- Email: [email protected]
N-Acetyl-Glucosaminidase Activity during Limb Regeneration in the Adult Newt
Differentiation
Differentiation (1981) 19: 121-123
0 Springer-Verlag 1981
Preliminary Reports N-Acetyl-Glucosaminidase Activity during Limb Regeneration in the Adult Newt M. RIVERA, J. R. ORTIZ', and E. ORTIZ Department of Biology, University of Puerto Rico, Rio Piedras, Puerto Rico 00931
N-Acetyl-glucosaminidaseactivity was measured during the first 25 days of limb regeneration. It was found that the enzyme is present in the normal limb. Following amputation a significant drop was obtained at day 3. A significant increase in enzyme activity was found at day 5 followed by a second drop by day 10. For days 12- 15 a second peak of enzyme activity was detected, followed by a third drop; by day 25, normal levels of enzyme activity were detected. Histochemical localization of the enzyme in tissue samples showing enzyme activity as detected biochemically (days 5 and 17 of regeneration) gave negative results. However, enzyme activity was found in the incubation medium, indicating that the enzyme is released from the cells. The peaks of enzyme activity coincide with the stages of limb regeneration where a high degree of tissue demolition and cell lysis occurs. The latter are important events in the regeneration process, cell dedifferentiation, and blastema formation.
IntrodUctioO
Methods
Limb regeneration in the adult newt comprises three consecutive events: wound healing, dedifferentiation (formation and growth of the blastema), and redifferentiation or morphogenesis of the limb. The regulatory mechanisms during regenerative processes are poorly understood. It has been suggested that metabolic gradients of chemical substances arranged in the cell surfaces play an important role in regeneration [l-31. In other biological systems there is evidence that macromolecules such as mucopolysaccharides are involved in morphogenesis. Hyaluronic acid, chondroitin sulphate, and heparine sulphate are found to be related with the morphogenesis of mouse salivary glands [4-71. In the chick embryo, these mucopolysaccharidesseem to be related with neural crest migration and differentiation [8] and with limb morphogenesis 19, 101. Studies of mucopolysaccharide metabolism have revealed that during the dedifferentiation phase of limb regeneration, the chondrocytes are released from their extracellular matrix [ll-131. It has also been shown that lysosomal enzyme activity and removal of macromolecules from the cell surface or extracellular matrix do alter the differentiated state [14-161. Studies of enzymes capable of removing components from the cell surface and extracellular matrix, such as N-acetyl-glucosaminidase, could contribute to the understanding of the mechanisms involved in differentiation and morphogenesis. The purpose of this preliminary report is to present measurements of glucosaminidase activity during limb regeneration in the adult newt.
Adult newts Notophthalrnus viridescem, obtained from Bill Lee's Newt Farm,Oak Ridge, Tennessee, USA,were used. The stages of regeneration were classified according to Iten and Bryant [17]. The animals were maintained in %gal tanks at 20" C. After amputation they were placed in 10-gal tanks in groups of 13-30 individuals and were fed Tubifex three or four times a week. Animals were anesthetized with 0.04% Tricaine (ethyl M-aminobenzoate, Sigma Chemical Co, St. Louis, MO, USA). Amputation was performed at two-thirds distal to the humerus, under the dissecting microscope. After amputation, the animals were placed briefly in urodele Ringer, and when completely recovered, they were moved back to water. Enzyme activity was measured at 0, 1,3,5,7, 10, 12,15, 17,20, 22, and 25 days after amputation, using the method described by Idoyaga-Vargas and Yamada [14]. The tissue samples were cut with scalpel and placed in 2.4 ml of phosphate buffer (PBS 80%). At day 0 enzyme measurements were made in the hands and in the region that corresponds to the distal part of the humerus, using 13 hands and 13 pieces of humerus 3 mm long. For days 1-10. 40 tissue samples containing mostly stump tissue were used for each measurement. For days 12-25, tissue samples consisted mostly of mesenchymal cells. Thirty-two tissue samples were used for each measurement for days 12 and 15 and twenty-four for days 17-25. The tissue samples were homogenized in a hand homogenizer with approximately 100 strokes. Enzyme measurements were made in triplicate. The reaction mixture consisted of 0.1 ml of homogenate, 0.33 ml of citrate (0.064 N) and phosphate (0.21 N) pH 4.2, and 0.4 ml of p-nitrophenyl N-acetyl glucosaminide solution 12 mM (Sigma) and was incubated 2 h at 37" C. To stop the reaction, 1 ml of cold 4% trichloroacetic acid (TCA) was added together with 0.7 ml of a 5 : 1 mixture of glycine (0.1 N) and carbonate buffer (0.8 N) with 0.5 N NaOH at pH 10.5. Enzyme activity was determined by reading the pnitrophenol absorbance in a Zeiss spectrophotometer at 412 nm, using a standard curve of p-nitrophenol absorbance versus concentration.
