Folic acid stimulation of the Gα4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium

Differentiation (1999) 64:195–204

© Springer-Verlag 1999

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

&roles:Jeffrey A. Hadwiger · Jaishree Srinivasan

Folic acid stimulation of the Gα4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium

&misc:Accepted in revised form: 11 February 1999

&p.1:Abstract In Dictyostelium discoideum, several G proteins are known to mediate the transduction of signals that direct chemotactic movement and regulate developmental morphogenesis. The G protein α subunit encoded by the Gα4 gene has been previously shown to be required for chemotactic responses to folic acid, proper developmental morphogenesis, and spore production. In this study, cells overexpressing the wild type Gα4 gene, due to high copy gene dosage (Gα4HC), were found to be defective in the ability to form the anterior prestalk cell region, express prespore- and prestalk-cell specific genes, and undergo spore formation. In chimeric organisms, Gα4HC prespore cell-specific gene expression and spore production were rescued by the presence of wildtype cells, indicating that prespore cell development in Gα4HC cells is limited by the absence of an intercellular signal. Transplanted wild-type tips were sufficient to rescue Gα4HC prespore cell development, suggesting that the rescuing signal originates from the anterior prestalk cells. However, the deficiencies in prestalk-specific gene expression were not rescued in the chimeric organisms. Furthermore, Gα4HC cells were localized to the prespore region of these chimeric organisms and completely excluded from the anterior prestalk region, suggesting that the Gα4 subunit functions cell-autonomously to prevent anterior prestalk cell development. The presence of exogenous folic acid during vegetative growth and development delayed anterior prestalk cell development in wild-type but not gα4 null mutant aggregates, indicating that folic acid can inhibit cell-type-specific differentiation by stimulation of the Gα4-mediated signal transducation pathway. The results of this study suggest that Gα4-mediated signals can regulate cell-type-specific differentiation by promoting prespore cell development and inhibiting anterior prestalk cell development.&bdy: J. Hadwiger (✉) · J. Srinivasan Department of Microbiology and Molecular Genetics, Oklahoma State University, 306 Life Science East, Stillwater, OK 74078-3020, USA Tel.: +1-405-744 9771, Fax: +1-405-744 6790 e-mail: [email protected]&/fn-block:

Introduction In many developmental programs, external signals can regulate cell fate by directing cell differentiation and localization. Identifying these signals and the molecular mechanisms of their reception are important steps in the characterization of cell fate and organization. During the developmental life cycle of the slime mold Dictyostelium discoideum, cell sorting plays a significant role in localizing cells to the prespore and prestalk regions that will eventually form the spore mass and stalk, respectively, of the fruiting body [5, 12, 43, 51]. This cell sorting process is initiated as nutrient-deprived cells aggregate (approximately 105 cells) in response to an extracellular cAMP signal that is relayed between cells [6]. As the aggregate forms, prespore and prestalk cells become segregated to the central and anterior regions, respectively, in a sorting process that is influenced by vegetative parameters (e.g., growth conditions and cell cycle progression) [2, 16, 17, 28, 30, 31, 48–50]. The movement of cells to the anterior prestalk region during tip formation of the aggregate is thought to be mediated by a chemotactic response to cAMP as indicated by the analysis of cAMP-chemotactic properties of cells moving to this region [12, 33, 37, 42–44, 46]. The role of cAMP in this process is also supported by the lack of tip formation in aggregates of cells that do not have a functional carB(cAR2) cAMP receptor gene [35]. While the movement of cells to the anterior prestalk region has been examined by many studies, substantially less is known about the localization of cells to the prespore region of developing aggregates. During multicellular development, prespore cells are localized to the central region of aggregates where they form a defined border with the anterior prestalk cell region [8, 18]. Cells in the prespore region display different patterns of cell movement compared to cells in the anterior prestalk cell region, suggesting that differences in chemotactic movement might contribute to the formation of these two regions [41]. Chemotactic movement of Dictyostelium cells, as well as many other eukaryotic cells, is often mediated by

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heterotrimeric G proteins that transduce signals from activated cell surface receptors to downstream regulatory proteins [6, 9, 29]. The analysis of Dictyostelium G protein genes has revealed that the Gα subunit encoded by the Gα4 gene is essential for chemotactic responses to folic acid [20]. The Gα4 subunit is not required for chemotactic responses to cAMP, but other signaling components such the Gβ subunit, adenylyl cyclase, and guanylyl cyclase are important for responses to both folic acid and cAMP [47, 52]. Expression of the Gα4 gene is increased upon cellular aggregation in a subset of cells that is distributed throughout the organism [19–21]. Developing gα4 null cells arrest as multicellular mounds with protruding tip structures that are composed primarily of prestalk cells [19]. These gα4 null aggregates are severely deficient with respect to spore production, implicating a role for Gα4 function in spore development. Cells overexpressing the wild type Gα4 gene, referred to as Gα4HC cells (due to high copy gene dosage), generally arrest in development as aggregates without a defined tip structure, indicating that excessive Gα4 function can inhibit dvelopmental morphogenesis. Gα4HC cells also accumulate higher levels of cGMP in response to folic acid as compared to wild type cells, consistent with Gα4 function in cellular responses to folic acid [20]. The requirement of the Gα4 subunit for chemotaxis and developmental morphology suggests that Gα4-mediated signal transduction pathway might play an important role in cell localization and differentiation during multicellular development. We present evidence in this report that Gα4-mediated signal transduction can cell-autonomously inhibit prestalk cell development and promote cell localization to the prespore cell region.

