Experimental and evolutionary reconstruction of developmental signalling pathways.

Background

Most biological processes are regulated by complex networks of interacting proteins and small molecules

Powerful tools are available to analyse these networks, such as:

Molecular genetics             –    to identify novel components and examine their order in a pathway

Biochemistry/cell biology   –    to examine how proteins work and interact

Mathematical modelling      –    to predict the regulatory potential of network

However none of these methods will reveal the underlying logic of the network because biological processes are not designed. They result from opportunistic recruitment of components that happened to be available at a particular moment in the course of evolution.

We are therefore using a fourth tool to dissect and understand complex signalling networks:

"Evolutionary reconstruction"

This is the only tool that can help us identify the deeply conserved core components of a network, to assign hierarchy in parallel processes and to understand why a particularly pathway is build up the way it is.

To quote the geneticist Theodosius Dobzhansky:

“Nothing in biology makes sense except in the light of evolution”

In the past 5 years we have demonstrated the feasibility and utility of this approach. We showed how the manifold roles of cAMP as secreted signal for chemotaxis and gene regulation, and as intracellular signal for a broad range of processes in the social amoeba Dictyostelium discoideum could be traced back to an original role in stress-induced encystation in solitary amoebas [1-3]. In current research we continue to expand on this approach and its integration with the more orthodox tools of experimental biology. We feel that incorporation of evolutionary history in the analysis of complex mechanisms will result in a much deeper, richer and more precise understanding than the more common single organism approach.

Dictyostelid social amoebas aggregate to form fruiting structures in response to starvation. This alternation between uni- and multicellular life styles offers unique opportunities to understand how multicellularity evolved and became increasingly more complex. Novel morphological features in multicellular organisms arise through changes in the developmental programmes that shape these features. These programmes are controlled by signals that are produced in a time- and space-dependent manner by the developing cells.  cAMP is the most deeply conserved signal molecule in living organisms. It mediates the regulation of metabolism and motility by environmental stimuli in bacteria and the action of  hormones in humans. In Dictyostelium discoideum, intracellular cAMP triggers the initiation of development and the maturation of spores and stalk cells in fruiting bodies. Extracellular cAMP acts as a chemoattractant to mediate aggregation of starving cells, and as a developmental signal to trigger the differentiation of prespore cells [4] and regulate the expression of multiple classes of genes throughout the developmental programme. 

We use an experimental approach to understand how cAMP is produced in a time- and space dependent manner to exert these diverse functions, and how the cAMP signal is processed to alter gene expression patterns. In addition we use a comparative approach to understand how cAMP signalling pathways evolved in the Dictyostelids and how modifications in the component genes  generated  phenotypic complexity during Dictyostelid evolution. Our recent research shows that the role of intracellular cAMP in initiation of development and maturation of spores is evolutionary derived from a role in mediating drought-induced encystation of solitary amoebas [3]. The role of secreted cAMP in coordinating aggregation of younger species, such as D.discoideum is evolutionary derived from a universal role in controlling fruiting body morphogenesis in all species [1]. An  additional and most likely the earliest  role of secreted cAMP in Dictyostelia was to direct cells to form spores and not cysts, once they were collected in aggregates [2].

Current projects

1. Molecular mechanisms of encystation and sporulation

The finding that the role of cAMP in spore formation in fruiting bodies is evolutionary derived from the same role in encystation of solitary amoebas has provided the first insight in the mechanisms that control amoebozaon encystation [2, 3]. This is of considerable medical importance, since 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 was thus far little information on the mechanisms controlling encystation. Now recent research achievements have brought the elucidating of these mechanisms is within reach:

1. The observation that like sporulation, encystation is mediated by cAMP and is likely to use signal transduction proteins of which have several have been identified in D.discoideum

2. The development of genetic tractability for an encysting Dictyostelid, Polysphondylium pallidum.

3. The completion of the genome sequence of P.pallidum

We now have outstanding opportunities for efficient genetic screens to identify all genes that are essential for encystation. We will study the function of these genes and investigate whether they also control encystation of pathogenic amoebas. Structure-function analysis of the cognate proteins can subsequently lead to design of therapeutics that inhibit encystation or cause excystation.

Because sporulation is evolutionary derived from encystation, the screen will also reveal novel genes involved in sporulation.

2. Regulation of cAMP synthesis: ACG and ACB.

The adenylate cyclases ACG and ACB play overlapping roles in synthesizing cAMP during Dictyostelium discoideum development. ACG is post-transcriptionally down-regulated during aggregation but re-appears in aggregates, where it induces the differentiation of prespore cells [5]. In fruiting bodies, ACG inhibits the germination of spores under the ambient conditions of high osmolarity, an effect that is mediated by its intrinsic osmo-sensor [6, 7]. 

ACB induces spore maturation and proper stalk formation in the final phase of development [8]. ACB is a complex protein with a sensor histidine kinase domain and two response regulator domains, which were recently found to have only marginal roles in the regulation its adenylate cyclase activity [9]. Similar to ACG, the ACB protein also appears to be down-regulated during aggregation, but unlike ACG, which re-appears in   prespore cells, ACB re-appears in prestalk cells. Evidently, at this developmental stage, regulation of protein levels may equally or more important than direct regulation of catalytic activity. 

