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Evolution of multicellularity and somatic cell specialization

Periodic Reporting for period 3 - EVOSOM (Evolution of multicellularity and somatic cell specialization)

Reporting period: 2020-05-01 to 2021-10-31

The evolution of multicellular organisms was a major event in the history of life, because it allowed cells to specialize into functions other than mere propagation. The specialized cells became organized into tissues and organs and thus greatly expanded the ability of the organism to perform a broad range of complex behaviours. In modern multicellular organisms the specialized cells that form the body (soma) of the organism vastly outnumber the germ cells that are responsible for its propagation.

While yielding immense gain of function, the organisation of the somatic cells into tissues and organs required novel intercellular communication systems to control the growth and differentiation of the specialized cell types at the correct time and place and in correct proportions within the organism.

We seek to identify the genetic changes that caused the transitions from uni- to multicellularity and that enabled cell-type specialization. Because modern organisms like ourselves are too complex to investigate this problem, we use the Dictyostelid social amoebas that maximally specialize into five cell types for this research.

Social amoebas aggregate together and form multicellular fruiting bodies when starved. They evolved from unicellular Amoebozoa and are subdivided into 4 major groups. Species in groups 1, 2 and 3 have at most 2 cell types, but 3 new cell types (basal disc, upper- and lower cup) appeared in group 4. Because species in all four groups have about 12000 genes, actually less than found in many unicellular amoebas, we know that neither the transition from uni- to multicellularity nor the appearance of novel cell types requires more genes.

We have a few clues what might be required: Firstly, we noted when comparing gene expression patterns between group 4 and groups 1,2 and 3, the patterns in group 4 were more complex, due to genes acquiring more regulatory sequences. Secondly, gene function also mattered, with cell-communication genes being particularly important for regulating multicellularity and cell specialization. Among about 500 genes known to be essential for Dictyostelium multicellular development, the genes encoding proteins inside the cell that process extracellular signals are also present in unicellular amoebas, and are mostly the same between the 4 groups of Dictyostelia. However, the genes that encode signal peptides or enzymes that synthesize signals, and the receptor proteins on the cell surface that detect signals are mostly different between unicellular and multicellular amoebas and between Dictyostelium groups.

Starting from the hypothesis that diversification of signals and receptors and diversification of gene regulation mechanisms are major drivers of multicellular evolution, we designed a series of experiments to investigate whether this is the case or whether other processes are also or more important. Overall the project has the following objectives
1. Understand the genetic changes that caused transitions to multicellularity.
2. Retrace the evolutionary history and genetic changes that caused somatic cell specialization.
Resolving these objectives will present a major breakthrough in our understanding of the evolution of multicellular life forms.

Significance for society:
Why are we here and where do we come from are humanity’s earliest and most profound questions. While most of the world’s religions provide individual mythologies about the origins of life and the universe, the only unifying explanation can come from science. By elucidating the molecular changes that caused a major evolutionary transition, we contribute to the great scientific endeavour to understand the world around us and to separate fact from fiction in retracing the history of life.

In more practical terms the project will highlight many novel genes and cell communication pathways important for regulating life cycle changes and cell differentiation in Amoebozoa, the sister group to Opisthokonta that also contains the animal lineage. Many of these genes are likely to be conserved in the animal lineage and lead to novel discoveries important for understanding human health and disease.
1. To understand the order in which novel cell types evolved it is essential to have a well-resolved family tree of the Dictyostelia and their closest Amoebozoan relatives. We constructed such a family tree by amplifying and sequencing the six conserved genes over 34 Dictyostelium species and combining this information with genes from 18 sequenced genomes, half of which were sequenced by us. By comparing the extent to which the conserved genes started to differ from each other by accumulating mutations over time, we derived a family tree for the Dictyostelia. The novel tree subdivides the Dictyostelia in two major branches, each containing two major groups, and with the positions of all smaller groups well resolved. This tree allows us to reconstruct the timeline at which novel cell-types appeared (Schilde et al., MPE 2019).
2. Cell differentiate by triggering RNA transcription from different groups of genes. The RNA transcripts then become translated into a range of novel proteins, which alter the appearance and function of the cells. To retrace the evolutionary progression of cell differentiation, we purified cell types from Dictyostelium species taken from each of the 4 major groups and sequenced their transcriptomes (all RNAs expressed in a particular species). In the initial analysis of the transcriptome of the model organisms D. discoideum, we found cup cells, a novel cell type in group 4 are derived from stalk cells. However, while stalk cells mostly express genes related to stalk wall synthesis, cup cells expressed novel genes related to signal processing and cell adhesion. (Kin et al. BMC Genomics 19:764, 2018).
3. Glycogen synthase kinase 3 (GSK3) regulates many cell fate decisions in human development. In multicellular structures of the group 4 species Dictyostelium discoideum, GSK3 promotes spore over basal disc differentiation. We investigated the role of GSK3 in the group 2 species Polysphondylium pallidum, which does not form a basal disc. However, unlike group 4, group 2 species can still encyst individually when starved. We found that deletion of the GSK3 gene had no effect on spore differentiation, but caused cells to choose encystation in preference to multicellular fruiting body formation. This indicated that the original role of GSK3 was to promote multicellularity over unicellular encystation. (Kawabe et al. EvoDevo 9:12. 2018).
4. Multicellular Dictyostelia which form spores in fruiting bodies when starved, evolved from unicellular amoebas that instead form cysts. To understand why spores are better than cysts, we stored both at a range of temperatures. It appeared that spores were frost resistant while cysts were not. EM studies showed that this was likely due to greater state of compaction and three-layered wall of the spores. We determined the time in evolution when Dictyostelia evolved from unicellular amoebas and this coincided with the neoproterozoid cryogenic period. It therefore seems likely the multicellular sporulation was triggered by a cooling climate (Lawal et al, SciRep 2020)
5. As part of a larger study to correlate evolution of novel cell types with molecular changes in genes involved in controlling cell differentiation, we investigated conservation and change in the presence, developmental regulation and cell-type specificity of transcription factors (Forbes GBE 2019). This provided hints for changes that might have mediated specific innovations. For one case, a group 4 specific gene duplication in the transcription factor cdl1 further experimentation showed that it was essential for the appearance of a novel cell type (Kin et al., in prep.)
6. We developed gene knock-out procedures for P. violaceum, a sister species to group 4 where most novel cell types appeared. We performed cell type specific and developmental transcriptomics and used the transcriptome data to annotate a draft P. violaceum genome. We used the novel data and procedures to investigate the ancestral role of DIF-1, a signal required for basal disc and stalk formation in group 4. We found that unexpectedly loss of DIF-1 synthesis led to excessive stalk formation indicating that its role in group 4 is relatively recent (Narita et al., 2019, GBE).
Work for the next 18 months involves
1. completion of several bioinformatics studies documenting evolutionary change in families of cell-cell communication and signal transduction proteins
2. using these studies to identify genetic change likely to given rise to cell-type innovation, and confirming by experimentation whether this was the case.
Combined this work will provide us with many proven instances and general principles how early multicellular organisms generate novel cell types in the course of evolution
3. exploring novel hypotheses suggested by our recent studies that the Dictyostelium transition to multicellularity and evolution of it somatic cell types centers around its adaptation to cold climate, with major changes being required to enhance the fitness of its dormant spores. We will particularly investigate the role of the somatic stalk, cup and disc cells in not only lifting the spore mass, but in self-sacrificing by autophagy to provide the maturing spores and their enveloping matrix with materials to increase their long-term survival.