Embracing the diversity of model systems to deconstruct the basis of regeneration and tissue repair.

The eighth EMBO conference in the series 'The Molecular and Cellular Basis of Regeneration and Tissue Repair' took place in Barcelona (Spain) in September 2022. A total of 173 researchers from across the globe shared their latest advances in deciphering the molecular and cellular basis of wound healing, tissue repair and regeneration, as well as their implications for future clinical applications. The conference showcased an ever-expanding diversity of model organisms used to identify mechanisms that promote regeneration. Over 25 species were discussed, ranging from invertebrates to humans. Here, we provide an overview of the exciting topics presented at the conference, highlighting novel discoveries in regeneration and perspectives for regenerative medicine.

The impact of Drew Noden's work on our understanding of craniofacial musculoskeletal integration.

The classical anatomist Drew Noden spearheaded craniofacial research, laying the foundation for our modern molecular understanding of development, evolution, and disorders of the craniofacial skeleton. His work revealed the origin of cephalic musculature and the role of cranial neural crest (CNC) in early formation and patterning of the head musculoskeletal structures. Much of modern cranial tendon research advances a foundation of knowledge that Noden built using classical quail-chick transplantation experiments. This elegant avian chimeric system involves grafting of donor quail cells into host chick embryos to identify the cell types they can form and their interactions with the surrounding tissues. In this review, we will give a brief background of vertebrate head formation and the impact of CNC on the patterning, development, and evolution of the head musculoskeletal attachments. Using the zebrafish as a model system, we will discuss examples of modifications of craniofacial structures in evolution with a special focus on the role of tendon and ligaments. Lastly, we will discuss pathologies in craniofacial tendons and the importance of understanding the molecular and cellular dynamics during craniofacial tendon development in human disease.

Blastemal progenitors modulate immune signaling during early limb regeneration.

Blastema formation, a hallmark of limb regeneration, requires proliferation and migration of progenitors to the amputation plane. Although blastema formation has been well described, the transcriptional programs that drive blastemal progenitors remain unknown. We transcriptionally profiled dividing and non-dividing cells in regenerating stump tissues, as well as the wound epidermis, during early axolotl limb regeneration. Our analysis revealed unique transcriptional signatures of early dividing cells and, unexpectedly, repression of several core developmental signaling pathways in early regenerating stump tissues. We further identify an immunomodulatory role for blastemal progenitors through interleukin 8 (IL-8), a highly expressed cytokine in subpopulations of early blastemal progenitors. Ectopic il-8 expression in non-regenerating limbs induced myeloid cell recruitment, while IL-8 knockdown resulted in defective myeloid cell retention during late wound healing, delaying regeneration. Furthermore, the il-8 receptor cxcr-1/2 was expressed in myeloid cells, and inhibition of CXCR-1/2 signaling during early stages of limb regeneration prevented regeneration. Altogether, our findings suggest that blastemal progenitors are active early mediators of immune support, and identify CXCR-1/2 signaling as an important immunomodulatory pathway during the initiation of regeneration.

Bringing tendon biology to heel: Leveraging mechanisms of tendon development, healing, and regeneration to advance therapeutic strategies.

Tendons are specialized matrix-rich connective tissues that transmit forces from muscle to bone and are essential for movement. As tissues that frequently transfer large mechanical loads, tendons are commonly injured in patients of all ages. Following injury, mammalian tendons heal poorly through a slow process that forms disorganized fibrotic scar tissue with inferior biomechanical function. Current treatments are limited and patients can be left with a weaker tendon that is likely to rerupture and an increased chance of developing degenerative conditions. More effective, alternative treatments are needed. However, our current understanding of tendon biology remains limited. Here, we emphasize why expanding our knowledge of tendon development, healing, and regeneration is imperative for advancing tendon regenerative medicine. We provide a comprehensive review of the current mechanisms governing tendon development and healing and further highlight recent work in regenerative tendon models including the neonatal mouse and zebrafish. Importantly, we discuss how present and future discoveries can be applied to both augment current treatments and design novel strategies to treat tendon injuries.

Pseudotyped retroviruses for infecting axolotl in vivo and in vitro.

Axolotls are poised to become the premiere model system for studying vertebrate appendage regeneration. However, very few molecular tools exist for studying crucial cell lineage relationships over regeneration or for robust and sustained misexpression of genetic elements to test their function. Furthermore, targeting specific cell types will be necessary to understand how regeneration of the diverse tissues within the limb is accomplished. We report that pseudotyped, replication-incompetent retroviruses can be used in axolotls to permanently express markers or genetic elements for functional study. These viruses, when modified by changing their coat protein, can infect axolotl cells only when they have been experimentally manipulated to express the receptor for that coat protein, thus allowing for the possibility of targeting specific cell types. Using viral vectors, we have found that progenitor populations for many different cell types within the blastema are present at all stages of limb regeneration, although their relative proportions change with time.

Endogenous Tenocyte Activation Underlies the Regenerative Capacity of Adult Zebrafish Tendon.

