Unraveling Tissue Regeneration Pathways Using Chemical Genetics

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. 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. The promise of regenerative medicine is that therapies will be devised to promote the repair or replacement of damaged or diseased tissues and organs. This emerging field is approached from two distinct lines of attack. In recent years, stem cell-based models have been developed to generate a suite of differentiated cells for therapeutic applications. The use of high throughput chemical genetic screening to identify modulators of stem cell fate offers great promise (1Ding S. Schultz P.G. Nat. Biotechnol. 2004; 22: 833-840Crossref PubMed Scopus (282) Google Scholar). The alternative approach exploits the inherent regenerative capacity of non-mammalian models to define the molecular events that permit tissue regeneration (2Brockes J.P. Kumar A. Science. 2005; 310: 1919-1923Crossref PubMed Scopus (295) Google Scholar). There are several regenerative animal models, including salamanders, newts, zebrafish, hydra, and flatworms, that are established to evaluate tissue regeneration (3Fujisawa T. Dev. Dyn. 2003; 226: 182-189Crossref PubMed Scopus (46) Google Scholar, 4Bader D. Oberpriller J.O. J. Morphol. 1978; 155: 349-357Crossref PubMed Scopus (71) Google Scholar, 5Akimenko M.A. Mari-Beffa M. Becerra J. Geraudie J. Dev. Dyn. 2003; 226: 190-201Crossref PubMed Scopus (249) Google Scholar, 6Mescher A.L. Int. J. Dev. Biol. 1996; 40: 785-795PubMed Google Scholar). What is currently lacking is the availability of a vertebrate regeneration model that is amenable to rapid throughput assessments. Zebrafish have the remarkable capability to regenerate their fins, optic nerve, scales, heart, and spinal cord (7Poss K.D. Keating M.T. Nechiporuk A. Dev. Dyn. 2003; 226: 202-210Crossref PubMed Scopus (323) Google Scholar). Adult caudal fin regeneration is the best studied model for dissecting the molecular signaling that controls regenerative growth and angiogenesis (7Poss K.D. Keating M.T. Nechiporuk A. Dev. Dyn. 2003; 226: 202-210Crossref PubMed Scopus (323) Google Scholar, 8Bayliss P.E. Bellavance K.L. Whitehead G.G. Abrams J.M. Aegerter S. Robbins H.S. Cowan D.B. Keating M.T. O'Reilly T. Wood J.M. Roberts T.M. Chan J. Nat. Chem. Biol. 2006; 2: 265-273Crossref PubMed Scopus (114) Google Scholar). Comparative genomics indicate significant genetic conservation between mammals and lower vertebrates, which begs the question: what are the molecular differences that permit tissue regeneration in zebrafish and make mammalian tissues recalcitrant to regeneration? Answers to this question will provide a path for comparative studies in mammals. Zebrafish recover the lost caudal fin tissue after amputation through a process of epimorphic regeneration, and this occurs in a stepwise manner with the formation of an epithelial wound cap, followed by blastema formation and finally the regenerative outgrowth (Fig. 1) (5Akimenko M.A. Mari-Beffa M. Becerra J. Geraudie J. Dev. Dyn. 2003; 226: 190-201Crossref PubMed Scopus (249) Google Scholar, 7Poss K.D. Keating M.T. Nechiporuk A. Dev. Dyn. 2003; 226: 202-210Crossref PubMed Scopus (323) Google Scholar). This complex regenerative process is orchestrated by sequential interactions between biomolecules and cells in a spatiotemporal manner. Global gene expression analysis on heart and fin regeneration in adult zebrafish illustrates the involvement of multiple signaling pathways mediated through the differential expression of hundreds of genes during this remarkable process (9Andreasen E.A. Mathew L.K. Tanguay R.L. Toxicol. Sci. 2006; 92: 254-269Crossref PubMed Scopus (62) Google Scholar, 10Schebesta M. Lien C.L. Engel F.B. Keating M.T. Sci. World J. 2006; 6: 38-54Crossref Scopus (84) Google Scholar, 11Lien C.L. Schebesta M. Makino S. Weber G.J. Keating M.T. PLoS Biol. 2006; 4: e260Crossref PubMed Scopus (222) Google Scholar). The identification of the signaling molecules that control these interactions will offer avenues to rapidly advance the field of regenerative medicine. The characterization of key regulators such as fibroblast growth factor and Wnt as critical factors during regeneration emphasizes the likely involvement of multiple signaling pathways in fin regeneration (12Poss K.D. Shen J. Nechiporuk A. McMahon G. Thisse B. Thisse C. Keating M.T. Dev. Biol. 2000; 222: 347-358Crossref PubMed Scopus (264) Google Scholar, 13Stoick-Cooper C.L. Weidinger G. Riehle K.J. Hubbert C. Major M.B. Fausto N. Moon R.T. Development (Camb.). 