Methods Strains and medium All D. discoideum strains used in this study were haploid, axenic, and isogenic to the wild type strain KAx-3 except at the loci noted. Construction of all strains, except for the G418-sensitive Gα4HC strain, has been previously described [19]. G418-sensitive Gα4HC cells were created by co-transforming a Gα4 expression plasmid pJH56 [19] and plasmid pJH60 into JH10 cells and selecting for thymidine prototrophs. Plasmid pJH60 contains a 3.2-kb fragment of the wild type THY1 gene inserted into the BamHI site of phagemid pT3T718U (Pharmacia, Piscataway, NJ, USA) that complements the thymidine auxotrophy of JH10 cells. Transformants with a high copy number of the Gα4 gene were identified by screening clonal aggregates for the aberrant developmental morphology associated with other Gα4HC strains. Verification of increased Gα4 gene dosage and expression was determined by genomic DNA blot [34] and RNA blot analyses, respectively, using previously described methods [27, 34]. Additional gα4 null strains were created by insertional mutagenesis of JH8 (pyr5–6 deficient strain) cells using a Gα4 segment disrupted with the PYR5–6 gene as described [19]. Gα4 gene disruption by homologous recombination was verified by genomic DNA blot analysis and gα4 null developmental morphology was confirmed by visual inspection. All DNA probes used in nucleic acid hybridizations were created by random primer DNA synthesis [14]. All strains were grown axenically in HL-5 medium. Plasmids were electroporated into Dictyostelium cells as previously described [11].

Analysis of cell sorting and gene expression in chimeric organisms Cells were grown to mid-log phase (2×106 cells/ml) and washed in phosphate buffer (12 mM NaH2PO4, adjusted to pH 6.1 with KOH), and then resuspended at 2×107 cells/ml in the phosphate buffer unless otherwise noted. Cell suspensions of the different strains were mixed in the described ratios before development on nonnutrient plates containing 1.5% granulated agar (DIFCO, Detroit, Mich., USA) in phosphate buffer. Development of cells for histochemical staining was carried out on nitrocellulose membrane filters (Millipore, Bedford, Mass., USA) that were placed over nonnutrient agar plates. Histochemical staining of β-galactosidase activity was performed as previously described [18] except that the cell aggregates were fixed for 5 min in Z buffer (80 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7) containing 2.5% glutaraldehyde and 0.1% Triton X-100 and then stained in Z buffer solution containing 1 mM K3[Fe(CN)6], 1 mM K4[Fe(CN)6], 0.1% Triton X-100, and 1 mg/ml Bluo-Gal (Sigma, St. Louis, Mo, USA; from stock of 20 mg/ml Bluo-Gal in N,N-dimethylformamide). The histochemical β-galactosidase substrate Bluo-Gal was used rather than X-Gal due to the greater contrast between staining and non-staining cells provided by Bluo-Gal. For quantitative β-galactosidase enzymatic assays, cell suspensions of 1×108 cells/ml were spotted onto Whatmann No. 50 paper filters that were then placed on nonnutrient plates. Measurement of β-galactosidase activity of Dictyostelium cell extracts was determined by a standard enzyme assay using o-nitrophenyl β-D-galactopyranoside (ONPG) as the substrate [10]. The cell extracts were also assayed for protein concentration using a dye binding assay [4]. Cell labeling with the fluorescent dye 5-chloromethylfluorescein diacetate (CellTracker Green; CMFDA), Molecular Probes, Eugene, Or, USA was performed as described [39]. Cells were washed in phosphate buffer and then incubated at 2×107 cell/ml for 20 min in 1 mM CMFDA solution (1/10 dilution of 10 mM CMFDA in dimethyl sulfoxide). After washing twice in phosphate buffer, the cells were mixed with unlabeled cells in a 1:9 ratio and then plated on nonnutrient plates for development. Labeling had no effect on developmental morphology and nonlabeled cells in the chimeras were prepared for development in an identical manner except for the absence of CMFDA. Labeled cells were developed in the dark until analyzed by fluorescence microscopy using a Nikon Optiphot-2 microsope. For tip transfer experiments, anterior cells from the tip region were transferred from labeled wildtype slugs under low illumination and then these chimeric organisms were returned to the dark for further development. Cell growth and development with exogenous folic acid were performed with cells previously grown to mid-log phase without exogenous folic acid. Cells were harvested by centrifugation and resuspended in fresh HL-5 medium (same volume) with or without 1 mM folic acid. After 4 h of growth, cells were harvested by centrifugation, washed in phosphate buffer, resuspended in phosphate buffer at 1×108 cells/ml, and plated on small (35-mm-diameter plate with 10 ml agar volume) nonnutrient agar plates supplemented with or without 100 µM folic acid. To minimize chemotactic movement across the agar surface, cells were spread over the entire surface of the agar during the plating. At specific times in development, cells were collected in phosphate buffer washes and then RNA was isolated for RNA blot analysis.

