Mef2A, a homologue of animal Mef2 transcription factors, regulates cell differentiation in Dictyostelium discoideum
© Galardi-Castilla et al.; licensee BioMed Central Ltd. 2013
Received: 14 November 2012
Accepted: 5 April 2013
Published: 11 April 2013
Transcription factors from the MADS-box family play a relevant role in cell differentiation and development and include the animal SRF (serum response factor) and MEF2 (myocyte enhancer factor 2) proteins. The social amoeba Dictyostelium discoideum contains four genes coding for MADS-box transcription factors, two of these genes code for proteins that are more similar to SRF, and the other two code for proteins that are more similar to MEF2 animal factors.
The biological function of one of the two genes that codes for MEF2-related proteins, a gene known as mef2A, is described in this article. This gene is expressed under the transcriptional control of two alternative promoters in growing cells, and its expression is induced during development in prespore cells. Mutant strains where the mef2A gene has been partially deleted were generated to study its biological function. The mutant strains showed reduced growth when feeding on bacteria and were able to develop and form fruiting bodies, but spore production was significantly reduced. A study of developmental markers showed that prespore cells differentiation was impaired in the mutant strains. When mutant and wild-type cells were set to develop in chimeras, mutant spores were underrepresented in the fruiting bodies. The mutant cells were also unable to form spores in vitro. In addition, mutant cells also showed a poor contribution to the formation of the tip-organizer and the upper region of slugs and culminant structures. In agreement with these observations, a comparison of the genes transcribed by mutant and wild-type strains during development indicated that prestalk gene expression was enhanced, while prespore gene expression decreased in the mef2A- strain.
Our data shows that mef2A plays a role in cell differentiation in D. discoideum and modulates the expression of prespore and prestalk genes.
Mef-2-related transcription factors belong to a family of proteins that are present in all eukaryotic organisms [1, 2]. These proteins share a very conserved DNA-binding and protein-dimerization domain, the MADS-box, named after the transcription factors MCM1 (from yeast), Agamous, Deficiens (from plants) and SRF (from animals) [3, 4]. Two subfamilies of MADS-box transcription factors have been defined according to the MADS-box sequence: type I and type II . Plants have a large number of types I and II MADS-box proteins while other organisms, such as fungi and animals, usually have one or more proteins of each subfamily . Animals, for example, have only one type I protein (serum response factor [SRF]) and four type II proteins (myocyte enhancer factor 2 A-D [Mef2 A-D]). The two types of factors recognize different A/T rich binding sites. SRF and related factors recognize the consensus sequence CC(A/T)6GG [7, 8], while Mef-2-related factors recognize the CTA(A/T)4TAG consensus sequence .
MADS-box transcription factors accomplish various biological functions. In plants, they are critically involved in floral formation and development . In yeasts, MCM1 participates in the regulation of pheromone expression, metabolism  and DNA replication . In animals, these transcription factors are mainly involved in the regulation of cell-differentiation processes. SRF deletion is lethal in mice because cells are impaired in cell adhesion and migration , and the embryo cannot complete gastrulation . Tissue-specific deletion has shown that SRF is also required for terminal differentiation of skeletal, cardiac and smooth muscle cells and for neural cell migration . Additional studies have demonstrated that SRF regulates the expression of a large number of genes coding for actin-cytoskeleton-related proteins [15, 16].
Mef-2 proteins are involved in regulating the expression of muscle-specific genes, both in Drosophila and in mammals , in collaboration with MyoD-related transcription factors . In mammals, there are four genes coding for very similar Mef-2 factors that appear to functionally complement each other, at least partially (Mef2A-D) . However, Mef2C-null mice die early in their development due to cardiovascular abnormalities , and Mef2A-null mice die perinatally from heart defects . In addition, numerous studies have shown that Mef-2 factors are also involved in the differentiation of several other cell types, such as neural crest cells, endothelial cells, chondrocytes, neurons and lymphocytes [23, 24].
