Comparative genome and transcriptome analyses of the social amoeba Acytostelium subglobosum that accomplishes multicellular development without germ-soma differentiation
- Hideko Urushihara1Email author,
- Hidekazu Kuwayama1,
- Kensuke Fukuhara1,
- Takehiko Itoh2,
- Hiroshi Kagoshima3,
- Tadasu Shin-I3,
- Atsushi Toyoda3,
- Kazuyo Ohishi3,
- Tateaki Taniguchi4,
- Hideki Noguchi2,
- Yoko Kuroki5,
- Takashi Hata1,
- Kyoko Uchi1,
- Kurato Mohri1,
- Jason S King6,
- Robert H Insall6,
- Yuji Kohara3 and
- Asao Fujiyama3, 7
© Urushihara et al.; licensee BioMed Central. 2015
Received: 12 August 2014
Accepted: 23 January 2015
Published: 14 February 2015
Social amoebae are lower eukaryotes that inhabit the soil. They are characterized by the construction of a starvation-induced multicellular fruiting body with a spore ball and supportive stalk. In most species, the stalk is filled with motile stalk cells, as represented by the model organism Dictyostelium discoideum, whose developmental mechanisms have been well characterized. However, in the genus Acytostelium, the stalk is acellular and all aggregated cells become spores. Phylogenetic analyses have shown that it is not an ancestral genus but has lost the ability to undergo cell differentiation.
We performed genome and transcriptome analyses of Acytostelium subglobosum and compared our findings to other available dictyostelid genome data. Although A. subglobosum adopts a qualitatively different developmental program from other dictyostelids, its gene repertoire was largely conserved. Yet, families of polyketide synthase and extracellular matrix proteins have not expanded and a serine protease and ABC transporter B family gene, tagA, and a few other developmental genes are missing in the A. subglobosum lineage. Temporal gene expression patterns are astonishingly dissimilar from those of D. discoideum, and only a limited fraction of the ortholog pairs shared the same expression patterns, so that some signaling cascades for development seem to be disabled in A. subglobosum.
The absence of the ability to undergo cell differentiation in Acytostelium is accompanied by a small change in coding potential and extensive alterations in gene expression patterns.
Morphogenesis and cell differentiation are the major components of multicellular development. In multicellular organisms, somatic cells, which are free from regenerative obligations, accomplish a variety of tasks to support complex body structures and functional integrity. The differentiation of mortal or sacrificial somatic cells from reproductive germ cells was the key event for the establishment and diversification of multicellular systems. How this was achieved in the history of life is an interesting and complex issue [1,2].
The social amoebae are unique organisms that exhibit conditional multicellularity and serve as an excellent model system to address this issue; they grow as solitary amoeba in the presence of sufficient food, but when starved, they gather together and form a multicellular fruiting body composed of a spore ball(s) and a supportive stalk(s). In many species, the stalk is filled with vacuolated stalk cells to stiffen it using osmotic pressure and cellulose walls that are deposited and polymerized on the extracellular matrix (ECM). While spores transmit their genetic information to their offspring, the stalk cells are no longer regenerative and represent one of the simplest forms of terminally differentiated somatic cells. In Dictyostelium discoideum, the most widely analyzed social amoeba species, cells in the migratory slug are committed to either the spore (prespore cells) or stalk lineage (prestalk cells) . The latter further diversifies to generate the prestalk subpopulations PstB, PstO, and PstU, which end up in the basal disk and upper and lower cup structures, in addition to PstA constituting the main stalk body. These developmental processes are mainly controlled by the external levels of chemical cues such as cyclic nucleotides, ammonia, polyketides, peptides, and steroids to activate the corresponding intracellular signaling cascades .
According to molecular phylogenetic analyses, D. discoideum belongs to the newest evolutionary clade (group 4), while the genus Acytostelium is in an older clade (group 2) [7,8] (Figure 1D). There are two possibilities for the lack of the ability to undergo cell differentiation in Acytostelium: this ability was either acquired in a later species or it had been acquired in a common ancestor and lost in the Acytostelium lineage. Since the species in the oldest clade, group 1, form fruiting bodies with cellular stalks, the latter possibility seems more likely, although it is still possible that cellular stalks arose independently multiple times, as pointed out by Swanson et al. . In either case, it is intriguing to determine what genetic information correlates with the ability to undergo germ-soma differentiation.