2007; 134: 479-489Crossref PubMed Scopus (420) Google Scholar). This again underscores the importance of a comprehensive approach to identify the full repertoire of molecular players required for tissue regeneration. Although adult zebrafish regeneration models have proven useful, many of the molecular and genetic tools that are useful for embryonic and larval studies are not easily applied to adult stage animals (7Poss K.D. Keating M.T. Nechiporuk A. Dev. Dyn. 2003; 226: 202-210Crossref PubMed Scopus (323) Google Scholar). Recent results indicate that these technical barriers may be overcome by using an early life stage regeneration model. Specifically, 2-day-old zebrafish larvae completely regenerate their fin primordia within 3 days following amputation (14Kawakami A. Fukazawa T. Takeda H. Dev. Dyn. 2004; 231: 693-699Crossref PubMed Scopus (86) Google Scholar, 15Mathew L.K. Andreasen E.A. Tanguay R.L. Mol. Pharmacol. 2006; 69: 257-265Crossref PubMed Scopus (69) Google Scholar, 16Nakatani Y. Kawakami A. Kudo A. Dev. Growth Differ. 2007; 49: 145-154Crossref PubMed Scopus (85) Google Scholar). Because this life stage is inherently amenable to molecular and genetic manipulations such as transient and stable transgenics, genetic mutant screens, and chemical genetics, this model offers a powerful new way to identify novel regulators of tissue regeneration. In vivo high throughput small molecule screening has the potential to target any biological process (17Zon L.I. Peterson R.T. Nat. Rev. Drug Discov. 2005; 4: 35-44Crossref PubMed Scopus (1099) Google Scholar, 18Peterson R.T. Shaw S.Y. Peterson T.A. Milan D.J. Zhong T.P. Schreiber S.L. MacRae C.A. Fishman M.C. Nat. Biotechnol. 2004; 22: 595-599Crossref PubMed Scopus (328) Google Scholar, 19MacRae C.A. Peterson R.T. Chem. Biol. 2003; 10: 901-908Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 20Shafizadeh E. Peterson R.T. Lin S. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2004; 138: 245-249Crossref PubMed Scopus (13) Google Scholar, 21Peterson R.T. Fishman M.C. Methods Cell Biol. 2004; 76: 569-591Crossref PubMed Google Scholar, 22Love D.R. Pichler F.B. Dodd A. Copp B.R. Greenwood D.R. Curr. Opin. Biotechnol. 2004; 15: 564-571Crossref PubMed Scopus (95) Google Scholar, 23Peterson R.T. Link B.A. Dowling J.E. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12965-12969Crossref PubMed Scopus (436) Google Scholar); however, this approach has not been applied in a vertebrate regenerative system. To probe tissue regeneration, an inhibitory screen was developed. The underlying premise is that if a chemical inhibits or modulates an essential molecular target, then regeneration will be impacted. The identification of the chemical target will thus help to reveal underlying molecular pathways that permit tissue regeneration. Previous larval fin regeneration studies demonstrated the feasibility of this general inhibitory approach; inhibition of fibroblast growth factor receptor-1 with SU5402 or activation of the aryl hydrocarbon receptor disrupted tissue regeneration (14Kawakami A. Fukazawa T. Takeda H. Dev. Dyn. 2004; 231: 693-699Crossref PubMed Scopus (86) Google Scholar, 15Mathew L.K. Andreasen E.A. Tanguay R.L. Mol. Pharmacol. 2006; 69: 257-265Crossref PubMed Scopus (69) Google Scholar, 16Nakatani Y. Kawakami A. Kudo A. Dev. Growth Differ. 