Chemotaxis assays All chemotaxis assays were conducted on cells grown to mid-log phase (2×106 cells/ml) and washed in phosphate buffer. Gradient assays were performed by placing 1-µl droplets of cell suspensions (1×106 cells/ml phosphate buffer) and 1-µl droplets of chemoattractant (10−2−10−5 M folic acid, 10−2−10−5 M monapterin or 10−3−10−5 M cAMP) solution 2 mm apart from each other on nonnutrient plates. Monapterin was obtained from Fluka (Ronkonkoma, NY, USA). For gradient chemotaxis assays at higher cell densities, the cell suspensions of 1×108 cells/ml were plated

197 on nonnutrient plates and allowed to develop for 10 h (mound stage of development). Cell aggregates were dispersed in phosphate buffer by repeated pipeting before being used for gradient chemotaxis assays. In assays with low cell density, chemotactic responsiveness was determined after 3 h by counting the number of cells that had migrated beyond the original perimeter of the cell droplet. These cell counts were corrected for nonchemotactic cell movement by subtracting the number of migratory cells observed in the absence of chemoattractant. ‘Spreading’ chemotaxis assays were performed as previously described [45] except that cell suspensions of 1×109 cells/ml were spotted onto nonnutrient plates for a prestarvation period of 10 h, resuspended in phosphate buffer at 1×108 cells/ml, and then spotted as 1-µl droplets on nonnutrient plates that contained chemoattractants (10−2−10−5 M folic acid or monapterin).

Results Developmental gene expression in clonal aggregates of Gα4HC and gα4 cells To make use of several reporter genes in the analysis of Gα4HC (overexpression of the Gα4 gene due to high gene-copy number) cells, we created a Gα4HC strain that is sensitive to the drug G418 because many of these reporter genes were available on vectors conferring resistance to this drug. This G418-sensitive Gα4HC strain was created by transforming Dictyostelium cells with a Gα4 expression vector that lacked the G418 resistance marker (see Methods for details). Morphological development of this new Gα4HC strain was more stringently blocked in tip morphogenesis as compared to previously described Gα4HC strains and genomic DNA blot analysis indicated that this increased stringency correlated with a higher copy number of the Gα4 gene (data not shown). This new Gα4HC strain also had no detectable spore production in clonal aggregates whereas other strains of Gα4HC cells have been shown to produce a very limited amount of spores (data not shown). All of the analyses of Gα4HC cells in this study were conducted with this new G418sensitive Gα4HC strain. RNA blot indicated that the Gα4 transcripts in these cells were similar in size to the Gα4 transcripts found in wild-type cells and the abundance of these Gα4 transcripts in the Gα4HC cells increased at the onset of multicellular development, which is similar to the pattern observed during the development of wildtype cells (Fig. 1). After aggregation, the Gα4 transcript level gradually decreases in these Gα4HC cells as the progression of developmental morphology is terminated. The overall level of the Gα4 transcript in the Gα4HC cells was much greater than that of wild-type cells at all stages of development. In contrast, gα4 null cells weakly express a smaller transcript that is not likely to provide any Gα4 function or contribute to the phenotypes of gα4 null cells due to the site of the insertional mutation [19]. The expression of prestalk specific genes, ecmA and ecmB, was severely reduced in Gα4HC cells compared to that observed for gα4 null and wild-type cells, consistent with the inability of Gα4HC aggregates to form tips. The expression of the prespore specific cotC (SP60) gene was also severely reduced in the Gα4HC cells compared

Fig. 1 Developmental expression of the Gα4, ecmA, ecmB, and cotC genes in wild-type, gα4 null and Gα4HC cells. Total RNA was isolated from cells at times indicated during development (10, 12, 14, and 16 h after starvation) and size-fractionated on horizontal formaldehyde gels. All lanes contained 4 µg total RNA, and loading consistency was verified by ethidium bromide staining of ribosomal RNA bands. The RNA was then blotted to a nylon membranes and hybridized with a Gα4, ecmA probe (also detects ecmB transcripts by cross hybridization), or cotC probe. Lanes marked (V) represent RNA isolated from vegetatively growing cells&ig.c:/f

