Periodic Reporting for period 1 - FlexAggon (Evolutionary origin of cell adhesion: the basis of multicellularity in the choanoflagellate Choanoeca flexa)
Reporting period: 2023-05-01 to 2025-04-30
Cell adhesion is a signature feature of animal multicellularity. Animal cells control and maintain stable cellular connections through direct or indirect contacts between cells using surface transmembrane receptors known as cell-adhesion molecules (CAMs). The activity of most CAMs – and thus cell adhesion – is primarily associated with their ligand binding and, in some cases, can also be regulated from inside the cell by proteins interacting with them or stabilised by extracellular molecules. Despite the importance of cell adhesion regulation in fundamental developmental processes, little is known about how cell adhesion evolved along the animal stem lineage.
Recently, efforts to reconstruct the mechanisms by which the first multicellular animals evolved have greatly benefited from comparisons of extant animals and their closest living unicellular relatives. Sequencing the genomes and transcriptomes of the closest unicellular relatives of animals revealed that they encode homologs of many genes involved in animal multicellularity previously considered exclusive to animals, including cell adhesion genes. This points to ancestral gene repurposing (co-option) as an important driving force in the emergence of multicellular animals. Moreover, investigations on the developmental modes in these organisms significantly changed our vision of the unicellular ancestor’s biological capabilities: many members in each unicellular lineage can temporarily transition to multicellular stages in their life cycles, making them powerful systems for the study of the early evolution of cell adhesion regulation and multicellularity in animals. However, whether homologs of animal “cell adhesion genes” and/or environmental regulators mediate multicellular development among unicellular relatives of animals remains unknown.
FlexAggon hypothesises that some of the molecular mechanisms for controlled cell adhesion in animals have preceded animals' evolution and might therefore still be found in their unicellular relatives. This hypothesis is suggested by comparative genomic studies and observations of the life history of extant unicellular relatives of animals, but remains to be tested on functional grounds. Thus, by systematically characterising and linking the extracellular (environmental) and endogenous (genetic) factors regulating cell adhesion during the formation of multicellular phenotypes in these extant unicellular species, we can clarify the cell adhesion mechanisms that permitted the unicellular-to-multicellular transition during animal evolution. The main aim of FlexAggon was to elucidate the cellular adhesion molecules and environmental regulators that govern multicellularity in one of the closest living relatives of animals, the recently discovered choanoflagellate Choanoeca flexa.
FlexAggon replicated a range of environmental conditions of C. flexa’s natural habitat in the laboratory and systematically tested their effect on C. flexa colony formation. Based on preliminary data, I focused on systematically varying salinity, which proved to influence C. flexa colony formation. This included standardizing lab conditions to induce and monitor C. flexa colony formation using live imaging, Confocal/Airyscan microscopy and inhibitory assays; as well as understanding C. flexa life cycle in its natural context in its original isolation site in the field. A significant discovery I contributed to was that (1) C. flexa develops sheets through a mixed mode of clonal-aggregative multicellularity; (2) salinity regulates transitions into and out of multicellularity in C. flexa under laboratory conditions. In addition, we explored how this phenomenon regulates C. flexa life cycle in its natural context. I, together with my collaborators, performed two independent fieldwork expeditions on the Caribbean island of Curaçao, which resulted in four additional unforeseen findings: (3) the life cycle of C. flexa is linked to the natural cycles of evaporation and refilling in splash pools; (4) gradual evaporation in splash pools triggers loss of multicellularity and differentiation into desiccation-resistant cysts in C. flexa; (5) rehydration of soil samples induce a cyst-to-flagellate transition and restores multicellularity; (6) aggregation in C. flexa is constrained by kin recognition; (7) the sequencing of C. flexa genome from various strains revealed genetic differences between strains, allowing us to identify candidate genes potentially involved in selective adhesion.
