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Identification of modules underlying the plastic evolution of NED

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In tunicates, the capacity of budding, and therefore re-growing of clonal copies of the original individual (zooid), occurs through different mechanisms of interaction between non-homologous epithelial tissues and/or putative stem cells circulating in the bloodstream. Our driving hypothesis is that emergence of non-embryonic development (NED) relies on homologous genetic modules repeatedly co-opted and rewired in different species regardless of the nature of cells and tissues triggering budding/regeneration. To explore the similarities and differences in the cellular and molecular mechanisms underlying different forms of NED, and to infer how these mechanisms have been gained and lost during evolution, we use keep study closely related suitable models belonging to the Styeliadae family: Botryllus schlosseri and Polyandrocarpa zorritensis. We test our hypothesis by approaching at two different levels: (A) by a comparison of the transcriptome profiles of the budding tissues at different developmental steps across the two different species, and (B) by exploring and functionally dissecting the mechanisms of budding focusing on Botryllus schlosseri, where protocols and functional approaches have been already developed.

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DEVOBLOOM - Study the effect of environmental factors on asexual development in salps

Salps are a group of gelatinous zooplankton present throughout the world's oceans (except the Arctic). Due to their complex life-cycle characterized by a very active phase of asexual proliferation (budding), salp populations seasonally grow exponentially and form very dense blooms that can cover several thousand Km2 of the ocean. Given the magnitude of these blooms, salps play a central role in oceanic trophic webs and global biogeochemical cycles. However, their biology has been surprisingly neglected in the last century.Salp blooms are deeply influenced by sea surface temperature and by food availability and are therefore sensitive to perturbations of the environment. In addition, the direct link between asexual budding and blooms makes salps a rare model to explore the direct effect of environmental inputs on the developmental dynamics, and up to the level of population demography. The present project combines developmental biology and ecological approaches to explore the links between environmental factors and asexual budding in two species of Mediterranean salps, Thalia democratica and Salpa fusiformis. We will first couple state of the art morphological and proliferation dynamics descriptions of the budding organ (stolon) with single-cell RNA sequencing to characterize the cells implicated in budding and investigate their spatio-temporal differentiation trajectories. In parallel we will study in laboratory-controlled conditions how variations of temperature and food concentration impact the budding mechanisms at the individidual, cellular and gene expression levels. These data will help to reinforce our current understanding of Mediterranean salps demography.

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EVOSOMA - Testing evolution through the accumulation of somatic mutations  using modular chordates

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Throughout the lifespan of multicellular species, somatic genetic variation continually emerges through the accumulation of somatic mutations (SM) that arise in cells and tissues, eventually making each individual a genetic mosaic. In many species, this mutational load is reset by a single-cell bottleneck through germline sequestration and sexual reproduction. Due to the separation between disposable soma and heritable germline, known as Weismann’s barrier, somatic mutations have historically been considered evolutionarily irrelevant. However, recent empirical studies and theoretical works strongly suggest that somatic genetic variations play an underestimated and even fundamental role in the evolution of long-lived modular organisms, including the majority of plants, fungi, and many animal species. Nearly half of the metazoan phyla contain species that propagate asexually via agametic reproduction, often forming colonies of genetically identical modules. Colonial growth and modular organization have important physiological and ecological consequences, as seen in many marine invertebrates such as sponges, Cnidarians, Lophophorates, Pterobranchs and Tunicates. Despite the abundance and adaptive success of modular species, evolutionary theories mainly rely on studies conducted in unitary, strictly sexually reproducing organisms. The evolutionary consequences of somatic variation are still widely unexplored, mainly due to the difficulty of tracking the typically low-frequency somatic mutations and reconstructing the sexual and asexual reproductive history of the colonial organism at issue. This project utilizes the modular chordate Botryllus schlosseri (Tunicata) as a laboratory model to investigate the interplay between somatically and meiotically generated genetic variation and to explore different levels of selection within and among modules.
B. schlosseri forms colonies through asexual budding of ramets (modules). Each colony belongs to one genet, which originates sexually from a single zygote. The propagation by budding occurs continuously on a weekly basis, corresponding to the succession of many asexual generations. Sexual reproduction is also fast, taking about a week. The hosting laboratory has the ability to control both the asexual and sexual cycles precisely, and genetically identifiable Botryllus colonies are available. The aim is to describe the extent, nature, and dynamics of genome-wide SM across several asexual generations, both intra- and inter-ramet, using deep-genome duplex sequencing. The goal is to obtain a spatial and temporal portrayal of SM propagative dynamics. Secondly, we will test whether selection or somatic genetic drift can increase the frequency of SM in the ramets and colony, and gather information on the potential adaptive value of these SM. Additionally, we will investigate whether SM can be sexually inherited by taking advantage of late germline segregation and plastic sexual fertility, thus breaking the Weissmann's Barrier. The potential transfer of somatic genetic variation to the sexual cycle suggests that SM can recombine into new genetic landscapes, increasing diversity and facilitating adaptation. Additionally, we will characterize the cellular and molecular dynamics of the somatic cell bottleneck associated with the budding onset. This will eventually help to infer intra-ramet mechanisms of cell selection.  
Together, these approaches will allow to explore, under laboratory conditions, the dynamics of multi-level selections in a modular chordate. Importantly, by providing empirical data appropriately collected based on theoretical expectations, we will be able, for the first time, to follow the fate of SM during an organism's growth and evaluate their role in evolution.

