SUMMARY Zebrafish heart regeneration occurs through the activation of cardiomyocyte proliferation in areas of trauma. Here, we show that within three hours of ventricular injury, the entire endocardium undergoes morphological changes and induces expression of the retinoic acid (RA)-synthesizing enzyme raldh2. By one day post-trauma, raldh2 expression becomes localized to endocardial cells at the injury site, an area that is supplemented with raldh2-expressing epicardial cells as cardiogenesis begins. Induced transgenic inhibition of RA receptors or expression of an RA-degrading enzyme blocked regenerative cardiomyocyte proliferation. Injured hearts of the ancient fish Polypterus senegalus also induced and maintained robust endocardial and epicardial raldh2 expression coincident with cardiomyocyte proliferation, while poorly regenerative infarcted murine hearts did not. Our findings reveal that the endocardium is a dynamic, injury-responsive source of RA in zebrafish, and indicate key roles for endocardial and epicardial cells in targeting RA synthesis to damaged heart tissue and promoting cardiomyocyte proliferation.

SUMMARYAdult teleosts rebuild amputated fins through a proliferation-dependent process called epimorphic regeneration, in which a blastema of cycling progenitor cells replaces the lost fin tissue. The genetic networks that control formation of blastema cells from formerly quiescent stump tissue and subsequent blastema function are still poorly understood. Here, we investigated the cellular and molecular consequences of genetically interfering with retinoic acid (RA) signaling for the formation of the zebrafish blastema. We show that RA signaling is upregulated within the first few hours after fin amputation in the stump mesenchyme, where it controls Fgf, Wnt/-catenin and Igf signaling. Genetic inhibition of the RA pathway at this stage blocks blastema formation by inhibiting cell cycle entry of stump cells and impairs the formation of the basal epidermal layer, a signaling center in the wound epidermis. In the established blastema, RA signaling remains active to ensure the survival of the highly proliferative blastemal population by controlling expression of the anti-apoptotic factor bcl2. In addition, RA signaling maintains blastema proliferation through the activation of growth-stimulatory signals mediated by Fgf and Wnt/-catenin signaling, as well as by reducing signaling through the growth-inhibitory non-canonical Wnt pathway. The endogenous roles of RA in adult vertebrate appendage regeneration are uncovered here for the first time. They provide a mechanistic framework to understand previous observations in salamanders that link endogenous sources of RA to the regeneration process itself and support the hypothesis that the RA signaling pathway is an essential component of vertebrate tissue regeneration.

Zebrafish restore amputated fins by forming tissue-specific blastema cells that coordinately regenerate the lost structures. Fin amputation triggers the synthesis of several diffusible signaling factors that are required for regeneration, raising the question of how cell lineagespecific programs are protected from regenerative crosstalk between neighboring fin tissues. During fin regeneration, osteoblasts revert from a non-cycling, mature state to a cycling, preosteoblastic state to establish a pool of progenitors within the blastema. After several rounds of proliferation, preosteoblasts redifferentiate to produce new bone. Blastema formation and proliferation are driven by the continued synthesis of retinoic acid (RA). Here, we find that osteoblast dedifferentiation and redifferentiation are inhibited by RA signaling, and we uncover how the bone regenerative program is achieved against a background of massive RA synthesis. Stump osteoblasts manage to contribute to the blastema by upregulating expression of the RA-degrading enzyme cyp26b1. Redifferentiation is controlled by a presumptive gradient of RA, in which high RA levels towards the distal tip of the blastema suppress redifferentiation. We show that this might be achieved through a mechanism involving repression of Bmp signaling and promotion of Wnt/β-catenin signaling. In turn, cyp26b1 + fibroblast-derived blastema cells in the more proximal regenerate serve as a sink to reduce RA levels, thereby allowing differentiation of neighboring preosteoblasts. Our findings reveal a mechanism explaining how the osteoblast regenerative program is protected from adverse crosstalk with neighboring fibroblasts that advances our understanding of the regulation of bone repair by RA.

Background: One of Darwin's chosen examples for his idea of sexual selection through female choice was the "sword", a colourful extension of the caudal fin of male swordtails of the genus Xiphophorus. Platyfish, also members of the genus Xiphophorus, are thought to have arisen from within the swordtails, but have secondarily lost the ability to develop a sword. The sustained increase of testosterone during sexual maturation initiates sword development in male swordtails. Addition of testosterone also induces sword-like fin extensions in some platyfish species, suggesting that the genetic interactions required for sword development may be dormant, rather than lost, within platyfish. Despite considerable interest in the evolution of the sword from a behavioural or evolutionary point of view, little is known about the developmental changes that resulted in the gain and secondary loss of the sword. Up-regulation of msxC had been shown to characterize the development of both swords and the gonopodium, a modified anal fin that serves as an intromittent organ, and prompted investigations of the regulatory mechanisms that control msxC and sword growth.

The zebrafish caudal fin consists of repeated units of bony rays separated by soft interray tissue, an organization that must be faithfully re-established during fin regeneration. How and why regenerating rays respect ray-interray boundaries, thus extending only the existing bone, has remained unresolved. Here, we demonstrate that a retinoic acid (RA)-degrading niche is established by Cyp26a1 in the proximal basal epidermal layer that orchestrates ray-interray organization by spatially restricting osteoblasts. Disruption of this niche causes preosteoblasts to ignore ray-interray boundaries and to invade neighboring interrays where they form ectopic bone. Concomitantly, non-osteoblastic blastema cells and regenerating blood vessels spread into the interrays, resulting in overall disruption of ray-interray organization and irreversible inhibition of fin regeneration. The cyp26a1-expressing niche plays another important role during subsequent regenerative outgrowth, where it facilitates the Shha-promoted proliferation of osteoblasts. Finally, we show that the previously observed distal shift of ray bifurcations in regenerating fins upon RA treatment or amputation close to the bifurcation can be explained by inappropriate preosteoblast alignment and does not necessarily require putative changes in proximodistal information. Our findings uncover a mechanism regulating preosteoblast alignment and maintenance of ray-interray boundaries during fin regeneration.

No abstract

The ability to regenerate injured or lost body parts has been an age-old ambition of medical science. In contrast to humans, teleost fish and urodele amphibians can regrow almost any part of the body with seeming effortlessness. Retinoic acid is a molecule that has long been associated with these impressive regenerative capacities. The discovery 30 years ago that addition of retinoic acid to regenerating amphibian limbs causes "super-regeneration" initiated investigations into the presumptive roles of retinoic acid in regeneration of appendages and other organs. However, the evidence favoring or dismissing a role for endogenous retinoids in regeneration processes remained sparse and ambiguous. Now, the availability of genetic tools to manipulate and visualize the retinoic acid signaling pathway has opened up new routes to dissect its roles in regeneration. Here, we review the current understanding on endogenous functions of retinoic acid in regeneration and discuss key questions to be addressed in future research.

A truly sustainable circular economy is more than just recycling, making it a complex matter. We argue that reducing complexity will ease the path to circular economy. Complexity reduction potential lies in lower material diversity, increasing accessibility and intuitive use for consumers, as well as simplifying business operations.