Growth factors are potentially important modulators of epimorphic regeneration. This study examined effects of intraperitoneal administration of selected growth factors on limb regeneration of adult newts, Notophthalmus viridescens. These agents stimulated regeneration, producing overlapping but nonidentical effects. Fibroblast growth factor-2 (FGF-2) and insulin-like growth factor I (IGF-I) stimulated bud emergence (8.3 ± 0.6 and 8.3 ± 0.7 days, respectively, vs 11.4 ± 1.1 days for controls). Progression to the cone stage was enhanced by both FGF-2 and transforming growth factor beta 5 ( TGF-~ 5) ; 14.6 ± 0.5 and 15.4 ± 0.4 days with FGF-2 and TGF-~5 , respectively, vs 16.5 ± 0.5 days in controls. Insulin accelerated attainment of the palette stage, 17.0 ± 0.7 days vs 19.0 ± 0.4 days for controls. No treatments affected attaining the digital stage; means between 22.4 and 23.4 days. Histological analysis revealed changes consistent with gross observations. In addition, regenerates from newts treated with FGF-2, TGF-~5. and insulin displayed signs of greater (or earlier) histogenesis than did control animals. These results are consistent with the notion that FGF-2, TGF-~5. and possibly lGF-I stimulate proliferation of blastema cells and that insulin, FGF-2, and TGF-~5 promote differentiation and histogenesis during forelimb regeneration. In conclusion, these results demonstrate that several polypeptide growth factors positively affect the progress of forelimb regeneration, that different growth factors influence the same or similar events of epimorphic regeneration, and that diverse growth factors have nonidentical effects on regeneration

Fahmy, Gehan H.

Sicard, Raymond E.