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Developing transgenesis in Botryllus schlosseri

In order to better test the function of specific genes, and to follow the dynamic of the cells involved in NED we aim to establish transgenic colonies of B. schlosseri, a technical challenge that will open new avenues for functional study and high-resolution live imaging of budding. We already produced preliminary data supporting the feasibility of the task, i.e. mastering of the in vitro fertilization, zygote microinjections, and expression of reporter RNA. We recently managed to express the photo-convertible protein Kaede under a Botryllus ubiquitous promoter (EEF1-alpha) and we aim to use this tool for live-imaging. Photo-conversion of budding tissues and cell will then allow to follow cell behaviors (proliferation, migration, EMT, etc.) and finely characterize budding dynamics. The data obtained from B.schlosseri, together with the differentially expressed transcriptomic datasets have the potential to help to infer the mechanisms of budding in the other species.

 

Role of haemoblasts during Polyandrocarpa zorritensis budding

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The aim of this project is to uncover the molecular mechanisms by which circulating mesenchymal cells are able to reconstruct a new individual. All ascidians possess undifferentiated mesenchymal cells named haemoblasts that circulate in their bloodstream. They participate in bud formation in different types of NED, and in the tissue and organ regeneration of solitary species. We showed that aggregates of hemoblasts proliferate at the onset of both B. schosseri vascular budding and preliminary data suggest their presence also in P. zorritensis NED. Hemoblasts are also present in solitary ascidians and are potentilly involved in their tissue regeneration. While an increasing amount of studies suggest that heamoblasts are toti/multipotent stem cells responsible of some forms of budding, no one assayed their degree of potency by tracking their fate and their dynamic during NED. We are currently describing (morphologically and molecularly) this cell population in P.zorritensis and we aim to test their potentiality by blocking and rescue of NED via single-cell transplantation.
 

Comparative genomics among solitary and colonial (NED) species

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To highlight genomic signatures linked to NED and to highlight other genotype-phenotype convergent associations related to characters linked to the capacity of undergoing NED, we are sequencing and comparing the genomes of four species of Styelid, two colonials (Botryllus schlosseri and Polyandrocarpa zorritensis) and two solitaries (Dendrodoa grossularia and Styela plicata). As an offshoot, we may be able to collect insights on the possible effects of asexual propagation on evolution of genome architecture, for example due to heritable mutations of the somatic pluripotent stem cells. We are currently sequencing the genomes via Oxford Millipore technology, we then assemble and optimize the scaffolding and: a) screen for gene loss and/or gene duplication and describe the expansion or contraction of multigene families, to identify orthologues groups of candidate genes involved in coloniality and budding, as well as linked to other sets of traits; b) explore the nature, distribution and potential conservation of cis-regulatory modules that drive gene expression during asexual budding, particularly focusing on the genes up-regulated in budding tissues and cells; c) we will be able to finely resolve the dynamic of chromatin accessibility linked to regulation of potential budding genes.
 