to gα4 null and wild-type cells and no mature spores were detected in dissected Gα4HC aggregates. The level of cotC expression in gα4 null aggregates contrasts earlier reports of reduced cotC expression but we created and examined several other gα4 null strains to confirm that the level of cotC expression in gα4 null aggregates is similar to that of wild-type aggregates [19]. All of the developmental phenotypes of the G418-sensitive Gα4HC cells were dependent on the elevated expression level of the Gα4 gene as cells reverted to wild-type development when Gα4 expression decreased due to losses in gene copy number (data not shown). Localization of G protein mutants in chimeric organisms To examine the role of Gα4 function in cell positioning, the spatial distribution of gα4 null and Gα4HC cells was monitored in chimeric organisms consisting primarily of wild-type cells. The mutant cells were distinguished from the wild-type cells in the chimeric organisms by transforming the mutants with a lacZ reporter gene regulated by the Dictyostelium actF(act6) promoter [10, 19]. The β-galactosidase produced from this actF::lacZ reporter gene can be detected in all regions of the developing aggregate when expressed in wild-type cells (Fig. 2A), although the β-galactosidase activity appears to be higher in the prestalk cells especially when the duration of the histochemical staining is limited. By using a low ratio (1:3) of mutant cells to wild-type cells and limiting the histochemical staining periods, we found that gα4 null cells are most abundant in the extreme posterior region and severely underrepresented in the prespore and prestalk regions (central and anterior, respectively) of the

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Fig. 3A–C Localization of mutant and wild-type cells labeled with 5-chloromethylfluorescein diacetate (CMFDA) in slugs containing a 1:9 ratio of labeled cells to unlabeled wild-type cells. Cells were labeled with CMFDA and then mixed with unlabeled wild-type cells as described in Methods. Cell mixtures were plated on nonnutrient agar plates and the labeled cells were detected throughout development using fluorescence microscopy. Slugs with labeled wild-type (A), gα4 null (B), or Gα4HC (C) cells at 18 h of development. All slugs are oriented with posterior ends to the left and anterior ends to the right&ig.c:/f Fig. 2A–E Histochemical staining of wild-type, gα4 null, or Gα4HC cells, carrying the actF::lacZ or ecmA::lacZ reporter gene, that have been developed with wild-type cells containing no reporter gene. Cells were grown as described in Methods and then mixed in a 1:3 ratio with wild-type cells lacking the reporter gene. Cell mixtures were developed to the slug stage (18 h) on nitrocellulose filters overlaying nonnutrient plates and then histochemically stained for β-galactosidase activity as described in Methods. A Wild-type cells (actF::lacZ). B gα4 null cells (actF::lacZ). C Gα4HC cells (actF::lacZ). D Wild-type cells (ecmA::lacZ). E Gα4HC cells (ecmA::lacZ). Organisms in A and B have been intentionally stained for a shorter period to emphasize the uneven pattern of reporter gene expression in wild-type cells and the uneven distribution of gα4 null cells, respectively&ig.c:/f

chimeric organisms (Fig. 2B). However, gα4 null cells are not excluded from the prespore and anterior prestalk cell regions. The higher accumulation of gα4 null cells in the posterior is similar to the distribution observed for anterior-like cells that can serve as precursors to anterior prestalk cells and possibly prespore cells [44]. The gα4 null cells eventually localize to the upper and lower cups of the chimeric fruiting bodies, similar to the pattern ob-

served for anterior-like cells at this stage in development (data not shown). In contrast to the gα4 null cells, Gα4HC cells were found in the prespore region with the majority of these cells positioned near the anterior border that separates the prespore and the anterior prestalk regions (Fig. 2C). The Gα4HC cells were not detected in the anterior prestalk region even after long periods of histochemical staining. In some chimeras, the Gα4HC cells could be detected near the posterior end of the slug but most Gα4HC cells were usually found in the central prespore region. During fruiting body formation, the Gα4HC cells remained localized in the prespore region and eventually developed into spores (data not shown). Gα4HC cells expressing the prestalk-specific ecmA::lacZ reporter gene were also found throughout the central and posterior regions but not in the anterior prestalk region, where this gene is primarily expressed in wild-type cells (Fig. 2D and E). Similar spatial distribution patterns were also observed for Gα4HC cells expressing the prestalk cell-specific ecmB::lacZ reporter gene (data not

