Identification of modules underlying the plastic evolution of NED

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.


 

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

 

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

 

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.


 

Cells and tissues of dormancy in ascidians: underlying developmental mechanisms and evolutionary origins

 

Colonial organisms, those that reproduce clonally and remain organically attached, can survive changing environmental conditions by adding/removing modules. Additionally, many colonial species survive adverse conditions by going dormant, where modules or whole colonies enter a state of physiological and morphological inactivity. Coloniality arose independently many times in the metazoans, and dormancy has evolved in almost all the classes of colonial animals. Yet the link between coloniality and dormancy remains unexplored. We hypothesize that dormancy arose in colonial lineages via co-option of mechanisms underlying asexual reproduction and regenerative potential present in colonial taxa. We then are exploring another similarly important developmental mechanism of colonial animals, i.e. dormancy. We examine the environmental, morphological, and molecular characteristics of dormancy in two ascidian species (Polyandrocarpa zorritensis and Clavelinna lepadiformis) that have independently evolved both coloniality and dormancy. We determined the environmental cues that induced and released dormancy in the lab and field. We described dormant tissues and cells using histology and TEM microscopy and we are now comparing the transcriptome profiles of dormant vs. non-dormant tissues at a different stages. Results from this work will provide the foundation for ascidians as a model for the origins of dormancy. Results will show if the origins of dormancy as a survival strategy display mechanistic links to coloniality, such as the deployment of circulatory stem cells.



 

Estimating the extent of functional foreign genetic material in Tunicates

 

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.

 

Art and Regeneration

 

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).

 

Project DEVODIVERSITY : Evolution of developmental strategies that generate diversity in marine chordate



 

Developing the pelagic Tunicate Salpa fusiformis and Thalia democratica as model organism for the study of Thaliaceans

Salps is the less studied class of the sub-phylum urochordata. They have a twofold scientific interest. From an ecological point of view, they can be the major constituent of the macro-zooplankton under favorable circumstances and from an evo-devo perspective, they are most likely closely related to the Ascidiacea, in particular the Aplousobranchia, although they underwent major morphological changes related to their pelagic lifestyle. In regard to the wealth of information that is now available about basal ascidians, acquisition of novel data on Salps would be very interesting concerning genetic, developmental and morphological evolution of novelties.

A. Transcriptome analyses of embryonic and blastogentic development of Salpa fusiformis In this project, led by Jacques Piette and Patrick Lemaire (University of Montpelier), samples of RNA from two blastozoids of S. fusiformis were submitted to RNAseq analysis. This allowed us to recover more than 6000 orthologues of Ciona intestinalis. Part of these data will be used by Emmanuel Douzery and Frédéric Delsuc (Montpellier) to refine ascidian phylogeny (together with RNAseq data obtained on Doliolum nationalis). The part of the project concerning our lab is focused on exploring the feasibility of whole mount in situ hybridization on Salp embryos.

B. Characterization of the Nervous system in the Thaliacean Thalia democratica Descriptions of the adult nervous systems among Tunicates are limited, at least from a molecular point of view, to the class Ascidiacea and Appendicularia. In Thaliaceans, a class of Tunicates represented by fragile and transparent pelagic species, the majority of the histological and anatomical studies of the neuronal architectures several decades old. This project, in collaboration with the Sordino Lab at the Anton Dhorn Zoological Station (Naples) we choose the salp Thalia democratica, as model to review the anatomy and the life cycle of this understudied class of tunicates.