2007; 49: 145-154Crossref PubMed Scopus (85) Google Scholar). We report here for the first time an in vivo vertebrate regeneration assay that employs a rapid small molecule library screening to identify pathways essential for tissue regeneration. We also demonstrate that this regenerative platform is well suited to identify the molecular targets of small molecules and to define the molecular and cellular mechanism underlying the chemical response. Screening for Inhibitors of Larval Fin Regeneration—Fertilized eggs were obtained from AB strain zebrafish (University of Oregon, Eugene, OR) for all of the experiments. All embryos were raised in our laboratory according to standard procedures. Two-day-old embryos were dechorionated and anesthetized with 3-aminobenzoic acid ethyl ester (tricaine). The larvae were laid on an agar plate, and the caudal fin primordia were amputated with a surgical blade just posterior to the notochord. Two amputated larvae were arrayed per well in 96-well plates containing 50 μl of E3 embryo buffer (5 mm NaCl, 0.17 mm KCl, 0.33 mm CaCl2, 0.33 mm MgSO4). The small molecules (2,000 bioactives from MicroSource Discovery Systems, Gaylordsville, CT) were added individually to the test wells a of The amputated larvae were for 3 days days days glucocorticoid days analysis of the larvae were anesthetized and to regenerative the was as a glucocorticoid receptor all was Adult Zebrafish zebrafish were for to or with from to The for the here is the were and their caudal were The were to the and were to or the of the The were larvae were amputated days and to or and the fin tissue was specifically was from the fin tissue using the per of larval fins, were to make an from the amputated larvae to or were 3 was from of embryos using the according to the were as the in the between embryos and embryos after to The the significant of between as using analysis of and To was from the control or The were to The as by all of the was from 3 of per using and in a was using with the μl of was for in the of using a according to the and were to formation of were as gene and of is required for inhibition of regeneration by control and were amputated and to or for 3 The were on 3 the the the gene with the The and indicate the and for to the The in the loss of to a the analysis of in control and after followed by is The the for the and the lower to the after the loss of expression was as the control. the results of analysis for the gene between control and are The of is and the significant differences repression of was using zebrafish was identified Because were on alternative a was an that was between The of was The of Pu.1 is were to 3 mm in mm NaCl, mm KCl, mm mm mm as A. Nat. 2000; PubMed Scopus Google Scholar). standard control was as the control of the was the embryos the The of the was to screen The control and were to or and raised for 3 days The control and Pu.1 were amputated and to for 3 days In in was on the fin time as K.D. Shen J. Keating M.T. Dev. Dyn. 2000; PubMed Scopus Google Scholar, Development (Camb.). PubMed Google Scholar). The and probes were obtained from Kawakami (14Kawakami A. Fukazawa T. Takeda H. Dev. Dyn. 2004; 231: 693-699Crossref PubMed Scopus (86) Google Scholar). The probe was a from and A. in by amputated larvae that were to or were with for from or The larvae were with in a 96-well with one in well of with the animals were with and in The larvae were with and then in the larvae were using a and The larvae were then with in for and then several with The larvae were in for and then several in This was followed by in then in for and with several The larvae were then with in for and then with with the larvae were with a Eugene, OR) for The larvae were then for in and by The cells were with the using the by amputated larvae to or were and in The larvae were in and in were with and by and larvae were anesthetized and amputated 3 as the time amputated larvae were from the of were C.A. S. 2006; PubMed Scopus Google and in the the of were the amputated larvae 3 were to a of for to The were and were the amputation as in and H. Development of Zebrafish regeneration is by an orchestrated of multiple pathways and signaling a vertebrate regeneration assay was developed to identify small molecules that specifically tissue regeneration. To demonstrate the of this a small molecule library was to identify of regeneration. larvae were to 96-well and to 3 the larvae were to regenerative of 17 small molecules of the inhibited tissue regeneration. These inhibitory several such as of and regeneration are (Fig. and Although a of small molecules the test to by the of the these animals completely their fin tissue (Fig. This that a be from