1 To whom reprint requests should be addressed
0301-4681/81/0019/0121/$01.00
M. Rivera et al.: Glucosaminidase Activity during Limb Regeneration
122
The unit of enzyme activity was expressed as micromoles of liberated pnitrophenohng of DNNh. DNA determination was made in triplicate through the use of the diphenylamine technique [18]. For histochemical localization of the enzyme, the Pugh-Walker method [19] was used in tissue samples at days 5 and 17. To determine if glucosaminidase was released to the incubation medium, the tissue samples were placed intact in the reaction mixture and incubated 1 h. The reaction was stopped and absorbance of pnitrophenol was read as described above. For each day a total of eight or ten determinations of enzyme activity were made. The mean and standard error were obtained and Student’st-test was used to determine statistical significance between some means, at the 99% confidence level.
Resalts and Discussion Figure 1 shows enzyme activity during the first 25 days after amputation. In the normal limb (day 0) enzyme activity was found to be the same in the hand as in the humerus. Between days 0 and 1, the activity showed a statistically significant drop of 4% (Pa0.001). From a level of 26.5 f 1.3 pmoledmg DNA/h at day 1,a threefold increase in activity was reached by day 5 (90.8 f 3.2 pmoles/mg DNA/h). After this, the activity decreased until day 10, reaching a value of 31.5 f 2.6 poles/mg DNA/h, representing a 65% drop. A second peak was observed at day 12 which was maintained until day 15 (66.6 f 3.5 p o l e d m g DNA/h, 65.7 f 4.0 pmoledmg DNA/h) and a subsequent drop followed until normal levels were reached by day 25 when measurements were terminated. These changes in glucosaminidase activity, when correlated with the stages of limb regeneration, show a decrease during wound healing, an initial peak followed by a drop and a second peak during the late dedifferentiation phase. The decrease observed at day 1 can be partly because of cell death [20] and partly because the tissue is mainly wound-healing epidermis which does not contribute to the formation of the blastema (211. The first increase in enzyme activity during the loo
0
r
early stages of limb regeneration could be partly explained in the first place by massive macrophage infiltration [20]. It has been shown in other systems that macrophages have high levels of enzyme activity and do release the enzyme into the extracellular space. Secondly, the increase could also represent endogenous activity in the cells at the amputation site. It is most likely that both factors contribute to this initial increase in enzyme activity, which is concomitant with the initiation of tissue demolition and cell lysis. Other lysosomal enzymes, such as alkaline phosphatase, have been shown to increase their activity during blastema formation in limb regeneration [22]. This has also been reported during the dedifferentiation phase of Wolffian lens regeneration, for N-acetyl glucosaminidase ~41. The sudden drop from days 5-10 coincides with maximum synthesis of hyaluronic acid (HA), which is needed for the formation of the blastema [9]. It is well known that N-acetyl glucosaminidase hydrolyzes HA residues [19]. If HA is necessary for morphogenesis of the blastema, one would expect the activity of the enzyme to be reduced, thus facilitating blastema formation. That HA is involved in morphogenetic processes is well documented [9, 10, 23, 241. According to Toole and Gross [9], the synthesis of HA decreases around day 12 of limb regeneration. This is concomitant with the second peak of