Tendons are essential, frequently injured connective tissues that transmit forces from muscle to bone. Their unique highly ordered, matrix-rich structure is critical for proper function. While adult mammalian tendons heal after acute injuries, endogenous tendon cells, or tenocytes, fail to respond appropriately, resulting in the formation of disorganized fibrovascular scar tissue with impaired function and increased propensity for re-injury. Here, we show that unlike mammals, adult zebrafish tenocytes activate upon injury and fully regenerate the tendon. Using a full tear injury model in the adult zebrafish craniofacial tendon, we defined the hallmark stages and cellular basis of tendon regeneration through multiphoton imaging, lineage tracing, and transmission electron microscopy approaches. Remarkably, we observe that the zebrafish tendon can regenerate and restore normal collagen matrix ultrastructure by 6 months post-injury (mpi). We show that tendon regeneration progresses in three main phases: inflammation within 24 hours post-injury (hpi), cellular proliferation and formation of a cellular bridge between the severed tendon ends at 3-5 days post-injury (dpi), and re-differentiation and matrix remodeling beginning from 5 dpi to 6 mpi. Importantly, we demonstrate that pre-existing tenocytes are the main cellular source of regeneration. Collectively, our work debuts the zebrafish tendon as one of the only reported adult tendon regenerative models and positions it as an invaluable comparative system to identify regenerative mechanisms that may inspire new tendon injury treatments in the clinic.

Endogenous tenocyte activation underlies the regenerative capacity of the adult zebrafish tendon.

Tendons are essential, frequently injured connective tissues that transmit forces from muscle to bone. Their unique highly ordered, matrix-rich structure is critical for proper function. While adult mammalian tendons heal after acute injuries, endogenous tendon cells, or tenocytes, fail to respond appropriately, resulting in the formation of disorganized fibrovascular scar tissue with impaired function and increased propensity for re-injury. Here, we show that, unlike mammals, adult zebrafish tenocytes activate upon injury and fully regenerate the tendon. Using a full tear injury model in the adult zebrafish craniofacial tendon, we defined the hallmark stages and cellular basis of tendon regeneration through multiphoton imaging, lineage tracing, and transmission electron microscopy approaches. Remarkably, we observe that zebrafish tendons regenerate and restore normal collagen matrix ultrastructure by 6 months post-injury (mpi). Tendon regeneration progresses in three main phases: inflammation within 24 h post-injury (hpi), cellular proliferation and formation of a cellular bridge between the severed tendon ends at 3-5 days post-injury (dpi), and re-differentiation and matrix remodeling beginning from 5 dpi to 6 mpi. Importantly, we demonstrate that pre-existing tenocytes are the main cellular source of regeneration, proliferating and migrating upon injury to ultimately bridge the tendon ends. Finally, we show that TGF-ß signaling is required for tenocyte recruitment and bridge formation. Collectively, our work debuts and aptly positions the adult zebrafish tendon as an invaluable comparative system to elucidate regenerative mechanisms that may inspire new therapeutic strategies.

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.

Midkine is a dual regulator of wound epidermis development and inflammation during the initiation of limb regeneration.

Formation of a specialized wound epidermis is required to initiate salamander limb regeneration. Yet little is known about the roles of the early wound epidermis during the initiation of regeneration and the mechanisms governing its development into the apical epithelial cap (AEC), a signaling structure necessary for outgrowth and patterning of the regenerate. Here, we elucidate the functions of the early wound epidermis, and further reveal midkine (mk) as a dual regulator of both AEC development and inflammation during the initiation of axolotl limb regeneration. Through loss- and gain-of-function experiments, we demonstrate that mk acts as both a critical survival signal to control the expansion and function of the early wound epidermis and an anti-inflammatory cytokine to resolve early injury-induced inflammation. Altogether, these findings unveil one of the first identified regulators of AEC development and provide fundamental insights into early wound epidermis function, development, and the initiation of limb regeneration.

Eya2 promotes cell cycle progression by regulating DNA damage response during vertebrate limb regeneration.

How salamanders accomplish progenitor cell proliferation while faithfully maintaining genomic integrity and regenerative potential remains elusive. Here we found an innate DNA damage response mechanism that is evident during blastema proliferation (early- to late-bud) and studied its role during tissue regeneration by ablating the function of one of its components, Eyes absent 2. In eya2 mutant axolotls, we found that DNA damage signaling through the H2AX histone variant was deregulated, especially within the proliferating progenitors during limb regeneration. Ultimately, cell cycle progression was impaired at the G1/S and G2/M transitions and regeneration rate was reduced. Similar data were acquired using acute pharmacological inhibition of the Eya2 phosphatase activity and the DNA damage checkpoint kinases Chk1 and Chk2 in wild-type axolotls. Together, our data indicate that highly-regenerative animals employ a robust DNA damage response pathway which involves regulation of H2AX phosphorylation via Eya2 to facilitate proper cell cycle progression upon injury.

A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors.

Mammals have extremely limited regenerative capabilities; however, axolotls are profoundly regenerative and can replace entire limbs. The mechanisms underlying limb regeneration remain poorly understood, partly because the enormous and incompletely sequenced genomes of axolotls have hindered the study of genes facilitating regeneration. We assembled and annotated a de novo transcriptome using RNA-sequencing profiles for a broad spectrum of tissues that is estimated to have near-complete sequence information for 88% of axolotl genes. We devised expression analyses that identified the axolotl orthologs of cirbp and kazald1 as highly expressed and enriched in blastemas. Using morpholino anti-sense oligonucleotides, we find evidence that cirbp plays a cytoprotective role during limb regeneration whereas manipulation of kazald1 expression disrupts regeneration. Our transcriptome and annotation resources greatly complement previous transcriptomic studies and will be a valuable resource for future research in regenerative biology.