In the present study, we analyzed the genome and transcriptome of A. subglobosum in comparison with other social amoebae making cellular stalks. The D. discoideum genome was reported initially in 2005 . Since then, the genomes of Dictyostelium purpureum (group 4) , Polysphondylium pallidum (group 2), and Dictyostelium fasciculatum (group 1)  have become available. The developmental transcriptome of D. purpureum was compared with that of D. discoideum to reveal their remarkable conservation . Our comparative analyses showed that dissimilarities in the gene repertoire between differentiating and non-differentiating species were limited, but that their transcriptomes had diverged. We suppose that the critical loss of early developmental genes relevant to cell-type specification affected the gene networks and led to the invention of a new developmental program where the entry of the entire amoeba to germ-line spores was traded off against the low efficiency of their dispersal due to short and fragile acellular stalks that were unable to support sizable spore balls.
Results and discussion
Structure and general features of the A. subglobosum genome
General features of A. subglobosum and other dictyostelid genomes
Genome size (Mbp)a
Genome (A + T) content (%)
Protein coding genes
Gene density (CDS/Mbp)
Simple sequence repeat (%)
Protein coding potential of A. subglobosum
We performed expressed sequence tag (EST) analysis of vegetative and developmental cDNA libraries to determine the protein coding potential of A. subglobosum. Altogether, 32000 clones were read from both ends (DDBJ:HY448297-HY508708), and the obtained sequences were clustered into 7439 non-redundant groups derived from 5749 genes, 98.4% of which were successfully mapped to the genome at an identity ≥ 95% and coverage ≥ 80%. Representative cDNA clones were chosen for each of these groups and re-sequenced. The transcript information thus obtained (Additional file 1: Figure S2) was incorporated into a gene prediction program based on dicodon analysis  (Additional file 1: Figure S3). We also constructed homology-based gene models using D. discoideum protein sequences. The three sets of gene models, actual transcript sequences, ab initio predictions, and homology based predictions were combined to generate 12722 non-redundant protein coding genes (Additional file 2: Table S2; Additional file 3). This gene number is smaller than D. discoideum, but larger than 3 other species (Table 1). Lack of stalk cells in A. subglobosum may therefore not be explained simply by the absence of gene sets required for cell differentiation and stalk-cell function. This supports the previous argument that the ability to undergo cell differentiation was lost in A. subglobosum rather than its acquisition in later species.
Orthology and gene family analyses
To analyze the lineage-specific expansion of gene families in D. discoideum, we combined the homology search results and OrthoMCL  data of D. discoideum. Namely, homologous genes for each D. discoideum gene were assigned to the same gene family. Clustering of D. discoideum proteins by OrthoMCL resulted in similar but slightly bigger families than those reported by Eichinger et al.  (Additional file 1: Figure S4). For the 5414 OrthoMCL OG-ids of D. discoideum , which did not exclude the unique genes in this species, 4582 A. subglobosum, 4468 P. pallidum, and 4445 D. fasciculatum gene families (and unique genes) were associated.
Lineage-dependent expansion of gene families
Family size in
Expansion in non- Dfas lineage
Zinc finger, B-box domain and FNIP repeat-containing protein
Expansion in non- Ddis lineage
IPT/TIG domain-containing protein, EGF-like domain-containing protein, C-type lectin domain-containing protein
Expansion in Group 2
Colossin D, Cna B-type domain-containing protein
Expansion in A. subglobosum lineage
N-terminal delta endotoxin domain-containing protein
Lack of expansion in A. subglobosum
Cellulose-binding domain-containing protein, putative extracellular matrix protein
Putative polyketide synthase, beta-ketoacyl synthase family protein
Search for genes associated with the ability to undergo cell differentiation
The main purpose of this study was to mine out genetic information that was correlated with the ability to undergo cell differentiation. It was noted, at the early stage of the study, that the major D. discoideum genes related to stalk-cell differentiation were present in A. subglobosum despite the absence of stalk cells in this species. Although this is seemingly contradictory, it is not irrational considering the fact that A. subglobosum does make stalks. Apparently, a simple loss of “stalk genes” cannot explain the unique developmental process of A. subglobosum.
