The molecular basis of phenotypic evolution in social amoebas

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

Abstract

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.

Technical Summary

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

Planned Impact

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.

Publications

10 25 50
 
Description Research outcomes in the same order as project objectives
I A. In collaboration with the C.J. Weijer lab, the optical density (OD) waves that are diagnostic for oscillatory chemoattractant secretion were recorded for 4 species each, from the 4 major Dictyostelid taxon groups. Most species showed OD waves during aggregation, except some smaller species, like D.lacteum. In group 4, wave propagation was blocked when aggregating cells were exposed to the chemoattractant cAMP. In groups 1 and, 2 and some group 3 species, this was the case for the chemoattractant glorin. This was a striking result indicating that glorin synthesis is also regulated by the feedback loops that generate spontaneous oscillations. There were some correlations between wave dynamics and aggregate morphology. This work is submitted for publication.
I B. To reconstruct the evolution of pattern formation in Dictyostelia, we used cell-type specific antibodies and promoter-reporter fusion constructs to record differentiation patterns in the 98 species that make up the 4 major and 3 minor groups of Dictyostelia. We in all groups, except group 4, cells differentiate into maximally two cell types, prestalk and prespore cells, with pattern formation being dominated by position-dependent transdifferentiation of prespore cells into stalk cells. Group 4 species set aside correct proportions of prestalk and prespore cells early in development, and differentiate into up to three more supporting cell types. This indicated that positional transdifferentiation is the ancestral mode of pattern formation. The early specification of a prestalk population equal to the number of stalk cells is a derived trait that emerged in group 4 and a few late diverging species in the other groups.
I C. Using 24 species representing all taxon groups, we tested spore and cyst survival after storage for 0, 1, 7, 30, 90, 180 and 365 days under four ecologically relevant conditions. Due to the heavy analysis workload at the early time points, the experiments were set up at one month intervals for 4 species at a time have now almost reached the final time points. We also analysed and morphometrically quantitated ultrastructural differences between spores and cysts (when present) of 28 species across the Dictyostelid phylogeny. In the remaining period of the grant, these features will be mapped onto the Dictyostelid phylogeny together with the spore survival data and previously analysed species characters to identify trends in character evolution
II A. Bioinformatics. We investigated conservation and change in 385 genes that are essential for multicellular development of D.discoideum across 4 taxon group representative Dictyostelid genomes and 3 genomes of unicellular amoebas. 305 developmentally essential genes are conserved throughout Dictyostelia and at least one unicellular amoebozoan. Developmental regulation and protein functional domain architecture were unchanged in 45% and 54% of conserved genes, respectively. When different, changes in gene expression correlated more strongly with phenotypic innovation than changes in protein functional domains. The genes common to both Dictyostelia and unicellular amoebozoa were enriched in intracellular proteins and protein kinases, while genes unique to Dictyostelia, mostly encoded cell surface and secreted proteins, with roles in sensing and cell recognition. Five genes, which crucially regulate cell-type specialization, were absent from unicellular amoebozoa, but had closest homologs in prokaryotes. The analysis showed that the transition from uni- to multicellularity required novel exposed signals and sensors, rather then novel signal transduction proteins].