English

University of Minnesota, Morris (UMM): Digital Well

Journal of the Minnesota Academy of Science Volume 63 Number 2 Article 6 1998 Acceleration of Amphibian Forelimb Regeneration By Polypeptide Growth Factors Gehan H. Fahmy University of Minnesota Raymond E. Sicard University of Minnesota Follow this and additional works at: https://digitalcommons.morris.umn.edu/jmas  Part of the Cell Biology Commons Recommended Citation Fahmy, G. H., & Sicard, R. E. (1998). Acceleration of Amphibian Forelimb Regeneration By Polypeptide Growth Factors. Journal of the Minnesota Academy of Science, Vol. 63 No.2, 58-60. Retrieved from https://digitalcommons.morris.umn.edu/jmas/vol63/iss2/6 This Article is brought to you for free and open access by the Journals at University of Minnesota Morris Digital Well. It has been accepted for inclusion in Journal of the Minnesota Academy of Science by an authorized editor of University of Minnesota Morris Digital Well. For more information, please contact skulann@morris.umn.edu. ACCELERATION OF AMPHffiiAN FORELIMB REGENERATION BY POLYPEPTIDE GROWTII FACTORS GEHAN H. FAHMYl AND RAYMOND E. SICARD2 Regeneration and Organogenesis Section Center for Wound Healing and Reparative Medicine University of Minnesota ABSTRACT Growth factors are potentially important modulators of epimorphic regeneration. This study examined effects of intraperitoneal administration of selected growth factors on limb regeneration of adult newts, Notophthalmus viridescens . These agents stimulated regeneration, producing overlapping but nonidentical effects. Fibroblast growth factor-2 (FGF-2) and insulin-like growth factor I (IGF-I) stimulated bud emergence (8.3 ± 0.6 and 8.3 ± 0.7 days, respectively, vs 11.4 ± 1.1 days for controls). Progression to the cone stage was enhanced by both FGF-2 and transforming growth factor beta 5 (TGF-~5) ; 14.6 ± 0.5 and 15.4 ± 0.4 days with FGF-2 and TGF-~5 , respectively, vs 16.5 ± 0.5 days in controls. Insulin accelerated attainment of the palette stage, 17.0 ± 0.7 days vs 19.0 ± 0.4 days for controls. No treatments affected attaining the digital stage; means between 22.4 and 23.4 days. Histological analysis revealed changes consistent with gross observations. In addition, regenerates from newts treated with FGF-2, TGF-~5. and insulin displayed signs of greater (or earlier) histogenesis than did control animals. These results are consistent with the notion that FGF-2, TGF-~5. and possibly lGF-I stimulate proliferation of blastema cells and that insulin, FGF-2, and TGF-~5 promote differentiation and histogenesis during forelimb regeneration. In co11dusion, these results demonstrate that several polypeptide growth factors positively affect the progress of forelimb regeneration, that different growth factors influence the same or similar events of epimorphic regeneration, and that diverse growth factors have nonidentical effects on regeneration. Regeneration of amphibian limbs has long been known to be under neural and endocrine influence. 1 Several investigators have suggested that polypeptide growth factors and hormones mediate these influences. For example, fibroblast growth factors (FGFs) have been shown to stimulate proliferation of blastema cells both in vivo2 and in vitro.3.4 More recently, Boilly and associates have demonstrated the presence of FGF-1 in and release by amphibian regeneration blastemas.5·6 In addition, Poulin and Chiu 7 have documented changes in the distribution of FGF receptors in regenerating newt limbs that suggest a potentially important role for FGFs. A dependence on insulin for progressive regeneration of appendages has also been demonstrated in vitro8·9 and in vivo.8, IO Other growth factors have not yet been implicated as modulators of amphibian limb regeneration. Nevertheless, certain growth factors are likely candidates in view of their roles in skeletal muscle regeneration 11 and wound healing. 12 These factors include the insulin-like growth factors (IGFs) and the transforming growth factor betas (TGF-~s). Previous studies with FGFs focused on modulating proliferation in bud or cone stage blastemas.2-4 The observation of widespread and changing distribution of FGF receptors in regenerating limbs7 suggests that FGFs might be playing a role at other times as well. Furthermore, there is limited or no information pertaining to the role of other polypeptide growth factors on regeneration of amphibian limbs. Accordingly, this study was undertaken to ascertain effects of FGF-2 (basic FGF), IGF-I , insulin , and TFG-~5 on overall progress of forelimb regeneration in adult Notophthalmus viridescens and to suggest events of regeneration that might be modulated or regulated by these factors. MATERIALS AND METHODS Adult newts, Notophthalmus viridescens (Charles Sullivan, Nashville, TN), were kept in aged deionized water (20 ± 2° C) and fed twice weekly. Regeneration was initiated by bilateral amputations through the distal third of the humerus. Protruding bone was trimmed back to the level of soft tissues. Amputations were performed following anesthetizing in MS-222 (0. 