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shown). These prestalk expressing Gα4HC cells might be analogous to wild-type anterior-like cells that express prestalk genes but do not reside in the anterior region [12, 51]. The localization of Gα4 mutants in mixed aggregates was also analyzed by labeling cells with the fluorescent dye CMFDA so that the detection of mutant cells would not be limited by the expression parameters of reporter genes. CMFDA-labeled wild type cells, mixed in an 1:9 ratio with unlabeled wild-type cells, maintained an even distribution within the mixed aggregates throughout development, indicating that the labeling conditions did not adversely affect cell localization (Fig. 3A). In contrast, CMFDA-labeled gα4 null cells were evenly distributed in the chimeric organisms only to the mound stage and then afterwards these cells began to accumulate near the posterior end as the mounds differentiated into slugs (Fig. 3B). This spatial pattern appeared to be very similar to that observed for gα4 null cells detected by lacZ reporter gene expression. Gα4HC cells labeled with CMFDA were also evenly distributed in chimeric mounds but then these cells were subsequently excluded from the anterior region as the mounds differentiated into slugs (Fig. 3C). The Gα4HC cells remained in the prespore region throughout development, consistent with the results obtained in lacZ reporter experiments. Chemotactic responses of G protein mutants after aggregation Previous studies have indicated the requirement of Gα4 function for folic acid chemotaxis prior to aggregation but the uneven distribution of Gα4 mutants in chimeric organisms suggests that Gα4 function might be important for cell movement after aggregation. To examine the potential role of Gα4 function in cell movement after aggregation, the chemotactic responsiveness of gα4 null and Gα4HC cells from aggregates was analyzed by measuring the relative number of cells that can chemotax to gradients of folic acid or cAMP (Table 1). Early Gα4HC aggregates (10 h of development) consistently had greater chemotactic responses to gradients of folic acid as compared to the chemotactic responses of wild-type aggregates, suggesting that increased Gα4 gene expression results in amplified responses to folic acid during multicellular development similar to that observed for cells assayed before aggregation. These amplified chemotactic responses to folic acid also resulted in greater distances traveled by the Gα4HC cells as compared to wild-type cells in the chemotaxis assays. In contrast, gα4 null cells from aggregates were not chemotactically responsive to gradients of folic acid consistent with the chemotactic properties observed before aggregation [20]. Similar results were also observed when chemotaxis assays were conducted on cells that had been plated at higher densities (data not shown). Gradient and ‘‘spreading’’ chemotaxis assays on Gα4 mutants were also used to examine cell movement responses to monapterin, a related pterin

Table 1 Chemotactic responsiveness of wild-type, Gα4HC, and gα4 null cells to gradients of folic acid and cAMP after 10 h of development (aggregate stage). Chemotactic responsiveness of these strains was assayed during development at the times indicated by counting the number of cells that had migrated toward gradients of folic acid or cAMP after 3 h (see Methods for details). The results of the chemotaxis assay were obtained from a typical experiment performed in duplicate. Error bars represent the deviations in the cell counts&/tbl.c:& A

Relative number of cells chemotactic to folic acid (moles) Strain

10−6

10−5

10−4

10−3

Wild-type Gα4HC gα4 null

2±2 18±6 0

28±8 142±12 0

74±6 269±46 0

209±27 449±97 0

B

Relative number of cells chemotactic to cAMP (moles) Strain

10−6

10−5

10−4

10−3

Wild-type Gα4HC gα4 null

0 101±3 4±1

4±4 478±17 15±6

6±0 943±100 88±10

3±1 278±66 6±2

&/tbl.:

compound that has been reported to only stimulate cells during multicellular development [20, 45]. Gradients of monapterin stimulated greater cell movement in Gα4HC cells compared to that observed for wild type cells and gα4 null cells displayed no cell movement response (data not shown). While monapterin was capable of stimulating cell movement, this movement was not directed toward the source of monapterin in the gradient assays, indicating that monapterin might not function as a chemoattractant. Furthermore, cell movement in response to monapterin noticeably decreased in both Gα4HC and wild-type cell droplets spotted from suspensions with low cell densities (<1×108 cells/ml), suggesting that monapterin responses might be dependent on cell density (data not shown). Chemotactic responsiveness to cAMP was observed in gα4 null, Gα4HC and wild-type cells after aggregation, indicating that Gα4 function, or lack of it, does not prevent this chemotactic response at this stage in development (Table 1). Intercellular signal rescues Gα4HC prespore-specific gene expression and spore production Spore production in gα4 null and Gα4HC cells can be increased by the presence of wild-type cells in chimeric organisms but whether this increase in spore development is due to changes in developmental gene expression or other developmental parameters (e.g., morphogenesis or spore dispersal) has not been determined [19]. To examine the developmental gene expression of gα4 null or Gα4HC cells in the presence of wild-type cells, the expression of developmentally regulated lacZ reporter genes (see Table 2 for list of reporter genes) was measured in the mutant cells

200 Table 2A, B Developmental gene expression of gα4 null and Gα4HC cells in chimeric aggregates. Cells were developed on filters overlaying nonnutrient plates for 19–21 h (slug stage for mutant/wild-type chimeras) and then assayed for β-galactosidase activity as described in Methods. The β-galactosidase activity of gα4 null cells carrying lacZ reporter genes was measured after cells were developed with either wild-type (gα4/wild-type aggregates) or gα4 null (gα4/gα4 aggregates) cells lacking the reporter gene (A). The β-galactosidase activity of Gα4HC cells carrying lacZ reporter genes was measured after these cells were developed with either wild-type (Gα4HC/wild-type aggregates) or Gα4HC (Gα4HC/Gα4HCaggregates) cells lacking the reporter gene (B). All aggregates contained a 1:2 ratio of cells carrying the reporter gene to cells lacking the reporter gene and both sets of aggregates (mutant/mutant and mutant/wildtype) were created concurrently using equal amounts of the same culture of cells with the reporter gene. Values of β-galactosidase activity are the means and standard deviations of three independent experiments. The β-galactosidase enzymatic activity is expressed in units of pmol substrate/(min·µg protein) using o-nitrophenyl β-D-galactopyranoside (ONPG) as the substrate. Basal levels of reporter gene expression in the gα4/gα4 and Gα4HC/Gα4HC aggregates result from a combination of promoter strength and copy number of reporter gene. Cell-type specificity of reporter gene promoters is as follows: cotC (SP60) – prespore cells; ecmA and ecmB – prestalk and anterior-like cells; rasD, ptpA (PTP1), and cprB (CP2) – prestalk and nonprespore/nonprestalk cells; and Gα4 – nonprespore/nonprestalk cells [13, 18, 19, 23, 32, 51]&/tbl.c:& A

