Transplanted hind limb-buds of chick embryos were treated with salicylic acid. Treatment with this chemical caused reduction in length, weight and volume of the developing limb-buds. A side effect observed was an apparent loosening of the basal membrane and examined with regard to length, weight, and volume. The above observations are discussed in relation to the different hypotheses of the ectoderm-mesoderm interaction. The physiological role of the basal membrane, as well as the possible implications of the basal findings to the question of limb development and morphogenesis, are also discussed

Baker, John R.

Suleiman, Suleiman A.

English

Name not available

Effect of Salicylic Acid on the Early Development of the Chick Limb-Bud

University of Northern Iowa

Proceedings of the Iowa Academy of Science Volume 78 Number 1-2 Article 9 1971 Effect of Salicylic Acid on the Early Development of the Chick Limb-Bud Suleiman A. Suleiman Iowa State University John R. Baker Iowa State University Copyright ©1971 Iowa Academy of Science, Inc. Follow this and additional works at: https://scholarworks.uni.edu/pias Recommended Citation Suleiman, Suleiman A. and Baker, John R. (1971) "Effect of Salicylic Acid on the Early Development of the Chick Limb-Bud," Proceedings of the Iowa Academy of Science, 78(1-2), 20-23. Available at: https://scholarworks.uni.edu/pias/vol78/iss1/9 This Research is brought to you for free and open access by the Iowa Academy of Science at UNI ScholarWorks. It has been accepted for inclusion in Proceedings of the Iowa Academy of Science by an authorized editor of UNI ScholarWorks. For more information, please contact scholarworks@uni.edu. 20 PRoc. low A AcAD. Ser. 78 ( 1971) Effect of Salicylic Acid on the Early Development of the Chick Limb-Bud SULEIMAN A. SULEIMAN1 and JOHN R. BAKER2 SULEIMAN A. SULEIMAN and JoHN R. BAKER. Effect of Salicylic Acid on the Early Development of the Chick Limb-Bud. Proc. Iowa Acad. Sci., 78( I) :20-23, 1971. SYNOPSIS. Transplanted hind limb-buds of chick embryos were treated with salicylic acid. Treatment with this chemical caused reduction in length, weight and volume of the developing limb-buds. A side effect observed was an apparent loosening of the and examined with regard to length, weight, and volume. Mitochondria are organelles found in all aerobic organisms in which the oxidation of pyruvic acid to carbon dioxide and water takes place. Several workers have isolated mitochondria to study their properties and find their role in embryogenesis. Carey and Greville ( 1959a,b) found that the mitochondria obtained from embryonic preparations appeared to be more fragile than their adult counter parts. McAlpino ( 1960) reported a marked increase in phosphate activity (which is related to mitochondria) in the ectodermal tips of th3 developing limb-buds of rat embryos. These observations and others (Bosund, 1957; Charnock, and Opit 1962a) show an essential mito-chondrial activity during early development. Salicylates are thought to be inhibitors of mitochondrial activity, which may act by disrupting or uncoupling the link between oxidation and phosphorylation. Brody ( 1956) was the first to show that salicylates in a concentration above 3 mg/100 ml. can decrease the P:O (phosphorylation: oxida-tion) ratio of rat liver and kidney mitochondria oxidizing a variety of substrates. The actual site of salicylate activity has not yet been found; but Charnock and Opit ( 1982b) have suggested that the locus of the action of salicylate in uncoupling oxidative phosphorylation is at the level of the mitochondrial membrane. It was therefore considered desir-able to observe the effect of Salicylic acid on the early de-velopment of the chick limb-buds and to find if Salicylic acid has another effect in addition to its suggested uncoupling factor. METHODS AND MATERIALS The hind limb-buds of white leghorn chick embryos at stage 19 of Hamilton and Hamburger, were extirpated and transplanted singly onto the chorioallantoic (CA) membrane of host embryos of stage 