Our group has approached the functional study of MADS-box transcription factors in the social amoeba Dictyostelium discoideum. These unicellular organisms live in forest soils, feeding on bacteria and other microorganisms and are able to develop as multicellular organisms under starvation conditions. In such conditions, up to 105 individual amoebas aggregate to form a fruiting body composed of a basal disk, stalk and sorus where up to 80% of the original amoeba differentiate into resistant forms called spores [25–27]. The initial step is the aggregation of the cells towards cAMP-secreting centers to form a mound. Cells within the aggregates initiate a differentiation process to form two main cell types: prestalk and prespore. Prestalk cells migrate to the top of the mound, emerging as a tip. A culmination process is later initiated by the migration of prestalk cells from the tip towards the substrate through the mass of prespore cells, piling up and terminally differentiating to form the stalk. The mass of prespore cells remains attached to the top of the forming stalk, rising from the substrate until culmination is completed. Migratory structures, called slugs, can be formed before culmination under adverse environmental conditions. In this case, the slugs migrate towards warmer and lighter places for culmination to facilitate the dissemination of the spores. By the end of culmination, prespore cells differentiate inside the sorus to form mature spores.
Analysis of the D. discoideum genome has shown that it contains four genes coding for MADS-box transcription factors, namely srfA, B, C and D. Previous studies in our laboratory have shown that srfA is required for the proper development of the fruiting body, including the slug migration and culmination steps, and is essential for spore terminal differentiation [28, 29]. The srfB gene is expressed earlier than srfA during development, and the encoded protein is involved in the initiation of the developmental process, cell migration and the initiation of culmination . The functional study of the srfC gene is described in this article. We present evidence demonstrating that srfC is more similar to animal Mef-2 genes and propose naming it mef2A. By analyzing the phenotype of mutant strains and gene expression levels during development, we clearly show that this protein is involved in D. discoideum development and, in particular, in the differentiation of prespore cells and one group of prestalk cells.
Characterization of the mef2A (srfC) gene
The transcriptional activity of both promoters was analyzed by the use of reporter vectors where Promoter 1 (Pr1), Promoter 2 (Pr2) or the complete promoter region (cPr) were cloned, thereby driving lacZ expression. Pools of transformed cells obtained for each promoter were analyzed for β-galactosidase activity. Promoter 1 drove lacZ expression in scattered cells at the mound and finger stages of development, but its activity markedly increased in the prespore region of slug, Mexican-hat and culminant structures (Figure 2C). Promoter 2 was active in scattered cells of aggregates and fingers, but the activity decreased almost completely at later developmental stages, except for a few cells in the basal disk of culminant structures. The activity of the complete promoter showed the sum of Pr1 and Pr2 and was maximal in the prespore region of developing structures.
Generation of mef2A-deficient strains
Production of spores by wild type and mef2A - mutant strains
100 ± 13.5
61.2 ± 12.5
100 ± 13.9
41.8 ± 15.8
The results obtained using pspA::lacZ as a cell marker are shown in the lower panel of Figure 5. Homogeneous mixtures of AX4/AX4 and mef2A- mutant/mef2A- mutant cells presented the same pattern of staining shown in Figure 4. In the case of the mutant cells, a reduced population of lacZ-expressing cells was also observed in the slugs and, to a lesser extent, the culminant structures. The mixture of pspA-labeled mef2A- mutant cells with wild-type unlabeled cells showed that very few mutant cells differentiated as prespore cells, and the cells that expressed pspA were found at the rear region of the slugs and the lower part of the sorus in the culminating structures. In contrast, intense lacZ staining was observed when pspA-expressing AX4 cells were mixed with mef2A- mutant cells.
