Understanding the re-deployment of gene regulatory networks underlying regeneration

Final Report Summary - EARN (Understanding the re-deployment of gene regulatory networks underlying regeneration)

Regeneration is the process that follows initial wound healing, during which a lost part of the animal body is restored to rebuild a fully functional organism. Regenerated body parts are essentially identical to the parts that were developed originally during embryogenesis. Taking into account that initial context of embryogenesis and regeneration are different, we hypothesize that core elements of the embryonic gene regulatory network are re-deployed and connected to regeneration specific elements in order to reform a functional organism
Modern functional genomics enables us today to use gene regulatory network (GRN) approaches to study regeneration and will provide us with a comprehensive molecular view of when, where and how the formation of the missing body parts is induced, specified and determined. The comparison of this regeneration GRN to the GRN underlying embryogenesis, within the same organisms, will allow us to identify the similarities as well as importantly the regeneration specific elements. One organism that is perfectly suited to compare the GRNs underlying embryogenesis and regeneration is the sea anemone Nematostella vectensis as i) a first cnidarian GRN underlying embryogenesis has been recently published and ii) this animal is able to regenerate missing body part in only five days after amputation. Thus, the proposed FP7-MC CIG project was articulated around three principal aims: 1) Identification and characterization of the temporal and spatial deployment of the regeneration GRN, 2) Functionally characterizing the wiring of individual components of this GRN and 3) in vivo characterization of regeneration in Nv.
Description of the work performed since the beginning of the project
Since the start of the FP7-MC CIG funded project, we made excellent progress in all three objectives mentioned above. We have been awarded additional grants and fellowships from private biomedical and cancer foundations to support this project. We put our initial efforts into a very detailed characterization of the regeneration process at the tissular and cellular levels, its limits, as well as in the development of new in vivo tools to assess wound-healing and pharynx reformation. This has lead to one publication (Amiel et al, 2015) and one is currently in revision (Amiel et al, in revision for Nature Communications). Importantly though, the detailed characterization of oral regeneration in Nematostella served as the basis for the proposed large scale RNAseq analysis that spans 16 time points (in three replicates) of this process (Warner et al, in press). The bioinformatics analysis is terminated and has been performed at two levels: 1) by looking at differential gene expression during regeneration, that highlighted 9 gene clusters corresponding to potential synexpression groups (Warner et al, in submission) and 2) at a more global level by comparing previous published developmental RNAseq data (Tulin et al. 2013, Helm et al. 2013) to our regeneration RNAseq analysis (Warner et al, in press). This global embryogenesis vs regeneration RNAseq comparison has revealed two important points: i) Regeneration is only a partial redeployment of regeneration and ii) embryonic network modules are rewired during regeneration. We have further confirmed latter observation using signaling pathway specific perturbation experiments followed by systematic downstream target analysis (Johnston et al, in prep, Warner et al in prep).
Description of the main results achieved so far
In line with our proposal, we have characterized in detail the oral regeneration process and developed a first set of in vivo tools to assess regenerative success. This analysis has revealed a very stereotyped behavior of the mesenteries (internal organs responsible for the reproduction and digestion) that enabled us to propose a chronological staging system for oral regeneration in Nematostella (Amiel et al. 2015). Using in vivo approaches, this study has shown that wound healing is terminated before 6 hours post amputation (hpa) and that endogenous auto-fluorescence present in the pharynx can be used to determine the timing of the initiation of pharynx reformation (72hpa) under normal regeneration conditions, and the effectiveness of the reformation of this structure in perturbation experiments (Amiel et al. 2015). Although highly regenerative, stem cell populations have yet to be described in anthozoan cnidarians (corals, sea anemones). Following up on the initial characterization of the regeneration process, we have further analyzed the morphological and cellular events underlying regeneration in Nv and highlighted that i) the mesenteries (digestive and reproductive tissues of anthzoans) are crucially required to induce regeneration and ii) that the amputation stress initiates a cellular response of two cell populations that are synergistically required to complete regeneration and that behave like potential stem cell populations (Amiel et al, in revision for Nature Communications).
Using the detailed characterization of oral regeneration in Nematostella, we defined 16 time points during regeneration that were used to perform RNAseq (Aim 1). The bioinformatics analysis is completed and we have developed a web-interface for accessing and mining all data developed in the course of the present project (nvertx.kahikai.org Warner et al, in press for Development, https://www.biorxiv.org/content/early/2018/01/04/242370). In addition to identifying genes that are part of the immediate stress response cluster, the comparison of the molecular dynamics of given genes (RNAseq) and the morphological and cellular dynamics enables us to predict potential connections that are currently being addressed by gene specific functional studies. Results of our in situ hybridization screen indicate the existence of at least five distinct expression domains within the amputation site and the combination of this information to the RNAseq data provides us with a first framework of the regeneration GRN. The broader comparative RNAseq analysis further highlighted that only a small fraction of developmental genes are reactivated and rewired during regeneration (Warner et al, in submission). Pathway specific perturbation experiments on Wnt and MEK/ERK signaling during regeneration, compared to data obtained during embetogenesis (Röttinger et al, 2012, Layden et al, 2016, Amiel & Johnston et al, 2017) further confirm this observation (Johnston et al in prep, Warner et al in prep). Importantly, this comparison also enabled us to identify “regeneration – specific” genes that are currently being analyzed and that are part of the major current research axis of the team. In addition to successfully creating CrispR/Cas9 mediated KI lines (e.g. Nvßcat::mCherry), the development of gene specific knock-down (hsp70::dnTCF) or conditional KO tools (e.g. CRE/Lox) have been carried out but require optimizations that are in progress. Constitutive KO of selected genes are ongoing and will provide important functional information in the near future.
Expected final results and their potential impact and use
Aging is a complex and multifactorial biological process that is tightly linked to regeneration and the development of age related diseases, such as cancer. In fact, in the aging organism the regenerative capacity of the tissues vanes and the risk of age-related diseases increases. Interestingly, certain marine invertebrates, such as the studies cnidarian Nematostella, possess extreme regenerative capacities, and undetermined but extended lifespan without the appearance of tumors. Thus understanding the process of extreme regeneration will provide crucial information about the mechanisms these animals deploy to maintain extended longevity in the absence of age-related diseases. The current project investigates the process of extreme regeneration at the GRN level combining modern RNAseq approaches and functional genomic tool in a uniquely suited new model system and will provide new insight into the morphological, cellular and molecular events underlying extreme regeneration. The comparison of the regeneration GRN to the one underlying embryogenesis will enable us to identify the similarities between these two developmental trajectories but importantly the regeneration specific stress response elements. In fact, we have identified over 120 genes that might be specifically involved in the regeneration process. The integration of my team at the Institute for Research on Cancer and Aging, provides a unique opportunity to carry out basic research on regeneration, whose results and concepts can be transferred directly to relevant regeneration models and other cancer and aging related topics such as autophagy, apoptosis, senescence, stem cells etc. This topic is of fundamental importance considering the aging European population that poses a great challenge to health and public finances. A major task to tackle in biomedical research is to enable an increased longevity while keeping the population healthy. Hence, the outcome of this FP7 MC CIG supported project will in the future participate in promoting an extended and healthy lifespan in humans.
Web resources:
The database we have developed for visualizing and mining all RNAseq data developed by the present project can be accessed at the following link: nvertx.kahikai.org

Final Report Summary - EARN (Understanding the re-deployment of gene regulatory networks underlying regeneration)

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