Multicellularity and development
- The genome of the social amoeba Dictyostelium discoideum

The evolution of multicellularity was arguably as significant as the origin of the eukaryotic cell in enabling the diversification of life. The common unicellular ancestor of the crown group of organisms must have posessed the basic machinery to regulate nutrient uptake, metabolism, cellular defense and reproduction, and it is likely that these mechanisms were adapted to integrate the functions of cells in multicellular organisms. Dictyostelium achieved multicellularity through a different evolutionary route from the plants and animals, yet the ancestors of these respective groups most likely started with the same endowment of genes and faced the same problem of achieving cell specialization and tissue organisation.

When starved, Dictyostelium develops as a true multicellular organism, organizing distinct tissues within a motile slug and producing a fruiting body comprised of a cellular, cellulosic stalk supporting a bolus of spores4. Thus, Dictyostelium has evolved differentiated cell types and the ability to regulate their proportions and morphogenesis. A broad survey of proteins required for multicellular development shows that Dictyostelium has retained cell adhesion and signalling modules normally associated exclusively with animals, while the structural elements of the fruiting body and terminally differentiated cells clearly derive from the control of cellulose deposition and metabolism now associated with plants. The Dictyostelium genome offers a first glimpse of how multicellularity evolved in the amoebozoan lineage. In the following sections, we consider some of the systems which are particularly relevant to cellular differentiation and integration in a multicellular organism.

Signal Transduction through G-protein coupled receptors
The needs of multicellular development add greatly to those of chemotaxis in demanding dynamically controlled and highly selective signalling systems. G-protein coupled cell surface receptors (GPCRs) form the basis of such systems in many species, allowing the detection of a variety of environmental and intra-organismal signals such as light, Ca2+, odorants, nucleotides and peptides. They are subdivided into six families which, despite their conserved secondary domain structure, do not share significant sequence similarity59. Until recently, in Dictyostelium only the seven CAR/CRL (cAMP receptor/ cAMP receptor-like) family GPCRs had been examined in detail60, 61. Surprisingly, a detailed search uncovered 48 additional putative GPCRs of which 43 can be grouped into the secretin (family 2), metabotropic glutamate/GABA B (family 3) and the frizzled/smoothened (family 5) families of receptors (Fig. 8; see also Supplementary Information). The presence of family 2, 3 and 5 receptors in Dictyostelium was surprising because they had been thought to be animal-specific. Their occurrence in Dictyostelium suggests that they arose before the divergence of the animals and fungi and were later lost in fungi and that the radiation of GPCRs predates the divergence of the animals and fungi. The putative secretin family is particularly interesting because these proteins were thought to be of relatively recent origin, appearing closer to the time of the divergence of animals62. The Dictyostelium protein does not contain the characteristic GPCR proteolytic site, but its transmembrane domains are clearly more closely related to secretin GPCRs than to other families (Fig. 8). Many downstream signalling components that transduce GPCR signals could also be recognized in the proteome, including heterotrimeric G-protein subunits (14 Gα, two Gβ and one Gγ proteins) and seven regulators of G-protein signalling (RGS), most similar to the R4 subfamily of mammalian RGS proteins.
SH2 domain signallingIn animals, SH2 domains act as regulatory modules of proteins in intracellular signalling cascades, interacting with phosphotyrosine-containing peptides in a sequence-specific manner. Dictyostelium is the only organism, outside the animal kingdom, where SH2 domain-phosphotyrosine signalling has been proven to occur63. What have been lacking in Dictyostelium are the other components of such signalling pathways - equivalents of the metazoan SH2 domain-containing receptors, adaptors and targeting proteins. Three newly predicted proteins are strong candidates for these roles (Fig. SI 15). One of them, CblA, is highly related to the metazoan cbl proto-oncogene product. This is entirely unexpected because it is the first time that a cbl homologue has been observed outside the animal kingdom. The Cbl protein is a “RING finger” ubiquitin-protein ligase that recognizes activated receptor tyrosine kinases and various molecular adaptors64. Remarkably, the Cbl SH2 domain went unrecognised in the protein sequence, but it was revealed when the crystal structure of the protein was determined65. Thus, although SH2 domain proteins are less prevalent in Dictyostelium, there is the potential for the kind of complex interactions that typify metazoan SH2 signalling pathways.


ABC transporter signalling
Dictyostelium, like other organisms, has adapted ABC transporters to control various developmental signalling events. Several ABC transporters, TagA, B and C, are used for peptide-based signalling, akin to that previously observed for mating in S. cerevisiae and antigen presentation in human T cells6668. The novel domain arrangement of the Tag proteins, a serine protease domain fused to a single transporter domain, suggests that they have been selected for improved efficiency in signal production. Additional ABC transporters are needed for cell fate determination in Dictyostelium, suggesting that this ubiquitous protein family may be used in similar developmental contexts within many different species69.


