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Periodic Report Summary 1 - EARN (Understanding the re-deployment of gene regulatory networks underlying regeneration)

Summary description of the project objectives

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 preparation and close to submission (Amiel et al, in prep). 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 (Aim 1). The bioinformatics analysis is currently ongoing and will be performed at two levels: 1) by looking at differential gene expression during regeneration, that highlighted 12 gene clusters corresponding to potential synexpression groups 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.
We are also actively working on Aim 2 and Aim 3 and have some very interesting results. For the functional characterization of the GRN components (Aim 2), we have begun with a pharmacological approach in blocking Wnt, FGF/MAPK as well as hypoxia signaling. We further initiated a screen using 80 pharmacological inhibitors targeting more than 20 families of Kinases (Jnk, Src, RTKs etc). To confirm the specificity of the pharmacological drugs used that affect MAPK signaling, we performed antibody immunostaining for the activated form of ERK-MAPK. For the other pathways we have begun to develop transgenic animals for reporter (eg. TOPflash, HRE) and fusion protein (Bcat:mCherry) constructs using CrispR/Cas9 approaches. In parallel, we are currently developing new tools for gene specific perturbation experiments in the regenerating animals (KO, inducible dominant negative and conditional KO’s). In line with these approaches we have also begun the assembly of the constructs that will enable us to follow in vivo cell proliferation, tested the use of photoconvertible proteins (Kaede) to follow the implication of certain regions in the regeneration process (Amiel et al. 2015). The development of these transgenic lines is at various progression levels spanning the assembly of constructs to stable heterozygous F1s.

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). Using the photoconvertible Kaede protein for tissue fate experiments during regeneration highlighted that the major part of the reformed pharynx originates from the oral part of the mesenteries (Amiel et al. 2015), which appears different from embryogenesis.
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 currently ongoing and the first results indicate that genes such as hif1a, ets and tcf are among the earliest genes to be activated after amputation. The comparison of the molecular dynamics of given genes (RNAseq) and the morphological and cellular dynamics enables us to predict potential connections that will be further addressed by gene specific functional studies. Results of our in situ hybridization screen indicate the existence of at least three 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 analysis of our regeneration RNAseq data and previously published embryonic RNAseq datasets is ongoing but already reinforces the idea that only a small fraction of developmental genes are reactivated during regeneration.
Pharmacological approaches to perturb the function of the Hypoxia, FGF/MAPK, cWnt signaling pathways have so far shown that a precise balance of hypoxia/normoxia, functional FGF/FGFR, ERK/MAPK and cWnt signaling are crucial for regeneration in Nematostella. Precise analysis of the resulting phenotypes at the morphological and cellular levels has revealed that Hypoxia and cWnt are important for early morphological and cellular events. Interestingly, we observed that ERK/MAPK signaling is crucial for early steps of regeneration while FGF/FGFR signaling appears only important for later processes such as pharynx and tentacle reformation. We therefore initiated a pharmacological screen using 80 inhibitors targeting over 20 families of kinases. Among the kinases targeted by the drugs that block regeneration, we identified RTKs (Receptor Tyrosine Kinases) and JNK that could potentially be the activators of ERK/MAPK during early regeneration steps. Using the genes of the embryonic GRN module controlled by cWnt, we performed qPCR analysis of the same set of genes after inhibiting cWnt signaling during regeneration. Interestingly, we observed that only a subset of these genes were affected during regeneration suggesting that only a subnetwork is reactivated after amputation. Using our regeneration RNAseq data we are defining potential regeneration specific downstream targets of the studied pathways that will be analyze by qPCR and in situ hybridization in the near future. The development of gene specific knock-down (e.g. inducible dominant negative version (dn) of TCF), KO, or conditional KO tools are in the process of development (e.g. F0 for dnTCF) in tight interaction with Dr. Aissam Ikmi (Stowers Institute, Kansas, USA). For this, we have optimized existing CrispR/Cas9 protocols for efficient use in Nematostella and we currently have heterozygous F1 ßcatenin:mCherry embryos expressing the tagged protein in all tissues. Additional constructs are underway and described in the project objectives.

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. 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:

We do not have an ad hoc web resource for the project FP7 MC-CIG, however, the lab’s webpage at the IRCAN can be reached at the following link:

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Life Sciences
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