Periodic Reporting for period 4 - reLIVE (Unraveling complex organ regeneration through live imaging and molecular profiling approaches)
Reporting period: 2021-07-01 to 2022-12-31
1) Discovering the so-called progenitor cells that contribute to re-making the regenerating leg. To identify these, we marked cells using fluorescent proteins and developed methods to track them under the microscope during the entire course of leg regeneration, which lasts approximately one week. This involved optimising microscopic methods and developing computer software that uses deep learning (a form of artificial intelligence) to facilitate the accurate tracking of cells over very long periods.
2) Discovering the cellular composition and microanatomy of Parhyale crustacean legs and testing whether regeneration produces faithful replicas of the original structures. We described at least 15 cell types with distinct molecular signatures and 8 different types of sensory organs on the surface of these legs, which are all regenerated with striking fidelity.
3) Describing how legs respond to injury, activating new sets of genes in different phases of regeneration. We explored parallels in the way different animals regenerate their legs, including distinct phases of wound healing and leg formation, and an active involvement of nerves.
4) Comparing how legs develop in the embryo with how they regenerate in adult stages. We were interested to find out whether these two processes, which generate identical structures, unfold in similar ways, or whether each is driven by distinct mechanisms. We performed these comparisons at two levels: we compared the sequence of activation of genes (transcriptional responses) and the behaviour of cells during the course of leg development (in embryos) and regeneration (in adults). Unexpectedly, we found that embryonic development and regeneration generate the same structure using different routes.
Genetic approaches: A significant part of our efforts was dedicated to developing new genetic tools, such as fluorescent markers for labelling different cell types and tools for stably labelling cell lineages. We tried several approaches which were ultimately unsuccessful (including CRE reporters, gene traps and CRISPR-mediated knock ins), and developed alternative strategies to overcome these failures (e.g. live imaging and post-staining using antibodies).
Transcriptional profiling approaches: To characterise the molecular changes that take place during regeneration, we applied transcriptional profiling techniques (RNAseq) on 120 individual leg samples covering the first 6 days of regeneration. To compare the transcriptional dynamics of develpment and regeneration, a similar dataset was obtained during the course of leg development in embryos. In parallel, we established single-cell transcriptional and chromatin profiling approaches, which served to characterise the diversity of cell types present in Parhyale legs, and to identify specific molecular markers (from snRNAseq) and putative regulatory elements (from snATACseq) for each cell type. The latter serve to develop fluorescent markers that will be used to track their regenerative progenitors by live imaging.
Nerve ablation: The nervous system has been implicated in the control of regeneration in several animals and we want to compare its role across different species. We used a pulsed UV laser to ablate the nerves of Parhyale legs and we studied the effects of this leg denervation on regeneration by live imaging. We found that nerves play distinct roles in early stages of wound healing and in later stages associated with cell division and the formation of the new leg.
1) We established a robust method for live imaging of regeneration at cellular resolution, which encompasses the entire time course of leg regeneration (up to 10 days post amputation).
2) We developed software for semi-automated cell tracking based on interactive deep learning (sparse manual annotation, followed by deep learning, and proofreading); the software is publicly available via github (https://elephant-track.github.io).
3) We established single-cell and bulk transcriptomics approaches in the context of Parhyale leg development and regeneration. The datasets that we generated, including an atlas of cell types and time series of single-limb transcriptional profiles covering 5-6 days of leg development and leg regeneration, are valuable resources for future studies in the field.
4) We demonstrated that regenerated legs are faithful replicas of the original legs they replace. They are indistinguishable in terms of cellular composition, the molecular profiles of constituent cell types, overall morphology, and the detailed microanatomy and function of the external sensory organs.
5) In spite of the high fidelity of regeneration, we showed that the transcriptional dynamics of leg development and regeneration differ, and cannot be aligned in a coherent manner. This suggests that development and regeneration follow different routes to generate the same structure. This is an unexpected and provocative idea which we will continue to explore at the level of gene regulation.
6) We demonstrated that nerves play an essential role in at least two distinct phases of regeneration, in early wound healing and at the onset of cell proliferation and morphogenesis.