The evolution of morphological complexity in the Dictyostelids

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences

Abstract

Biologists want to understand how complex multicellular organisms like ourselves have evolved from their simple single-celled ancestors. We know in theory how this 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. However, when starved, they come together and form a multicellular fruiting body, in which a proportion of cells is preserved as spores. The other cells are sacrificed to form a structure that aids spore dispersal. This life style depends on mutual collaboration and specialization of cells. In the course of evolution the social amoebae have progressed from basal species that formed structures with 10-100 cells and only two cell-types, to species that form large complex structures with over 100.000 cells and up to five cell types. 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. Because its genome has been sequenced, we have access to all the genes that control these processes. D.discoideum uses cyclic AMP (cAMP) as the major signal molecule for cell-cell communication. It acts as a chemoattractant to bring starving cells together. It then continues to guide cells to move coherently and build a fruiting body. cAMP also decides which cells should differentiate into spores. D.discoideum is one of 75 known social amoeba species. These species display large differences in the size and shape of their fruiting structures. To understand how these species gradually became mor complex and different from each other, we first need to know how they are related to each other and to their ancestors, the solitary amoebas. In previous research we used DNA data to construct a family tree of the social amoebas and we now know that there are four major groups of social amoebas. D.discoideum belongs to the most evolved group 4. We also found that many of the genes that are necessary for cAMP signalling are present in all four groups. However, between species, there are differences in the stage of development at which these genes are active. In addition, some genes have duplicated and started to assume novel roles. In this project we will reconstruct in what order all changes in shape and size occurred during social amoeba evolution. We will do this by measuring a large number of characters that determine the typical size and shape of all 75 species and by plotting these characters on the family tree. This will allow us to conclude which character was there first and how it gradually changed into greater or sometimes lesser complexity. We will also plot the presence of specific cAMP signalling genes to the family tree and the changes in these genes. This allows us to conclude whether a specific change in a gene was accompanied by a specific change in character. By manipulating the gene in question and observing its effect on species character we will be able to prove that a particular genetic change was the actual cause for a specific change in character. In this manner we will be able to unravel the genetic mechanisms that have been used by evolution to generate species diversity and complexity.

Technical Summary

We use the Dictyostelids as a model system to understand the genetic mechanisms that generated diversity and morphological complexity during the evolution of multicellular organisms. The project is based on recent construction of a molecular phylogeny of the Dictyostelids and on current data that a deeply conserved mechanism for cAMP signalling regulates cell-type diversification and coordinated cell movement in the Dictyostelids. We will use the following strategies to achieve three objectives: Objective I. Reconstruct the evolution of morphological complexity in the Dictyostelids The entire repertoire of morphological traits of all known Dictyostelid species will be mapped tot the molecular phylogeny in order to infer the basal state of each trait and its history of change. We will use gross morphological traits that are available from species descriptions, such as the size and shape of growing amoebae, spores, stalks and spore heads, the habit and branching patterns of fruiting structures and the morphology and phototactic behaviour of slugs. Where necessary we will curate and complement these data by microphotography and morphometric quantitation. In addition, we will obtain detailed information on the major hallmarks of multicellular life i.e. the traits that mark cell-type divergence and the coordination of cell movement. To do so we will visualize the presence of a prestalk-prespore tissue pattern in all species with an antispore antibody. We will measure proportions of prestalk-, prespore- and anterior-like cells in dissociated structures. The latter cell-type is the progenitor of multiple support structures in derived species. We will also visualize the dynamics of cell movement. cAMP oscillations mediate aggregation in D.discoideum and are also emitted by organizer regions in multicellular structures. The oscillations give rise to complex spiral cell movement patterns which are considered to shape slugs and fruiting bodies. We will use time-lapse videomicroscopy to record cell movement during and after aggregation in a large number of representative species and we will plot the presence and dynamics of cell movement waves to the phylogeny. Objective II. Develop genetic tractability for representative species. The ability to modify or disrupt genes is essential to be able to establish causality between genetic and morphological change. For the model system D.discoideum, mutants are available that can grow in liquid media. Under these conditions modified genetic material can readily be introduced using plasmids that are either of bacterial or D.discodeum origin. Transfection procedures have been developed for species, such as P.pallidum that grow on the natural food source, bacteria. We will test and optimize these procedures for transfection of one or two species from each of the four taxon groups. Objective III. Determine whether the evolution of specific morphological traits is caused by modifications in cAMP signalling genes. We will use both PCR and biochemical assays to test whether the cAMP signalling genes ACA, PdsA, PdeE and cAR have been conserved throughout the phylogeny. We will study the spatio-temporal expression pattern of conserved genes to assess whether gene regulation has been altered during evolution. For one species from each taxon group, we will obtain full length coding sequence for conserved genes by library screens or inverse PCR to complement a null mutant in its D.discoideum ortholog. This will tell us whether gene function is altered. By plotting the observed changes in gene function and/or regulation to the phylogeny we can infer whether the history of morphological change is correlated with the history of change in cAMP signaling genes. By disrupting the function of the altered genes in a genetically tractable species we will establish whether the observed correlation results from causality.