Percentage of fluorescent spores in wild-type/mef2A-mutant chimeric structures
42.4 ± 6.5
54.9 ± 6.8
91.5 ± 11.8
94.4 ± 8.9
7.8 ± 2.3
5.3 ± 2.1
42.6 ± 7.8
41.7 ± 5.6
Gene expression profile of mef2Amutant cells
Genes that showed a significant difference in their expression between wild-type and mef2A-mutant structures developed for 16 hours, as determined by mRNA sequencing
Increased expression in the mutant
Decreased expression in the mutant
hssA-related genes (prestalk-specific)
G0267936 -93 (93–1)
G0293362 10.74 (26–279)
G0268400 -80 (160–2)
G0281013 11.95 (19–227)
G0277741 -55.6 (1724–31)
G0281001 -31.2 (780–25)
G0281189 -134.75 (539–4)
G0281191 0 (79–0)
G0281195 -35.33 (106–3)
G0281197 -55.5 (111–2)
G0282307 -106.67 (320–3)
G0283713 -72.75 (291–4)
G0293356 -11.24 (281–25)
hssA. -10 (1550–155)
Small proteins (57–59 aa) (prestalk specific)
G0283421 -34.33 (515–15)
G0283465 -118.67 (356–3)
G0283501 -25.19 (3929–156)
G0283503 -41.42 (994–24)
G0283505 -33.95 (1935–57)
G0283507 -38.25 (1224–32)
G0283511 -9.55 (2015–211)
G0283515 -14.28 (2313–162)
G0283519 -55.88 (950–17)
G0272188 -15.28 (2119–139)
G0269674 -15.57 (794–51)
G0271888 -40.5 (81–2)
G0269672 -7.46 (574–77)
Small proteins (69–72 aa) (prespore specific)
G0285863 28.07 (208–7523)
G0284623*c 35.48 (29–1029)
G0271110* 13.91 (11–153)
Tiger family proteins
tgrF1. -39.85 (518–13)
tgrC5*c 18.9 (10–189)
Polyketide synthase family
Pks32 -6.62 (1205–182)
mybC −10.86 (228–21)
comH*ca 11.53 (184–2123)
G0288967* 17.07 (96–1639)
G0290847*a 21.74 (80–1739)
G0290855*c 32.39 (41–1328)
G0271438* 65.5 (4–262)
srfC. 0 (0–73)
St15 -7.69 (1084–171)
psiI* 26.06 (469–12220)
psiN −41.51 (5645–136)
Omt12 -19.79 (277–14)
hspC*ca 15.69 (26–408)
arrK −10.65 (213–20)
osbH −38.33 (115–3)
fhbB 6.54 (525–3433)
G0278647 -23.43 (164–7)
Putative Membrane proteins
G0275535 -6.03 (3197–530)
G0285697* 451.2 (5–2256)
G0289143 -9.79 (1116–114)
G0267564* 16.32 (41–669)
G0287195 -10.05 (774–77)
G0272714* 10.99 (73–802)
G0284683 -11.87 (273–23)
G0270342* 10.81 (1679–18158)
G0272042 8.53 (459–3915)
Rpl32* 79.86 (7–559)
R52*a 10.44 (195–2035)
G0277795 -23.55 (259.11)
G0284969* 12.87 (93–1197)
cog2* 12.41 (539–6687)
G0276325* 32.62 (8–261)
G0290965*c 12.31 (13–160)
The study of the biological function of the mef2A gene was approached by generating deletion mutants in D. discoideum. Several mutants were generated in the AX2 and AX4 strains, and similar phenotypes were observed for all strains, as shown in Figure 3 and Tables 1 and 2. Over-expression of mef2A caused developmental defects that were morphologically similar to those observed in the mutants. Pools of mef2A over-expressing cells were similar to the mutant strains in the formation of more structures, more heterogeneous in size, and fewer slugs with reduced migration (data not shown), which prevented mutant complementation studies. Similar results were obtained when mef2A was expressed from integrative vectors using the constitutive Act15 promoter or the endogenous, prespore-specific mef2A promoter. A possible explanation for these results might be that over-expressed MefA proteins bind to co-factors or activating molecules outside the chromatin environment, thus impairing their regulatory function on DNA-bound Mef2A molecules.
