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Single Cell Sequencing Analysis of Lizard Phagocytic Cell Populations and Their Role in Tail Regeneration.

ABSTRACT: Lizards are the closest relatives of mammals capable of tail regeneration, but the specific determinants of amniote regenerative capabilities are currently unknown. Macrophages are phagocytic immune cells that play a critical role in wound healing and tissue regeneration in a wide range of species. We hypothesize that macrophages regulate the process of lizard tail regeneration, and that comparisons with mammalian cell populations will yield insight into the role phagocytes play in determining an organism's regenerative potential. Single cell RNA sequencing (scRNAseq) was used to profile lizard immune cells and compare with mouse counterparts to contrast cell types between the two species. Treatment with clodronate liposomes effectively inhibited lizard tail stump tissue ablation and subsequent regeneration, and scRNAseq was used to profile changes in lizard immune cell populations resulting from tail amputation as well as identifying specific cell types affected by clodronate treatment. ScRNAseq analysis of lizard bone marrow, peripheral blood, and tissue-resident phagocyte cell populations was used to trace marker progression during macrophage differentiation and activation. These results indicated that lizard macrophages are recruited to tail amputation injuries faster than mouse populations and express high levels of matrix metalloproteinases (MMPs). In turn, treatment with MMP inhibitors inhibited lizard tail regeneration. These results provide single cell sequencing data sets for evaluating and comparing lizard and mammalian immune cell populations, and identifying macrophage populations that are critical regulators of lizard tail regrowth.

SUBMITTER: Londono R

PROVIDER: S-EPMC7182306 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Introduction:Human cartilage is an avascular tissue with limited capacity for repair. By contrast, certain lizards are capable of musculoskeletal tissue regeneration following tail loss throughout all stages of their lives. This extraordinary ability is the result of a complex process in which a blastema forms and gives rise to the tissues of the regenerate. Blastemal cells have been shown to originate either from dedifferentiated tissues or from existing progenitor cells in various species, but their origin has not been determined in lizards. As reptiles, lizards are the closest relatives to mammals with enhanced regenerative potential, and the origin of blastemal cells has important implications for the regenerative process. Hence, the aim of this study is to determine the cellular origin of regenerated cartilage and muscle tissues in reptiles using the mourning gecko lizard as the regenerative model. Methods:To trace the fate and differentiation potential of cartilage during tail regeneration, cartilage cells pre-labeled with the fluorescent tracer Dil were injected into lizard tails, and the contribution of cartilage cells to regenerated tail tissues was assessed by histologic examination at 7, 14, and 21?days post-tail amputation. The contribution of muscle cells to regenerated tail tissues was evaluated using muscle creatine kinase promoter-driven Cre recombinase in conjunction with the Cre-responsive green-to-red fluorescence shift construct CreStoplight. 21 days after amputation, tail tissues were analyzed by histology for red fluorescent protein (RFP)-positive cells. Results:At 7?days post-amputation, Dil-labeled cartilage cells localized to the subapical space contributing to the blastema. At 14 and 21?days post-amputation, Dil-labeled cells remained in the subapical space and colocalized with Collagen type II (Col2) staining in the cartilage tube and myosin heavy chain (MHC) staining in regenerated muscle. Lineage tracing of myocytes showed colocalization of RFP with Col2 and MHC in differentiated tissues at 21?days post-amputation. Conclusion:This study demonstrates that differentiated cartilage cells contribute to both regenerated muscle and cartilage tissues following tail loss, and in turn, differentiated muscle cells contribute to both tissue types as well. These findings suggest that dedifferentiation and/or transdifferentiation are at least partially responsible for the regenerative outcome in the mourning gecko.

Project description:Protemics of anolis carolinensis tails undergoing regeneration. As amniote vertebrates, lizards are the most closely related organisms to humans capable of appendage regeneration. Lizards can autotomize, or release their tails as a means of predator evasion, and subsequently regenerate a functional replacement. Green anoles (Anolis carolinensis) can regenerate their tails through a process that involves differential expression of hundreds of genes, which has previously been analyzed by transcriptomic and microRNA analysis. To investigate protein expression in regenerating tissue, we performed whole proteomic analysis of regenerating tail tip and base. This is the first proteomic data set available for the green anole lizard. We identified 976 proteins only in the regenerating tail base, 796 only in the tail tip, and 874 in both tip and base. For 90% of these proteins in these tissues, we were able to assign a clear orthology to gene models in either the Ensembl or NCBI databases. For 20 proteins in the tail base (2.5%), 7 proteins in the tail tip (0.9%), and 7 proteins in both regions (0.8%), the gene model in Ensembl and NCBI matched an uncharacterized protein, confirming that these predictions are present in the proteome. Ontology and pathways analysis of proteins expressed in the regenerating tail base identified categories including actin filament-based process, ncRNA metabolism, regulation of phosphatase activity, small GTPase mediated signal transduction, and cellular component organization or biogenesis. Analysis of proteins expressed in the tail tip identified categories including regulation of organelle organization, regulation of protein localization, ubiquitin-dependent protein catabolism, small GTPase mediated signal transduction, morphogenesis of epithelium, and regulation of biological quality. These proteomic findings confirm pathways and gene families activated in tail regeneration in the green anole as well as identify uncharacterized proteins whose role in regrowth remains to be revealed.

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Single Cell Sequencing Analysis of Lizard Phagocytic Cell Populations and Their Role in Tail Regeneration.

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