1% aqueous methane tricaine sulfonate, pH 7.0; Sigma Chemical Corp., St. Louis, MO). Experimental treatments consisted of intraperitoneal injections (I 00 J.!L) of growth factors every three days from days 6 through 21 postamputation. Treated animals received either recombinant human IGF-I (2000 or 200 ng/injection), bovine brain-derived FGF-2 (340 or 34 ng/injection), recombinant Xenopus TFG-~5 (25 ng/injection) [all from R & D Systems, Minneapolis, MN] or recombinant human insulin (500 ng humulin/injection; Ely Lilly, Indianapolis, IN). Control animals received no treatment. Gross morphology of regenerating limbs was monitored, with the aid of a dissecting microscope, every other day between days 7 and 25 postamputation. Definition of stages of regeneration was based on descriptions by Iten and Bryant. 13 Representative regenerates from animals in each group were removed for histological study. These tissues were fixed in I 0% buffered formalin and decalcified for 7 days in Jenkin's decalcifying mixture. Ten micron paraffin sections were stained with hematoxylin and eosin. Statistical analysis of data consisted of unpaired t-tests with differences considered significant at p < 0.05 . Procedures in this study conform to NIH guidelines for animal welfare and were 1 Present address: Department of Zoology, Faculty of Science, Alexandria University, Moharrem Bey 21511, Alexandria, EGYPT 2 Address for correspondence: Raymond E. Sicard, Ph.D., Department of Surgery, Box 120 UMHC, University of Minnesota, Minneapolis, MN 55455. Tel: (6 12) 625-1 169; e-mail: Raymond.E.Sicard-1 @tc.umn.edu 58 journal of the Minnesota Academy of Science Polypeptide Growth Factors Accelerate Limb Regeneration reviewed by the University Animal Care and Use Committee (University of Minnesota) . RESULTS The growth factors used positively affected regeneration as expressed by accelerated progression from one stage of regeneration or another (Table l ). While some similarities occurred, effects of the growth factors were not identical. For example, IGF-I and FGF-2 appeared to accelerate bud emergence, causing 78 - 81% and 71 - 88%, respectively, of treated animals to display early buds by II days postamputation while only 56% of controls had emerging buds. In addition, 47% of TGF-~5-treated newts had palette stage regenerates by 17 days postamputation whereas only 22% of controls had reached this stage. Moreover, by 19 days postamputation, 89% of limbs of insulin-treated animals had reached the palette stage (and 28% already had progressed to digital stage) as compared to only 72% of control animals displaying palettes and none with imminent digit formation. Within I - 2 days of initiating treatment, limbs of animals receiving IGF-1 or FGF-2 appeared to have enhanced dedifferentiation. This was not seen in limbs of newts receiving TGF-~5 or insulin. By 15 days postamputation, limbs from animals injected with FGF-2 or TGF-~5 displayed larger blastemas than controls. More importantly, these limbs seemed to show more differentiation and histogenesis. Limbs from IGF-1-and insulin-treated newts did not display these features. However, limbs from insulin-treated cases displayed similar evidence of histogenesis by 17 days postamputation. In all these latter cases, the degree of redifferentiation and histogenesis exceeded that of control animals. No treatment-related alterations on later histogenesis or patterning were seen during the course of this study. DISCUSSION The present study suggests that FGF-2, IGF-1, insulin, and TGF-~5 can stimulate regeneration of amphibian limbs. However, the influences of these factors on the progress of regeneration were not identical. FGF-2 and IGF-1 seemed to cause blastemas to emerge about two days earlier than controls (Table 1). This appeared to be accompanied by early dedifferentiation. In their study examining the potential of growth factors to substitute for the wound epithelium in sustaining and promoting growth of dedifferentiated cells, Chew and Cameron 14 reported stimulation of mitosis in axolotl limbs implanted with FGF-impregnated beads. Our results are consistent with this effect of FGF. Moreover, our data suggest that IGF might also be able to promote proliferation of dedifferentiated blastema cells. On the other hand, apparent acceleration of blastema emergence also could result from promotion of dedifferentiation itself. However, the design of our study only enables us to raise the questi~n at this time. FGF-2 and TGF-~5 appeared to accelerate progression to the cone stage (Table I), stimulating formation of larger and more developed blastemas than in controls. While it cannot be proved from our data, it is reasonable to suggest that FGF-2 and TGF-~5 stimulated proliferation to produce these effects. Others have shown that FGFs can stimulate proliferation in blastemas. 2-6 However, our results suggest that TGF-~5 also might promote proliferation of blastema cells. Further studies are required to confirm this. Moroever, limbs of newts treated with FGF-2 and TGF-~5 showed signs of precocious histogenesis . While FGFs are recognized mitogens, they are not generally associated with promoting differentiation. Nevertheless, Chew and Cameron 14 reported precocious chondrogenesis following implantation of FGF-impregnated beads into axolotl limbs. In contrast, TGF-~s have been shown to influence differentiation and histogenesis. For example, TGF-~ 1 can promote osteogenesis by mammalian cells in culture; however, it appears to antagonize myogenesis of mammalian myoblasts in vitro. 