Gene modification. We deleted 18 genes that are essential for D.discoideum (Dd) sporulation in the group 2 species P.pallidum (Pp). Suprisingly, 8 knock-outs (bzpF-, srfA-, yakA-, crlA-, dokA-, alxA-, stkA- and splA-) showed no defects in development or sporulation, indicating that despite deep conservation, their roles in sporulation may be recent. DcsA, essential for Dd spore and stalk formation, was duplicated in Pp. The duplicate gene dcsB was required for stalk formation, while Pp dcsA- cells were defective in both sporulation and encystation. Encystation is the survival strategy of unicellular amoebozoa, which is still present in Pp, but lost in Dd. Pp null mutants in regA (a cytosolic cAMP phosphodiesterase) showed similar accelerated development and defective spore germination as Dd regA- cells, but additionally showed precocious encystation and blocked cyst germination]. The adenylate cyclases AcrA and AcgA are essential for Dd spore formation. Both activate PkaC, which in addition to sporulation, is also required for aggregation. Pp pkaC- mutants were similarly defective in aggregation and sporulation, but also lacked encystation. Surprisingly, Pp acrA and acgA were not required for sporulation, but were essential for encystation. This work provided strong support for the hypothesis that the essential role for cAMP and PKA in sporulation is derived from a similar role in encystation, but also showed that there has been considerable refunctionalization of pathway components. The third Dd adenylate cyclase ACA was duplicated twice in Pp. Similar to Dd ACA, all three Pp ACAs are highly expressed at the organizing tip, but ACA2 and ACA3 are also expressed in prespore cells, possibly performing the role in spore formation that acrA and acgA have in Dd. All single null mutants in the ACAs have normal development, but an aca1-/aca2- double null mutant does not form the typical side-branches of Pp fruiting bodies (Kawabe, submitted). Finally, knockout of gsk3, essential for prespore differentiation in D.discoideum, had no effect on P.pallidum prespore differentiation, but caused amoebas to encyst under conditions where they normally aggregate and form fruiting bodies. This suggests that here gsk3 mediated signalling determines life cycle choice (Kawabe, submitted.)
II B. Analysis of conservation and change in gene families encoding G-protein coupled receptors and histidine kinases across five Dictyostelid genomes are completed, while analysis of protein kinases and transcription factor families is in progress.
II C. Conservation and change in 80 randomly selected Dd genes without gene ontology terms across five genomes is completed, but proved to be very time-consuming. Except for the manually curated Dd genome, there are only computationally predicted gene models for the other 4 genomes and therefore labour-intensive manual prediction or correction was often needed. To improve gene models, we performed a de novo cDNA assembly from available RNA deep sequencing experiments for the group 1, 2 and 3 genomes, and have now performed extensive gene model correction.
Supporting project. To resolve the root and positions of group intermediate clades of dictyostelia, we sequenced the genomes of 6 species with disputed positions and used a concatenated set of 47 proteins from these and 6 available genomes to construct a robust core phylogeny of Dictyostelia.
Exploitation Route We have 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 s project we have performed extensive curation and annotation of gene models, which have thus far only been computationally predicted. The availibility of orthologous gene models will 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 benefit research in the UK and worldwide in other ways:
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.
Additionally our research has opened 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 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 established a genetically tractable model for studying amoebozoan encystation. Our research has already yielded a several potential drug targets for inhibition of Acanthamoeba encystation
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description ERC Advanced Grant
Amount € 2,128,602 (EUR)
Funding ID 742288 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 04/2017 
End 03/2022
 
