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Animal evolution from a cell type perspective: multidisciplinary training in single-cell genomics, evo-devo and in science outreach

Periodic Reporting for period 1 - EvoCELL (Animal evolution from a cell type perspective: multidisciplinary training in single-cell genomics, evo-devo and in science outreach)

Reporting period: 2018-01-01 to 2019-12-31

Throughout the evolution of animals, the cells that compose their bodies have become increasingly diverse; each cell type distinguished by the unique set of genes it expresses. Yet, the processes through which cells acquired such diverse roles remain poorly understood. Currently we do not even know how many distinct cell types animals possess, how new cell types arise in evolution, how many are in common between different animal groups and how many unique cell types have evolved in different animal lineages. Composed by a network of 12 laboratories across Europe and 20 ESRs, EvoCELL is studying these fundamental questions in animal evolution and development using single cell sequencing.
We are training a new generation of multidisciplinary scientists skilled in exploring the vast breadth of animal differentiation. We have jointly sampled data from all major animal lineages and are developing new tools for comparative analyses, through which we will together pioneer three branches of cell evo-devo: evolution of stem cells; emergence of animal life cycles, and the stunning diversity of neural cell types.
EvoCELL’s main aim is to identify the different cell types in several marine invertebrates and vertebrates and in different developmental stages. One focus is on the evolution of neurons and nervous systems. We are identifying and characterizing the cell types in vertebrates’ brain to shed light on its cellular origins and regulatory evolution, a fundamental question in biology that is also of public appeal. We are also looking into the brains and nervous systems of invertebrate species, such as the sea urchin, the spider, or the marine annelid Platynereis, to characterize their neuron types and how they relate to those of the vertebrates. Another focus is on regeneration. We are trying to determine to which extent cell content is faithfully restored after amputation and regeneration in specific animals. This will allow us to better understand the complex and sub-explored process of regeneration that is found in many species but not in most classical animal models. Related to this we investigate the role of piRNAs and PIWI proteins in stem cell maintenance, differentiation and cancer. This will be helpful in gaining a better understanding of the piRNA pathway during tumorigenesis to help identify molecules that might serve as new prognostic markers and therapeutic targets. In addition, we are interested in the life cycle of marine invertebrates and how the cell type complements change between larval and adult stages. The aim is to identify cell types at different life cycle stages in the cnidarians, using the molecular model species Clytia hemisphaerica and the coral Pocillopora acuta. EvoCELL is also conducting research on the function of interesting cell types driving the behaviour of marine invertebrates. Cilia are conserved whip-like cellular organelles with several important functions in animal physiology and behaviour. They beat in a coordinated fashion to drive fluid in the body or move small animals in water. In small marine larvae that swim with multiple cilia, these phenomena of coordination can be easily observed.
To communicate our combined genomics, evo-devo and neuroscience research, we are putting a considerable effort into the development of new strategies for basic science outreach by blending classical dissemination formats with new virtual exhibitions.
We have developed and optimized successful dissociation protocols for the species of the network at different developmental stages. We have generated single-cell and single-nucleus sequencing data and are developing bioinformatics pipelines for the identification of cell types. We have developed and optimised methods for gene expression analysis in order to assign cell identity once cell markers are characterised via single cell sequencing.
In particular, we have almost completed the establishment of a cellular brain atlas for the vertebrate sea lamprey and initiated comparison with other vertebrates to reveal an ancestral vertebrate cell type complement. For sea urchin, we have already produced a large single cell transcriptome dataset that shows how a phenotypically simple animal is composed of complex cell types. For the marine annelid Platynereis, we have so far focused on the characterization of neural cell types in larvae and young worms. This will yield insight into the evolution of neurons within the bilaterian clade. We have also characterised the effect of different neurotransmitters on the ciliomotor pacemaker system of the Platynereis larva. With a combination of immunostaining and in situ hybridisation methods, we have mapped in detail various neurotransmitter markers and receptors to the ciliomotor neurons. For the oyster, we have identified cell types present in the swimming larvae and compared them to those of the Muller’s larvae of flatworms. This will deepen our understanding of how cell types evolved that produce the shell. For the crustacean Parhyale hawaiensis cells that are present in the limbs have been transcriptome-sequenced and are now analysed. They will be compared to the cell types found in the crustacean limb after regeneration. Outside bilaterians, for the cnidarian molecular model species Clytia hemisphaerica and the coral Pocillopora acuta, large single-cell datasets have already been generated and are currently analysed. The aim is to identify cell types at different stages of the cnidarian life cycle, and to learn how these cell types relate between stages and across the evolutionary tree. We have also developed a bioinformatics pipeline to analyse data from smallRNA-seq dedicated to the identification and annotation of somatic piRNAs. In addition, we have demonstrated the physical association between PIWIL1 and several piRNAs and their predicted mRNA targets, suggesting that the PIWI/piRNA pathway may actively contribute to the establishment and/or maintenance of clinicopathological features of CRCs.
The single-cell sequencing data generated in this network will provide insight into cell type and animal evolution. For example, the nervous system of the sea urchin larva appears to share a common gene regulatory signature with the endocrine cells of vertebrates. We are studying cell differentiation to identify progenitor cells and understand the cellular basis of regeneration. The project will reveal the contribution of different conserved neurotransmitters and their receptors in the generation of rhythmic activity by a simple and fully mapped nervous system. This will inform our understanding of pacemaker systems in general and is expected to stimulate further work on the neuronal control of cilia, both in invertebrates and vertebrates. Our single-cell transcriptomic data for the lamprey brain are unique and will provide a first detailed overview of the cellular origins and evolution of the vertebrate brain. These results should be of wide public interest, given that the results will shed light on the origins of a key organ of humans - the brain. After the characterization of the PIWIL1/piRNA pathway in CRC cells, we are extending our analysis to other tissues and cancer types, integrating also data at single cell levels. We aspect to identify tumor-specific piRNA expression patterns and to understand the role of piRNA machinery in the specification and maintenance of differentiated cell type and in cancer formation.
A hypothetical cell type evolutionary tree in animals (from Arendt et al., 2019)