the regeneration response. of as of were of chemical and using a cluster of compounds was identified as glucocorticoids of the Although the of this was to demonstrate the feasibility of using small molecules to probe tissue regeneration, we also to demonstrate the ability to identify small molecule targets rapidly during early life We glucocorticoids for studies this was the cluster of and specifically inhibited regeneration a fin (Fig. This occurs the tissue to the amputation to and the amputation are that of their by the J. J. Biochem. PubMed Scopus Google Scholar, J. S. M. M. Y. A. Rev. PubMed Scopus Google Scholar, J. Biochem. Mol. Biol. PubMed Scopus Google Scholar). mammalian studies is that are several of of differential and alternative of which and are the J. Biochem. Mol. Biol. 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Because the adult fin regeneration model is was to whether chemical identified in the larval screen be for the adult fin regeneration the regeneration to glucocorticoid on fin regeneration was also in adult zebrafish. to the inhibited adult caudal fin regeneration, and small were the of amputation (Fig. The inhibitory that a underlying molecular target was by this of the of glucocorticoids for regeneration was in the is likely that the glucocorticoids are activation of the To test whether the target genes were including and S. Y. A. M. J. PubMed Scopus Google Scholar, D.B. C. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar, Mol. 2006; PubMed Scopus Google Scholar, D.B. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). To the expression of these was using from the larval fin tissue in the and of All genes were in to that the is by glucocorticoids in this regeneration model (Fig. analysis was in the embryo 3 after to and the expression of genes was in to the fin tissue These results that is the and that the inhibitory on regeneration be mediated through for to whether the was the molecular target of and to whether the was for the inhibition of regeneration, a was to block of the the well (Fig. knockdown of not early that the is not essential for early embryonic development not The standard control larvae and were amputated and for 3 days to or is that the control and to completely their fin that is not required for larval fin regeneration (Fig. to to knockdown of completely regenerative capacity in the of (Fig. These results indicate that regeneration by is mediated through To was with in control and The was in the control but was not in the knockdown of (Fig. The was also as a lower (Fig. and the of this is with the loss of the analysis significant in the between control and (Fig. These results indicate that the molecular target for glucocorticoids identified in the small molecule screen is the Collectively, tissue regeneration, and the was to to identify the events of that this response. of significant of chemical genetics is that the and of the chemical be the screen be to probe any of this complex We have that aryl hydrocarbon receptor block regeneration early and in adult and larval zebrafish L.K. Andreasen E.A. Tanguay R.L. Mol. Pharmacol. 2006; 69: 257-265Crossref PubMed Scopus (69) Google Scholar, J.M. Tanguay R.L. Toxicol. Sci. 2003; 76: PubMed Scopus Google Scholar). To define the regenerative stage that is to was added a of distinct time that were to following amputation for just to regenerate (Fig. and However, larvae to and then 3 were as completely their These indicate that glucocorticoids target early of regeneration, which wound healing/blastema in larvae (Fig. C and In adult zebrafish, for just was not sufficient to block regeneration not differences in the regenerative of The differential in adult zebrafish be to in the and in the complex adult regeneration system. and the of in larval zebrafish is within the regeneration, we in with a molecular that the wound which is the first of regeneration that occurs after amputation of the fin The expression of the wound M. Lien C.L. Engel F.B. Keating M.T. Sci. World J. 2006; 6: 38-54Crossref Scopus (84) Google Scholar, A. Fukazawa T. Takeda H. Dev. Dyn. 2004; 231: 693-699Crossref PubMed Scopus (86) Google was to whether the formation of an wound