N-acetyl glucosaminidase in our findings. In addition, hyaluronidase activity is first detected during limb regeneration between days 12 and 15 [lo]. The latter enzyme is known to hydrolyze HA into residues, which serve as a substrate for glucosaminidase. An increase in glucosaminidase activity would not permit an accumulation of HA residues, which in turn would interfere with the events leading to the formation of the new limb [9, 101. The gradual decrease in glucosaminidase activity after day 15 coincides with the initial events of redifferentiation. According to Hay [12], day 18 marks a 50% reduction in DNA synthesis which is related to morphogenesis of the new limb. Histochemical localization of the enzyme in tissue samples of days 5 and 17 gave negative results. However, when the incubation medium of tissue samples of the same days were analyzed biochemically, pnitrophenol was liberated at the rate of 35.2 p o l e d m l at day 5 and 16.2 pmoles/ml at day 17. These values are within the range of those obtained in tissue homogenates: 39.1 pmoledml at day 5 and 26.8 pmoledml at day 17. Thus, the failure to localize the enzyme histochemically could be explained by its release into the incubation medium. That glucosaminidase and other lysosomal glycosidases are selectively released into the extracellular matrix or milieu has been shown in several biological systems [14, 25-27]. Idoyaga-Vargas and Yamada [14] have measured glucosaminidase activity during lens regeneration in the newt. They found that activity of the enzyme increases during dedifferentiation and begins to decrease during the early stages of redifferentiation. This pattern of enzyme activity is similar to our findings for the limb regeneration system. In the lens regeneration system it is known that cell surface alterations are an integral part of the dedifferentiation phase. This involves loss of cytoplasmiccomponents, fragments of membranes, and removal of specific molecules from the cell surface [15, 16,28, 291. Based on their results, Idoyaga-Vargas and Yamada [ 141 suggested that glucosaminidase is actively involved in cell
1 5 10 15 20 25 30
Days after amputation Fig. 1. N-Acetylglucosaminidaseactivity during the first 25 days of limb regeneration.Vertical bars indicate standard errors. Significant change when compared with day 0 (P Q 0.001); ** Significant change when compared with day 1 (P 4 0.001); *** Significant change when compared with day 5 (Pe0.001); **** Significant change when compared with days 12-15 (P 4 0.001)
M. Rivera et al.: Glucosaminidase Activity during Limb Regeneration surface alterations necessary for altering the differentiated state of iris epithelial cells during lens regeneration. Our results indicate that glucosaminidase activity may play a similar role during limb regeneration, specifically during the dedifferentiation phase of the regeneration process. Further studies, however, are needed to elucidate the definite role of the enzyme during limb and lens regeneration. Acknowledgements. This research was sponsored by NIH Grant RR-8102, NSF Grant RIM-7816334,and the Office of Coordination of Graduate Studies and Research, University of Puerto Ria, Rio Redras Campus. The authors thank Dr. L. R. Otero, Medical Science Campus, University of Puerto Rico, San Juan, P.R. for instruction during histochemical studies; Dr. Thomas G. Connelly of the Department of Anatomy, University of Michigan Medical Science 11, Ann Arbor Michigan 48109, USA, and Dr.Tuneo Yamada, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland, for critically reading the manuscript.