The results indicate that mef2A is involved in the determination or differentiation of prespore cells and of a group of prestalk cells in D. discoideum. Mutant cells do not differentiate to spores in vitro and, in vivo, produce approximately half the number of spores than wild-type cells produce. These defects are cell-autonomous because the presence of wild-type cells is not able to induce differentiation of the mef2A- mutant cells. The proposed role would represent a conserved function for Mef2 proteins during evolution, given that plant and animal homologous proteins also play important roles in cell differentiation, as mentioned in the Introduction. Mef2A mutants also show impaired growth when feeding on bacteria. This defect does not appear to be due to a reduced phagocytic capacity, as determined by incubation with fluorescent microspheres (Fluoresbrite, PolySciences, data not shown). Differences in cell motility could also explain the smaller size of the colonies, but these possibilities have not been further studied. However, mef2A (srfC) has been previously identified as one of the genes whose expression is regulated depending on the growth substrate of the D. discoideum cells, bacteria or axenic media .
As mentioned above, mef2A appears to be involved in the determination or differentiation of a population of prestalk cells located at the tip of the culminant structures and at the anterior-most region of the slugs. These cells express the ecmB gene and include the tip-organizer cells that regulate the culmination of the structures . The evidence for this function is that mef2A- mutant cells expressing ecmB::lacZ do not participate in the formation of the tip in mixed developmental processes (Figure 5). In addition, mef2A mutant cells differentiate poorly to ecmB-expressing prestalk cells in vitro in the presence of DIF and cAMP  (data not shown). However, we have not detected mef2A expression in these cells. The cell-autonomous function of mef2A during differentiation may be due to a cell-type determination process taking place at the previous mound stage of development where mef2A is expressed (Figure 2). Alternatively, the expression of mef2A in ecmB-expressing prestalk cells might be too low to be detected by gene reporter expression analyses.
Other populations of prestalk cells, however, are enlarged in mutant structures, as shown in Figure 4. Differences in cell type specification could explain the phenotypes observed. For example, the existence of a larger number of prestalk cells that adhere more strongly to each other might contribute to the breakage of the streams. In addition, a number of the genes that are misregulated in mef2A mutants, such as the tgr family of genes (tgrF1, tgrC5), are involved in cell adhesion . However, no differences in Ca-dependent cell adhesion could be determined experimentally using the method described by Parkinsons et al.  (data not shown). The slug structures also showed a highly altered proportion and distribution of prestalk and prespore cells (Figure 4), which could explain their smaller size and limited motility.
Despite the developmental defects discussed above, it seems clear that mef2A is not absolutely required for D. discoideum development given that a large number of fruiting bodies and spores are formed in the mutant strains. We would like to suggest that mef2A participates in a network of transcription factors that regulate cell differentiation and that could compensate, at least partially, for the absence of mef2A. For example, mutations of histone deacetylases  and chromatin-binding proteins  affect prespore differentiation or cell-type patterning. In addition, the expression of several genes coding for putative transcriptional regulators is regulated by mef2A. For example, comH codes for a GATA-binding transcription factor expressed in prespore cells. G0288967 codes for a putative β-sandwich domain transcription factor, and G0290847, G0290855 and G0271438 code for proteins containing domains possibly involved in DNA binding and transcription regulation. rblA, a retinoblastoma homolog, also controls the preference of the cells for stalk or spore differentiation . This gene is expressed in the prespore region, and, in chimera with wild-type cells, rblA mutant cells show a strong preference for stalk differentiation, as was also demonstrated here for mef2A mutants.
Mef2A might play a cell-autonomous regulatory role in cell differentiation in response to extracellular signals analogous to those described in other biological systems. The activity of vertebrate Mef2 transcription factors is tightly regulated by extracellular signals. One of the best-known regulatory pathways involves the regulated association of Mef2 with class II histone deacetylases in a process mediated by protein phosphorylation . Mef2 can also be directly phosphorylated through MAP kinase pathways, regulating its transcriptional activity . In this respect, the D. discoideum MAP kinase ErkB is required for spore differentiation [44, 45]. It would be of interest to determine whether mef2A participates in this ErkB-mediated spore differentiation pathway, especially given that Mef2A presents three consensus ERK phosphorylation sites.