Title Core phylogeny of Dictyostelia 
Description Sequencing of six genomes of early diverging Dictyostelid social amoebas. Construction of a multigene phylogeny using sequence data from these novel and eight published genomes. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact The core phylogeny was the cornerstone for the subsequent inference of a phylogeny across 52 species and current studies are in progress to include all known Dictyosteium species. An accurate phylogeny is foundation for all evolutionary comparative studies in this group and for accurate inference of ancestral states of genes and phenotypic traits. 
URL https://www.ebi.ac.uk/ena/data/view/PRJEB14640
 
Description Analysis of the role of crlA as a receptor for the effect of MBPD on sporulation and early development 
Organisation Sophia University Japan
Department Graduate School of Science and Technology
Country Japan 
Sector Academic/University 
PI Contribution Analysed effects of MBPD on aggregation and cell signalling in wildtype and crla null mutants.
Collaborator Contribution Analysed effects of MBPD on growth and sporulation in wildtype and crla null mutants.
Impact A manuscript describing the results of the study is published: doi: 10.1093/femsle/fnx022
Start Year 2014
 
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 Continued with comparative transcriptome analysis and sequencing of Protostelium genomes
Collaborator Contribution The Jena team performed most of the genome and transcriptome sequencing, sequence assembly and gene model prediction. The Jena and Cologne also contributed to protein annotation.
Impact The sequences of two genomes have been published doi:10.1101/gr.121137.111. The publication of sequence of the third Dictyostelium genome was incorporated into a broader evolutionary analysis of multicellularity genes (doi: 10.1038/ncomms12085). The comparative transcriptomic analysis was also published (DOI: 10.1186/s12864-016-3223-z) and the data were also used for improved gene model annotation (doi: 10.1186/s12864-017-3505-0) A manuscript for one Protostelium genome is in preparation. The available genome sequences form a cornerstone of our evolutionary work and are also widely used by the Dictyostelium community.
Start Year 2013
 
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 Continued with comparative transcriptome analysis and sequencing of Protostelium genomes
Collaborator Contribution The Jena team performed most of the genome and transcriptome sequencing, sequence assembly and gene model prediction. The Jena and Cologne also contributed to protein annotation.
Impact The sequences of two genomes have been published doi:10.1101/gr.121137.111. The publication of sequence of the third Dictyostelium genome was incorporated into a broader evolutionary analysis of multicellularity genes (doi: 10.1038/ncomms12085). The comparative transcriptomic analysis was also published (DOI: 10.1186/s12864-016-3223-z) and the data were also used for improved gene model annotation (doi: 10.1186/s12864-017-3505-0) A manuscript for one Protostelium genome is in preparation. The available genome sequences form a cornerstone of our evolutionary work and are also widely used by the Dictyostelium community.
Start Year 2013
 
Description Sequencing and annotation of the Physarum polycephalum genome 
Organisation The Otto-von-Guericke University Magdeburg
Country Germany 
Sector Academic/University 
PI Contribution Annotation of signal transduction genes in genome of Physarum polycephalum
Collaborator Contribution Sequencing assembly and gene model prediction in the Physarum genome
Impact no outcomes yet
Start Year 2013
 
Description Sequencing and annotation of the Physarum polycephalum genome 
Organisation U.S. Department of Energy
Country United States 
Sector Public 
PI Contribution Annotation of signal transduction genes in genome of Physarum polycephalum
Collaborator Contribution Sequencing assembly and gene model prediction in the Physarum genome
Impact no outcomes yet
Start Year 2013
 
Description Vist to P5/P6 Golfhill Primary School in Glasgow 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Visit by postdoctoral researcher to P5/P6 Golfhill Primary Schoolin Glasgow to participate in an "Ask the scientist" Q&A session in March 2014.
Year(s) Of Engagement Activity 2014
 
Description Family fun day at Charleston Community Centre 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We offered one of five stands at a Science Fair with hands on experiments, an instruction video and a poster about the use of model organisms to study questions in evolutionary biology at the Charleston Community Centre in Dundee
Year(s) Of Engagement Activity 2017
URL https://www.dundee.ac.uk/news/2017/women-in-science-festivals-family-fun-day.php
 
Description Open day for high school pupils 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact We participate in yearly open days for high school pupils and their parents with a stand with information on the Cell and Developmental Biology teaching programme and research carried out by the PI and members of the research team.
Year(s) Of Engagement Activity 2015
 
Description Open doors day College of Life Sciences Dundee 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Members of the public participated enthousiastically in hands-on experiments and gained information on the life cycles of social and pathogenic amoebas

Increased understanding of science in the general public
Year(s) Of Engagement Activity 2012,2014,2015
URL http://www.dundee.ac.uk/revealingresearch/newsandevents/dod/
 
Description Public Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Presentation of a public lecture on 16 December 2014 at the Darcy Thompson museum for Natural History with the title: The road to multicellularity - lifting the veil on the emergence of multicellular life forms, which was followed by a lively discussion with the audience over drinks and nibbles.
Year(s) Of Engagement Activity 2014
 
Description Publication of research summary for general audience 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Popular scientific paper "From So Simple a Beginning - The Origins of Multicellularity" in Scientia global
Year(s) Of Engagement Activity 2018
URL https://www.scientia.global/professor-pauline-schaap-from-so-simple-a-beginning-the-origins-of-multi...