35 of Hamilton and Hamburger. Immediately after transplantation, 0.08 ml. of salicylic acid were dropped on top of the transplanted buds with a micro-dropper. There were seven groups of transplants correspond-ing to seven concentrations. The host eggs were again incu-bated at 38° C. The transplanted limb-buds were recovered at intervals of 6 hours, 1, 2, 3, 4, and 5 days after operation lSuleiman A. Suleiman, 253 Science Building, Iowa State Univer-sity, Ames, Iowa 50010 2John R. Baker, Ph.D., Assistant Professor of Zoology, 351 Science Building, Iowa State University, Ames, Iowa 50010 basal membrane. The above observations are discussed in relation to the different hypotheses of the ectoderm-mesoderm interaction. The physiological role of the basal membrane, as well as the possible implications of the basal findings to the question of limb development and morphogenesis, are also discussed. INDEX DESCRIPTORS: Salicyclic acid, chick limb bud, ectoderm-mesodern interaction, limb bud growth. Some of them were then fixed in 4 per cent neutral formalin and others in Helley's fluid, washed in tap water, dehydrated, and embedded in paraffin. The sections were cut at 4 u by AP-121 Microtome, and stained either by Novelli's method for demonstration of mitochondria or with eosine and haema-toxylin (E and H). Controls were done with each group of eX!perimental concentrations in which 0.08 ml. of Pannet-Compton solution were added to the transplant. For thin sectioning, limb-buds were fixed in chilled gluter-aldehyde (4°C) for 4 hours, and post fixed in osmium tet· roxide (in phosphate buffer) for 45 minutes. After dehydra-tion with graded alcohols ( 35, 70, 90 per cent and three changes of absolute alcohol) the tissues were transfered to the Araldite mixture, the Araldite was changed twice or thrice during this time. The sections (Jf - \!4 µ thick) were cut by glass knives on LKB ultra-microtome, and were stained with acid fuschin. OBSERVATIONS The group of limb-buds treated with 250 ,_,,gm/ml and 200 p.gm/ml concentration showed a pronounced growth inhibi-tion during the whole time of transplantation, and especially during the first 4 days of transplantation, compared to the controls (Figure 1). The groups of limb-buds treated with the concentrations of 170, 150, and 135 p.gm/ml also showed growth inhibition but to a lesser degree and in the period of the first 3 days of transplantation (Figure 2). The groups of limb-buds treated with the concentration of 125 and 100 µ.gm/ml did not show any significant growth inhibition; (Fig-ure 3) and the growrh of the group treated with 100 p.gm/ml was very similar to the controls. The only drfference between the groups of limb-buds treated with the last two concentra-tions and the control was less growth in the first day (Figure 3). In all groups of transplanted limb-buds (except for those treated with the first two concentrations) the growth (length, weight, and volume) tends to become normal after the third day of treatment. In the groups treated with the last two concentrations the growth is very close to the normal. In all the transplanted limb-buds there was no significant dif-ference in growth during the first 6 hours of transplantation compared to the controls. After this the growth behavior differed according to the concentration. 1Suleiman and Baker: Effect of Salicylic Acid on the Early Development of the Chick LiPublished by UNI ScholarWorks, 1971LIMB Buo DEVELOPMENT 21 17 16 15 13 12 11 E10 "" E .9 ~ 8 "" " ..., 4 -----Leng. of control __________ Wt. of control ___ Experimental ,' DAYS / , , I , , I , I / , I I I / , 4 Fig. 1. Growth of transplanted limb-buds treated with 250µgm/ ml of salicylic acid. In the untreated limb-buds (Figure 4) the basal side of the ectoderm is sharply demarcated from the mesoderm. It was observed that in Salicylic acid treatment with the higher con-centrations (250 µ,gm/ml and 200 µ,gm/ml) the basal mem-brane looked loose under the light microscope, and it was easy to separate an almost intact ectoderm from