Publications

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Chen ZH (2010) Functional dissection of adenylate cyclase R, an inducer of spore encapsulation. in The Journal of biological chemistry

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Kawabe Y (2009) Activated cAMP receptors switch encystation into sporulation. in Proceedings of the National Academy of Sciences of the United States of America

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Kawabe Y (2009) Evolutionary Biology

 
Description One of the major goals of biology is to understand how species diversity and complexity was generated through adaptive evolution. We investigate this problem in the social amoebas, which live as single cells when they are feeding, but come together when starved to form a multicellular fruiting body, in which a proportion of cells is preserved as spores.
In this grant we aimed to achieve the following objectives
I. Obtain a comprehensive overview of the evolution of morphological complexity in the Dictyostelids by mapping the
morphological traits of all 75 species to the phylogenetic tree.
II. Develop genetic tractability for one or two species in each of the four major taxon groups. Gene modification will allow
us to assess causality for observed correlations between genetic and morphological changes.
III. Understand how modifications in cAMP signalling genes during Dictyostelid evolution have contributed to the
generation of morphological complexity.
We obtained the following results:
We measured morphological traits in 100 taxa and we
measured new traits such as pattern formation, robustness of growth and development, use of attractant, phototaxis and phototropism. Phylogeny based statistical analysis of the traits revealed that the last common ancestor (LCA) of Dictyostelia probably erected small fruiting structures directly from aggregates. It secreted cAMP to coordinate fruiting body morphogenesis, and another compound to mediate aggregation. This phenotype persisted up to the LCAs of three of the four major groups of Dictyostelia. The group 4 LCA co-opted cAMP for aggregation and evolved much larger fruiting structures. However, it lost encystation, the survival strategy of solitary amoebas that is retained by many species in groups 1-3. Large structures, phototropism and
a migrating intermediate 'slug' stage coevolved as evolutionary novelties within most groups.

2. We established full genetic tractability for the group 2 taxon P.pallidum. We made LoxP-Neo vectors that enable reiterated knockout of an unlimited number of genes in P.pallidum cells.
3. We established conservation of key cAMP signalling genes, such as cARs, ACG, ACB, ACA,PdsA and RegA throughout the Dictyostelid phylogeny. Functional analysis of a subset yielded
the first plausible scenario for the evolution of developmental signalling from a protist stress response. Our work indicates that cAMP signaling in social amoebas evolved from cAMP mediated encystation in solitary amoebas
Exploitation Route Our finding that cAMP acts as the signalling intermediate for stress-induced encystation is currently being followed up with genetic screens to identify essential proteins in the cAMP signalling pathways that can act as targets for screens to find drugs that inhibit encystation of pathogenic amoebas. We constructed a database of 30 phenotypic traits over 100 Dictyostelid species, which can be exploited by other researchers to study phenotypic evolution and identify genotype-phenotype relationships.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description We used the finding that cAMP mediates amoebozoan encystation to initiate genetic screens to uncover essential genes in encystation, which have up to date yielded three candidate protein. In collaboration with the Dundee drug discovery unit we are performing compound screens to identify nhibitors for these proteins
First Year Of Impact 2012
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Comparative genome sequencing of Dictyostelia 
Organisation Friedrich Schiller University Jena (FSU)
Country Germany 
Sector Academic/University 
PI Contribution Comparative genome sequencing of three Dictyostelid species. Genome sequencing, fosmid mapping and gene annotation of three Dictyostelium genomes
Collaborator Contribution The Jena team performed most of the genome sequencing and sequence assembly. The Jena and Cologne contributed to gene annotation
Impact The sequences of two genomes has been published doi:10.1101/gr.121137.111 a manuscript for the third is in preparation. The available genome sequences form a cornerstone of our evolutionary work and resulted in many secondary publications.
Start Year 2007
 
Description Comparative genome sequencing of Dictyostelia 
Organisation University of Cologne
Country Germany 
Sector Academic/University 
PI Contribution Comparative genome sequencing of three Dictyostelid species. Genome sequencing, fosmid mapping and gene annotation of three Dictyostelium genomes
Collaborator Contribution The Jena team performed most of the genome sequencing and sequence assembly. The Jena and Cologne contributed to gene annotation
Impact The sequences of two genomes has been published doi:10.1101/gr.121137.111 a manuscript for the third is in preparation. The available genome sequences form a cornerstone of our evolutionary work and resulted in many secondary publications.
Start Year 2007
 
Description RSE Masterclass 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact High school pupils participated with enthousiasm and obtained a certificate for successfully completing experiments

The Masterclasses continue to be highly popular with schools in the regions
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2012
URL http://www.royalsoced.org.uk/cms//files/youngpeople/sciencemasterclass/DundeeAutum14flyer.pdf
 
Description Sharing science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The exhibit received a lot of visitors, mostly parents and their children, who performed experiments with great enthousiasm

No notable impact
Year(s) Of Engagement Activity 2007