References 1. French B, Bryant P, Bryant S (1976) Pattern regulation in epimorphic fields. Science 193: 969 2. Stocum DL (1968) The urodele limb regeneration blastema: A self-organizing system. Dev Biol 18: 144 3. Stocum DL (1978) Organization of the morphogenetic field in regenerating Amblystoma limbs. Am Zoo1 18: 883 4. Walker BE (1961) The association of mucopolysaccharides with morphogenesis of the palate and other structures of the mouse embryo. Embryo1 Exp Morphol 9:22 5. Banerjee SD, Cohn RH, Bernfield MR (1977) Basal lamina of embryonic salivary epithelia. J Cell Biol 73: 445 6. Bernfield MR, Cohn RH, Banerjee SD (1977) Glycosaminoglycans and epithelial organ Eonnation. Am Zoo1 13: 1067 7. Cohn RH, Banerjee SD, Bernfield M R (1977) Basal lamina of embryonic salivary epithelia. Nature of glycosaminoglycans and organization of extracellular materials. J Cell Biol 73: 469 8. Derby MA (1978) Analysis of glycosaminoglycans within the extracellular components encountered by migrating neural crest cells. Dev Biol 66:321 9. Toole BP, Gross J (1971) The extracellular matrix of the regenerating newt limb. Synthesis and removal of hyaluronilate prior to differentiation. Dev Biol 25: 57 10. Toole BP (1973) Hyaluronilate and hyaluronidase in morphogenesis and differentiation. Am Zoo1 13: 1061 11. Hay ED (1959) Electron microscopic observation of muscle dedifferentiation in regenerating Amblystoma limb. Dev Biol 1:555
123
12. Hay ED (1965) Metabolic pattern of limb development and regeneration. In: DeHaan RH, Ursprung H (eds) Organogenesis. Holt, Rinehart & Winston. New York, p 315 13. Mailman ML, Dresden MH (1976) Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: Synthesis, accumulation and maturation. Dev Biol 50: 378 14. Idoyaga-Vargas V, Yamada T (1974) Glucosaminidase and dedifferentiation of newt iris epithelium. Differentiation 2:91 15. Zalik SE,Scott V (1973) Sequential disappearence of cell surface components during dedifferentiation in lens regeneration. Nature New Biol 244: 212 16. Zalik SE, Scott V, Dimitrov E (1976) Changes at cell surface during in vivo and in vitro dedifferentiation in cellular metaplasia. Progress in differentiation Research. North Holland Publishing Co., Amsterdam, p 361 17. Iten LE,Bryant SV (1973) Forelimb regeneration from different levels of amputation in the newt Notophthalmus viridescem: Length rate and stages. Wilhelm Roux Arch Entwicklungsmech Org 173: 263 18. Burton K (1965) A study of condition and mechanism of diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62: 315 19. Pugh N, Walker PG (1960) The localization of N-acetyl glucosaminidase in tissues. J Histochem Cytochem 9: 242 20. Thornton CS (1938a) The histogenesisofmusclein the regenerating forelimb of larval Amblystomu mexicanurn. J Morphol62: 17 21. Reddiford LM (1960) Autoradiographic studies of tritiated thymidine infused into the blastema of the early regenerate in the newt triturus. J Exp Zoo1 144: 25 22. Schmidt AJ, Weary M (1963) Localization of acid phosphatase in the regenerating forelimb of the adult newt Diemictylus viridescem. J Exp Zoo1 152: 101 23. Toole BP (1972) Hyaluronilate rate turnover during chondrogenesis in the developing chick limb and axial skeleton. Dev Biol 29 :321 24. Solursh M, Fisher M, Siglev CT (1979) The synthesis of hyaluronic acid by ectoderm during early organogenesisin the chick embryo. Differentiation 14: 77 25. Ashworth JM, Qwance J (1972) Enzyme synthesis in mixoamaebae of the cellular stime mold Dictyostelium discoideum during growth in axenic culture. Biochem J 126: 601 26. Muller M (1972) Secretion of acid hydrolases and its extracellular source in Tetrahymena pirijormh. J Cell Biol 52: 472 27. Loomis WF (1969) Acetylglucosaminidase, an early enzyme in the development of Dictyostelium discoideum. J Bacteriol 97: 1149 28. Zalik S, Scott V (1972) Cell surface changes during dedifferentiation in the metaplastic transformation of iris into lens. J Cell Biol 55: 134 29. Yamada T (1977) Control mechanism in cell-type conversion in newt limb regeneration. Monogr Dev Biol 13: 1
Received November 19WAccepted February 1981
Differentiation (1981) 19: 121-123
0 Springer-Verlag 1981
Preliminary Reports N-Acetyl-Glucosaminidase Activity during Limb Regeneration in the Adult Newt M. RIVERA, J. R. ORTIZ', and E. ORTIZ Department of Biology, University of Puerto Rico, Rio Piedras, Puerto Rico 00931