The main regulatory pathway described as inducing prespore cell differentiation is initiated by extracellular cAMP and requires protein kinase A (PKA) activation . The in vitro spore differentiation study shown in Figure 6 was induced by Br-cAMP treatment, resulting in direct PKA activation. The mef2A- mutant cells were unable to differentiate under these conditions. This result indicates that Mef2A regulation takes place downstream of PKA. In fact, a PKA consensus phosphorylation site is present close to the C-terminal end of Mef-2, indicating the possibility that this protein might be a substrate for PKA.
Massive-sequencing analyses of gene expression indicate that Mef2A can play a role in cell differentiation through the regulation of gene expression. There are 32 genes whose expression decreases in mef2A mutants, and many of these genes are specifically expressed in prespore cells, as determined by the mRNA expression analysis available at Dictybase (DictyExpress ) and by the in situ hybridization analysis . A number of prespore specific proteins, often used as prespore markers, also showed differences in expression between AX4 and the mef2A mutant strain but did not reach the filter requirements set up in the analysis of the sequencing data (more than 3 times the difference in the expression level and a p-value smaller than 0.01). For example, cotA was expressed 2.12 more times in AX4 than in the mutant, cotC was expressed 2.02 times, cotD 1.48 times, pspD 1.95 times, pspB 2.52 times and pspA 1.39 times. In contrast, many of the genes that are over-expressed in the mef2A mutant have been identified as prestalk-specific, as shown in Table 3. The prestalk-specific gene ecmA was also expressed at higher levels in the mutant (1.51 times). However, ecmB was expressed 1.53 times more in AX4, in agreement with the reduced and more disorganized distribution of ecmB-expressing cells in the mutant at 16 hours of development (Figure 4).
Further studies are required to determine the mechanism involved in the transcriptional regulation of the genes whose expression is altered in mef2A mutants. Several of the genes that are under-expressed in mef2A mutants appear to be almost completely dependent on this transcription factor for their expression. The regulation of the expression of these genes could be mediated by the direct binding of Mef2A to their regulatory regions. The DNA binding site of Mef2-related factors has been conserved through evolution and corresponds to the consensus sequence CTA(A/T)4ATG. We looked for the presence of this sequence on the 1000 nucleotide-long fragments located upstream of the 21 genes down-regulated in the mutant using the oPOSSUM program . As shown in Table 3, 6 of these genes contained this consensus sequence in the region analyzed. Thirteen additional genes contained related sequences that differed in the C or the G nucleotides. In addition, 4 genes presented the consensus binding site of a related MADS-box transcription factor (ARG80 from S. cerevisiae) . These data indicate that a number of these genes could be direct regulatory targets of Mef2A. Alternatively, mef2A could regulate the expression of other transcription factors controlling the expression of these genes.
The initial analysis of the structure of the mef2A gene detected the existence of alternative promoters that drive the expression of the gene at different times of development and in distinct structures. It is remarkable that the related srfA and srfB genes are also transcribed from alternative promoters, specific for different cell types and developmental stages [30, 51]. Other developmental regulatory genes are also transcribed from alternative promoters in D. discoideum, such as pdsA (extracellular phosphodiesterase) carA (cAMP receptor)  and acaA (adenylyl cyclase A) . The existence of alternative promoters might have been an evolutionary adaptation that regulates the expression of a gene under different conditions of growth and/or different developmental processes.
mef2A, which codes for a protein homologous to myocyte enhancer factor 2 transcription factors, is required for several of the steps of the D. discoideum biological cycle, including growth on bacteria and multicellular development. In particular, mef2A is involved in the regulation of the determination or differentiation of prespore cells and a group of prestalk cells during the developmental process of fruiting body formation.
Cell culture, transformation and development
D. discoideum cells were cultured axenically in HL5. Transformation by electroporation was performed as described previously . Transformed cells were selected by treatment with blasticidin  or neomycin (G418). Filter development was induced by spreading 1–2 × 107 cells (0.6-1.2 × 106 cells/cm2) on nitrocellulose filters (Millipore Co., Bradford, MA, USA) . The phosphate-based PDF buffer was used to obtain finger, Mexican-hat and culminant structures. Slug structures were obtained by conducting the development in water-soaked filters under directional light. Submerged development was induced by incubation of the cells in 2 ml of PDF phosphate-based buffer on 37-mm diameter cell-culture dishes at 5 × 105 cells/ml.
The amino acid sequences of MADS-box regions from various organisms were obtained from public databases and compared to those of the four D. discoideum proteins containing MADS-box-related sequences, obtained from DictyBase (http://www.dictybase.org). Amino acid sequences were compared using the ClustalW program at the online Biology Workbench facilities from the San Diego Supercomputer Center (http://workbench.sdsc.edu) and the ClustalX program . Phylogenetic trees were determined using the neighbor-joining method . A random generator seed of 111 and 1000 bootstrap trials were calculated. Trees were drawn using the NJplot program.
Rapid amplification of cDNA ends
RNA was isolated from AX4 cells at 8 hours of multi-cellular development on nitrocellulose filters. The SMART™ cDNA amplification kit from Clontech (Clontech Laboratories, Inc, Mountain View, CA, USA) was used for the amplification of the 5′ untranslated region of mef2A mRNA according to the manufacturer’s instructions. The oligonucleotide TGTTGCCTGTCTATTTCTTTCATTAG, complementary to nucleotides 145 to 164 of the gene, was used as the primer. Amplification products were cloned in the pGEM®-T Easy Vector System (Promega Co, Madison, WI, USA), and the insert of at least 10 different colonies of each product were sequenced.
Determination of mef2Aexpression by RT-PCR
RNA was isolated from 2 × 107 cells, either during growth or after development on nitrocellulose filters for 2 to 24 hours, using the TRI reagent (Sigma-Aldrich Inc., St. Louis, MO, USA) according to the manufacturer’s instructions. RNA was further purified using the RNeasy Mini Kit (Quiagen). cDNAs were generated from 2 μg of total RNA using gene-specific oligonucleotides as primer. cDNAs were used as substrates for PCR reactions using as primers the oligonucleotides used for cDNA synthesis and upstream oligonucleotides designed from the coding region of the transcripts. The oligonucleotides GGACTAGTTTCCATTGAACCAATTGGGTGAGCG and CTGATAATACAGATAATACTCGC were used for mef2A cDNA synthesis. The large mitochondrial rRNA was amplified as a control, using oligonucleotides GGGTAGTTTGACTGGGGCGG and CACTTTAATGGGTGAACACC.
Vectors for the generation of knockout strains
Flanking regions of the mef2A gene, including nucleotides −4069 to −2692 and 596 to 1298, in relation to the A of the translation initiation codon were generated by PCR and cloned on both sides of the blasticidin resistance gene in the pLPBLP plasmid vector . The ligonucleotides GGCCGCGGCCATTCCCAGCAACGCTGGTAATC, GGTCTAGACCTGGAAAACTGGAAAACCAATTG, GGATCGATCCACCCACACTAACACACACC and GGGTCGACGGTGGTGGTGATTGGTGCTG were used for these amplifications. The oligonucleotides TGGGAAGGAATAAAATTACAATTGAAAAG and GCGAGTATTATCTGTATTATCAG were used to test for the deletion in AX4 strains, and GTTGCCTGTCTATTTCTTTC and CACTCACTTACATATCACACACC were used in AX2 strains.
Construction of the reporter and expression vectors
The two mef2A promoter regions were amplified by PCR from D. discoideum genomic DNA and cloned in the reporter vector PsA-ialphaGal  in substitution of the XbaI/BglII pspA promoter fragment. The oligonucleotides GGTCTAGAGCACAAGATTATACTTGCCA and GGAGATCTCATGGTGTGTGATATGTAAGTGAGTG were used to amplify the −2201 to −489 region of the gene, corresponding to Promoter 1. The oligonucleotides GGTCTAGACACTCACTTACATATCACACACC and GGAGATCTTGTTGCCTGTCTATTTCTTTCATTAG were used to amplify the −511 to 164 region, corresponding to Promoter 2. The complete promoter region (−2201 to 164) was amplified using the first and last oligonucleotides described above. Previously described lacZ reporter vectors were used to determine the expression of the developmental markers pspA, ecmA and ecmB.
The mef2A gene was expressed using the pDV-CGFP-CTAP vector , under control of the Actin15 promoter. The region coding amino acids 4 to 1046 of the protein, including the third intronic region of the gene, was amplified from genomic DNA using the Pfx DNA Polymerase (Invitrogene™). Two overlapping fragments were obtained using the oligonucleotide pairs GGATCCAGGAATAAAATTACAATTGAAAAG/GGAGATTGATGCTGTGGTTG and CAACAACAAAGCGCCAATCC/ACTAGTAGGTTCCATTGATTTTCTTTTTCGG. The two fragments were joined together using the overlapping HaeII restriction site and the resultant fragment cloned between the BamHI and SpeI restriction sites of the vector. A second expression vector where mef2A was expressed under control of his own promoter was constructed by substituting the Actin15 promoter of the pDV-CGFP-CTAP/mef2A vector by the mef2A promoter. The Actin15 promoter was excised by SalI and BamHI digestion and replaced by the complete mef2A promoter previously cloned in the PsA-ialphaGal vector, as described above. The mef2A promoter was isolated by XbaI/BglII digestion. The SalI end of the vector and the XbaI end of the promoter were converted to blunt ends before ligation.
Histochemistry and determination of β-galactosidase activity in developmental structures
Cells transformed with the different reporter vectors were allowed to develop on nitrocellulose filters for the time periods indicated in each experiment. The structures were fixed and permeabilized, and β-galactosidase activity was detected by hydrolysis of X-gal (5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside), as previously described .
Cell tracking experiments
Growing cells were collected by centrifugation and resuspended in phosphate-based PDF buffer containing 5 μl/ml of 100 mM CellTracker™ Blue CMHC (4-chloromethyl-7-hydroxycoumarin) (Invitrogen, Eugene, Oregon, USA) or a vehicle (DMSO) and incubated for 1 hour in shaking cultures. Cells were then washed, resuspended in free PDF buffer and mixed in a 1:1 proportion. A total of 6.6 × 106 cells from each mixture were spread out on nitrocellulose filters for 24–36 hours, after which several sori from each mixture were harvested and dissociated in water. Spores were visualized in a Zeiss Axiophot fluorescence microscope and counted.
