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A histochemical study on the differentiating vertebral column of chick
Acta histochem. Bd. 56, S. 100 -106 (1976)
Zoology Department, Kalyani University, Kalyani, Nadia, W. B.,India
A histochemical study on the differentiating vertebral column of chick By ARUN CHATTERJEE, LAKSHMI CHATTERJEE, SACHI NANDAN KUNDU and ASOKE BOSE (Received August 15, 1975)
Summary 1. The differentiation of the vertebral elements of the cervical region of chick has been studied from 4 to 23 day age group counted from the day of the commencement of incubation. 2. From the histochemical test.s it appears that chondrogenesis does not take place simultaneously in all the areas of the vertebra. 3. Histochemical changes usually follow histological differentiation. 4. The findings have been discussed in the light of inducing principles by the spinal cord.
References The chondrification of different vertebral elements in birds has not been properly studied (ROMANOFF 1960). The process, according to GOODRICH (1930), does not occur simultaneously in all the parts of the spinal column. Moreover, the maturation of different chondrogenic foci (arcualia) may occur at different periods of epigenesis {O'HARE 1972a). Along with histogenesis, there is also a biochemical differentiation in the cartilage (AMPRINO 1955a, b, MALINSKY 1959). Among many other substances that are involved with cartilage differentiation, chondroitin sulphate has been found to be an essential component (CABRINI 1961, KOBAYASHI 1971). The occurrence of this substance in a vertebral zone, may indicate chondrification of the tissue of that region. At the same time, the biogenesis of chondroitin sulphate depends on the availability of glycogen as the starting material. After its intracellular synthesis (DzIEWIATKOWSKI and LEWIS 1944), the mucopolysaccharide molecule is released in the extracellular matrix where it helps in the precipitation of collagen fibres (GOULD 1961, KEECH 1961). In this step of collagen precipitation, the role of ascorbic acid has been shown to be of great importance (GOULD 1961, 1963, REYNOLDS 1966). The extracellular matrix thus becomes much organised. The significance of extracellular matrix in tissue differentiation has already been explained by different authors (ELSDALE and JONES 1963; GROBSTEIN 1959, 1967, O'HARE 1972a, b). With a view to these facts, a histological and histochemical study on differentiation of cervical vertebra of chick has been undertaken. The findings may reveal the relationship between the chemical and tissue differentiation of the various components of a cervical vertebra at its various phases of epignesis.
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Material and Methods The differentiating cervical vertebrae of chick embryo of Rhode Island Red breed at 4 to 20 days of incubation as also those of 2-uay old chicks are the materials of observations. The embryos at specific age groups were dissocted out and the cervical vertebrae were fixed in suitable fixatives for histological and histochemical studies.
Histological observation Tissues fixed in BOUIN'S fluid and embedded in paraffin after the routine process of dehydration, were sectioned at 8 p.m thickness. The sections were stained in Haematoxylin and Eosin.
Detection Chondroitin sulphate For detection of chondroitin sulphate, the tissues were fixed in ZENKER'S fluid and metachromatic reactions with 0.1 % Toluidine blue in paraffin embedded tissue sections were taken into consideration. As it is generally agreed (STEINBERG 1951, SYLVEN 1959, KOBAYASHI 1971) that sites of metachromasia may contain some other substances apart from chondroitin sulphate, a control series of slides treated with hyaluronidase (L. Light & Co., U. K.) was also run. The enzyme is expected to remove chondroitin sulphate A and C (CABRINI 1961, PEARSE 1968). The slides after being deparaffinised and hydrated, were treated for 60 min with citrate buffer at pH = 4.5 to remove trace of calcium that may interfere the staining reaction with toluidine blue (LORCH 1946, GREEN et al , 1947). They were brought to pH = 7.0 and stained with Toluidine blue. Further, as there has been much debate as to the nature of the metachromatic substances visible after staining with Toluidine blue, sections were studied in water and then their alcohol fastness was studied by dehydrating quickly in grades of butyl alcohol (PEARSE 1968). However, after dehydration very little change in colour intensity or spectral shift was noticed by visual comparison.
Detection of Glycogen Glycogen was studied cytochemically in the sections of BODIN'S fixed tissue by PAS method with and without a prior treatment with 2% diastase solution (GLEGG et al. 1952).
Detection of Ascorbic acid Tissues were fixed and impregnated with 5 % silver nitrate. The paraffin sections at 8 pm thickness were developed in thiosulphate after the routine process (PEARSE 1968). Each observation was based on tissues of 10 different embryos.
Observations In 4-day embryos, the cells in the middle of the centrum show vacuolations. The mesenchyme round the perichordal tube are elongated, compact and parallel in distribution; but they are spherical and loose in the intervertebral region. The reaction for glycogen is intense in the membranes of mesenchyme cells and in the perichordal tube but it is less in the cells of the intervertebral area. The reactions for ascorbic acid are very feeble. There is no display of metachromasia which may indicate that the cartilage matrix has not yet been established in the 4-day embryos. The differentiation of the matrix as evidenced by the manifestation of metachromasia is seen in the middle of the centrum of 6.day embryos. In this period of development, the glycogen becomes increased particularly in the membrane of mesoderm cells. In the centrum, the peripheral cells display more reactions for glycogen and
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ascorbic acid than the central cells. The neural arch differentiates with a sign of chondrification. The chondroblast s, in the middle of the neural arch , are loosely arranged and they are sur rounded by cells oriented parallel with the nerve tube. The base of t he arch shows deep localizations for ascorbic acid while the peripher al part show s less reaction. In thc intervertebral area, there is a wedge shaped intrusion of some cells arranged irregularly with less reaction for glycogen . Th e ventral portion of the spinal cord shows a deep react ion for ascorbic acid than that in other areas. In 8-day embryos, as more mat rix begins t o appea r in t he cent ru m and in the n eural arch, t he concentrat ions of both glycogen and ascorbic acid become increased . The mesen chyme in the intervert ebral area is m ore compact and basophilic. The neural arch shows less metachr omasia than the cent rum. Th e react ions become en hanced in the cells of the middle of the arch of the IO-day embryos. In this phase of development, the localizations for glycogen in the cent rum, the neural arch and in the intervertebral area become decreased. The lev el of ascorbic acid remains more or less same like that of the 8-day embryos and it is observed particularly in relation of the chondroblasts in the ventral portion of the spinal cord. When examin ed at 12-day of development, the matrix in the centrum is found t o be well esta blished with in creased metachromasia but less glycoge n . The formation of the neural arch is complet e. I n the centrum, the rea cti ons for ascorbic acid becomes decreased. Som e reactions a re, however, obser ved in t he periphery of the centrum and that of the neural arch . In the ventral part of the spinal cord, the localiz ations for ascorbic acid become remarkably decreased . The cent rum of I4-day embry os displays more or less uniform metachromasia an d glyc ogen localizations at the two ends but they are ve ry feeble in the middle part. The chondroblasts are few in t he centrum. The ap ex of the neural arch sho ws some metachromasia and glycoge n localization but t he base shows less metachromatic property. The distribution of asco rbic acid shows a similar patt ern a nd it is distributed mainly with the chondroblasts . The spina l cord shows some localizati on for ascorbic acid. There is an overall decrease in the metachromatic property and glycog en localizations in the vertebral area of 16-day embryos. Some of the cells of the centrum and of the neural arch display some metachromatia but those within the lacunae and in the periphery of the centrum are elongat ed and nonmetachromatic. The apex of the neural arch with the neural spine do es not show any metachr omasia. There is a slight ris e in the ascorbic acid concentration in the centrum and in the spinal cord but its level is very high in the cho ndroblasts of the neural a rch . Ther e is a further diminution of metachromasia in the vert ebral area of IS-day em bryos except in the ba se of the neural arch whi ch displays a metachromatic stain . Both glycogen and ascorbic acid show a decline in t hei r localizations in the neural arch a nd in the cent ru m . I n the spina l cord also ther e is a decrease in conce nt ration of ascorbic acid. In 20-day embryos and in 2-day old chick t he centrum do es n ot sho w any metachromasia but cells with deep Toloudine blue st ain are observ ed . The lacunae are without any colouration. The neural arch sh ows a simil ar reaction. Ther e is practi cally no reaction for
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glycogen. The cell membranes which so long showed some stain practically present a negative reaction. The distribution pattern of ascorbic acid in 20-day embryos is very much similar with that of IS-day ones with a slight increase in concentration of the substance in the peripheral portion of the neural arch. In 2-day chick, there is a depletion of ascorbic acid concentration in different portions of the vertebral elements.
Discussion The essential outcome of this study is that in chick chondrification of a cervical vertebra does not take place simultaneously throughout its area and the process is intimately associated with histochemical changes of the structure. As evident from metachromatic reactions, chondrification, starts from the centrum. Though the mechanism and causal factors involved in the process are with much controversy (HOLTZER 1951, 1968, WATTERSON et aJ. 1954, AVERY et aJ. 1956, SENO and BUYUKOZER 1958, STOCKDALE et aJ. 1961, LASH et aJ. 1962, LASH 1963, STRUDEL 1962, 1963, ELLISON et aJ. 1969a, b, KOBAYASHI 1971, O'HARE 1972a, b) as regards the synthesis of macromolecules connected with chondrification, it is generally accepted that a limited period of association between spinal cord and somite is necessary for induction of cartilage (LASH et aJ. 1957, KABAYASHI 1971, O'HARE 1972a). This inducing process must have some effects over the surrounding mesenchyme tissue. It is evident from the present study that on the 4th day of development, the cells of the centrum and notochord are basophilic and non metachromatic. On thc 6th day, metachromasia begins to appear in the middle region of the centrum, in the basal cells of the neural arch and also in the somites, This is indicative of the synthesis of chondroitin sulpahte in these areas. There upon the accumulation of metachromatic substance goes on increasing upto 14 daya of incubation. The appearance of chondroitin sulphate in the centrum on the 6th day of development deserves an explanation. It is known that the central nerVOUB system in birds becomes differentiated at 48 h of incubation (WADDINGTON 1952, 1956, ROMANOFF 1960). The spinal cord at this stage of development and with high mitotic index (MEDDAand BOSE 1969) appears to start a "general cartilage promoting activity" (O'HARE 1972c) that results in the establiehment of basiventral cartilage on the 6th day (WILLIAMB 1942, ROMANOFF 1960) along with the appearance of metachromasia in the vertebral area as evidenced in the present study. This fits well with the observations of LASH (1963) that synt.hesis of mucopolysaccharide in a cartilage starts precisely on the fourth day of its induction. As the spinal cord mainte.ins the mitotic index more than I % level upto lO-day of incubation (MEDDA and BOBE 1969) metachromatic substance increases in the vertebral zone upto 14 day of incubation. The occurrence of chondroitin sulphate in the subsequent phases of development in relation to the neural arch may, however, be related to the "somite specif'ic activity" of the spinal cord (MOBS 1960, KOBAYASHI 1971, O'HARE 1972a). On the other hand, the localization of glycogen shows a somewhat different picture. It is observed that though metaohromaaia is not demonstrated in 4-day embryos, glycogen becomes concentrated in different cell typos particularly in the membrane area. Moreover, the concentration of glycogen becomes increased prior to the increase of chondroitin sulphate. This may be explained as due to the contribution of this carbohydrate in the formation of amino sugar which is an essential component of the mucopolysaccharide (MALINSKY 1959, ROMANOFF 1960, CABRINI 1961, THROP and DORFMAN 1967). ThB concentration of glycogen becomes decreased after 12 days of incubation when localization of chondroitin sulphate also becomes less demonstrable indicating the commencement of ossification. This gradual diminution of chondroitin sulphate may be correlated with the activity of alkaline phosphatase associated with the formation of bones in the vertebral area that starts after 10 days of incubation (BOSE 1960) and in the mighbourhood of 12 days of incubation (ROMANOFF 1960). The significance of the identical picture in the distribution of glycogen in the present study and alkaline phosphatase as recorded previously in the vertebral elements of chick embryos at dif-
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ferent stages of development (BOSE 1960) is possibly due to utilization of glucose in the formation or ossification of chondrocytes by the break down of either glucose-B-phosphate or phosphopyruvate due to the activity of alkaline phosphatase. It may be presumed that the liberated hydrogen phosphate might diffuse to the matrix to form calcium phosphate. Thus, though the role of glycogen in chondrogenesis has led to controversial findings (HAY 1958, ANDERSON 1964, MATUKAS et a!. 1967, DAVIES et a!. 1962, GOEL and JURAND 1972), the simultaneous occurrence of glycogen and chondroitin sulphate in the differentiating vertebral elements deserves attention. At the same time, the localization of ascorbic acid in differentiating vertebral column shows a pattern which is very much similar to that of the chondroitin sulphate and it is always found to be associated with the chondroblasts. Whenever, the matrix appears to be laid down, the ascorbic acid is found to occur in that area. As the chondroblasts gradually recede from the middle portion of the centrum, the localization of ascorbic acid also becomes decreased in the area. Same is the condition with other portion of the vertebral body undergoing differentiation. This may indicate that in the synthesis of extracellular matrix and its deposition, ascorbic acid may play some role to maintain the normal polysaccharide/collagen ratio (GOULD 1961, 1963, ROBERTSON 1961, SCHNEIDER and STAUDINGER 1965, JEFFREY and MARTIN 1964, REYNOLDS 1966) possibly by stimulating collagen synthesis as also by inhibiting abnormal hydration of chondroblasta in the vertebral area (GREEN and GOLDBERGER 1964, SCHIMIZU et a!. 1965, PETERKOFSKY and UDENFRIEND 1965, REYNOLDS 1966). The importance of extracellular matrix material synthesised in association with spinal cord and somite in enhancing chondrogenesis has been stressed previously (O'HARE 1972b). In the deposition of extracellular matrix and in the process of chondrogenesis (GROBSTEIN 1959, COHEN and HAY 1971), the spinal cord, thus, may have some role.
Literature AMPRINO, R., Distribution of S-35 sodium sulphate in early chick embryos. Experientia 11, 19-21 (1955a). - On the incorporation of radiosulphate in cartilage. Experientia 11, 65- 67 (1955 b). ANDERSON, D. R., The ultra structure of elastic and hyaline cartilage of rat. Am. J. Anat. 114, 403-433 (1964). AVERY, G., CHOW, M., and HOLTZER, H., An experimental analysis of the development of the spinal column. V. Reactivity of chick somites. J. Expt!. Zoo!' 132, 409-425 (1956). BOSE, A., Localization of alkaline phosphatase in development of vertebral column in chick. Experientia 16, 4, 144 (1960). CABRINI, R. L., Histochemistry of ossification. Int. Rev. Cyto!. Ed. G. H. Bourne and J. F. DANIELLI, Academic Press, N. Y., 11, 283-304 (1961). COHEN, A. M., and HAY, E. D., Production of connective t issue matrix by isolated embryonic neural tuba in virto. Anat. Roo. 169, 229 (1971). DAVIES, D. V., BARNELL, C. J., COKRANE, W., and PALFREY, A. J., Electron microscopy of articular cartilage in young adult rabbit., