Roles of retinoic acid in the regeneration of the zebrafish larval and adult caudal fin
Final Report Abstract
Using the zebrafish caudal fin as a model, we examined the roles of endogenous retinoic (RA) signaling during regeneration. We first developed a transgenic line, hsp70:cyp26a1, which efficiently inhibits RA signaling, and used it as a genetic tool to deplete amputated fins of RA at different stages of regeneration. We found that RA controls cell proliferation in the amputated stump and is required for the formation of the blastema. We investigated its function in advanced regenerates and found that RA signaling also controls proliferation in the mature blastema, by integrating growth-stimulatory signals from the Fgf- and Wnt/βcatenin signaling pathways and inhibitory signals from the non-canonical Wnt signaling pathway. In addition, RA is required to maintain the fast cycling blastemal population in the adult fish by protecting them from cell death. Several diffusible signals have been implicated in zebrafish fin regeneration. This raised the question of how cell-lineage specific programs are protected from adverse effects of regenerative crosstalk between neighboring fin tissues. Embryonic osteoblasts display overossification in the presence of increased RA. Yet, during fin regeneration, when osteoblasts switch from a mature, non-cycling state to a cycling, less differentiated state, RA concentrations are maximal to promote blastema formation. This raised the question how mature osteoblasts achieve dedifferentiation in stump tissue flooded by RA synthesis. We examined osteoblasts throughout all stages of fin regeneration and found that two key steps of the osteoblast regenerative program, osteoblast dedifferentiation and subsequent redifferentiation, are adversely affected by RA from neighboring tissues. We found that RA is essential for promoting the proliferation of dedifferentiated osteoblasts. Nevertheless, osteoblasts will dedifferentiate only when they autonomously protect themselves from RA by upregulating expression of the RA-degrading enzyme Cyp26b1. Redifferentiation is achieved when RA concentrations drop below a threshold in more proximal parts where fibroblasts act as a RA-sink. Finally, we discovered an important role for bone resorption in fin regeneration, leading to the proposal that removal of excess matrix by osteoclasts is required to generate the final hemiray shape. These findings provide a conceptual framework for understanding how the osteoblast regenerative program is achieved against a background of massive RA synthesis during regeneration. A major unresolved question in regeneration is how an amputated organ manages to rebuild an exact copy of the lost structures. Accordingly, the principles that compel regenerating fin rays to respect ray-interray boundaries are still unknown. We could show that a RA-free niche is established by Cyp26a1 in the proximal basal epidermal layer that orchestrates rayinterray organization by allowing preosteoblasts to align in the lateral parts of the nascent blastema. Disruption of the RA-free 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 RA-free niche plays another important role during subsequent regenerative outgrowth, where it facilitates the Shha-promoted proliferation of preosteoblasts. Finally, we show that the previously observed distal shift of ray bifurcations in regenerating fins upon RA treatment, or amputation close to the bifurcation, is caused by inappropriate preosteoblast alignment rather than by assumed changes in proximodistal information. These findings uncover the mechanism regulating preosteoblast alignment and maintenance of ray-interray boundaries during fin regeneration. Our results were covered by online media (Spiegel Online, ZEIT Online). http://www.spiegel.de/wissenschaft/natur/nachwachsende-organe-das-geheimnis-des-zebrafischs-a-804216.html http://www.zeit.de/wissen/umwelt/2011-12/unterschaetztes-tier-zebrafisch
Publications
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2012 Retinoic acid signaling controls the formation, proliferation and survival of the bastema during adult zebrafish fin regeneration. Development 139:107-116
Blum N, Begemann G
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2013 The roles of endogenous retinoid signaling in organ and appendage regeneration. Cell Mol Life Sci. 70(20):3907-27
Blum N, Begemann G
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2015. Osteoblast de- and redifferentiation is controlled by a dynamic response to retinoic acid during zebrafish fin regeneration. Development 142 (17):2894-903
Blum N, Begemann G
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2015. Retinoic acid signaling spatially restricts osteoblasts and controls ray-interray organization during zebrafish fin regeneration. Development 142 (17):2888-93
Blum N, Begemann G