D. discoideum developmental genes lacking orthologs in the A. subglobosum lineage a
Mutant information c
Involved in cell fate-determination
ABC transporter B family protein, serine protease
Multiple tips in mound (partial KO); Aberrant fruiting body morphology (KO)
Involved in stalk-cell diversification
Lipid-translocating exporter family protein
Expressed in PstU cells (lacZ fusion)
Putative homeobox transcription factor
Development arrests at slug stage, decreased prespore cell differentiation, and increased PstO cell differentiation (KO)
Involved in terminal differentiation
Aberrant culminant morphology (OE)
Aberrant culmination (KO)
Other genes in Table 3 are expressed at the later stages of D. discoideum development and their relevance to cell-fate determination is less likely. Two genes, rtaA and warA, are concerned with prestalk-cell diversification. The rtaA gene encodes a putative GPCR [28,29] expressed in PstU cells , which finally populate the upper cup of a fruiting body whose function is to lift up the spore mass . The latter gene, warA, is a homeodomain-containing putative transcription factor expressed in prestalk cells and determines the proportion of PstO cells in the slug, which occupy the zone between the prestalk and prespore regions . Since the corresponding cell populations do not exist even temporarily in A. subglobosum, the mechanistic aspect of culmination seems different in this species, as pointed out by Bonner . The expl7 gene encodes a member of the expansin-like protein family and is homologous to the plant cellulose-binding protein expansin . Although the over-expression of expl7 resulted in an anomaly of stalk morphology, its disruption did not affect the fruiting body morphology in D. discoideum , suggesting complementation by its paralog(s). The disruption mutant of rsc12 resulted in aberrant culminant at the final stage of development , but detailed analysis has not been carried out.
Loss of function can also be caused by the acquisition of new genes with suppressive effects. Although it is possible that some such genes may act to suppress the A. subglobosum counterparts of developmental genes in stalk-cell-making species, their functions in relation to fruiting body formation are elusive without molecular biological analyses.
Comparative transcriptome analysis
The above mentioned analysis on the gene repertoires demonstrated that the majority of genes involved in fruiting body formation in D. discoideum have orthologs in A. subglobosum and only few of them are without counterparts. To clarify whether the conserved genes are actually expressed, the developmental transcriptome of A. subglobosum was analyzed. Approximately 4.5 Gb of cDNA reads, generated at each of 0, 8, 16, and 24 h of development, were combined, clustered, and mapped to the A. subglobosum genome contigs (Additional file 1: Figure S7), resulting in the association of at least 9067 gene models. For the 5961 A. subglobosum orthologs to D. discoideum genes with a significant level of expression, expression data for 5062 (85%) genes were obtained. As for the developmental gene orthologs, 70 had no clear evidence of expression during growth and asexual development.
D. discoideum developmental genes with altered expression in A. subglobosum
Cell-type specificity in D. discoideum
aprA*, cf50-1*, cmfA, ctnA*
mgp2, mppA1, sglA, tmem184C, DG1060
DG1112 , gsr, spkA*
alg9, midA , ppp4C, sgcA, DG1104, DDB_G0287723
clc, phlp1, DG1040
cshA*, ctr9, Dd5P4, nfaA, snfA, DDB_G0278945
kif12, lvsD, DDB_G0269680, DDB_G0285083, DDB_G0288007
amtA*, dymA*, srfA
atg5, dgkA, DDB_G0270344
adprt3 , tipC
captC , ifkA, DG1003
alrA, dokA, phyA, vmp1
In addition to the differential expression time course, altered mRNA levels, both increased and decreased, were noticed in a substantial number of ortholog pairs (Additional file 1: Figure S9). It caught our attention that counting factor and related components involved in determination of aggregate size (cfn50-1, ctnA, and cf60) were greatly repressed. Only one of the related genes, cfaD, is expressed at a comparable level and in the same pattern, but this gene is presumed to control the growth-development transition. Since inactivation of these genes in D. discoideum results in larger aggregates, the implications of the above finding on the small fruiting bodies of A. subglobosum are unclear. It may be related to the fact that A. subglobosum development is possible only at lower cell densities than for D. discoideum development . It is also interesting that the expression of genes for G protein α subunits 6 (gpaF) and 9 (gpaI) were altered in mutually opposite directions. These transcriptome differences should exert significant influences on the signal-response cascades and gene networks during development.
Signaling cascades for cell differentiation and morphogenesis
The spore lineage cells induced by a high concentration of extracellular cAMP in turn induce uncommitted cells to the prestalk lineage in D. discoideum. The important gene at this step, tagA, is missing, as mentioned above, and orthologs of two cAMP receptors, cAR3 and cAR4 (encoded by carC and carD), are also absent in A. subglobosum, corresponding to the lack of cell-type differentiation in this species. The specific substrate of TagA is suspected to be the acyl coenzyme A binding protein (AcbA) from the genetic evidence . The facts that lack of the transporter domain of tagA caused the multi-tip phenotype in D. discoideum but that complete disruption of this gene resulted in a single gnarled stalk suggest the dual function of TagA during development.
Stalk-cell diversification is triggered by DIF-1, which is secreted from prespore cells in D. discoideum. Despite the fact that A. subglobosum does not make the basal disk or upper and lower cup structures of the fruiting body, genes for the components of the DIF-1 signaling cascade do exist and are expressed at more or less similar timings. On the other hand, we were unable to detect DIF-1 production in A. subglobosum either biochemically or biologically . Therefore, it is possible that a similar but different substance is produced in A. subglobosum and induces a modified version of the polyketide signaling cascade to activate ECM genes and the cellulose synthase required for stalk formation.
The molecular mechanisms of synchronized and rapid spore encapsulation caused by exocytosis of spore-coat materials in the prespore vesicles are relatively well understood. They employ peptides called spore differentiation factor 1 (SDF-1) and SDF-2, which are secreted as precursors from prespore cells and processed by the serine protease-ABC transporters TagB and TagC, respectively [39,40]. The whole process is accelerated by GABA- and MPBD-mediated cascades, the former being triggered by a steroid type SDF (SDF-3) . The accumulation of SDF precursors in the prespore cells is controlled by intracellular cAMP levels via protein kinase A . Overlaying A. subglobosum orthology and expression data suggested that only the core cascade involving SDF-2 is intact in A. subglobosum (Figure 6B); enhancement by the SDF-3-GABA route is probably disabled by the lack of its essential gene, gadA. The SDF-1 cascade seems to be hampered by the absence of tagB. Considering the small size of the A. subglobosum sorus, the synchronous encapsulation of spore precursor cells may not require multiple cascades, although the possibility of functional complementation by paralogous genes still exists.
Overall developmental signalling is compared between the 2 species in Figure 5. We already showed that a sequential gene expression of “prespore” and “prestalk” genes was observed in individual cells . The results presented here suggest that this finding can be extended: In contrast to the developmental process of D. discoideum achieved by 2 types of cells in parallel, the developmental program of A. subglobosum seems to depend largely on sequential gene expression, which is regulated cell-autonomously, and on a few cell-cell interactions.
Our genome analysis of A. subglobosum revealed the unexpected conservation of D. discoideum stalk-specific genes. However, alterations in developmental transcriptomes were extensive. This suggests that non-differentiating species utilize fundamentally different developmental programs, even though their final morphologies appear similar. Since gene losses at the early stages of cell-fate determination must disturb the later developmental processes enormously, they are likely to have been compensated by differential gene regulations.
Cell culture and asexual development
The clonal line of A. subglobosum strain LB-1/A1 was described previously (6). A1 cells were grown in shaking HL5 medium at 22°C. For asexual development, the cells were harvested at their early growth phase (1.0–3.0 × 106 cells/mL), washed twice with KK2 buffer (20 mM K2HPO4/KH2PO4, pH 6.8), and spread on a cellulose ester membrane (48 mm in diameter) (Advantech) at a density of 2.5 × 105 cells/cm2. This was the upper limit for efficient fruiting body formation in A. subglobosum. Ten membranes were put on a 20 cm × 20 cm plate of plain agar containing charcoal to enhance development, and incubated at 22°C.
Genome size determination
Approximately 1 × 108 cells were harvested from the HL5 culture, washed in phosphate-buffered saline (PBS) and pelleted by centrifugation. Their nuclei were prepared using a nuclei extraction kit NE-PER (Pierce), resuspended in 2 mL PBS containing 1 mM EDTA, 200 μg/mL RNase A, and 50 μg/mL propidium iodide, and then analyzed on a FACS Calibur platform (Becton Dickinson) using an excitation wavelength of 488 nm. To ensure single-nucleus measurement, the gate was set using the FLS-A and FL2-W parameters of the doublet discrimination module.
Chromosome number determination
Approximately 5 × 106 cells were seeded in a 5 cm dish containing acid-washed coverslips and incubated for 2 h in 5 ml HL5 medium to allow cells to adhere. The culture medium was replaced with fresh HL5 containing 33 μM nocotazole. After incubation for 4 h, the coverslips were placed in chilled distilled water for 10 min and fixed for 1 h in ice-cold 3:1 ethanol/glacial acetic acid, followed by 10 min re-fixation in the fresh fixative. The coverslips were air dried and mounted on glass slides in 3 μL DAPI/Vectashield and observed under a wide-field fluorescent microscope using a 100 × 1.4 NA objective.
Genome sequencing and assembly of A. subglobosum
Genomic DNA was extracted from the nuclei of growth phase A1 cells and processed for nucleotide sequencing. We constructed a hybrid de novo assembly based on Sanger pair-end whole genome shotgun (WGS) sequences from plasmid clones with a ~3 Kb insert and supplemented with Illumina WGS sequences. The Sanger sequence data were assembled into sequence contigs using PCAP  and the subsequently independently assembled Illumina contigs using Platanus  were used to extend them and to close gaps between Sanger-based contigs. The transcriptome data (see below) were also used to fill the contig gaps where possible. A fosmid library was also constructed and its 6912 clones were end-sequenced to aid scaffold construction. Some contig gaps were filled by manual walking-in.
