The molecular basis of phenotypic evolution in social amoebas

Biologists want to understand how complex multicellular organisms have evolved from simple single-celled ancestors. We know in theory what happened: Spontaneous mutations in the genes of earlier organisms caused small changes in the developmental program of their offspring. This sometimes resulted in an improved adult that more successfully reproduced, and therefore gradually replaced the earlier form. However, to really understand this process and prove that it actually occurred, we have to trace back which genes were mutated and how this mutation changed gene function. We also need to know which developmental mechanisms were regulated by the mutated genes and how the altered developmental mechanism eventually produced the improved adult form.
Because it is not possible to obtain such detailed information for highly evolved animals like ourselves, we investigate this problem in the social amoebas. These organisms live as single cells when they are feeding, but aggregate when starved to form a multicellular fruiting body, in which a proportion of cells is preserved as spores. The other cells form a stalk and other structures to support the spore mass. This life style depends on mutual collaboration and specialization of cells. One species, D.discoideum, is used by many laboratories as a model system to understand how cells move, eat, propagate and communicate with each other.
In previous research, we constructed a family tree of all 100 known social amoeba species, which showed that there are four major groups of social amoebas. For each of the 100 species, we have measured 30 properties (traits), which describe their behaviours, the size and shape of their component parts and the number of cell types in which they can differentiate. By combining this information with the family tree, we have gained information in what order these traits evolved and which traits are always seen together. The earliest social amoeba formed very small fruiting bodies directly from aggregates. All cells first differentiated into prespore cells and then some changed again to form the stalk. These early species probably used a compound called glorin to aggregate and, like their ancestors the solitary amoebas, they could still form cysts from single cells to survive starvation.
The ability to form large fruiting bodies appeared together with an intermediate migratory "slug" stage that could bring the aggregates to the soil surface. Inside the slug prestalk and prespore cells differentiated in the same proportions as needed in the fruiting body. Cells also formed new structures to support the stalk and used cAMP pulses to aggregate. However, they lost the ability to form cysts. In the new project we want to understand how these traits evolved and why they evolved together. What is the connection between them and what novel mechanisms were needed to obtain more cell types and build larger structures. Secondly, we want to understand how the genes of the more advanced species were altered to make these changes possible.
In collaboration with a German team, we have recently sequenced the genomes of species that represent groups 1,2 and 3 of social amoebas. The genome of D.discoideum in group 4 was already sequenced before. We can now, in theory, identify changes in all the genes that occurred during evolution. However, due to the large number of genes in each organism (~12.000) this requires at first a computational approach to identify the most likely genes to be involved in the mechanisms that we want to study. Once candidate genes have been selected, we can replace the gene of a more evolved species with that of an earlier form and see whether this results in the loss of the more advanced property. The reverse is also possible. In this manner we will be able to determine the genetic mechanisms that have been used by evolution to generate the enormous variety of multicellular organisms that we see today.

Multicellular organisms evolved several times from single-celled protozoa and achieved high levels of organisational complexity and an immense range of forms. Novel adult phenotypes result from genetic changes in the developmental processes that generated the earlier forms. Using Dictyostelid social amoebas as a model, we try to understand the underlying genetic changes that altered developmental control mechanisms and thereby allowed multicellular organisms to achieve ever greater complexity.
We constructed a molecular phylogeny of all 100 known Dictyostelid species, which subdivides species into four major groups. We measured 30 phenotypic traits over all species and inferred ancestral states and trait co-evolution. The results show co-evolution of large fruiting structures, light-oriented migration, cell-type proportioning, use of cAMP pulses as attractant and loss of encystation. In collaboration with a German team, we have sequenced the genomes of species representative of groups 1, 2 and 3, which, with the previously sequenced group 4 genome of D.discoideum, now represent the entire breadth of the phylogeny.
In the new project we will investigate the mechanisms underlying the evolution of oscillatory chemoattractant signalling, cell type proportioning and pattern formation and their connection and contribution to spore fitness, the ultimate outcome of the developmental program. We will also initiate a large scale bioinformatic analysis aimed at identifying evolutionary changes in genes that control the above mentioned processes, followed by gene replacement to prove causality between genotypic and phenotypic change. Bioinformatics combined with reverse genetics will also be used to identify novel genes that regulate morphogenesis, pattern formation and sporulation. Lastly, we will use comparative bioinformatics to assign putative functions to the large fraction of D.discoideum genes that have as yet no assigned protein function or biological role

Benificiaries from our research are:
1. The entire Dictyostelium and broader research community
We have already provided the research community with three completely finished genomes, which, with the D.discoideum genome, systematically represent the Dictyostlid phylogeny. In the course of the analysis proposed in this project we will perform extensive curation and annotation of gene models, which have thus far only been computationally predicted. The availibility of orthologous gene models will also aid to improve gene model curation for the D.discoideum genome that at least 10 UK labs and hundreds of labs worldwide use as the basis for their research. The comparative genome information will also in many other ways benefit research in the UK and worldwide:
In the narrowest sense comparative gene and gene family information will allow investigators of any fundamental biological problem to select and concentrate on deeply conserved genes and ignore genes that result form recent duplications.
In the broadest sense, comparative analysis allows researchers to retrace the evolutionary history of the process under study. This raises the characterization of a process from not only knowing its component parts, but from also understanding why the process is built up the way it is.

2. Researchers studying protist borne diseases and protist biology in a broader sense
D.discoideum is widely used to study fundamental processes related to human disease, but our research is opening up new opportunities to use dictyostelids as genetically tractable models for understanding protist-borne disease.
Most unicellular protists, including many pathogens, form dormant cysts when exposed to stress. This severely impairs treatment, since the cysts are resistant to antibiotics and immune clearance. Due to limited genetic tractability of pathogenic protists, there is little information on the mechanisms controlling encystation. Our previous BBSRC funded research showed that sporulation in Dictyostelids is evolutionary derived from encystation, with both being triggered by cAMP acting on PKA. This is the case for both the encysting Dictyostelid P.pallidum and in pathogen Acanthamoeba castellani. By sequencing its genome and developing tools for molecular genetic analysis of P.pallidum, we have now established a genetically tractable model for studying amoebozoan encystation. Our research has already yielded a conserved potential drug target for inhibition of Acanthamoeba encystation and in this project we continue to study the mechanisms that control this important process.

3. The pharmaceutical industry
Together with the Drug Development Unit in our institute we are currently performing a compound search for inhibitors of an adenylate cyclase that mediates Acanthamoeba encystation. A commercially available inhibitor was already detected that inhibits both recombinant purified Acanthamoeba adenylate cyclase as well as encystation. However the activity of the compound was not high enough for therapeutic application. Once effective compounds are identified and patented, pharmaceutical companies will be approached for further development of the product.

4. Training of researchers
Research in my laboratory encompasses a very broad range of approaches, such as molecular cloning, gene disruption and gene replacement, analysis of mutant and species phenotypes with a range of microscopic and cell-biological techniques, biochemical characterization of signal transduction proteins, and more recently genomics, molecular phylogeny and phylogeny based statistical procedures and data analysis. Postdoctoral researchers leave my laboratory as highly competent molecular biologists and biochemists with a range of transdisciplinary skills and the greater majority have either directly or after continued postdoctoral research found employment as principal investigators, lead investigators in biotechnology companies or in management.