Determination of mRNA levels by quantitative RT-PCR
RNA was isolated from 2 × 107 cells, either during growth or after development on nitrocellulose filters for the times indicated in each experiment, using the TRI reagent (Sigma-Aldrich Inc., St. Louis, MO, USA) according to the manufacturer’s instructions. RNA was further purified using the RNeasy Mini kit (Quiagen). cDNAs were generated from 2 μg of total purified RNA using random primers (Promega Co., Madison, WI, USA). cDNAs were used as substrate for quantitative real-time PCR reactions using the following gene-specific oligonucleotides: hssA gene (DDB_G0280999) GTGCTATTACCTCAATTTCAAG and GGCAACCACATGAACCACTTG; DDB_G0283503 gene CAAATCATTACAATCAATCACAAGTG and GGGCTACAGCAGCAACTG; prS1 gene (DDB_G0285863) CCAATAATTCTTTGAAGGCCC and CAATAGCTTGGCCCATAGTAGC; tgrF1 gene (DDB_G0292732) CCCACCATTTACTCCAATACTC and GTAGAGATGGTGTTGATGGAG; tgrC5 gene (DDB_G0281407) GCTGGCTTAGCACTTTCATCAG and GAGACCAACGGCAGCGACAC; pks32 gene (DDB_G0292732) CAACTCCAGTCACAACTATAGC and GATTATCATGAATGTGGAATGCTG; mybC gene (DDB_G0281563) GGTGGAGGTAAAACTGGTGC and CATCCATCCAACTAATATCACG; DDB_G0290847 gene CAGTACTGAACAAGCATTATCAAG and GTTAACATAACCTTGTTGAGAATC; DDB_G0271438 gene GTCATGAAATTGGAGATCGAAG and CATGAGATGATGTTGATTTGG; psiI gene (DDB_G0288919) GGTTGTACACTTGTACCACG and GAGGTGCTTCAAAGAGAGC; DDB_G0285697 gene GGTAAGGCAGTTGTCAATGC and GCCTACCAGCTGAGACTTCAGC. A region of the large mitochondrial ribosomal RNA was amplified as a loading control using the oligonucleotides CACTTTAATGGGTGAACACC (used as a reverse oligonucleotide) and GGGTAGTTTGACTGGGGCGG (used as a forward oligonucleotide). The StepOnePlus Real-Time PCR System (Life Technologies Co., Applied Biosystems, Carlsbad, CA, USA) was used in these experiments. PCR products were labeled with SYBR Green using the Power SYBR® Green PCR Master Mix reaction mix (Applied Biosystems) following the manufacturer’s instructions. The final volume of the reaction was 20 μl, using a 0.2 μM concentration of each primer. PCR conditions were as follows: 95°C, 10 m; (95°C, 15 s; 45°C, 30 s; 62° 1 m) × 30–40 cycles.
In vitro spore differentiation
Exponentially growing cells were washed in KK2 buffer (16.5 mM KH2PO4, 3.9 mM K2HPO4, 2 mM MgSO4, pH 6.2), plated on culture dishes at a concentration of 106 cells/ml in spore buffer (10 mM MOPS, 20 mM KCl, 20 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 6.2) and supplemented with 12.5 mM 8-Br-cyclic-AMP and 20 μM CdCl2[32, 65]. Cells were incubated in the dark for 30 hours and then observed under a TS100 Eclipse Nikon microscope (Nikon, Tokyo, Japan). Pictures were taken with a Leica DFC420 camera (Leica Microsystems, Wetzlar, Germany).
RNA was isolated from structures developed on nitrocellulose filters for 16 hours using the TriReagent and purified with an RNeasy Mini kit, as described previously. Poly(A)-containing RNA was isolated and converted to cDNA. The cDNA was fragmented, amplified by PCR and the nucleotide sequences determined using an Illumina Genome Analyzer IIx massive sequencer at the Parque Científico de Madrid. Sequencing data were analyzed at Sistemas Genómicos, S.L. (Valencia, Spain). The generated sequences were mapped to the D. discoideum genome using the TopHat v1.1.3 software . Transcripts were identified and quantified using the Cufflinks v1.0.3 program. The total number of reads per gene was determined using the HTSeq package (http://www-huber.embl.de). Statistical analyses of the results was performed using the DESeq package , using an FDR of 0.01. A minimal difference of three times in expression levels was considered.
The authors are indebted to the Dictybase organization for information and reagents. The collaboration of the Parque Cientifico de Madrid with RNA sequencing experiments is also acknowledged. LS was funded by grant BFU2008-02249 from the Spanish Ministry of Science and Innovation (Ministerio de Ciencia e Innovacion). MGC was supported by a JAE predoctoral fellowship from the High Council on Scientific Research (Consejo Superior de Investigaciones Cientificas). We acknowledge the financial support for publication by the CSIC Open Access Publication Support Initiative through its Research Information Resources Unit (URICI).
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