Exploring cell diversity and fidelity in crustacean limb regeneration using single-nucleus RNA sequencing
The ability to regenerate injured or amputated body parts is widespread in the animal kingdom. Highly regenerative species can restore organs with complex architectures and multiple differentiated cell types, such as external appendages, internal organs or, in some cases, the entire body from tiny tissue fragments. Single-cell sequencing technologies have opened a new way to answer old questions on the cellular composition and dynamics of regenerating tissues at single cell resolution. During my thesis, I implemented this method in the emerging model system Parhyale hawaiensis to investigate the catalogue of cell types found within the limbs and to assess the fidelity of regeneration. Pahryale limbs are small but complex tissues surrounded by an impermeable cuticle, which presents a challenge for extracting intact cells. After several trials and optimisation, I established a single-nucleus RNA sequencing (snRNA-seq) approach which diminishes the bias in recovering heterogeneous cell populations compared with single-cell methods. Sequencing the nuclear transcriptome of intact limbs allowed me to identify distinct clusters of cells, based on their transcriptional profile, reflecting a diversity of putative cell types. The comparison of datasets from uninjured and post-regenerated limbs revealed that the diversity and proportions of the cell types are faithfully restored during regeneration. Additionally, these datasets revealed a distinct cluster with a strong signature related to the moulting cycle. This work establishes the basis for exploring the cell trajectories during regeneration using snRNA-seq, characterising their dynamics and identifying the progenitor cells.
Thesis defense of Mrs. ALMAZAN Alba, under the supervision of Mr. AVEROF Michail