the examined transplanted limb-buds in the period of the third day of transplantation. In the case of the lower concentrations the loosening was less significant and it was more difficult to separate the ecto-de1m (Figure 6). In the groups receiving 150 µ,gm/ml and 100 µ,gm/ml concentrations there was no loosening ex-cept after six hours treatment. Whether any change has oc-cured in the chemical constitution of the basal membrane is not known. DLSCUSSION During the past twenty years, much experimental work has been directed to the study of ectoderm-mesoderm interaction in limb morphogenesis in avian embryos. It has been debated whether the ectoderm or the mesoderm of the limb-bud is the site of developmental factors. Balinsky ( 1956) suggested that both mesoderm and ectoderm take an active part in the de-velopment of an induced as well as normal limb. Recently two different hypotheses have been formulated to account for the development of limbs in tetrapod vertebrates. In both hypotheses the mesoderm is claimed to be the essen-tial site for limb development. The area of disagreement is in the role played by limb-bud ectoderm, particularly the apical ridge. An increasing body of evidence has accumulated which 17 16 15 11 13 12 11 S1o "" s .9 ~8 "" 7 " ..., 4 3 2 -----Leng. of control ________ ._cwt. of control ____ Experimental ------2 DAYS 3 4 I I Fig. 2. Growth of transplanted limb-hud treated with 170 µgm/ ml of salicylic acid. 17 16 15 14 13 11 -----Leng.of control ------------Wt. of control ____ Experime~tal I, ,!/ ,., ...... ·,· /,.. / _..., I , I I , .__... _...- /,// ::_:~~·:••~:-DAYS , , , , I , I , I I I I , , ' I ,' /I ,' // / ;' . , I / / '/ Fig. 3. Growth of transplanted limh-h11ds tre:itPrl with 100 µgm/ ml of salicylic acid. 2Proceedings of the Iowa Academy of Science, Vol. 78 [1971], No. 1, Art. 9https://scholarworks.uni.edu/pias/vol78/iss1/922 PROC. low A AcAD. Sci. 78 ( 1971 ) Fig. 4. Portion of sagittal section of untreated limb-bud recovered after one day; section at 1/ 2 µ and stained with acid fuschin. x 960. Fig. 5. Portion of sagittal section of limb-bud treated with 250 µgm/ml of salicylic acid recovered after two days; sectioned at 3 µ and stained by Novelli's method. Note the loosening of the basal membrane. X 1170. indicates an active participation of ectoderm in limb develop-ment (Zwilling, 1961; Amprino, 1962; D eH aan and Ebert, 1964 ) especially the thickened portion which is morphologi-cally prominent in birds. According to our observation of Fig. 6. Portion of sagittal section of limb-bud treated with 170 µgm/m l of salicyclic acid recovered after one day; section at 3 µand stained by Novelli's method. X 1110. loosening in the basal membrane, sp ecial attention must be given to this seemingly important intermediate zone between the ectoderm and m esoderm in the limb-bud of the chicken. It is observed that any effect on this is reflected on the limb-bud as growth inhibition. The above observation would also stress the importance of ectoderm (not just a covering ), since the loosening of the basal membrane and its tendency to stay with the ectoderm reflects inhibition of growth. In various publications the term basal membrane is ap-plied to various structures, in this paper the basal m embrane is defined as the zone present at the dermo~epidermal junc-tion . In electron microscopy the term is usually applied to a con tinuous m embrane covering the basal m embrane cells (Sjostrand, 1953; Jurand, 1965 ). It has b een suge;ested that the basal m embrane composed mainly of tropocollagen fin e filaments whkh are either em-bedded in an am orphous matrix of the same density (prob-ably mucopoly-saccharides), or are very closely compacted so that the individual fiber can not b e resolved (F awcett, 1966) . Although the exact function of the basal m embrane is still uncertain , it is b elieved to b e a diffusion barrier (Balinsky, 1956, 1957 ) . Caeser and Edward ( 1957) suggested a pro-tection fun ction from too rapid ion concentrtation changes. It is not known if salicylic acid acts on the tropocollagen in th e basal m embrane, but it interferes with the metabolism of collagen (Bellamy, 1963). It affects the biosynthesis of mu-copolysaccharides in connec tive tissues (Bostrum, 1955. 