Final Report Summary - PLURIPOTENCY (A new model for study relationship between pluripotency and tumorogenesis: molecular insights from basal chordates)
Analyzing the relationship between pluripotency and tumorogenesis: molecular insights from a new basal chordate model
The main aim of this research proposal was to develop a newly emerging chordate experimental model, the colonial ascidian Botryllus schlosseri (Urochordata), in order to study the cellular and molecular bases of its regenerative plasticity and in particular to explore the relationship between cell pluripotency and tumorogenesis.
Botryllus schlosseri is one of the closest relatives to vertebrates that, beside embryogenesis, can adopt distinct developmental pathways to regenerate their entire body. In particular, through a process known as vascular budding, an adult is regenerated from pluripotent cells in a sequence of morphologically abnormal developmental stages which pass through a teratoma-like structure. The teratoma maintains a population of cells that preserve their pluripotency and eventually regain positional identity and differentiate “correctly” into a functional body via asexual development (blastogenesis). In this proposed project we established a new research laboratory that uses B. schlosseri as chordate model; we took advantage of its in vivo developmental and regenerative pathways to explore the early developmental steps that lead to the formation of a functional body through a teratoma-like structure. We applied two complementary approaches: (1) To undertake unbiased high-throughput gene screening in order to define the transcriptome implicated in the blastogenesis and vascular budding; (2) to target candidate genes involved in maintenance of pluripotency and cell differentiation.
In the first 24 months, we first set up a marine-culture system in order to breed different genotypes of B. schlosseri. This allowed us to select clones from collected wild-type animals and to establish lines of genetically identical colonies. In order to identify discrete regenerative stages we re-described the early steps of vascular budding from anatomical and molecular point of view, we selected candidate colony clones and optimized the sample preparation in order to improve the process of RNAseq screening. This allowed us to prepare a set of RNA samples ready to be linearly amplified and sequenced. Concerning the second approach, we cloned 30 candidate genes involved in maintenance of “stemness”, endomesoderm differentiation, plus chromatin modifiers and other markers related to cell fate determination, we then started to screen the temporal and spatial pattern of expression during blastogenesis and in vascular budding. In addition we developed and published a methodology that help to track phagocytic cells in vivo and to describe their role during the regenerative processes.
In the last 24 months we accomplished the first approach and provided a complete assembled transcriptome of Botryllus schlosseri, an overall analyses of the transcriptome landscapes during the onset of blastogenesis and on key steps of the vascular budding, highlighting the transcriptomic shift of a series of potential candidate gene and pathways. From the second approach we described the co-option of conserved developmental pathways and genes during a regenerative, non-embryonic development and analyzed it from an evolutionary and developmental point of view.
In addition to the scientific goals, the IRG provided a successful kick-start for the newly established research team. Firstly assuring to the fellow an independent permanent position and integrating him in the French academic system (CNRS-UPMC). Then, allowing him to obtain further extramural funding from different agencies (AFM, FRM, ANR, TEFOR, UPMC, Sorbonne Universities). During the 4-years the fellow established and consolidated an international collaborative network with former and new partners, he attended national and international meeting and contributed to eight scientific manuscripts, both published and on preparation.
While the nature of this study can be classified as basic research, the phylogenetic position, the robust orthology of the main molecular pathways, combined with the high regenerative plasticity that characterize colonial ascidians, makes Botryllidae unique models to uncover conserved basic molecular mechanisms that regulate cell fate in a non embryonic context, like blastogenesis and vascular budding. In particular, the latter allows in vivo analysis of the cell dynamics that drive from a state of pluripotency towards a differentiated state. The formation of teratoma due to transplantation of undifferentiated pluripotent stem cells is considered to be a downside in regenerative medicine, which aims to employ stem cells to differentiate into lost tissues or organs. Identification of basic and conserved molecular differences between pluripotent stem cells that lead to a teratoma-like and pluripotent stem cells that differentiate correctly might allow better understanding of the in vivo behavior of transplanted teratoma-forming stem cells and provide insight into why teratoma and teratocarcinoma form. This project aims to start to understand how a closely related, highly plastic, organism can use conserved molecular tools to recapitulate so accurately the embryonic development, and how these pathways are wired together, in order to compare with the scenario in vertebrate chordates, included humans
The main aim of this research proposal was to develop a newly emerging chordate experimental model, the colonial ascidian Botryllus schlosseri (Urochordata), in order to study the cellular and molecular bases of its regenerative plasticity and in particular to explore the relationship between cell pluripotency and tumorogenesis.
