The salamander limb: a perfect model to understand imperfect integration during skeletal regeneration.

Limb regeneration in salamanders is achieved by a complex coordination of various biological processes and requires the proper integration of new tissue with old. Among the tissues found inside the limb, the skeleton is the most prominent component, which serves as a scaffold and provides support for locomotion in the animal. Throughout the years, researchers have studied the regeneration of the appendicular skeleton in salamanders both after limb amputation and as a result of fracture healing. The final outcome has been widely seen as a faithful re-establishment of the skeletal elements, characterised by a seamless integration into the mature tissue. The process of skeletal integration, however, is not well understood, and several works have recently provided evidence of commonly occurring flawed regenerates. In this Review, we take the reader on a journey through the course of bone formation and regeneration in salamanders, laying down a foundation for critically examining the mechanisms behind skeletal integration. Integration is a phenomenon that could be influenced at various steps of regeneration, and hence, we assess the current knowledge in the field and discuss how early events, such as tissue histolysis and patterning, influence the faithful regeneration of the appendicular skeleton.

Methods for Studying Appendicular Skeletal Biology in Axolotls.

The axolotl is a great model for studying cartilage, bone and joint regeneration, fracture healing, and evolution. Stainings such as Alcian Blue/Alizarin Red have become workhorses in skeletal analyses, but additional methods complement the detection of different skeletal matrices. Here we describe protocols for studying skeletal biology in axolotls, particularly Alcian Blue/Alizarin Red staining, microcomputed tomography (µCT) scan and live staining of calcified tissue. In addition, we describe a method for decalcification of skeletal elements to ease sectioning.

Osteoclast-mediated resorption primes the skeleton for successful integration during axolotl limb regeneration.

Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven, and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency, and treatment with zoledronic acid resulted in osteoclast function inhibition and failed tissue integration. Moreover, we identified the wound epithelium as a regulator of skeletal resorption, likely releasing signals involved in recruitment/differentiation of osteoclasts. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration.

In vivo assessment of mechanical properties during axolotl development and regeneration using confocal Brillouin microscopy.

In processes such as development and regeneration, where large cellular and tissue rearrangements occur, cell fate and behaviour are strongly influenced by tissue mechanics. While most well-established tools probing mechanical properties require an invasive sample preparation, confocal Brillouin microscopy captures mechanical parameters optically with high resolution in a contact-free and label-free fashion. In this work, we took advantage of this tool and the transparency of the highly regenerative axolotl to probe its mechanical properties in vivo for the first time. We mapped the Brillouin frequency shift with high resolution in developing limbs and regenerating digits, the most studied structures in the axolotl. We detected a gradual increase in the cartilage Brillouin frequency shift, suggesting decreasing tissue compressibility during both development and regeneration. Moreover, we were able to correlate such an increase with the regeneration stage, which was undetected with fluorescence microscopy imaging. The present work evidences the potential of Brillouin microscopy to unravel the mechanical changes occurring in vivo in axolotls, setting the basis to apply this technique in the growing field of epimorphic regeneration.

Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum.

BACKGROUND: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging. RESULTS: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation. CONCLUSIONS: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth.

Single-cell revolution unveils the mysteries of the regenerative mammalian digit tip.

The digit tip is an exciting model for studying regeneration in mammals, but the precise mechanisms and the populations of cells involved in the formation and remodeling of the blastema remain unknown. In an exciting new work, Storer et al. take advantage of single-cell RNAseq combined with Pdgfra+ â€‹lineage-tracing to open the way into the enigmatic world of mammalian tissue regeneration.

Expression of CTGF/CCN2 in response to LPA is stimulated by fibrotic extracellular matrix via the integrin/FAK axis.

Fibrosis is a common feature of several chronic diseases and is characterized by exacerbated accumulation of ECM. An understanding of the cellular and molecular mechanisms involved in the development of this condition is crucial for designing efficient treatments for those pathologies. Connective tissue growth factor (CTGF/CCN2) is a pleiotropic protein with strong profibrotic activity. In this report, we present experimental evidence showing that ECM stimulates the synthesis of CTGF in response to lysophosphatidic acid (LPA).The integrin/focal adhesion kinase (FAK) signaling pathway mediates this effect, since CTGF expression is abolished by the use of the Arg-Gly-Asp-Ser peptide and also by an inhibitor of FAK autophosphorylation at tyrosine 397. Cilengitide, a specific inhibitor of αv integrins, inhibits the expression of CTGF mediated by LPA or transforming growth factor ß1. We show that ECM obtained from decellularized myofibroblast cultures or derived from activated fibroblasts from muscles of the Duchenne muscular dystrophy mouse model ( mdx) induces the expression of CTGF. This effect is dependent on FAK phosphorylation in response to its activation by integrin. We also found that the fibrotic ECM inhibits skeletal muscle differentiation. This novel regulatory mechanism of CTGF expression could be acting as a positive profibrotic feedback between the ECM and CTGF, revealing a novel concept in the control of fibrosis under chronic damage.

SMAD3 and SP1/SP3 Transcription Factors Collaborate to Regulate Connective Tissue Growth Factor Gene Expression in Myoblasts in Response to Transforming Growth Factor ß.

Fibrotic disorders are characterized by an increase in extracellular matrix protein expression and deposition, Duchene Muscular Dystrophy being one of them. Among the factors that induce fibrosis are Transforming Growth Factor type ß (TGF-ß) and the matricellular protein Connective Tissue Growth Factor (CTGF/CCN2), the latter being a target of the TGF-ß/SMAD signaling pathway and is the responsible for the profibrotic effects of TGF-ß. Both CTGF and TGF are increased in tissues affected by fibrosis but little is known about the regulation of the expression of CTGF mediated by TGF-ß in muscle cells. By using luciferase reporter assays, site directed mutagenesis and specific inhibitors in C2C12 cells; we described a novel SMAD Binding Element (SBE) located in the 5' UTR region of the CTGF gene important for the TGF-ß-mediated expression of CTGF in myoblasts. In addition, our results suggest that additional transcription factor binding sites (TFBS) present in the 5' UTR of the CTGF gene are important for this expression and that SP1/SP3 factors are involved in TGF-ß-mediated CTGF expression.