Periodic Reporting for period 1 - EvoMorphoCell (From cell shape to organism shape: the cellular basis for the evolutionary origin of animal morphogenesis)
Période du rapport: 2022-08-01 au 2025-01-31
My work concerns a critical question: How did the cellular mechanisms underpinning animal morphogenesis first evolve? While the first multicellular ancestors of modern animals have left limited fossil traces, insights can be gained by studying their closest living relatives: the choanoflagellates. These microeukaryotes have several features of unique relevance to animal origins, including temporal cell differentiation, facultative multicellularity, and a metazoan-like ‘developmental gene toolkit’. Moreover, they have become amenable to functional genetics in the past few years. We will study the molecular and cellular mechanisms of three morphogenetic transitions in choanoflagellates: (1) the formation of the “collar complex”, a ring of microvilli surrounding the flagellum, which represents an example of complex single-cell morphogenesis and plays a central role in hypotheses on early animal evolution; (2) the molecular control of the transdifferentiation of choanoflagellates into amoeboid cells under confinement, which I recently discovered and whose mechanisms remain unknown; (3) the cellular basis of adhesion and inversion in sheet colonies of the multicellular species Choanoeca flexa, which I co-discovered. These 3 processes will be characterized by omic approaches (transcriptomics, proteomics, phosphoproteomics and lipidomics) which will allow unbiased comparisons with the growing dataset of molecular atlases for animal cell types. We will perform knockout, chemical inhibition, and fluorescent tagging of defined candidate genes identified by omics and/or known to play important roles in animals – including both structural genes (such as cytoskeletal and adhesion molecules) and signalling molecules. This project has the potential to illuminate long-standing questions on the pre-metazoan function of developmental genes and to inform the mechanistic basis of the transition from cells to organisms in both development and evolution.
Two years after the beginning of the project, the progress has been the following:
* We have established a new KO method for S. rosetta, which has notably allowed us to identify a new molecular regulator of multicellular rosette size: Warts kinase, a component of the Hippo pathway. This has potentially far-reaching evolutionary implications, as the Hippo pathway plays a crucial role in setting multicellular size in animals - but was so far not known to do so outside animals. More broadly, this new method might be of help to the choanoflagellate research community in accelerating functional studies.
* We have identified key molecular players and cytoskeletal remodeling events of the flagellate-to-ameoboid transition in S. rosetta.
* We have reconstituted the life history of Choanoeca flexa in its natural environment. We have shown it develops multicellularity by a novel mechanism - mixed clonal-aggregative multicellularity - controlled by salinity in its native environment.
* We have established a new KO method for S. rosetta, which has notably allowed us to identify a new molecular regulator of multicellular rosette size: Warts kinase, a component of the Hippo pathway. This has potentially far-reaching evolutionary implications, as the Hippo pathway plays a crucial role in setting multicellular size in animals - but was so far not known to do so outside animals. More broadly, this new method might be of help to the choanoflagellate research community in accelerating functional studies.
* We have identified key molecular players and cytoskeletal remodeling events of the flagellate-to-ameoboid transition in S. rosetta.
* We have reconstituted the life history of Choanoeca flexa in its natural environment. We have shown it develops multicellularity by a novel mechanism - mixed clonal-aggregative multicellularity - controlled by salinity in its native environment.
We have developed a novel and improved gene knockout method for S. rosetta, relying on targeted insertion of a puromycin resistance cassette in the loci of interest. Compared to earlier methods, that relied on manual isolation and screening of clones, our new protocol is faster, more robust, more affordable, and less labor-intensive. We have described our method in a manuscript we uploaded on biorXiv and which is currently under review at Cell Reports (Combredet & Brunet 2024). This new KO method has been transformative for our lab, and has the potential to significantly accelerate choanoflagellate functional genetics.
On a different note, our reconstitution of the C. flexa life history in its natural environment represents an unusual synthesis of field biology and laboratory experiments, which had rarely been achieved so far in other unicellular relatives of animals. Indeed, most choanoflagellates and other unicellular holozoans which have been studied at the cellular or molecular level so far have been laboratory strains whose natural environment was poorly characterized or unknown. Our work thus positions C. flexa as a rare emerging model for the "eco-evo-devo" of multicellularity among close animal relatives.
On a different note, our reconstitution of the C. flexa life history in its natural environment represents an unusual synthesis of field biology and laboratory experiments, which had rarely been achieved so far in other unicellular relatives of animals. Indeed, most choanoflagellates and other unicellular holozoans which have been studied at the cellular or molecular level so far have been laboratory strains whose natural environment was poorly characterized or unknown. Our work thus positions C. flexa as a rare emerging model for the "eco-evo-devo" of multicellularity among close animal relatives.