β-Galactosidase activity

Reporter gene

gα4/gα4 Aggregates

gα4/Wild-type Aggregates

cotC::lacZ ecmA::lacZ ecmB::lacZ rasD::lacZ ptpA::lacZ cprB::lacZ Gα4::lacZ

39.2±2.9 2.15±0.03 82.3±11.2 86.3±3.4 41.4±4.1 97.4±0.5 64.2±4.4

55.3±2.7 2.12±0.08 67.1±10.9 95.5±10.4 45.7±13.9 95.2±4.0 60.3±4.1

B

β-Galactosidase activity

Reporter gene

Gα4HC/Gα4HC Aggregates

Ratio of activity Gα4HC/Wild-type Gα4HC/Wild-type Gα4HC/Gα4HC Aggregates

cotC::lacZ ecmA::lacZ ecmB::lacZ rasD::lacZ ptpA::lacZ cprB::lacZ Gα4::lacZ

3.08±0.58 0.37±0.03 7.78±0.43 62.2±9.1 280±17 251±2 29.3±3.4

23.6±0.56 0.48±0.07 10.7±1.8 109±11 288±26 418±44 30.2±2.1

Ratio of activity gα4/Wild-type gα4/gα4 1.41 1.17 0.81 1.10 1.10 0.98 0.94

7.66 1.30 1.38 1.76 1.03 1.67 1.03

&/tbl.:

during development with wild-type or mutant cells that lack the reporter genes. The reporter genes in gα4 null cells were expressed at similar levels regardless of whether these cells were mixed with wild-type or other gα4 null cells (Table 2A). However, Gα4HC cells displayed an 8fold increase in the expression of the prespore-specific cotC::lacZ reporter gene, when developed in the presence of wild-type cells (Table 2B). This observation is consistent with the ability of wild-type cells to rescue spore development in Gα4HC cells [19]. Only minor changes in gene expression were observed for the other reporter genes in Gα4HC cells when development occurred in the presence of wild-type cells. However, none of these other

Fig. 4A, B Distribution of CMFDA-labeled anterior cells from wild-type slugs after transfer to mounds of Gα4HC cells. Wild-type cells were labeled as described in Methods and then developed synchronously but separately from Gα4HC cells on nonnutrient agar plates. After 16 h of development, anterior cells (approximately 20% of total aggregate) from wild-type slugs were transferred to Gα4HC aggregates (without mixing) under low illumination. The spatial distribution of the labeled wild-type cells in the chimeras during subsequent development was examined by fluorescence microscopy. A A chimeric slug 4 h after tip transfer. B An early culminant 8 h after tip transfer&ig.c:/f

reporter genes (ecmA::lacZ, ecmB::lacZ, rasD::lacZ, ptpA::lacZ, cprB::lacZ, and Gα4::lacZ) are specific to prespore development [13, 19, 23, 32, 51]. Two of these reporter genes, ecmA::lacZ and ecmB::lacZ, are highly expressed in the anterior prestalk cells of wild-type aggregates, but Gα4HC cells expressing these genes are absent from the anterior prestalk region in chimeric organisms (previously described in Fig. 2). The ability of wild-type cells to increase the presporespecific gene expression in Gα4HC cells suggests that wild-type cells are capable of rescuing Gα4HC spore development by providing an intercellular signal. We tested the ability of cells in the anterior region of wild-type organisms to provide this signal since the anterior prestalk cell region is absent from the development of Gα4HC aggregates. Cells from the anterior region of wild-type slugs were transplanted on to aggregates of Gα4HC cells, resulting in wild-type (anterior)/ Gα4HC chimeras that developed into fruiting bodies with large spore masses. The distribution of the transplanted cells in the chimeric organisms was examined by detecting the fluorescence of wild-type cells pre-labeled with CMFDA (Fig. 4). We found that the transplanted cells remained segregated to the anterior end of the developing chimeric organisms.