Recently, efforts to reconstruct the mechanisms by which the first multicellular animals evolved have greatly benefited from comparisons of extant animals and their closest living unicellular relatives. Sequencing the genomes and transcriptomes of the closest unicellular relatives of animals revealed that they encode homologs of many genes involved in animal multicellularity previously considered exclusive to animals, including cell adhesion genes. This points to ancestral gene repurposing (co-option) as an important driving force in the emergence of multicellular animals. Moreover, investigations on the developmental modes in these organisms significantly changed our vision of the unicellular ancestor’s biological capabilities: many members in each unicellular lineage can temporarily transition to multicellular stages in their life cycles, making them powerful systems for the study of the early evolution of cell adhesion regulation and multicellularity in animals. However, whether homologs of animal “cell adhesion genes” and/or environmental regulators mediate multicellular development among unicellular relatives of animals remains unknown.
FlexAggon hypothesises that some of the molecular mechanisms for controlled cell adhesion in animals have preceded animals' evolution and might therefore still be found in their unicellular relatives. This hypothesis is suggested by comparative genomic studies and observations of the life history of extant unicellular relatives of animals, but remains to be tested on functional grounds. Thus, by systematically characterising and linking the extracellular (environmental) and endogenous (genetic) factors regulating cell adhesion during the formation of multicellular phenotypes in these extant unicellular species, we can clarify the cell adhesion mechanisms that permitted the unicellular-to-multicellular transition during animal evolution. The main aim of FlexAggon was to elucidate the cellular adhesion molecules and environmental regulators that govern multicellularity in one of the closest living relatives of animals, the recently discovered choanoflagellate Choanoeca flexa.
FlexAggon replicated a range of environmental conditions of C. flexa’s natural habitat in the laboratory and systematically tested their effect on C. flexa colony formation. Based on preliminary data, I focused on systematically varying salinity, which proved to influence C. flexa colony formation. This included standardizing lab conditions to induce and monitor C. flexa colony formation using live imaging, Confocal/Airyscan microscopy and inhibitory assays; as well as understanding C. flexa life cycle in its natural context in its original isolation site in the field. A significant discovery I contributed to was that (1) C. flexa develops sheets through a mixed mode of clonal-aggregative multicellularity; (2) salinity regulates transitions into and out of multicellularity in C. flexa under laboratory conditions. In addition, we explored how this phenomenon regulates C. flexa life cycle in its natural context. I, together with my collaborators, performed two independent fieldwork expeditions on the Caribbean island of Curaçao, which resulted in four additional unforeseen findings: (3) the life cycle of C. flexa is linked to the natural cycles of evaporation and refilling in splash pools; (4) gradual evaporation in splash pools triggers loss of multicellularity and differentiation into desiccation-resistant cysts in C. flexa; (5) rehydration of soil samples induce a cyst-to-flagellate transition and restores multicellularity; (6) aggregation in C. flexa is constrained by kin recognition; (7) the sequencing of C. flexa genome from various strains revealed genetic differences between strains, allowing us to identify candidate genes potentially involved in selective adhesion.
The main discovery derived from this action is that (1) C. flexa develops sheets through a mixed mode of clonal-aggregative multicellularity. The main experiments that supported this finding were a combination of live imaging using widefield microscopy, inhibitory assays, and dual-labelling experiments coupled with live imaging and Airyscan microscopy. This action also explored the impact of abiotic factors, particularly salinity, on C. flexa colony formation. This led to the discovery that (2) salinity regulates transitions into and out of multicellularity in C. flexa under laboratory conditions. The main experiments that supported this finding were monitoring the effects of the addition of increasing concentrations of salts on colony formation and integrity in live C. flexa cultures, as well as assessment of its effects on cell growth and cellular architecture using live imaging and Airyscan microscopy.