The larvae to a expression of in the wound of the the larvae to to (Fig. and This that the wound is not in the larvae and that the wound be the target of which the of the critical of early To whether blastema formation is by we the expression of two blastema (14Kawakami A. Fukazawa T. Takeda H. Dev. Dyn. 2004; 231: 693-699Crossref PubMed Scopus (86) Google and and A. in by in The expression of in the blastema just the of amputation was in the larvae but expression was in the larvae (Fig. C and the blastema which was in the blastema of the larvae was completely in larvae (Fig. and that activation by inhibits blastema which is essential for fin regeneration. we the formation of blastema by analysis of of or In the a blastema was to the amputation (Fig. In the cells were and by to the amputation in the formation of blastema (Fig. and In addition, many of the epithelial cells in larvae and the was from cell these results indicate that activation signaling molecules critical for wound and blastema formation. Cell by the of activation on early regeneration stage cellular studies were and of and formation in larval fin many cells were just the of amputation in the was significant in the of cells in the larvae (Fig. There was also a significant in cellular in the larvae with (Fig. and not to we also a cluster of cells the posterior and of the that is likely to to the development of the caudal fin (14Kawakami A. Fukazawa T. Takeda H. Dev. Dyn. 2004; 231: 693-699Crossref PubMed Scopus (86) Google Scholar). of the chemical was in the of cells the of the that the growth of the caudal fin is not by the inhibitory is to the fin these results indicate that activation results in the inhibition of cell multiple regenerative and for the multiple of glucocorticoids, the best is the on the inflammatory J. Biochem. Mol. Biol. PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar). is that amputation of the caudal fin in zebrafish inflammation S. Chem. 2003; PubMed Scopus Google Scholar, C.A. S. 2006; PubMed Scopus Google and from model that and molecules, such as growth factors and that are for wound and tissue We that the action of glucocorticoids on regeneration might inhibition of or to the of To whether the of or to the amputation we the which the control of the C.A. S. 2006; PubMed Scopus Google Scholar). larvae were amputated and to or as There was a in the of the amputation of the larvae and with the that the glucocorticoid be mediated by a small in (Fig. and the of that to the amputation was by and was significant in the of between and larvae (Fig. C and a that the block in regeneration by glucocorticoids was mediated by a in the amputation or by the of regenerative by inflammatory To the importance of cells in larval fin regeneration, we antisense to target a factor required to permit cell development D. A. J. L.I. J.P. 2004; PubMed Scopus Google Scholar, G.J. M.A. L.I. J.E. Dev. Biol. PubMed Scopus Google Scholar, J. A. M. J.P. Dev. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Pu.1 completely their caudal fin from control that and are not required for larval fin regeneration (Fig. and This the of wound studies in the Pu.1 tissue repair in the of and Wood B. Biol. Sci. 2004; PubMed Scopus Google Scholar). The of Pu.1 was by the cells for and for and were in the control but were completely the amputation in the Pu.1 (Fig. These results for the first time that acute inflammation through and is not required for the of regeneration in zebrafish. the in the of the Pu.1 gene during wound between and zebrafish underscores the function of genes vertebrate be if therapies that tissue regeneration were available. the of in vivo chemical genetics to identify novel compounds and their molecular targets that function to modulate tissue regeneration. The genetic and molecular of this early life stage regeneration model such as transient and stable transgenics, genetic mutant screens, rapid antisense and the use of chemical genetics make this vertebrate regeneration model an The results be rapidly in regeneration By the of comparative the will be a molecular for the differences in regenerative capacity and will reveal pathways for therapeutic We and for technical with