Bone Regenerates via Dedifferentiation of Osteoblasts in the Zebrafish Fin

Knopf, Franziska; Hammond, Chrissy L.; Chekuru, Avinash; Kurth, Thomas; Hans, Stefan; Weber, Christopher; Mahatma, Gina; Fisher, Shannon; Brand, Michael; Schulte‐Merker, Stefan; Weidinger, Gilbert

doi:10.1016/j.devcel.2011.04.014

Cited by 344 publications

(486 citation statements)

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“…Alizarin red staining in tiny larva is a fine and complicated procedure, and it only reflects bone mineralization. In tg(sp7:egfp), osteoblasts are specifically marked by GFP, which make osteoblasts visible [24][25][26] . Osteoblast differentiation and bone formation in tg(sp7:egfp) can be directly monitored.…”

Section: Discussionmentioning

confidence: 99%

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Salvianolic acid B stimulates osteogenesis in dexamethasone-treated zebrafish larvae

et al. 2016

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Aim: Our previous studies show that salvianolic acid B (Sal B) promotes osteoblast differentiation and matrix mineralization. In this study, we evaluated the protective effects of Sal B on the osteogenesis in dexamethasone (Dex)-treated larval zebrafish, and elucidated the underlying mechanisms. Methods: At 3 d post fertilization, wild-type AB zebrafish larvae or bone transgenic tg (sp7:egfp) zebrafish larvae were exposed to Sal B, Dex, or a mixture of Dex+Sal B for 6 d. Bone mineralization in AB strain larval zebrafish was assessed with alizarin red staining, and osteoblast differentiation in tg (sp7:egfp) larval zebrafish was examined with fluorescence scanning. The expression of osteoblastspecific genes in the larvae was detected using qRT-PCR assay. The levels of oxidative stress markers (ROS and MDA) in the larvae were also measured. Results: Exposure to Dex (5-20 μmol/L) dose-dependently decreased the bone mineralization area and integral optical density (IOD) in wild-type AB zebrafish larvae and the osteoblast fluorescence area and IOD in tg (sp7:egfp) zebrafish larvae. Exposure to Dex (10 μmol/L) significantly reduced the expression of osteoblast-specific genes, including runx2a, osteocalcin (OC), alkaline phosphatase (ALP) and osterix (sp7), and increased the accumulation of ROS and MDA in the larvae. Co-exposure to Sal B (0.2-2 μmol/L) dosedependently increased the bone mineralization area and IOD in AB zebafish larvae and osteoblast fluorescence in tg (sp7:egfp) zebrafish larvae. Co-exposure to Sal B (2 μmol/L) significantly attenuated deleterious alterations in bony tissue and oxidative stress in both Dex-treated AB zebafish larvae and tg (sp7:egfp) zebrafish larvae. Conclusion: Sal B stimulates bone formation and rescues GC-caused inhibition on osteogenesis in larval zebrafish by counteracting oxidative stress and increasing the expression of osteoblast-specific genes. Thus, Sal B may have protective effects on bone loss trigged by GC.

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Section: Discussionmentioning

confidence: 99%

“…Zebrafish of the AB wild line and bone transgenic osterix:nlsGFP line, named tg(sp7:egfp) [26] , were used. The embryos of the AB line and tg(sp7:egfp) line zebrafish were, respectively, collected from the natural mating and spawning of wild-type AB line zebrafish and tg(sp7:egfp) transgenic zebrafish.…”

Section: Zebrafish Husbandrymentioning

confidence: 99%

Salvianolic acid B stimulates osteogenesis in dexamethasone-treated zebrafish larvae

et al. 2016

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“…Upon amputation, muscle, cartilage, and connective tissue cells underneath the injury site lose their differentiated characteristics and reenter the cell cycle to give rise to the blastema (5)(6)(7)(8). This mechanism has also been observed during zebrafish heart and fin regeneration (9,10). In contrast, reversals of the differentiated state are rarely observed in mammalian tissues, which led to the suggestion that inability to undergo dedifferentiation could contribute to the failure of regeneration in mammals (11).…”

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confidence: 97%

Regulation of p53 is critical for vertebrate limb regeneration

Yun

Gates

Brockes

2013

Proc. Natl. Acad. Sci. U.S.A.

