mg/ml gelatine, pH 8.3) and 1.5 units Taq DNA polymerase (Boehringer-Mannheim, Lowes, U.K.). Promoter sub-fragments generated by PCR were phenol:chloroform extracted, ethanol precipitated and cut with the appropriate restriction enzyme. Following restriction, the fragments were phenol:chloroform-extracted, ethanol-precipitated, ligated into the lacZ vectors pDdGal17 [8] or actin15∆Bam:gal [1] and transformed into Eschericia coli. In order to guard against artefactual results because of the PCR step, two independent isolates of each construct were used for Dictyostelium transformation. In order to avoid problems with clonal variation due to copy number and/or integration site of the construct, pooled populations of individual transformants were analysed. Analysis of β-galactosidase gene expression Aggregates at the desired developmental stage were fixed in 1% glutaraldehyde in Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 2 mM MgCl2) for 20 min, after which time the fixative was removed and the samples were washed twice in Z buffer. Samples were then incubated in staining solution (Z buffer containing 5 mM K3(Fe(CN)6), 5 mM K(Fe(CN)6) and

Fig. 2 For southern transfer analysis of genomic DNA using pDd26 as a probe, 10 µg of Dictyostelium genomic DNA was digested with BglII, subjected to agarose gel electrophoresis and transferred to a nylon membrane. The filter was probed with an asymmetric polymerase chain reaction (PCR) product covering nucleotides 21–456 of the pDd26 coding region, i.e. almost the entire coding sequence [17]). The blot was washed under conditions (0.1 × SSC at 65°C) predicted to be near the Tm. The pDd26 gene does not contain a site for BglII, and therefore a single band is expected if there is a single-copy gene. The presence of two bands suggests there to be two closely related genes, one of which is presumably staB. The alternative explanation, that there is a BglII site in an intron within the pDd26 gene cannot be definitively ruled out, but seems relatively unlikely because introns in Dictyostelium genes are very short and extremely AT-rich. Also, the staB and pDd26 genes are so closley related, with only five nucleotides different, they would definitely cross-hybridise under these conditions so we predict at least two hybridising bands&ig.c:/f

Marker Size (kb) Origin

6.5

3.5

2.0

1.6 1.4

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B

A

C Fig. 3A–D Staining patterns of lacZ fusion gene constructs. A The staining pattern when the entire 1.6-kb upstream region of staB was fused to lacZ and cells were developed on nitrocellulose filters. The entire staB promoter region of 1.6 kb was isolated by PCR using a primer that annealed to the staB coding region creating a BglII site immediately downstream of the intiation codon. The PCR fragment was cloned as an XbaI-BglII fragment into the Dictyostelium G418 resistance vector pDdGal17, to create an inframe fusion with the E. coli lacZ gene [8]. The vector was introduced into Dictyostelium and a stable transformant that expresses the gene was selected, allowed to develop on nitrocellulose filters until culmination was complete and stained overnight at 37°C with X-gal [3]. B The base of a culminant containing cells transformed with a lacZ fusion to the entire staB upstream region. This trans-

D formant contains an analogous construct to that shown in A, except that the lacZ protein contains a nuclear localisation signal at its extreme N terminus [12]. It was allowed to develop and analysed as described in A. C Culminants containing cells transformed with a lacZ fusion to the entire staB upstream region and allowed to develop on agar. Transformant cells containing the construct described in A were developed on 2% water agar plates in the dark and allowed to migrate as slugs for approximately 12 h. They were then induced to culminate, by exposure to overhead light, and fixed and stained at the very end of culmination. Much more of the stalk is stained than in A, and there is a stained structure outside the stalk (top left) that we term the apical disc. D A high-power view of the top of a structure similar to that shown in C&ig.c:/f

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A

Fig. 4 Summary of lacZ fusion gene constructs and deduced organisation of the staB promoter Constructs A and B were made by a strategy similar to that described for the entire promoter (legend to Fig. 3A) while constructs C and D were made by cloning PCR fragments upstream of the TATA box and cap site of an actin 15:lacZ fusion gene (as described in [1]). In each case two separately isolated constructs were transformed into Dictyostelium to safeguard against PCR artefacts&ig.c:/f 1 mM X-gal) at 37°C in a humid chamber, for various times, before mounting and photography.