1963). The loosening of this m embrane observed in our ex-p eriments may b e a ttributed to this effect, or it might sug-gest an effect of salicylic acid on other components not yet known. The inhibition of growth by salicylic acid disappears especially in the groups treated with 100-170 µ.gm /ml con-centrations (Figures 1, 2, 3) . This disa ppearance may be explained by diffusion of salicylic acid into the surround-ing tissue. It is also likely that it affects an interaction be-tween the ectoderm and m esoderm through this m embrane. ing tissue. It is also likely that it affects an interaction be-lts diffusion and reduction of its effectiveness allows a refor-mation of an "inte'racting substance" in this membrane, so a normal or very close to normal growth is maintained again. More bioch emical studies must b e done to determine the precise make-up of the basal m embrane, and the exact mode of action of salicylic acid on this m embrane. 3Suleiman and Baker: Effect of Salicylic Acid on the Early Development of the Chick LiPublished by UNI ScholarWorks, 1971LIMB BUD DEVELOPMENT 23 LITERATURE CITED AMPRINO, R. 1962-1963. Aspetti della Morphogenesi della Estre-mita nei Vertebrati. Mon. Zool. Ital. Suppl. 70-71:1-130. BALINSKY, B. I. 1956. A new theory of limb induction. Proc. Acad. Sci., U.S.A. 42:781-785. ____ . 1957. New method on the mode of action of the limb inductor. ]. Exp. Zool. 134:239-274. BELLAMY, A., A. K. HUGGINS, & M. J. H. SMITH. 1963. Salicylate and glutamate metabolism. ]. Pharm. Pharmacol. 15:559-560. BosTRUM, H. and B.· MAANSSON. 1955. The action of salicylates and related compounds on the sulphate exchange of chondroitin sulphuric acid. ]. Pharm. Pharmacol. 7:185-190. , A. MoRETT & M. WHITEHOUSE. 1963. Studies on the bio-_c_h_e_m_i-stry of heart valves, 1. on the biosynthesis of mucopoly-saccherides in bovine heart valves. Biohem. Biophys. Acta 74: 213-221. BosuND, I. 1957. The effects of salicylic acid, benzoic acid and some of their derivatives on oxidation phosphorylation. Acta, Chem. Scand. 11 :541-544. BRODY, T. M. 1956. Action of sodium salicylate and related com-pounds on tissue metabolism in vitro. ]. Pharm. Expt. Therap. 117:39-51. CAESER, R. & G. A. EDWARD. 1957. The physiologic significance of the basement membrane. Anat. Rec. 128: 530. CAREY, H. H. & G. D. GREVILLE. 1959a. Mitrochondria from embryonic tissues of the chick. 1. preparation, characterization, and some enzymic properties. Biochem. ]. 71: 159-166. ____ & . 1959b. Mitochondria from embryonic tis-sues of the chick II. Metabolic activities. Biochem. ]. 71: 166-176. CHARNOCK, J. S. & L. J. 0PIT. 1962a. Effect of salicylates on ATP activity of rat liver mitochondria. Biochem. ]. 83:596-602. & . 1962b. An evaluation of the effect of salicylate on oxidative phosphorylation in rat liver mitochondria. Biochem. ]. 83: 602-606. DEHANN, R. L. & J. D. EBERT. 1964. Morphogenesis. Ann. Rev. Physiol. 26: 15-46. FAWCETT, D. W. 1966. An atlas of fine structure. The Cell. Phila-delphia and London. Saunders. JuRAND, A. 1965. Ultrastructural aspects of early development of the fore-limb buds in the chick and the mouse. Proc. R. Soc. B. 162:387-405. McALPINO, R. J. 1955. Alkaline phosphatase in the developing thyroid, parathyroid and thymus of the albino rat. Am. ]. of Ant. 96: 191-227. SJOSTRAND, F. S. 1953a. Ultrastructure organization of retinal rods and cones.]. Applied Physics 24:117. ZwrLLLING, E. 1961. Limb morphogenesis. 1. Advances in morpho-genesis. New York Academic Press: 301-330. 4Proceedings of the Iowa Academy of Science, Vol. 78 [1971], No. 1, Art. 9https://scholarworks.uni.edu/pias/vol78/iss1/9

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Effect of Salicylic Acid on the Early Development of the Chick Limb-Bud

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