Some animals have the capacity to regenerate their organs with a high degree of fidelity during adult life; the regenerated organs are precise replicas of those originally produced during embryonic development. Does this capacity result from the re-use of embryonic gene regulatory networks (GRNs), or have these animals evolved GRNs that are unique to regeneration to produce the same structure? Our project will address this question in the crustacean Parhyale hawaiensis, an emerging experimental model for studying leg regeneration. Parhyale can regenerate their legs with high fidelity, throughout their lifetime. First, we will collect data on the chromatin accessibility and gene expression profiles from tens of thousands of cells at different time points during the course of leg development and regeneration. In parallel, we will determine the DNA binding preferences of the entire repertoire of transcription factors expressed at relevant stages. These data will serve as the basis for inferring the GRNs that underpin leg development and regeneration, by correlating the expression of transcription factors with patterns of chromatin accessibility and expression of putative target genes, using established methods. We will compare the predicted GRNs of development and regeneration to identify shared and divergent elements. We will validate key nodes of these GRNs experimentally using transgenic approaches. Discovering whether regeneration recapitulates development is a key for understanding the genetic underpinnings and the evolutionary dynamics of regeneration.

The vertebrate mesoderm is divided into several compartments: the cranial mesoderm, the axial mesoderm (prechordal plate, notochord), the paraxial mesoderm which is segmented into somites, and the lateral mesoderm. The latter is involved in the formation of several vertebrate-specific structures, including a closed circulatory system, a chambered heart, or paired appendages (limbs/fins) in jawed vertebrates. The notochord and somites are ancestral structures in chordates. On the other hand, the origin of the lateral mesoderm, and the evolution of the structures derived from it, remain enigmatic. We propose to decipher the evolutionary and developmental trajectory of the lateral mesoderm using two model species having a key phylogenetic position: the cephalochordate amphioxus and the lamprey, a jawless vertebrate, through a detailed morphological description paralleled by a single-cell transcriptional analysis approach.

In our project, we will determine the gene regulatory networks (GRNs) triggered by an injury and that control whole-body regeneration by dissecting the 1) transcriptional dynamics and 2) genome-wide chromatin accessibility at the tissue and single-cell levels. We will also 3) infer and 4) validate the predicted cellular trajectories and regulatory elements using genetic approaches. This study is based on data from the lab obtained over the past five years and will be carried out using the emerging whole-body regeneration model - the sea anemone Nematostella vectensis (Cnidaria, Anthozoa) - developed and mastered by the lab. After injury, the capacity to regenerate varies drastically from poorly regenerating mammals to whole-body regenerating aquatic organisms, such as cnidarians. Determining the gene regulatory networks underlying whole-body regeneration in order to reveal the similarities and in particular the differences with mammals are thus necessary. Yet comprehensive regeneration GRNs especially from non-bilaterian animals with whole-body regenerative capacities (i.e. cnidarians) are in their infancy. So far, establishing whole-body regeneration gene regulatory networks in cnidarians, especially at the single cell levels, has been hampered by a lack of suitable models and sufficiently sensitive techniques. We believe that it is now possible by using the state-of-the-art scRNAseq and (sc)ATACseq approaches mastered by the consortium in combination with Nematostella, a cnidarian whole-body regeneration model that we’re experts in and that is suitable for genetically dissecting gene function. In order to determine the GRNs underlying whole-body regeneration in Nematostella, we have three specific objectives: 1. Determine the transcriptional dynamics and regulatory elements active at the tissue and single-cell levels using scRNA-seq and ATAC-seq approaches. 2. Infer the cellular dynamics and blueprints of the regeneration GRNs and the factors that control them using state-of-the art trajectory reconstruction and motif enrichment analysis. 3. Experimentally validate core elements and wiring of the regeneration gene regulatory network by testing the regeneration specific enhancer elements and gene-specific functional approaches using CRISPR/Cas9. These objectives will enable us to reveal the precise cellular dynamics, i.e. stem cell trajectories that follow injury and the underlying regulatory mechanisms that are involved in the activation of regeneration-specific core modules. This in turn is crucial for evolutionary/comparative studies to gain insight into why some animals regenerate while others don’t (or do less) and to further develop biomedical approaches that foster the re-initiation of regenerative program(s) in organs or tissues that lost this capacity during evolution or during aging.