Osteoblast de- and redifferentiation is controlled by a dynamic response to retinoic acid during zebrafish fin regeneration

Abstract

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.

Transcriptomic, proteomic, and metabolomic landscape of positional memory in the caudal fin of zebrafish

“…For example, proximal rays have wider hemirays, accommodating more fibroblasts, than distal rays. Thus, the abundance of proximally enriched factors stimulating RA signaling (e.g., aldh1a2) could be directly due to the increased fibroblast population and bone matrix production that are prevalent in the proximal rays (31). Accordingly, cyp26a1 and cyp26b1 are distally enriched, indicative of their roles in osteoblast proliferation and positioning as observed during life-long growth at the distal end of the fin (31,82).…”

“…Thus, the abundance of proximally enriched factors stimulating RA signaling (e.g., aldh1a2) could be directly due to the increased fibroblast population and bone matrix production that are prevalent in the proximal rays (31). Accordingly, cyp26a1 and cyp26b1 are distally enriched, indicative of their roles in osteoblast proliferation and positioning as observed during life-long growth at the distal end of the fin (31,82). However, it is also possible that these patterns are retained from development, because the same expression patterns for aldh1a2 and cyp26b1 are seen in developing limb buds (83).…”

“…Fish treated with exogenous RA during fin regeneration suffer dramatic bone patterning defects, but not an overall change in fin size (29,30). However, these patterning defects may be due to RA-induced proximalization of regenerating tissue or to the role of RA signaling in the differentiation of preosteoblasts (31). In addition, it is unclear how RA signaling would set up a pattern of positional memory before injury because no gradient of endogenous RA signaling has been demonstrated in intact salamander limbs or zebrafish fins.…”

Retinoic acid signaling spatially restricts osteoblasts and controls ray-interray organization during zebrafish fin regeneration

“…1G) (Blum and Begemann, 2012). Although proximal fibroblast-derived blastema cells express cyp26b1 (Blum and Begemann, 2015), it is unlikely that RA diffusion into adjacent epidermal cells is efficiently prevented. We thus suspected that cyp26a1-expressing cells provide niches of low RA levels that might facilitate expression of RA-sensitive genes.…”

“…Moreover, we did not detect TUNEL + osteoblasts in control or cyclopamine-treated fish (data not shown). Hence, even though RA signaling promotes osteoblast proliferation (Blum and Begemann, 2015), prolonged experimental RA exposure should cause downregulation of shha and consequently lead to a reduction of osteoblast proliferation. Indeed, osteoblast proliferation was reduced 12 h after RA injection at 3 dpa (Fig.…”