To construct the growth phase cDNA libraries, mRNA was extracted using Oligotex-dT30 < super > (TAKARA), reverse transcribed, and ligated with (asgl library) or without (asgs library) size fractionation (>1 Kb) to pSPORT1 using the SuperScript Plasmid System with Gateway Technology (Invitrogen) and transformed into Escherichia coli DH10B ElectroMax (Invitrogen). For the preparation of developmental RNA, the developing cells were detached from the membranes by incubation in cold PBS containing 5 mM EDTA for 5 min followed by vigorous shaking, and washed twice with cold PBS. A full-length developmental cDNA library (asdv) was constructed from the 3 combined preparations of 20 h cells by the SMART method (TAKARA) using pDNR-LIB as a vector. The inserts of randomly chosen clones from these 3 cDNA libraries were sequenced from both ends using an ABI 3730 DNA Analyzer (Applied Biosystems). The obtained EST data were assembled by the CAP3 program to obtain non-redundant sequences. We selected and re-sequenced 700 clones with an unfilled internal sequence to generate high quality cDNA sequences.
For mRNA massive sequencing, we combined 6 independent preparations of total RNA from 0, 8, 16, and 24 h of development. The cDNA templates for Solexa sequencing were synthesized using an mRNA-Seq RNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions. The sequence data were assembled using ABySS  and mapped to the genome contigs using the exonerate assembly program .
Gene model construction
A. subglobosum gene models were constructed by the following 3 methods. 1) Acyto_CDS: the cDNA sequences and CAP4 assembly of ESTs were aligned to the genome contigs and those with an identity ≥ 95% and coverage ≥ 80% were selected. Where it was appropriate, the forward and reverse sequences of each singlet were joined. The longest open reading frames starting with the initiation codon were adopted. 2) Dicty_Pept: the translated A. subglobosum genome sequences homologous to D. discoideum protein sequences at a similarity ≥ 30% and coverage ≥ 50% were extracted and joined where appropriate. 3) Ab initio prediction: a gene prediction program based on dicodon analysis  was used employing the real transcript information obtained here. The final gene models were constructed by unifying the above 3 models and, in part, by manual curation.
Genome information and gene models of other dictyostelid species
The genome sequences and gene models of D. discoideum, D. purpureum, P. pallidum, and D. fasciculatum were downloaded from dictyBase . For D. discoideum, the genes located on the duplicated region of chromosome 2  were eliminated. D. discoideum “developmental genes” were extracted from published reports summarized in the Dicty Stock Center website .
Gene orthology and family assignment
Orthologous gene pairs were determined between 2 species by the bidirectional best hit approach setting the blastp threshold to an e-value of 1E-10. Genes of non-orthologous hits were regarded as paralogs in each species. We used OrthoMCL  to cluster the proteins of D. discoideum and manually supplemented the results using information from the dictyBase gene list and reports by Sucgang et al.  and Heidel et al. . Orthologous genes in other species and their paralogs were assigned to the same gene family.
The mRNAseq data of A. subglobosum obtained as the mean of 6 biological replicates, excluding contaminating rRNA sequences, were converted to reads per kilobase per million as in the case of D. discoideum. For the downloaded D. discoideum data of Parikh et al. , those from 0, 8, 16, and 24 h were extracted and the mean of 2 biological replicates was obtained. To normalize the data of the 2 species, each value was multiplied by [107/the sum of the relative expression levels for each time point of each species]. Genes that were not expressed throughout development were eliminated, and all remaining data of the 2 species were combined. K-means clustering was performed using Orange software  with distance measure, Pearson correlation, initialization, random, and restart 100 times. Cluster number 8 was employed after trials using larger and smaller numbers.
Availability of supporting data
The data sets supporting the results of this article were deposited to DDBJ under project ID PRJDG1513. Their accessions are HY448297-HY508708 for 60412 EST, BAUZ01000001-BAUZ01000371 for 371 WGS and DF837573-DF83768 for 106 CON (Contiguous sequence) entries. CON entries include 11687 CDS loci (locus_tag: SAMD00019534_000010- SAMD00019534_126860; protein_id: GAM116827-GAM29510). Protein sequences of gene models are also supplied in Additional file 3.
This work was supported by a Grant-in-aid for Scientific Research on Priority Areas (#20017004) and a Grant-in-Aid for Scientific Research (B) (#17310112) to H. Urushihara from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We acknowledge Mr. Ryuji Yoshino for his assistance in setting up A. subglobosum culture and experiments.
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