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Extensive regeneration of the vertebrate body plan is found in salamander and fish species. In these organisms, regeneration takes place through reprogramming of differentiated cells, proliferation, and subsequent redifferentiation of adult tissues. Such plasticity is rarely found in adult mammalian tissues, and this has been proposed as the basis of their inability to regenerate complex structures. Despite their importance, the mechanisms underlying the regulation of the differentiated state during regeneration remain unclear. Here, we analyzed the role of the tumor-suppressor p53 during salamander limb regeneration. The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its upregulation is necessary for the redifferentiation phase. Importantly, we show that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation. In addition, we have uncovered a potential mechanism for the regulation of p53 during limb regeneration, based on its competitive inhibition by ΔNp73. Our results suggest that the regulation of p53 activity is a pivotal mechanism that controls the plasticity of the differentiated state during regeneration. myogenesis | chondrogenesis | p73 | carcinogenesis U nlike mammals, which exhibit limited regenerative abilities, the urodele amphibians-or salamanders-are capable of regenerating an extraordinary range of body structures, including ocular tissues, tail, sections of the heart, parts of the nervous system, and entire limbs (1). In salamanders, such as the newt and axolotl, limb regeneration depends on the formation of a blastema, a mound of progenitor cells of restricted potential that arises after amputation (2-4). Following a period of proliferation, blastema cells redifferentiate and restore the structures of the limb.Extensive evidence indicates that limb regeneration depends on reprogramming of cells in mature limb tissues. Upon amputation, muscle, cartilage, and connective tissue cells underneath the injury site lose their differentiated characteristics and reenter the cell cycle to give rise to the blastema (5-8). This mechanism has also been observed during zebrafish heart and fin regeneration (9, 10). In contrast, reversals of the differentiated state are rarely observed in mammalian tissues, which led to the suggestion that inability to undergo dedifferentiation could contribute to the failure of regeneration in mammals (11). Despite their significance, the mechanisms underlying regulation of the differentiated state during vertebrate regeneration remain poorly understood.Recently, the tumor suppressor p53, whose best-characterized functions are in the maintenance of genome stability (12), has been implicated in the suppression of artificial cell reprogramming to pluripotency (13-17) and the promotion of differentiation pathways in mammals (18). In addition, it has been observed that inhibiting p...

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“…Understanding how salamanders regenerate limbs should provide critical insight into efforts to stimulate these processes in vertebrates that do not regenerate limbs, yet the list of modern molecular genetic tools that can be applied to salamanders is currently very short. Other model systems with a much more sophisticated experimental toolkit, such as zebrafish, have provided valuable clues to the molecular underpinnings of vertebrate appendage regeneration (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). However, fin regeneration in teleost fish (such as zebrafish) is not completely analogous to limb development or regeneration.…”

mentioning

confidence: 99%

Inducible genetic system for the axolotl

Whited

Lehoczky

Tabin

2012

Proc. Natl. Acad. Sci. U.S.A.

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Transgenesis promises a powerful means for assessing gene function during amphibian limb regeneration. This approach is complicated, however, by the need for embryonic appendage development to proceed unimpeded despite the genetic alterations one wishes to test later in the context of regeneration. Achieving conditional gene regulation in this amphibian has not proved to be as straightforward as in many other systems. In this report we describe a unique method for obtaining temporal control over exogenous gene expression in the axolotl. Based on technology derived from the Escherichia coli Lac operon, uninduced transgenes are kept in a repressed state by the binding of constitutively expressed Lac repressor protein (LacI) to operator sequences within the expression construct. Addition of a lactose analog, IPTG, to the swimming water of the axolotl is sufficient for the sugar to be taken up by cells, where it binds the LacI protein, thereby inducing expression of the repressed gene. We use this system to demonstrate an in vivo role for thrombospondin-4 in limb regeneration. This inducible system will allow for systematic analysis of phenotypes at defined developmental or regenerative time points. The tight regulation and robustness of gene induction combined with the simplicity of this strategy will prove invaluable for studying many aspects of axolotl biology.

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Bone Regenerates via Dedifferentiation of Osteoblasts in the Zebrafish Fin

Cited by 344 publications

References 53 publications

Salvianolic acid B stimulates osteogenesis in dexamethasone-treated zebrafish larvae

Salvianolic acid B stimulates osteogenesis in dexamethasone-treated zebrafish larvae

Regulation of p53 is critical for vertebrate limb regeneration

Inducible genetic system for the axolotl

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