15 A more detailed understanding of differences between regenerating amphibian limbs and these other models is required in order to reconcile these apparent inconsistencies . While it is premature to suggest specific influences of FGF-2 and TGF-~5 as direct inducers of skeletogenesis and myogenesis in limb regeneration, such roles would not be uncharacteristic for these factors . Insulin's influence on regeneration in this study was subtle and late (Table I) . Our results were surprising in light of previous Table 1 : Effects of growth factors on progressive forelimb regeneration TREATMENT DOSE REGENERATION STAGE (mean days ± 1 sem) (ng/injection) BUD CONTROL 11.4±1.1 IGF-I 2000 8.3 ± 0.7* 200 8.7 ± 0.7* FGF-2 340 9.7 ± 0.7 34 8.3 ± 0 .6* TGF-~5 25 11.8 ± 0 .8 Insulin 500 9.6 ± 0.9 N = 8 to 10 newts per treatment group (and measurement). * p < 0.05 vs controls. Special Issue: Vol. 63, No. 2, 1998 CONE PALETTE 16.5 ± 0.5 19.0 ± 0.4 16.1 ± 0.6 18.5 ± 0 .6 16.6 ± 0.3 19.4 ± 0 .5 16.7 ± 0 .3 19.2 ± 0.4 14.6 ± 0.5* 19.0 ± 0.2 15.4 ± 0.4* 18.6 ± 0 .7 15.0 ± 0.7 17.0 ± 0 .7* DIGITAL 23.0 ± 0.4 23.1 ± 0.9 23 .1±1.0 22.4 ± 0.4 23.4 ± 0.5 23.1 ± 0.9 22.4 ± 0.8 59 Polypeptide Growth Factors Accelerate Limb Regeneration work8·9•16 suggesting an important role for insulin in promoting blastema( growth and development. Our data suggest a less important role for insulin in promoting blastema( growth than suggested by these other studies. Differences in the source of insulin used, the dose and method of administration, and other physiological factors could account for this. Additional studies are clearly warranted to reconcile these differences and better determine insulin's role in forelimb regeneration. In summary, FGF-2, IGF-I, insulin, and TGF-~5 appeared to promote the progress of forelimb regeneration. Each factor was distinctive in when it seemed to act, alternatively accelerating bud emergence or progression to the cone or palette stages of regeneration. In addition, several events of regeneration (e.g., dedifferentiation, blastema! growth, and histogenesis) appeared to be influenced by one or more of these growth factors. These results underscore that regulation of forelimb regeneration is complex and multifactorial. ACKNOWLEDGEMENTS Support for this work was provided by the Egyptian Ministry of Higher Education (GHF) and the Minnesota Medical Foundation (RES). REFERENCES I. Sicard RE (ed). 1985. Regulation of Vertebrate Limb Regeneration New York:Oxford University Press. 2. Mescher AL, Gospodarowicz D. 1979. Mitotic effect of a growth factor derived from myelin in denervated regenerates of newt forelimbs. J Exp Zoo/207:497-503. 3. Carlone RL, Ganagarajah M, Rathbone MP. 1981. Bovine pituitary fibroblast growth factor has neurotrophic activity for newt limb regenerates and skeletal muscle in vitro. Exp Cell Res 132: 15-21. 4. Albert P, Boilly B, Courty J, Barritault D. 1987. Stimulation in cell culture of mesenchymal cells of new limb blastemas by EDGF I or II (basic or acidic FGF). Cell Diff2! :63-68. 60 5. Boilly B. 1989. Production of growth factors by the blastema during limb regeneration of urodeles (Amphibia). In: V Kiortsis , S Koussoulakos, H Wallace (eds) Recent Trends in Regeneration Research, pp 81-96, New York:Plenum Press. 6. Zengari C, Boilly B, Hondermarck H, Boilly-Marer Y. 1997. Nerve-blastema interactions induce fibroblast growth factor-! release during limb regeneration in Pleurodeles walt/. Develop Growth Diff 39: 15-22. 7. Poulin M, Chiu I. 1995. Re-programming of expression of KGFR and bek variants of fibroblast growth factor receptor 2 during limb regeneration in newts (Notophthalmus viridescens). Dev Dynam 202:378-387. 8. Vethamany S. 1970. In vivo and in vitro studies of the influence of hormones on limb and tail regeneration in adult Diemictylus viridescens. PhD thesis, University of Toronto, Toronto, Canada. 9. Globus M, Vethamany-Globus S. 1985. In vitro studies of controlling factors in newt limb regeneration. In: RE Sicard (ed) Regulation of Vertebrate Limb Regeneration, pp 106-127, New York:Oxford University Press. 10. Cowan BJ, Foty RA, Liversage RA. 1995. Insulin receptors in Xenopus laevis liver and forelimb regenerates and the effects of local insulin deprivation on regeneration. J Exp Zoo/273: 130-141. II. Allen RE, Rankin LL. 1990. Regulation of satellite cells during skeletal muscle growth and development. Proc Soc Expt Bioi Med 194:8 1-86. 12. Clark RAF (ed). 1996. The Molecular and Cellular Biology of Wound Repair. second edition. New York: Plenum Press. 13. Iten L, Bryant SV. 1973. Forelimb regeneration from different levels of amputation in the newt, N viridescens: length, rate and stages. Raux's Arch Dev Bio/173:263-282. 14. Chew KE, Cameron JA. 1983. Increase in mitotic activity of regenerating axolotl limbs by growth factor-impregnated implants. J Exp Zoo/226:325-329. 15. Roberts AB, Sporn MB . 1991. The transforming growth factor-j3s. In: MB Sporn, AB Roberts (eds) Peptide Growth Factors and Their Receptors vol I, pp 419-472, New York:Springer. 16. Albert P, Boilly B. 1986. Evolution in vitro de cellules de blastemes de n5generation de membre d' Axolotl: influence de l'insuline et d'extraits nerveux sur Ia proliferation cellulaire. Bioi Cell 58:251-262. journal of the Minnesota Academy of Science 

Acceleration of Amphibian Forelimb Regeneration By Polypeptide Growth Factors

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Acceleration of Amphibian Forelimb Regeneration By Polypeptide Growth Factors

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