B

Results 1. Isolation and DNA sequence determination of the staB gene We screened a genomic library with the insert from the pDd26 cDNA clone and isolated a very closely related gene that we call staB (stalkB). The predicted coding region of the staB gene contains only five nucleotide differences from that of the pDd26 gene but the two genes differ considerably in the sequences of their 3’ non-coding regions (Fig. 1 and [17]). These data suggested the existence of at least two closely related genes. Hence we performed Southern transfer analysis, using pDd26 as a probe and cleaving Dictyostelium genomic DNA with a restriction enzyme that does not cut within the pDd26 coding region. This analysis was performed at high stringency and is consistent with there being two, highly related genes within the Dictyostelium genome (Fig. 2 and legend). Because of the extreme similarity of its coding region, we assume that the gene that cross-hybridises to the pDd26 probe is staB. 2. The expression pattern of the staB gene inferred from a promoter fusion to the lacZ gene In order to generate a reporter fusion, the 5’ non-coding region and 1.6 kb upstream sequence from staB were fused to the lacZ gene. Using cells stably transformed with this construct there was no detectable staining prior to culmination (data not shown). When development was

C Fig. 5A–C Analysis of expression directed by subregions of the staB promoter. A The base of a culminant containing cells transformed with construct A. A pooled population of transformants containing construct A (Fig. 4) was developed on nitrocellulose filters and analysed as described in Fig. 3A. B The base of a culminant containing cells transformed with construct C. A pooled population of transformants containing construct C was developed on nitrocellulose filters and analysed as described in Fig. 3A. C Culminants containing cells transformed with construct D. A pooled population of transformants containing construct D was developed on nitrocellulose filters and analysed as described in Fig. 3A. The top and bottom halves of two separate culminants have been aligned in this figure, but this low-level, scattered staining is typical of all structures observed&ig.c:/f

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performed on nitrocellulose filters, conditions that are not permissive for migratory slug formation, β-galactosidase expression directed by staB:lacZ occurred in the bottom half of the stalk and in the basal disc (Fig. 3A, B; N.B. In the structure shown in Fig. 3B the β-galactosidase protein contains a nuclear tag to make the distribution of expressing cells clearer). When development was performed on agar, conditions that are permissive for slug formation, most of the stalk was stained (Fig. 3C, D). N.B. The region of the stalk within the spore head of the structure in the centre of Fig. 3C was very weakly stained because the spores interfere with fixation and staining (V. Robinson, unpublished observations) but in the structure at the right, where most of the spores were lost during fixation, staining extends all the way through the stalk). A structure we propose terming the apical disc, which is comprised of the very last cells to enter the stalk, was also stained when development is performed on agar (Fig. 3D). 3. Dissection of the staB promoter We next analysed expression directed by subfragments of the staB promoter, using filter development, the condition where the intact promoter directs expression in the lower stalk and the basal disc. This pattern of expression was retained when the promoter was deleted from its 5’ end such that only 527 nucleotides remained (Fig. 4 construct A, Fig. 5A). The sequence of this minimal promoter region is presented in Fig. 1. When a further 150 nucleotides were removed the promoter was rendered entirely inactive (Fig. 4 construct B; data not shown). Thus there are sequences between –527 and –377 (Fig. 4, promoter region 1) that are essential for expression in both the stalk and the basal disc. When 184 nt were deleted from the 3’ end of the minimal promoter region, defined by 5’ deletion (i.e. from a promoter fragment with its 5’ end at –527), expression within the basal disc was eliminated while expression within the stalk was retained (Fig. 4 construct C, Fig. 5B). A further 3’ deletion, to nucleotide –347 (Fig. 4 construct D) completely eliminated cell-type-specific expression but a few stained cells were present, scattered throughout the fruiting body (Fig. 5C).

