Mapping the cellular basis of limb regeneration: new approaches in an emerging model organism – ReMODEL

Task 1 involves the development of genetic resources, specifically of cell-specific markers and tools. Two complementary approaches will be followed, a gene-trapping screen to generate a large number of transgenic lines with tissue and cell-type specific expression during regeneration (Task 1a, months 1-24) and a targeted approach to generate a Pax3/7 reporter (Task 1b, months 1-9). These will serve as a resource for generating diverse markers and drivers for lineage analysis, cell ablation and cell sorting, to be used in Tasks 2-4.
Task 2 involves the development of genetic tools for lineage analysis (Task 2a, months 1-24) and for inducible cell ablation (Task 2b, months 1-24) that will allow us to rigorously identify the progenitor cells that give rise to regenerated tissues, and to map their degree of lineage commitment (Objective 2). We will use conditional cell ablation, with specific drivers from Task 1, to assess the lineage and role of different cell populations during limb regeneration.
Task 3 will establish live imaging of regeneration at single cell resolution (Task 3a, months 1-12), allowing us to visualize the behaviour of progenitor cells, and to record their proliferation and cell genealogy during regeneration (Task 3b, months 13-24) (Objective 3).
Task 4 involves the development of cell sorting and mRNA tagging techniques to isolate mRNA from specific cell types (Task 4a, months 1-12). That will allow us to study the transcriptional responses of progenitor cells during regeneration (Task 4b, months 13-24), leading to the identification of key molecular players underpinning regulatory events in regeneration (Objective 4). This task opens the way for future gene-centred approaches.
Task 5 Dissemination.

The project has been successfully launched. The Averof team in now fully established at the IGFL, researchers with appropriate experience have been recruited on the project, and the scientific tasks of the project have been initiated as planned.
Preliminary experimental results are very encouraging:
- the gene-trap screen (Task 1) has started to yield the first traps
- cell transplantation experiments suggest that we have identified a progenitor for muscle regeneration (Task 2)
- BAC recombineering and CRISPR-mediated gene targeting are being tested as alternative approaches for generating cell-type-specific markers and drivers (all Tasks)

The aim of ReMODEL is to launch a new model organism, providing powerful genetic tools and approaches for in-depth studies of complex organ regeneration.
In particular, we anticipate the following direct and long-term impacts:
1) ReMODEL will allow us to address 3 key issues in the field of regenerative biology: (a) the identification of progenitor cells and determination of their degree of plasticity / commitment to specific cell lineages, (b) the rigorous study of the proliferative behaviour and cell lineage of these progenitors during regeneration, and (c) the identification of molecular players involved in regeneration, using an inbiassed approach.
2) These key questions are also being pursued in other regeneration models, such a zebrafish, planarians, hydra and salamanders. Aproaching these questions in a crustacean will provide the material for future comparative studies on the mechanisms of limb regeneration, which may provide information on conserved mechanisms and insights for regenerative medicine.
3) The gene-traps generated will provide a unique resource for the wider research community. We will ensure that this resource is exploited by the Parhyale community by inviting members of other laboratories to participate in the screen and select lines of interest for different aspects of Parhyale development.
4) Our lab will continue to exploit the genetic tools and approaches developed in ReMODEL, to gain a deep understanding of the mechanisms underpinning regeneration. Following on from this project, we anticipate undertaking projects to identify signalling events that trigger and co-ordinate regeneration, to identify the role of key molecular players and to analyse their regulatory interactions.
5) By training a new generation of young researchers and hosting visiting fellows who will move on to independent careers in research, we hope to create an active scientific community focused on Parhyale as an experimental model.

On track.

In spite of great medical and biological interest in regeneration, the molecular and cellular mechanisms underlying this process are poorly understood. This is in part due to the lack of suitable model organisms and genetic tools. ReMODEL aims to establish the crustacean Parhyale hawaiensis as a powerful model organism where the cellular and molecular basis of limb regeneration can be studied in a rigorous and unbiased fashion. Parhyale is attractive because it combines extensive regenerative abilities with advanced genetic approaches.
ReMODEL is structured around four complementary Tasks:
In Task 1 we will conduct a large exon-trap screen to generate transgenic lines with tissue and cell-type specific expression during regeneration. These will serve as a resource for generating diverse markers and drivers for gene expression, recombination and cell ablation, using a transgene-replacement technique that we recently established in Parhyale.
In Task 2 we will use clonal analysis to rigorously identify the progenitor cells that give rise to regenerated tissues, and to map their degree of lineage commitment. We will use conditional cell ablation, driven by specific gene traps, to assess the lineage and functions of different cell populations during limb regeneration.
In Task 3 we will use live imaging at single cell resolution to visualize cell proliferation and cell genealogy during regeneration.
In Task 4 we will use cell sorting and RNA-seq to study the transcriptional responses of specific cell types during regeneration. This unbiased approach will lead to the identification of key molecular players underpinning regulatory events in regeneration, opening the way for future gene-centred studies.
These approaches provide a rigorous framework for exploring the cellular and molecular basis of limb regeneration.