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Fig. 6 Developmental gene expression in wild-type and gα4 null mutant cells in the absence (−FA) or presence (+FA) of exogenous folic acid. Cells were grown and developed in the absence or presence of exogenous folic acid as described in Fig. 5. At the times indicated (8, 11, or 14 h after starvation) during development, RNA was isolated and examined by blot analysis as described in Fig. 1. RNA blots were probed with ecmA (upper panel) or cotC (lower panel) cDNA sequences. Lower bands on the upper panel are ecmB transcripts that cross-hybridize to the ecmA probe&ig.c:/f

Fig. 5A–D Development of wild-type and gα4 null mutant cells in the presence or absence of exogenous folic acid. Folic acidtreated cells were grown in fresh medium with 1 mM folic acid 4 h before plating for development on nonnutrient agar containing 100 µM folic acid, as described in Methods. Control cells (without folic acid treatment) were grown and developed under identical conditions except for the absence of exogenous folic acid in the growth medium and nonnutrient agar. Developing structures were photographed, at the same magnification, after 15 h of development. A Wild-type cells without folic acid treatment. B Wild-type cells treated with folic acid. C gα4 null cells without folic acid treatment. D gα4 null cells treated with folic acid. The morphology of wild-type aggregates in B is less defined due to the low profile of the ‘‘loose’’ aggregation state&ig.c:/f

These transplanted cells later formed the stalk of the chimeric fruiting body without significantly contributing to the collection of cells in the spore mass. The lack of mixing between the mutant and wild-type cells suggests that the rescuing signal might be a diffusible factor produced by prestalk cells. The production of this factor does not require Gα4 function as gα4 null cells are also capable of rescuing Gα4HC spore development [19]. Other studies have reported that extracellular cAMP can induce prespore gene expression in suspensions of wild-type cells so we conducted similar tests to determine if extracellular cAMP could rescue spore development in Gα4HC aggregates [18]. However, we observed no dramatic increase in prespore gene expression or any spore production when slowly shaken suspensions of Gα4HC aggregates were treated with exogenous cAMP (data not shown). Folic acid delays the formation of the anterior prestalk cell region The inhibition of tip morphogenesis in Gα4HC aggregates suggested that stimulation of the Gα4-mediated

pathway in wild-type cells with exogenous folic acid might also produce similar developmental aberrations. The addition of folic acid to the growth medium and to the nonnutrient agar substratum delayed the formation of tips in wild-type aggregates for at least 5 h as compared to wild-type aggregates that were developed without the folic acid treatment (Fig. 5). This delay was in part due to an approximate 1-h delay in the onset of aggregation but the greatest delay in development appeared to be in the formation of tips. The effect of folic acid on tip formation was dose-dependent, as concentrations less than 1 µM folic acid in the growth medium and agar substratum did not significantly delay tip formation (data not show). Eventually, many of the folic acid-treated wildtype aggregates developed tips and continued unimpeded through to the fruiting body stage. Regions of higher cell densities were able to undergo tip formation earlier than regions of lower cell density, consistent the ability of Dictyostelium cells to inactivate folic acid [7]. Delays in tip formation were shorter in duration if folic acid was only added to the growth medium or the nonnutrient agar substratum, indicating that folic acid affects cells in both growth and development (data not shown). The delay in wild-type cell development was most consistent when fresh medium was provided to the cells at the same time as the exogenous folic acid but fresh medium alone was not sufficient to cause the delayed morphogenesis (data not shown). In contrast to wild-type cell development, gα4 null cell development was not delayed by the presence of folic acid indicating the requirement of Gα4 function for the folic acid-mediated developmental delay. Delays in cell-type-specific developmental gene expression were observed in wild-type cells treated with folic acid, consistent with the delays in morphogenesis (Fig. 6). Expression of the prestalk genes ecmA and ecmB was severely delayed in folic acid-treated wild-type cells

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whereas this expression in gα4 null cells was not affected by folic acid. Prespore gene expression, as indicated by cotC expression, was also delayed by the folic acid treatment in wild-type aggregates but his delay might result from the lack of anterior prestalk cells, analogous to Gα4HC cell development.

Discussion The lack of cell-type-specific gene expression in developing Gα4HC cells is consistent with the termination of morphogenesis at the mound stage and the absence of spore production. However, prespore cell-specific gene expression appears to be limited due to the absence of an intercellular signal that can be produced by anterior prestalk cells, as indicated by the rescue of prespore gene expression and spore production in chimeric organisms. Other recent studies also indicate that spore differentiation signals can arise from prestalk cells [1, 40]. In contrast, the loss of prestalk cell-specific gene expression cannot be rescued by intercellular signaling, implying that the Gα4 subunit functions cell autonomously to inhibit prestalk cell differentiation. The loss of Gα4 function in gα4 null cells does not result in increased levels of prestalk gene expression, compared to wild-type cells, suggesting that mechanisms independent of Gα4 function also regulate prestalk cell-specific gene expression. This result is consistent with the requirement of both cAMP and differentiation inducing factor (DIF) to induce prestalk cell gene expression and differentiation [3]. The absence of Gα4HC cells in the anterior prestalk regions of chimeric organisms implies that signal transduction by the Gα4 subunit cell autonomously inhibits cell movement into this region and/or promotes cell localization in other regions of the aggregate. After aggregation, Gα4HC cells retain the ability to chemotax to cAMP, a chemoattractant of anterior prestalk cells, but this responsiveness might not be adequate or compatible with the amplitude or frequency of cAMP signaling that mediates the localization of cells to the anterior region. Other studies have revealed that multiple cAMP receptors, with varying affinities to cAMP, are differentially expressed during multicellular development leading to many potential forms of cAMP signaling [15, 24, 26, 35, 36, 53]. Gα4HC cells that display some prestalk cell characteristics, such as the expression of the prestalk specific reporter gene ecmA::lacZ, are also restricted from the anterior prestalk cell region, indicating that the ability to weakly express prestalk cell-specific genes does not confer cell localization to the anterior region. While expression of the ecmA and ecmB genes is not sufficient for cell localization to the anterior prestalk region, the overexpression of the Gα4 gene might possibly block some other step in prestalk cell differentiation that is required for the localization of these cells to the anterior region. Overexpression of the Gα4 gene might also promote cell localization to the prespore region through chemotactic