In addition, we explored how this phenomenon regulates C. flexa life cycle in its natural context. I, together with my collaborators, performed two independent fieldwork expeditions on the Caribbean island of Curaçao, which resulted in four additional unforeseen findings: (3) the life cycle of C. flexa is linked to the natural cycles of evaporation and refilling in splash pools; (4) gradual evaporation in splash pools triggers loss of multicellularity and differentiation into desiccation-resistant cysts in C. flexa; (5) rehydration of soil samples induce a cyst-to-flagellate transition and restores multicellularity; (6) aggregation in C. flexa is constrained by kin recognition; (7) the sequencing of C. flexa genome from various strains revealed genetic differences between strains, allowing us to identify candidate genes potentially involved in selective adhesion. The main activities that supported these findings were the correlation of the presence of C. flexa colonies in a narrow salinity range by inspecting more than 400 splash pool samples directly collected from the field. Moreover, daily monitoring of the presence of C. flexa colonies in 15 different splash pools for eight days revealed that C. flexa colonies cannot be observed when salinity reaches a certain threshold, yet they reappear in naturally rehydrated splash pools as well as artificially rehydrated soil samples. These results were later validated in the laboratory using an artificial splash pool experimental set-up designed to coincide with an evaporation rate within the range empirically observed in splash pools. Live imaging and Airyscan microscopy of C. flexa colonies under these conditions confirmed loss of multicellularity and differentiation into desiccation-resistant cysts. Finally, we compared whole-genome sequencing (WGS) data from three different C. flexa strains. We identified coding regions with signs of diversifying selection based on the highest ratio between synonymous and non-synonymous mutations, from which we selected a list of candidates potentially involved in selective adhesion.
In addition, we explored how this phenomenon regulates C. flexa life cycle in its natural context. I, together with my collaborators, performed two independent fieldwork expeditions on the Caribbean island of Curaçao, which resulted in four additional unforeseen findings: (3) the life cycle of C. flexa is linked to the natural cycles of evaporation and refilling in splash pools; (4) gradual evaporation in splash pools triggers loss of multicellularity and differentiation into desiccation-resistant cysts in C. flexa; (5) rehydration of soil samples induce a cyst-to-flagellate transition and restores multicellularity; (6) aggregation in C. flexa is constrained by kin recognition; (7) the sequencing of C. flexa genome from various strains revealed genetic differences between strains, allowing us to identify candidate genes potentially involved in selective adhesion. The main activities that supported these findings were the correlation of the presence of C. flexa colonies in a narrow salinity range by inspecting more than 400 splash pool samples directly collected from the field. Moreover, daily monitoring of the presence of C. flexa colonies in 15 different splash pools for eight days revealed that C. flexa colonies cannot be observed when salinity reaches a certain threshold, yet they reappear in naturally rehydrated splash pools as well as artificially rehydrated soil samples. These results were later validated in the laboratory using an artificial splash pool experimental set-up designed to coincide with an evaporation rate within the range empirically observed in splash pools. Live imaging and Airyscan microscopy of C. flexa colonies under these conditions confirmed loss of multicellularity and differentiation into desiccation-resistant cysts. Finally, we compared whole-genome sequencing (WGS) data from three different C. flexa strains. We identified coding regions with signs of diversifying selection based on the highest ratio between synonymous and non-synonymous mutations, from which we selected a list of candidates potentially involved in selective adhesion.
FlexAggon involves fundamental research that contributes to society by expanding the general knowledge base and supporting future innovation. It offers insights into the evolutionary history of animals and the surrounding ecological diversity. However, it does not directly address climate change, regional industrial or societal challenges, or generate market opportunities. As such, it does not impact company competitiveness or growth, and there are no innovation activities or expected users outside the scientific community. Thus, this action does not directly contribute towards European policy objectives and strategies or impact policy-making. The potential users of the results derived from the action would be other researchers working with close animal relatives and other protists, or interested in evolution of multicellularity, evolutionary cell biology and ecology. Thanks to all dissemination activities derived from the action, there has been suitable communication with the aforementioned interested parties.