Project description:Treatment with midkine inhibitor iMDK inhibits limb regeneration. To elucidate the transcriptional mechanisms of iMDK treatment, we sequenced regenerating limbs treated with either DMSO or iMDK (bulk) in biological triplicate at 11 days post-amputation (dpa).
Project description:Regeneration of complex multi-tissue structures, such as limbs, requires the coordinated effort of multiple cell types. In axolotl limb regeneration, the wound epidermis and blastema have been extensively studied via histology, grafting, and bulk-tissue RNA-sequencing. However, studying the contributions of these tissues is hindered due to limited information regarding the molecular identity of the cell types in regenerating limbs. By performing unbiased single-cell RNA-sequencing on over 25,000 cells from axolotl limbs, we identify a plethora of cellular diversity within epidermal, mesenchymal, and hematopoietic lineages in homeostatic and regenerating limbs. We identify regeneration-induced genes, develop putative trajectories for blastema cell differentiation, and propose the molecular identity and origin of fibroblast-derived blastema progenitor cells residing in homeostatic limbs. This work will enable application of molecular techniques to assess the contribution of these populations to limb regeneration. It will also facilitate work aimed at identifying transcripts and cells critical for limb regeneration.
Project description:Salamanders are capable of regenerating amputated limbs by generating a mass of lineage-restricted cells called a blastema. Blastemas only generate structures distal to their origin unless treated with retinoic acid (RA), which results in proximodistal (PD) limb duplications. Little is known about the transcriptional network that regulates PD duplication. In this study, we identified expression patterns that explain PD duplication including upregulation of proximal homeobox gene expression and silencing of distal-associated genes whereas limb truncation was associated with disrupted skeletal differentiation. Overall, mechanisms were identified that regulate RAR’s multifaceted roles in the salamander limb including regulation of skeletal patterning during epimorphic regeneration, skeletal tissue differentiation during regeneration, and homeostatic regeneration of intact limbs.
Project description:Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus in which the BMP inhibitor Noggin can be over-expressed at any time during development. We have previously shown that activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles. In the current study, we have taken advantage of this transgenic line to directly compare gene expression in same stage regenerating vs. non-regenerating hind limb buds. Using Affymetrix gene chip analysis, we have identified genes whose expression levels are linked to regenerative success. These include the BMP inhibitor Gremlin and the stress protein Hsp60 (no blastema in zebrafish). Analysis of overrepresented Gene Ontology functional groupings suggests that successful regeneration in the Xenopus hind limb depends on induction of stress response pathways. Furthermore, as expected, genes involved in embryonic development and growth are also significantly over-represented in regenerating early hind limb buds. Keywords: Differential expression, regeneration
Project description:Lizards cannot naturally regenerate limbs but are the closest known relatives of mammals capable of epimorphic tail regrowth. However, the mechanisms regulating lizard blastema derivation and chondrogenesis remain unclear. We utilized single-cell RNA sequencing analyses of regenerating lizard tails throughout the course of regeneration to assess diversity and heterogeneity in regeneating tail cell populations.
Project description:Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether the process of regeneration mirrors development is an open question in most regenerative species. Here we take a transcriptomics approach to examine to what extent leg regeneration shows the same temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, global patterns of gene expression during leg regeneration show a high degree of variation, related to the physiology of individual animals. A major driver of this variation is the molting cycle. After dissecting the transcriptional signals of individual physiology from regeneration, we obtain temporal signals that mark distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although both processes use largely the same genes, the temporal patterns in which these gene sets are deployed are different and cannot be systematically aligned.