Kinases and transcription factors
Much cellular signal transduction involves the regulation of protein function through phosphorylation by protein kinases, often leading to the reprogramming of gene transcription in response to extracellular signals. The Dictyostelium proteome contains 295 predicted protein kinases, representing as wide a spectrum of kinase families as that observed in metazoa (Tables SI 14–16; Fig. SI 16). Given the presence of SH2 domain-based signalling it was surprising that no receptor tyrosine kinases could be recognized in the genome. However, Dictyostelium has a number of other receptor kinases such as the histidine kinases and a group of eight novel putative receptor serine/threonine kinases, which are involved in nutrient and starvation sensing70. Most of the ubiquitous families of transcription factors are represented in Dictyostelium, with the notable exception of the otherwise ubiquitous basic helix-loop-helix proteins (Table SI 17; Fig. SI 17). Compared to other eukaryotes, Dictyostelium appears to have fewer transcription factors relative to the total number of genes, suggesting that many transcription factors are yet to be defined, or that the activities of a smaller repertoire of factors are combined and controlled to achieve complex regulation (Table SI 18; Fig. SI 18).


Cell adhesion
Throughout Dictyostelium development, cells must modulate their adhesiveness to the substrate, to the extracellular matrix and to other cells in order to create tissues and carry out morphogenesis. To accomplish this, Dictyostelium uses a surprising number of components that have been normally only associated with animals. For example, disintegrin proteins regulate cell adhesiveness and differentiation in a number of metazoa and at least one Dictyostelium disintegrin, AmpA, is needed throughout development for cell fate specification71. We also identified distant relatives of vinculin and α-catenin – normally associated with adherens junctions - that support the idea that the epithelium-like sheet of cells that surrounds the stalk tube contains such junctions72. Consistent with this, the Dictyostelium genome encodes numerous proteins previously described as components of adherens junctions in metazoa like β-catenin (Aardvark), α-actinin, formins, VASP and myosinVII.

In animals, tandem repeats of immunoglobulin, cadherin, fibronectin III or E-set domains are often present in cell adhesion proteins, although their common protein fold predates the emergence of eukaryotes. EGF/Laminin domains are also found in adhesion proteins but, prior to the analysis of the Dictyostelium genome, no non-metazoan was known to have more than two EGF repeats in a single predicted protein. Dictyostelium has 61 predicted proteins containing repeated E-set or EGF/Laminin domains and many of these contain additional domains that suggest they have roles in cell adhesion or cell recognition, such as mannose-6-phosphate receptor, fibronectin III, or growth factor receptor domains and transmembrane domains (Fig. 9). In support of this idea, four of these proteins, LagC, LagD, AmpA and ComC, have been shown to be required for cell adhesion and signalling during development71, 7375.

Cellulose-based structures
During development, Dictyostelium cells produce a number of cellulose-based structural elements. Dictyostelium slugs synthesize an extracellular matrix, or sheath, around themselves that is comprised of proteins and cellulose. Several of the smaller sheath proteins bind cellulose and are believed to have a role in slug migration, while the larger, cysteine-rich EcmA protein is essential for full integrity of the sheath and for establishing correct slug shape76, 77. During terminal differentiation, cellulose is deposited in the stalk and in the cell walls of the stalk and spore cells7880. The first confirmed eukaryotic gene for cellulose synthase was discovered in Dictyostelium and this gene has since been recognized in many plants, N. crassa and the ascidian Ciona intestinalis,81. The fungal and urochordate enzymes are more closely related to the Dictyostelium homologue than to plant or bacterial cellulose synthases, indicating that the common ancestor of fungi and animals carried a gene for cellulose synthase that was subsequently lost in most animals. The Dictyostelium genome encodes more than 40 additional proteins that are likely to be involved in cellulose synthesis or degradation and probably are involved in the production and remodelling of cellulose fibres of the slug sheath, stalk tube and cell walls (see Supplementary Information).

The fundamental similarities in cellular cooperation found in Dictyostelium and in the metazoa clearly resulted in a parallel positive selection for structural and regulatory genes required for cell motility, adhesion and signalling. Dictyostelium uses a set of signals and adhesion proteins that are distinct from those employed for similar purposes in metazoa but, like the metazoa, Dictyostelium has maintained a diversity of GPCRs, protein kinases and ABC transporters which enable it to respond to those signals. Dictyostelium has also retained and modified an organizational strategy perfected in plants, basing several structural elements on cellulose. At one level Dictyostelium has achieved multicellularity by employing strategies that are similar to plants and metazoa, but the differences between them suggest convergent evolution, rather than lineal descent from an ancestor with overt or latent multicellular capacities.

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