We are currently studying the mechanisms that are responsible for ACG and ACB down-regulation and the signals that regulate this process.

3. Comparative genomics

The completion of the first molecular phylogeny of all known Dictyostelia in 2006 [10], and the completion of the D.discoideum genome in 2005 [11], provided a good starting point for projects aimed to sequence the genomes of species that represent each of the 4 major taxon groups of Dictyostelia. A US team sequenced the genome of D.purpureum, a group 4 species like D.discoideum,  we collaborated with Dr. Gloeckner in Jena (now Berlin) to sequence the genomes of the group 1 species D.fasciculatum and the group 2 species P.pallidum, and we are now in the process of sequencing the genome of the group 3 species D.lacteum. The first analysis of the genomes has already given us remarkable insights in evolutionary changes in genome structure and gene content, but we are only at the start of a large endeavour to retrace how the genomes  of the social amoeba have changed throughout their a billion years of multicellular evolution.

4. Evolution of form and function in social amoebas

Interesting as the study of genes and genomes may be, they are in essence only a passive blueprint of the organism. The really interesting part is how novel genes and changes in existing genes gave rise to novel forms and functions in the living organism. To adress these questions we have embarked on measuring a broad variety of phenotypic traits across all species of Dictyostelia. All traits will be mapped onto the phylogenetic tree to gain insight into the order in which these traits evolved. This has already been done for the morphological traits that were used for the original species descriptions [10] and will continue for a range of traits under investigation. Advanced statistical methods will be used to assess how the evolution of phenotypic traits is correlated with the evolution of genotype as evident from analysis of the different sequenced genomes. Because genes can be readily replaced and altered in Dictyostelia, we can test whether such correlations are actually based on causal relationships. Modification of a candidate gene in either late or early diverging species to respectively a more ancestral or more derived version will be used to test whether the change in the candidate gene caused the novel phenotype.

5. Evolution of developmental signalling

Our earlier PCR-based approach for gene identification across the Dictyostelium phylogeny is now superceded by the convenience of a few minutes search in completed genomes. This will allow us to extend our research from the well-conserved cAMP signalling genes to other developmental signalling pathways.  However in initial studies we will follow up on an initial intriguing finding that existing cAMP signalling genes appeared to obtain novel roles in development by addition of distal promoters [1]. This is the case for cAMP receptors and the extracellular cAMP phosphodiesterase PdsA, which are only used and expressed after aggregation in early diverging species, and which gained distal promoters for expression during aggregation in the youngest group 4. The first questions to address are whether this is the case for all genes that group 4 species for aggregation to (no species in other groups use cAMP) and whether this is unique for this set, or a common mechanism for elaboration of gene function in Dictyostelia.

1.   Alvarez-Curto E, Rozen DE, Ritchie AV, Fouquet C, Baldauf SL, Schaap P. (2005) Evolutionary origin of cAMP-based chemoattraction in the social amoebae. Proc. Natl. Acad. Sci. USA, 102, 6385-6390.

2.   Kawabe Y, Morio T, James JL, Prescott AR, Tanaka Y, Schaap P. (2009) Activated cAMP receptors switch encystation into sporulation. Proc Natl Acad Sci U S A, 106, 7089-7094.

3.   Ritchie AV, van Es S, Fouquet C, Schaap P. (2008) From drought sensing to developmental control: evolution of cyclic AMP signaling in social amoebas. Mol Biol Evol, 25, 2109-2118.

4.   Saran S, Meima ME, Alvarez-Curto E, Weening KE, Rozen DE, Schaap P. (2002) cAMP signaling in Dictyostelium - Complexity of cAMP synthesis, degradation and detection. Journal of Muscle Research and Cell Motility, 23, 793-802.

5.   Alvarez-Curto E, Saran S, Meima M, Zobel J, Scott C, Schaap P. (2007) cAMP production by adenylyl cyclase G induces prespore differentiation in Dictyostelium slugs. Development, 134, 959-966.

6.   Saran S, Schaap P. (2004) Adenylyl cyclase G is activated by an intramolecular osmosensor. Mol. Biol. Cell, 15, 1479-1486.

7.   Van Es S, Virdy KJ, Pitt GS, Meima M, Sands TW, Devreotes PN, Cotter DA, Schaap P. (1996) Adenylyl cyclase G, an osmosensor controlling germination of  Dictyostelium spores. J. Biol. Chem., 271, 23623-23625.

8.   Soderbom F, Anjard C, Iranfar N, Fuller D, Loomis WF. (1999) An adenylyl cyclase that functions during late development of Dictyostelium. Development, 126, 5463-5471.

9.   Chen Z-H, Schilde C, Schaap P. (2010) Functional dissection of ACB, a deeply conserved inducer of spore encapsulation. submitted.

10.  Schaap P, Winckler T, Nelson M, Alvarez-Curto E, Elgie B, Hagiwara H, Cavender J, Milano-Curto A, Rozen DE, Dingermann T, Mutzel R, Baldauf SL. (2006) Molecular phylogeny and evolution of morphology in the social amoebas. Science, 314, 661-663.

11. Eichinger L, Pachebat JA, Glockner G, et al.,  (2005) The genome of the social amoeba Dictyostelium discoideum. Nature, 435,   43-57