movement, since Gα4HC cells retain chemotactic responsiveness to folic acid and cAMP, even after cellular aggregation. Interestingly, the aberrant morphological development of gα4 null aggregates is characterized by the inability of most prespore cells to follow the prestalk cell region as it extends away from the mound [19]. Furthermore, gα4 null cells are not evenly distributed in chimeric organisms, suggesting that a cell-autonomous defect in cell localization results from the absence of Gα4 function. If Gα4 function is necessary for chemotactic movement to the prespore region, then this function is likely to occur early in the sorting process since Gα4 gene expression is not very prominent in the central prespore region [19, 20]. The increased responsiveness to folic acid and monapterin displayed by Gα4HC cells, compared to wild-type cells, and the complete absence of this responsiveness in gα4 null cells are both consistent with the requirement for Gα4 function in pterin-stimulated cGMP accumulation [20]. Both of these pterin compounds have also been demonstrated to stimulate actin association with the cytoskeleton [45]. Our studies also confirmed that responsiveness to monapterin arises several hours after the onset of starvation, supporting the existence of multiple pterin receptor classes. However, responsiveness to monapterin did not result in direct cell movement in gradient chemotaxis assays, leaving open the possibility that monapterin stimulates increased cell movement rather than chemotaxis. Evidence of folic acid chemotaxis during multicellular development has also been previously reported in a related slime mold, Dictyostelium minutum, where chemotaxis to folic acid rather than cAMP is thought to facilitate the aggregation phase of development [7]. In contrast to D. discoideum development, virtually all the cells within a D. minutum aggregate initially possess prespore characteristics [37, 38]. While differences exist between the developmental life cycles of D. minutum and D. discoideum, folic acid signaling might provide similar functions in both organisms by promoting prespore development. The ability of exogenous folic acid to delay tip morphogenesis in wild-type cells suggests that the developmental Gα4HC phenotypes arise from increased signaling of the Gα4-mediated signal transduction pathway. Further support for this idea comes from the observation that exogenous folic acid treatments produce developmental gene expression patterns similar to those of Gα4HC cells. The delay in anterior prestalk cell development caused by the folic acid treatments must be mediated through the Gα4 pathway, as opposed to other pathways, since gα4 null mutants do not appear to be affected by the presence of folic acid. However, the overexpressed Gα4 subunit could potentially compete with other Gα subunits and this competition might contribute to the phenotypes observed in Gα4HC cells. Interestingly, the D. discoideum Gα5 subunit shares the greatest identity with the Gα4 subunit and the loss of Gα5 function results in a delay of tip formation [22]. Therefore, the tip

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formation defect of Gα4HC might be in part mediated by interference with the Gα5 signal transduction pathway. However, the Gα5 subunit is not essential for tip formation and so the Gα4HC cells are not likely to be only defective in the Gα5-mediated signal transduction pathway. The aberrant developmental phenotypes of Gα4HC cells appear to be specific to the Gα4 subunit since these phenotypes are not observed when other structurally related Gα subunits are overexpressed [22, 25]. While the levels of folic acid or other pterin compounds have not been extensively characterized during multicellular development, the transduction of signals by the Gα4 subunit is clearly important for proper developmental morphology and cell differentiation based on the phenotypes of gα4 null and Gα4HC mutants. The cell autonomous functions of the Gα4 signal transduction pathway appear to promote spore development and inhibit prestalk cell development, implying that folic acid or other pterin compound signals might serve as a mechanism to regulate cell fate choice. Folic acid signaling could potentially target cells with high energy reserves from recent feedings on bacterial sources to undergo spore development. Further support for this idea comes from the observation that delays in development caused by folic acid are most effective when cells have been recently supplied with nutrients. Other studies have shown that cells grown in the absence of glucose respond to cAMP earlier and that these cAMP-responsive cells sort to the anterior prestalk cell regions of chimeric organisms [30, 48]. While long regarded as a signal to seek out bacterial food sources, folic acid can also serve as a signal that affects multicellular development with respect to morphogenesis and cell differentiation by stimulating the Gα4-mediated signal transduction pathway. &p.2:Acknowledgements We thank C. Ashley for her technical assistance in these studies, R. Firtel, P. Howard, C. Gaskins, L. Haberstroh, K. Esch, and J. Williams for the construction of reporter gene vectors, and M. Maeda for helpful discussions. This research was supported by an American Cancer Society grant to J.A.H. (DB-83284).

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