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 first step in the formation of the vertebrate central nervous system (CNS) during embryonic development is called neural induction (NI). It is the instructive process by which naive ectodermal cells are committed to a neural fate. This concept was established by Mangold and Spemann who showed that the dorsal blastopore lip of a newt gastrula (i.e. organizer), when grafted to the ventral side of a host gastrula, was able to induce the formation of a Siamese twin embryo in which part of the secondary CNS developed from the host and not from the graft. Concerning the nature of neural inductive signals coming from the organizer contrasting views exist and no unifying scheme can be drawn. Several cellular signals appear to be important in this process. Thus, while inhibition of BMP signaling is necessary for NI in vertebrates, the emerging view is that NI requires a sequence of signals in a specific order. Several signals seem to play a role in this process such as FGF, Wnt and Notch. An additional important question concerns the evolutionary origin of the NI process, in other words, how this process appeared and evolved in the chordate lineage. Chordates represent a group of deuterostome metazoans including the vertebrates but also invertebrate groups such as the cephalochordates (i.e. amphioxus), and the urochordates (i.e. ascidians). The morphological and developmental characteristics of cephalochordates show many similarities with vertebrates, and it is considered that amphioxus is vertebrate-like but simpler. On the other hand, urochordates show some developmental modalities and genetic characteristics that are derived. Concerning NI, urochordates have lost the organizer, the inhibition of BMP does not appear to play a major role, and the endogenous neural inducer is FGF9/16/20. However, in amphioxus, we have shown that there is a functional organizer which is able to induce a complete secondary axis when grafted onto a host embryo, and which is able to induce neural fate in an ectodermic explant that acquires epidermal fate by default in absence of the organizer. We have also shown that i) the activation of the BMP signal ventralises the embryo and induces epidermal fate in the whole ectoderm, in a similar way to vertebrates; ii) inhibition of BMP generates an undifferentiated ectoderm, and that therefore a neural inducing signal is necessary; iii) the FGF signal does not seem to play an essential role in neural induction; iv) the neural inducing signal is Nodal/Activin, and v) using an ATAC-seq approach we have been able to construct a virtual GRN controlling NI in amphioxus downstream of Nodal/Activin. Therefore, the main objective of this project is to understand the different steps that lead an undifferentiated ectodermal cell to become a neural cell during the early development of chordates. Our goal is focused on both a cellular and a molecular level. By using a single cell RNA-seq (scRNA-seq) approach, we intend to decipher in a dynamic way how some undifferentiated ectodermal cells of the cephalochordate (i. e. amphioxus) embryo become neural, and also how the different genes implicated in the neural induction gene regulatory network (GRN) are expressed in each of these cells during early developmental stages. Insights into the temporal and spatial factors essential for neural induction will be crucial to understand how such a complex process occurs. In addition, our study on amphioxus embryos will allow us to perform comparisons with known data in vertebrates and will contribute to our understanding of the evolution of this developmental process.

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.