Estimating the extent of functional foreign genetic material in Tunicates

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Horizontal gene transfer (HGT) is the acquisition of genetic material from non-parent organisms of different species, by opposition with vertical gene transfer where the genetic material is inherited from the parents. While this process is widely described in prokaryotes where it has been shown to accelerate their evolution, the impact of HGT on the evolution of eukaryotes, and particularly on metazoans, remains a relatively unexplored field of research. In collaboration with the team of Dr. Simon Blanchoud (University of Fribourg, Switzerland) we began to investigate the extent and the nature of HGTs in Tunicates. We will take advantage of a recent and significant increase of genomic data that covers almost the whole taxon, as well as of the development of novel algorithmic tools, to perform an exhaustive and consistent analysis of HGT. Tunicates present one of the best-documented case of active HGT in metazoans, cellulose synthase. Preliminary proteomic studies have identified chemical compounds similar to some known plant secondary metabolites, suggesting a co-option of vegetal biosynthetic pathways, potentially through HGTs. Therefore, Tunicates provides a unique opportunity to study the distribution and the potential function of HGTs in chordates and eventually to integrate these results with the metabolic, physiologic, and developmental specifics of these animals.
 

DORMANSEA - Dormancy in tunicates: environmental, morphological and molecular bases of a survival strategy

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In variable environments, organisms can experience conditions that are limiting their survival, growth, or reproduction. One of the strategies that many animal species adopt to persist in variable environments is to enter a state of dormancy, a curtail of the metabolic activity often linked to drastic morphological changes. The ability to undergo and survive in a dormant state is pervasive, yet irregularly distributed among invertebrates, suggesting that dormancy has evolved independently many times. Despite its prominence, dormancy is investigated only in a handful of invertebrate taxa and the mechanisms that link the environmental signals and translate into the organismal physiological and developmental changes are very poorly studied.  In this project, we will compare the dynamics of entrance and release from a state of dormancy in three widespread species of tunicate (Botryllus schlosseri, Polyandrocarpa zorritensis, and Clavelina lepadiformis). First, we will establish the abiotic clues that trigger and release dormancy, both in laboratory conditions and in a native environment. Then, the two processes will be staged and the morphological changes will be analyzed via histology, enzymatic assays, immunohistochemistry, and electron microscopy. The phenotypic changes observed in each stage will be coupled with their relative transcriptomic profiles to describe their molecular shifts by RNAseq differential expression analyses. The anatomical atlas obtained, associated with their corresponding molecular dataset, will provide the basis to dissect cellular and molecular signals that connect an environmental shift with a drastic developmental change. The results obtained will set the foundation for tunicates as a model to study the evolution of dormancy. More in general, this work will directly contribute to understanding the mechanisms of a survival strategy that allows facing sudden environmental changes.
 

Art and Science

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Funded by the Advanced Research Program at the Université Côte d'Azur, the project ‘ If we were to look at regeneration with a different eye’ has been initiated as an artistic-scientific collaboration with the artist Irene Kopelman and the Röttinger (IRCAN – UCA, CNRS, INSERM), and is driven by Irene long-term engagement with scientific research which underlies her artistic practice. This interest has led Iren to find a number of parallels between the practices of science and art, both in terms of objectives and methodologies. Looking for points of convergence between the two fields has become one of the main features of Irene’s work. Irene will work with the researchers at both labs to develop responses to their work and make new observations through drawing, as a mode of investigation into the visual characteristics of their vital field of study. As an artist, Irene ultimate goal is to create a body of art works (including drawings, paintings, sculptures and graphic material). The aim is to focus on a part of nature that has been overlooked in the context of contemporary art: marine invertebrates. The art-works produced during the project will shared with the public in various and different formats at Musée d'art moderne et d’art contemporain (MAMAC).

 

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