Discussion Analysis of Dictyostelium genomic DNA by high-stringency hybridisation using pDd26 as a probe suggests there to be a second, very closely related gene, and we describe its isolation and characterisation. There are only five nucleotide differences between the coding portions of the pDd26 (staA) and staB genes, suggesting either a very recent gene duplication or a very high rate of intergenic recombination. Given this high degree of nucleotide homology, and assuming a similar transcript size, it would be very difficult to determine the time courses and

cell-type specificities of expression of the two genes by Northern transfer. Indeed all previous results obtained using pDd26 as a probe must be re-evaluated, because the gene being analysed may very well have been staB. However, this is likely not to be a major defect of previous studies, because a promoter fusion in which the staB promoter was coupled to the lacZ gene shows that the pattern of expression of staB is very similar to that inferred for pDd26. Northern transfer suggested that the pDd26 gene is expressed late during development and selectively in the stalk [11]. This also holds true for staB, but the abilty to obtain spatial information from the β-galactosidase analysis allowed us to show that, when cells are developed on nitrocellulose filters, the staB gene is expressed only in the bottom half of the stalk. When slugs are allowed to migrate before entering culmination the top of the stalk is also stained. Under these conditions there is also staining in a structure we propose terming the apical disc, which presumably corresponds to the button of apical staining that is very often seen with neutral-red-stained cells. The top half of the stalk tube derives from the pstO cells [4] and this presumably also holds true for the apical disc. It would seem, therefore, that newly differentiated pstO cells are not competent to transcribe the staB gene, but they acquire this competency during slug migration. In contrast, the pstA cells seem to be able to transcribe the staB gene immediately after they differentiate. Interestingly, by deleting the binding site for a transcriptional activator, it is possible to generate a construct from the ecmB gene that phenocopies the staB gene in being expressed selectively in the bottom of the stalk. G boxes are short GT-rich sequences that are present in the promoters of many genes expressed late during aggregation and thereafter (reviewed in [18]). They act to amplify transcription that is directed by other, cell-type-specific, elements and they form binding sites for G-box binding factor C (GBF), a zinc-finger-containing protein [18]. The cap-site-proximal 877 nt of the ecmB promoter directs expression throughout the stalk, but deletion of a promoter-proximal region that contains a G box leads to expression only within the bottom of the stalk [2]. While weakening the ecmB promoter, by deleting a G box, results in a restriction of expression to the bottom of the stalk, we do not believe that this implies an identical mechanism of activation for the staB gene, because we cannot detect any homology to elements known to be important in ecmB gene regulation. Expression of the ecmB gene within the stalk is regulated by a repressor protein, but there is no good fit to the consensus binding site for the ecmB repressor (TTGnCAA, where n = 2 or 4, [7]) within the 527 nucleotides that direct stalk-specific expression in the staB gene. Activation of ecmB gene expression also requires a GA-rich sequence (A. Ceccarelli, J.A. Kirk, J.G. Williams manuscript in preparation) but again we can find no homology to this sequence within the minimal region of the staB promoter. The lack of obvious homology between the ecmA, ecmB and staB promoters is perhaps not too surprising

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because the staB gene differs in being very much more slowly induced by DIF [11]. We interpret this to mean that staB is expressed within a cell as an eventual consequence of its becoming a stalk cell, rather than as a direct consequence of its initial commitment to stalk cell differentiation. The fact that de novo protein synthesis is needed before cells treated with DIF can express the pDd26 gene is consistent with this idea [20]. Irrespective of the precise mechanism of activation, the staB gene and the promoter construct that is expressed only in stalk cells (Fig. 4, construct C) will both be valuable as markers of terminal differentiation. Finally, our results show that a different combination of sequence elements is required for stalk-specific gene expression than is required for basal-disc-specific gene expression. Promoter regions 1 and 3 are necessary for expression within the stalk while basal disc expression requires sequences within region 1 and region 4 (Fig. 4, lower part). This data is consistent with previous evidence suggesting that different signalling pathways may be active in these two cell types. The signalling pathway that directs pre-basal disc differentiation contains gskA, the Dictyostelium homologue of glycogen synthase kinase 3 (GSK3) [6], and cAMP-dependent protein kinase lies on the signalling pathway that directs differentiation within the stalk [9, 10, 15, 19]. Again, however, the fact that activation in the pstB cells is a late event implies that expression of staB is likely to be a secondary event in the inductive process. &p.2:Acknowledgements This work was intially supported by the Imperial Cancer Research Fund and was completed with the support of the Wellcome Trust through Program Grant 039889 to J.G.W.

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