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J. Biol. Chem., Vol. 282, Issue 48, 35202-35210, November 30, 2007

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Unraveling Tissue Regeneration Pathways Using Chemical Genetics^*

Lijoy K. Mathew^¶, Sumitra Sengupta^¶, Atsushi Kawakami^||, Eric A. Andreasen^¶, Christiane V. Löhr^**, Catherine A. Loynes, Stephen A. Renshaw, Randall T. Peterson, and Robert L. Tanguay^¶1

From the Department of Environmental and Molecular Toxicology, Environmental Health Sciences Center, ^¶Marine and Freshwater Biomedical Sciences Center, and ^**Department of Veterinary Medicine, Oregon State University, Corvallis, Oregon 97331, the ^||Department of Biological Information, Tokyo Institute of Technology, Yokohama 226-8501, Japan, the Medical Research Council Centre for Developmental and Biomedical Genetics, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom, and the Developmental Biology Laboratory, Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.

Received for publication, August 9, 2007 , and in revised form, September 7, 2007.

^* This work was supported in part by NIEHS Grants ES10820, ES00210, and ES03850 from the National Institutes of Health, an Oregon Medical Research Foundation grant, a predoctoral fellowship from the American Heart Association (to L. K. M.), and National Science Foundation Grant 0641409. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence and reprint requests should be addressed: Dept. of Environmental and Molecular Toxicology, Oregon State University, 1007 ALS, Corvallis, OR 97331. Tel.: 541-737-6514; Fax: 541-737-7966; E-mail: robert.tanguay{at}oregonstate.edu.

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