N-Acetyl-glucosaminidaseactivity was measured during the first 25 days of limb regeneration. It was found that the enzyme is present in the normal limb. Following amputation a significant drop was obtained at day 3. A significant increase in enzyme activity was found at day 5 followed by a second drop by day 10. For days 12- 15 a second peak of enzyme activity was detected, followed by a third drop; by day 25, normal levels of enzyme activity were detected. Histochemical localization of the enzyme in tissue samples showing enzyme activity as detected biochemically (days 5 and 17 of regeneration) gave negative results. However, enzyme activity was found in the incubation medium, indicating that the enzyme is released from the cells. The peaks of enzyme activity coincide with the stages of limb regeneration where a high degree of tissue demolition and cell lysis occurs. The latter are important events in the regeneration process, cell dedifferentiation, and blastema formation.
IntrodUctioO
Methods
Limb regeneration in the adult newt comprises three consecutive events: wound healing, dedifferentiation (formation and growth of the blastema), and redifferentiation or morphogenesis of the limb. The regulatory mechanisms during regenerative processes are poorly understood. It has been suggested that metabolic gradients of chemical substances arranged in the cell surfaces play an important role in regeneration [l-31. In other biological systems there is evidence that macromolecules such as mucopolysaccharides are involved in morphogenesis. Hyaluronic acid, chondroitin sulphate, and heparine sulphate are found to be related with the morphogenesis of mouse salivary glands [4-71. In the chick embryo, these mucopolysaccharidesseem to be related with neural crest migration and differentiation [8] and with limb morphogenesis 19, 101. Studies of mucopolysaccharide metabolism have revealed that during the dedifferentiation phase of limb regeneration, the chondrocytes are released from their extracellular matrix [ll-131. It has also been shown that lysosomal enzyme activity and removal of macromolecules from the cell surface or extracellular matrix do alter the differentiated state [14-161. Studies of enzymes capable of removing components from the cell surface and extracellular matrix, such as N-acetyl-glucosaminidase, could contribute to the understanding of the mechanisms involved in differentiation and morphogenesis. The purpose of this preliminary report is to present measurements of glucosaminidase activity during limb regeneration in the adult newt.
Adult newts Notophthalrnus viridescem, obtained from Bill Lee's Newt Farm,Oak Ridge, Tennessee, USA,were used. The stages of regeneration were classified according to Iten and Bryant [17]. The animals were maintained in %gal tanks at 20" C. After amputation they were placed in 10-gal tanks in groups of 13-30 individuals and were fed Tubifex three or four times a week. Animals were anesthetized with 0.04% Tricaine (ethyl M-aminobenzoate, Sigma Chemical Co, St. Louis, MO, USA). Amputation was performed at two-thirds distal to the humerus, under the dissecting microscope. After amputation, the animals were placed briefly in urodele Ringer, and when completely recovered, they were moved back to water. Enzyme activity was measured at 0, 1,3,5,7, 10, 12,15, 17,20, 22, and 25 days after amputation, using the method described by Idoyaga-Vargas and Yamada [14]. The tissue samples were cut with scalpel and placed in 2.4 ml of phosphate buffer (PBS 80%). At day 0 enzyme measurements were made in the hands and in the region that corresponds to the distal part of the humerus, using 13 hands and 13 pieces of humerus 3 mm long. For days 1-10. 40 tissue samples containing mostly stump tissue were used for each measurement. For days 12-25, tissue samples consisted mostly of mesenchymal cells. Thirty-two tissue samples were used for each measurement for days 12 and 15 and twenty-four for days 17-25. The tissue samples were homogenized in a hand homogenizer with approximately 100 strokes. Enzyme measurements were made in triplicate. The reaction mixture consisted of 0.1 ml of homogenate, 0.33 ml of citrate (0.064 N) and phosphate (0.21 N) pH 4.2, and 0.4 ml of p-nitrophenyl N-acetyl glucosaminide solution 12 mM (Sigma) and was incubated 2 h at 37" C. To stop the reaction, 1 ml of cold 4% trichloroacetic acid (TCA) was added together with 0.7 ml of a 5 : 1 mixture of glycine (0.1 N) and carbonate buffer (0.8 N) with 0.5 N NaOH at pH 10.5. Enzyme activity was determined by reading the pnitrophenol absorbance in a Zeiss spectrophotometer at 412 nm, using a standard curve of p-nitrophenol absorbance versus concentration.