Botryllus schlosseri is one of the closest relatives to vertebrates that, beside embryogenesis, can adopt distinct developmental pathways to regenerate their entire body. In particular, through a process known as vascular budding, an adult is regenerated from pluripotent cells in a sequence of morphologically abnormal developmental stages which pass through a teratoma-like structure. The teratoma maintains a population of cells that preserve their pluripotency and eventually regain positional identity and differentiate “correctly” into a functional body via asexual development (blastogenesis). In this proposed project we established a new research laboratory that uses B. schlosseri as chordate model; we took advantage of its in vivo developmental and regenerative pathways to explore the early developmental steps that lead to the formation of a functional body through a teratoma-like structure. We applied two complementary approaches: (1) To undertake unbiased high-throughput gene screening in order to define the transcriptome implicated in the blastogenesis and vascular budding; (2) to target candidate genes involved in maintenance of pluripotency and cell differentiation.
In the first 24 months, we first set up a marine-culture system in order to breed different genotypes of B. schlosseri. This allowed us to select clones from collected wild-type animals and to establish lines of genetically identical colonies. In order to identify discrete regenerative stages we re-described the early steps of vascular budding from anatomical and molecular point of view, we selected candidate colony clones and optimized the sample preparation in order to improve the process of RNAseq screening. This allowed us to prepare a set of RNA samples ready to be linearly amplified and sequenced. Concerning the second approach, we cloned 30 candidate genes involved in maintenance of “stemness”, endomesoderm differentiation, plus chromatin modifiers and other markers related to cell fate determination, we then started to screen the temporal and spatial pattern of expression during blastogenesis and in vascular budding. In addition we developed and published a methodology that help to track phagocytic cells in vivo and to describe their role during the regenerative processes.
In the last 24 months we accomplished the first approach and provided a complete assembled transcriptome of Botryllus schlosseri, an overall analyses of the transcriptome landscapes during the onset of blastogenesis and on key steps of the vascular budding, highlighting the transcriptomic shift of a series of potential candidate gene and pathways. From the second approach we described the co-option of conserved developmental pathways and genes during a regenerative, non-embryonic development and analyzed it from an evolutionary and developmental point of view.
In addition to the scientific goals, the IRG provided a successful kick-start for the newly established research team. Firstly assuring to the fellow an independent permanent position and integrating him in the French academic system (CNRS-UPMC). Then, allowing him to obtain further extramural funding from different agencies (AFM, FRM, ANR, TEFOR, UPMC, Sorbonne Universities). During the 4-years the fellow established and consolidated an international collaborative network with former and new partners, he attended national and international meeting and contributed to eight scientific manuscripts, both published and on preparation.
While the nature of this study can be classified as basic research, the phylogenetic position, the robust orthology of the main molecular pathways, combined with the high regenerative plasticity that characterize colonial ascidians, makes Botryllidae unique models to uncover conserved basic molecular mechanisms that regulate cell fate in a non embryonic context, like blastogenesis and vascular budding. In particular, the latter allows in vivo analysis of the cell dynamics that drive from a state of pluripotency towards a differentiated state. The formation of teratoma due to transplantation of undifferentiated pluripotent stem cells is considered to be a downside in regenerative medicine, which aims to employ stem cells to differentiate into lost tissues or organs. Identification of basic and conserved molecular differences between pluripotent stem cells that lead to a teratoma-like and pluripotent stem cells that differentiate correctly might allow better understanding of the in vivo behavior of transplanted teratoma-forming stem cells and provide insight into why teratoma and teratocarcinoma form. This project aims to start to understand how a closely related, highly plastic, organism can use conserved molecular tools to recapitulate so accurately the embryonic development, and how these pathways are wired together, in order to compare with the scenario in vertebrate chordates, included humans