Deciphering the gene regulatory network logic underlying whole-body regeneration – RENEW

The current state-of-the-art for understanding the evolution of regeneration is to build gene-regulatory networks (GRN) that comprehensively represent genetic interactions underlying a biological process, with the goal of comparing them across phyla to highlight similarities or identify elements that might be specific to induce regeneration. Several recent studies have demonstrated the value of performing single-cell RNA-seq (scRNA-seq) and ATACseq to uncover cell-type diversity and regulatory programs that underlie cell type specification and differentiation in whole embryos and animals. These approaches have also proven to be insightful to understand the molecular and cellular dynamics during injury or disease-induced regeneration in vertebrates.

However, scRNA and/or ATAC-seq approaches to understand whole-body regeneration or stem cell biology in marine/aquatic invertebrates remain in their infancy, despite the power of these systems to study regeneration. In planarians, bulk RNA-seq and scRNA-seq has been used to create a catalogue of adult cell types and to identify a specific pluripotent stem cells sub-type whose transcriptional response differs during homeostasis and regeneration 28. In acoels, a recent study has combined bulk RNA-seq and ATAC-seq to propose a whole-body regeneration GRN2. Finally, In the cnidarians Nematostella and Hydra, scRNA-seq and ATAC-seq studies have revealed a high diversity of cell-types and stem cell differentiation trajectories in homeostatic adults. Overall, though, the trajectories of cell differentiation during whole-body regeneration, as well as their underlying GRNs, remain largely unknown.

In this project, we propose to combine ATAC-seq and scRNA-seq approaches to determine the GRNs underlying the initiation and subsequent cellular dynamics during Nematostella regeneration, and to experimentally validate key elements using CRISPR/Cas9-based genome manipulation. Nematostella is among the leading cnidarian models in which these state-of-the art technologies have been established – and the methods are well-established within the labs of the coordinator and partner teams. Overall, our multidisciplinary consortium has strong expertise in these techniques and their associated analytical & statistical approaches, as well as the research model and its functional tools, and we believe that we’re uniquely positioned to tackle this project with success.

We have selected FO founders for endogenously tagged piwi and mycB transgenice lines enabling us to follow their expression (flags) and fate (P2A::mOrange tag). Genotyping of F1 offspring is not conclusive yet, however visual analysis of the F1 animals indicate that germ-line transmission of the transgene may have happened. We’re currently pursuing their genotyping with optimized primer pairs as well as visual screens to select positive F1 animals.

P1 and P2 have optimized the cell dissociation and nuclei extraction protocols to yield sufficient gDNA and tested efficient library preparation for ATAC-sequencing. Mapping of the obtained sequence reads to the available genome browser, validated our experimental pipeline.

P1 and P3 have optimized the cell dissociation and single-cell mRNA extraction protocols. Enough cells and sample quality have been validated using standard quality controls. Using the same cell dissociation protocol, a morphological cell type analyses using the AMNIS Imagstream enabled us to begin to develop a morphological cell type atlas in uncut polyps as well as during regeneration. Those data will complement the molecular data obtained from the scRNA-seq analyses.

We have hired a Master student to develop a database / UI for mining existing and to be developed genetic Nematostella resources (WT strains, KO, KI), some of which will be produced during the project. A first draft of the database is available on an internal server and another master student will be hired in 2023 to finalize this database and make it publicly available.

Finally, and with support from the IDEX UCAjediwe have developed novel outreach activities with the Artist, Irene Kopelman, that performed her residency partly in the coordinators team at the IRCAN and that worked on a project entitled – if we were to look at regeneration with a different eye -. In fact, towards the end of the residency, a 2-week workshop was organized at the contemporary Museum in Nice, France (MAMAC), enabling pupils and the general art-public to be introduced to whole body regeneration in marine organisms. A 6-months art show will begin in September 2022 featuring the regenerative capacity of Nematostella vectensis.

The present project will enable us to gain unprecedented insight into the cellular and molecular mechanisms underlying whole body regeneration in Nematostella and provide important information to increase our understanding of the evolution of this process across metazoans. A systematic comparison of GRNs deployed during regeneration across whole-body regeneration models will ultimately enable us to identify a “regeneration kernel”, i.e. a minimal GRN module required and sufficient to trigger a regenerative response. In addition, comparison of such data with less-regenerative models, may provide novel insight into why certain organisms possess this fascinating capacity while others don’t.

Data generated by the present project will be published in leading international peer-reviewed journals, selected to maximise the scientific impact of our results, and favouring open-access publications where appropriate. Finally, we foresee to make our scRNA-seq data readily available to the scientific community upon publication of our results via a freely accessible online database, as well as standard deposition in publicly-accessible scientific repositories.

1. Röttinger, E. Nematostella vectensis, an Emerging Model for Deciphering the Molecular and Cellular Mechanisms Underlying Whole-Body Regeneration. Cells 2021, 10, 2692. doi.org/10.3390/cells10102692

2. Croce O, Röttinger E. Creating a User-Friendly and Open-Access Gene Expression Database for Comparing Embryonic Development and Regeneration in Nematostella vectensis. Methods Mol Biol. 2022;2450:649-662.

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.