1 To whom reprint requests should be addressed
0301-4681/81/0019/0121/$01.00
M. Rivera et al.: Glucosaminidase Activity during Limb Regeneration
122
The unit of enzyme activity was expressed as micromoles of liberated pnitrophenohng of DNNh. DNA determination was made in triplicate through the use of the diphenylamine technique [18]. For histochemical localization of the enzyme, the Pugh-Walker method [19] was used in tissue samples at days 5 and 17. To determine if glucosaminidase was released to the incubation medium, the tissue samples were placed intact in the reaction mixture and incubated 1 h. The reaction was stopped and absorbance of pnitrophenol was read as described above. For each day a total of eight or ten determinations of enzyme activity were made. The mean and standard error were obtained and Student’st-test was used to determine statistical significance between some means, at the 99% confidence level.
Resalts and Discussion Figure 1 shows enzyme activity during the first 25 days after amputation. In the normal limb (day 0) enzyme activity was found to be the same in the hand as in the humerus. Between days 0 and 1, the activity showed a statistically significant drop of 4% (Pa0.001). From a level of 26.5 f 1.3 pmoledmg DNA/h at day 1,a threefold increase in activity was reached by day 5 (90.8 f 3.2 pmoles/mg DNA/h). After this, the activity decreased until day 10, reaching a value of 31.5 f 2.6 poles/mg DNA/h, representing a 65% drop. A second peak was observed at day 12 which was maintained until day 15 (66.6 f 3.5 p o l e d m g DNA/h, 65.7 f 4.0 pmoledmg DNA/h) and a subsequent drop followed until normal levels were reached by day 25 when measurements were terminated. These changes in glucosaminidase activity, when correlated with the stages of limb regeneration, show a decrease during wound healing, an initial peak followed by a drop and a second peak during the late dedifferentiation phase. The decrease observed at day 1 can be partly because of cell death [20] and partly because the tissue is mainly wound-healing epidermis which does not contribute to the formation of the blastema (211. The first increase in enzyme activity during the loo
0
r
early stages of limb regeneration could be partly explained in the first place by massive macrophage infiltration [20]. It has been shown in other systems that macrophages have high levels of enzyme activity and do release the enzyme into the extracellular space. Secondly, the increase could also represent endogenous activity in the cells at the amputation site. It is most likely that both factors contribute to this initial increase in enzyme activity, which is concomitant with the initiation of tissue demolition and cell lysis. Other lysosomal enzymes, such as alkaline phosphatase, have been shown to increase their activity during blastema formation in limb regeneration [22]. This has also been reported during the dedifferentiation phase of Wolffian lens regeneration, for N-acetyl glucosaminidase ~41. The sudden drop from days 5-10 coincides with maximum synthesis of hyaluronic acid (HA), which is needed for the formation of the blastema [9]. It is well known that N-acetyl glucosaminidase hydrolyzes HA residues [19]. If HA is necessary for morphogenesis of the blastema, one would expect the activity of the enzyme to be reduced, thus facilitating blastema formation. That HA is involved in morphogenetic processes is well documented [9, 10, 23, 241. According to Toole and Gross [9], the synthesis of HA decreases around day 12 of limb regeneration. This is concomitant with the second peak of