Periodic Reporting for period 3 - reLIVE (Unraveling complex organ regeneration through live imaging and molecular profiling approaches) Reporting period: 2020-01-01 to 2021-06-30 Summary of the context and overall objectives of the project Many animals have the ability to regenerate parts of their body following injury or amputation. While there is great biological and medical interest in this process, many fundamental questions remain unanswered because complex organ regeneration is poorly represented among the organisms classically studied in research laboratories. Flies, nematodes and mice have limited regenerative abilities, in contrast to flatworms, crustaceans and fish which can regenerate complex organs throughout their lifetime.This project explores fundamental questions on regeneration in the crustacean Parhyale hawaiensis, an experimental model that combines extensive regenerative abilities, genetic tools and live imaging. The project addresses the following centuries-old questions on regeneration: 1) Which are the progenitor cells that underpin complex organ regeneration? Do epidermis, tendons, neurons, glia and muscle arise de novo from undifferentiated adult stem cells, or do they emerge from differentiated cell types? Are the progenitors committed to specific cell fates or multipotent? Which are their molecular responses and behaviors during the course of regeneration? 2) Do diverse animals regenerate in similar ways? Do the regenerative progenitor cells of crustaceans have common molecular and functional properties with those of vertebrates and flatworms? Are the molecular responses similar? Do they have a shared evolutionary history? 3) How does regeneration differ from embryonic development when organs are first formed? Are these processes operating on comparable temporal and spatial scales? How similar are the transcriptional responses and cell behaviors that underpin embryonic and regenerative morphogenesis of the limb? Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far To answer these questions we have taken advantage of the unique opportunities offered by Parhyale as an experimental system, namely the ability to genetically manipulate this species, to mark cells using fluorescent proteins, and to track their behavior during regeneration. We have also applied transcriptional profiling approaches to study the molecular responses that take place during regeneration. We track cells using fluorescence microscopy on live regenerating animals, covering the entire time course of regeneration from amputation to the appearance of fully regenerated limbs. This requires continuous live imaging over 5-10 days, which we can now perform reliably on a dedicated confocal microscope. To efficiently track cells in these long live recordings, we developed software for semi-automated cell tracking based on artificial intelligence complemented by manual curation. To identify different cell types and to track their progenitors, a significant part of our work has focused on generating fluorescent markers for specific cell types. Our initial approach based on CRISPR knock-in was unsuccessful, so we have turned to alternative approaches, including cis-regulatory sequence reporters and antibody staining. To characterize the molecular responses that take place during regeneration we applied transcriptional profiling techniques (RNAseq) on 120 individual limb samples covering the first 6 days of regeneration. In parallel, we are establishing single-cell profiling approaches, which will serve to characterize the molecular changes and fate transitions that occur within individual cell types. The nervous system has been implicated in the control of regeneration in several animals and we want to compare its role across different species. Using laser ablation and live imaging, we are starting to investigate the role of nerves in Parhyale limb regeneration, particularly on the onset of cell proliferation and morphogenesis. To compare leg regeneration with embryonic leg development, we are developing computational tools that allow us to quantify and compare the contributions of different cell behaviors (cell division, cell shape changes, cell rearrangements, etc.) to leg morphogenesis in embryos and adults. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The early phases of the project focused on establishing experimental approaches, tools and resources that are essential for achieving our objectives. The main achievements so far are: 1) Establishing a robust method for live imaging of regeneration at cellular resolution, which encompasses the entire time course of regeneration. 2) Establishing software for semi-automated cell tracking based on deep learning complemented by manual curation. 3) Generating transcriptional profiles of single limbs covering the first 6 days of regeneration at high resolution.By the end of the project we expect to have gained a comprehensive understanding of the cellular basis of leg regeneration in our experimental model. In particular, we expect to have identified the progenitors and cell lineages of major cell types, such as epidermis, muscles, neurons and glia, to have characterized the behavior of these cells and how they contribute to leg formation, and to have characterized their molecular responses. Comparing this detailed knowledge with what we find in developing embryonic limbs of the same species will reveal to what extent regeneration resembles (perhaps even recapitulates) development in the embryo. Comparing this knowledge with what is found in other regenerating animals will help us to understand whether common mechanisms underpin regeneration in evolutionarily distant species. vmp1630.jpg