N-acetyl glucosaminidase in our findings. In addition, hyaluronidase activity is first detected during limb regeneration between days 12 and 15 [lo]. The latter enzyme is known to hydrolyze HA into residues, which serve as a substrate for glucosaminidase. An increase in glucosaminidase activity would not permit an accumulation of HA residues, which in turn would interfere with the events leading to the formation of the new limb [9, 101. The gradual decrease in glucosaminidase activity after day 15 coincides with the initial events of redifferentiation. According to Hay [12], day 18 marks a 50% reduction in DNA synthesis which is related to morphogenesis of the new limb. Histochemical localization of the enzyme in tissue samples of days 5 and 17 gave negative results. However, when the incubation medium of tissue samples of the same days were analyzed biochemically, pnitrophenol was liberated at the rate of 35.2 p o l e d m l at day 5 and 16.2 pmoles/ml at day 17. These values are within the range of those obtained in tissue homogenates: 39.1 pmoledml at day 5 and 26.8 pmoledml at day 17. Thus, the failure to localize the enzyme histochemically could be explained by its release into the incubation medium. That glucosaminidase and other lysosomal glycosidases are selectively released into the extracellular matrix or milieu has been shown in several biological systems [14, 25-27]. Idoyaga-Vargas and Yamada [14] have measured glucosaminidase activity during lens regeneration in the newt. They found that activity of the enzyme increases during dedifferentiation and begins to decrease during the early stages of redifferentiation. This pattern of enzyme activity is similar to our findings for the limb regeneration system. In the lens regeneration system it is known that cell surface alterations are an integral part of the dedifferentiation phase. This involves loss of cytoplasmiccomponents, fragments of membranes, and removal of specific molecules from the cell surface [15, 16,28, 291. Based on their results, Idoyaga-Vargas and Yamada [ 141 suggested that glucosaminidase is actively involved in cell
1 5 10 15 20 25 30
Days after amputation Fig. 1. N-Acetylglucosaminidaseactivity during the first 25 days of limb regeneration.Vertical bars indicate standard errors. Significant change when compared with day 0 (P Q 0.001); ** Significant change when compared with day 1 (P 4 0.001); *** Significant change when compared with day 5 (Pe0.001); **** Significant change when compared with days 12-15 (P 4 0.001)
M. Rivera et al.: Glucosaminidase Activity during Limb Regeneration surface alterations necessary for altering the differentiated state of iris epithelial cells during lens regeneration. Our results indicate that glucosaminidase activity may play a similar role during limb regeneration, specifically during the dedifferentiation phase of the regeneration process. Further studies, however, are needed to elucidate the definite role of the enzyme during limb and lens regeneration. Acknowledgements. This research was sponsored by NIH Grant RR-8102, NSF Grant RIM-7816334,and the Office of Coordination of Graduate Studies and Research, University of Puerto Ria, Rio Redras Campus. The authors thank Dr. L. R. Otero, Medical Science Campus, University of Puerto Rico, San Juan, P.R. for instruction during histochemical studies; Dr. Thomas G. Connelly of the Department of Anatomy, University of Michigan Medical Science 11, Ann Arbor Michigan 48109, USA, and Dr.Tuneo Yamada, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland, for critically reading the manuscript.
References 1. French B, Bryant P, Bryant S (1976) Pattern regulation in epimorphic fields. Science 193: 969 2. Stocum DL (1968) The urodele limb regeneration blastema: A self-organizing system. Dev Biol 18: 144 3. Stocum DL (1978) Organization of the morphogenetic field in regenerating Amblystoma limbs. Am Zoo1 18: 883 4. Walker BE (1961) The association of mucopolysaccharides with morphogenesis of the palate and other structures of the mouse embryo. Embryo1 Exp Morphol 9:22 5. Banerjee SD, Cohn RH, Bernfield MR (1977) Basal lamina of embryonic salivary epithelia. J Cell Biol 73: 445 6. Bernfield MR, Cohn RH, Banerjee SD (1977) Glycosaminoglycans and epithelial organ Eonnation. Am Zoo1 13: 1067 7. Cohn RH, Banerjee SD, Bernfield M R (1977) Basal lamina of embryonic salivary epithelia. Nature of glycosaminoglycans and organization of extracellular materials. J Cell Biol 73: 469 8. Derby MA (1978) Analysis of glycosaminoglycans within the extracellular components encountered by migrating neural crest cells. Dev Biol 66:321 9. Toole BP, Gross J (1971) The extracellular matrix of the regenerating newt limb. Synthesis and removal of hyaluronilate prior to differentiation. Dev Biol 25: 57 10. Toole BP (1973) Hyaluronilate and hyaluronidase in morphogenesis and differentiation. Am Zoo1 13: 1061 11. Hay ED (1959) Electron microscopic observation of muscle dedifferentiation in regenerating Amblystoma limb. Dev Biol 1:555
123
12. Hay ED (1965) Metabolic pattern of limb development and regeneration. In: DeHaan RH, Ursprung H (eds) Organogenesis. Holt, Rinehart & Winston. New York, p 315 13. Mailman ML, Dresden MH (1976) Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: Synthesis, accumulation and maturation. Dev Biol 50: 378 14. Idoyaga-Vargas V, Yamada T (1974) Glucosaminidase and dedifferentiation of newt iris epithelium. Differentiation 2:91 15. Zalik SE,Scott V (1973) Sequential disappearence of cell surface components during dedifferentiation in lens regeneration. Nature New Biol 244: 212 16. Zalik SE, Scott V, Dimitrov E (1976) Changes at cell surface during in vivo and in vitro dedifferentiation in cellular metaplasia. Progress in differentiation Research. North Holland Publishing Co., Amsterdam, p 361 17. Iten LE,Bryant SV (1973) Forelimb regeneration from different levels of amputation in the newt Notophthalmus viridescem: Length rate and stages. Wilhelm Roux Arch Entwicklungsmech Org 173: 263 18. Burton K (1965) A study of condition and mechanism of diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62: 315 19. Pugh N, Walker PG (1960) The localization of N-acetyl glucosaminidase in tissues. J Histochem Cytochem 9: 242 20. Thornton CS (1938a) The histogenesisofmusclein the regenerating forelimb of larval Amblystomu mexicanurn. J Morphol62: 17 21. Reddiford LM (1960) Autoradiographic studies of tritiated thymidine infused into the blastema of the early regenerate in the newt triturus. J Exp Zoo1 144: 25 22. Schmidt AJ, Weary M (1963) Localization of acid phosphatase in the regenerating forelimb of the adult newt Diemictylus viridescem. J Exp Zoo1 152: 101 23. Toole BP (1972) Hyaluronilate rate turnover during chondrogenesis in the developing chick limb and axial skeleton. Dev Biol 29 :321 24. Solursh M, Fisher M, Siglev CT (1979) The synthesis of hyaluronic acid by ectoderm during early organogenesisin the chick embryo. Differentiation 14: 77 25. Ashworth JM, Qwance J (1972) Enzyme synthesis in mixoamaebae of the cellular stime mold Dictyostelium discoideum during growth in axenic culture. Biochem J 126: 601 26. Muller M (1972) Secretion of acid hydrolases and its extracellular source in Tetrahymena pirijormh. J Cell Biol 52: 472 27. Loomis WF (1969) Acetylglucosaminidase, an early enzyme in the development of Dictyostelium discoideum. J Bacteriol 97: 1149 28. Zalik S, Scott V (1972) Cell surface changes during dedifferentiation in the metaplastic transformation of iris into lens. J Cell Biol 55: 134 29. Yamada T (1977) Control mechanism in cell-type conversion in newt limb regeneration. Monogr Dev Biol 13: 1
Received November 19WAccepted February 1981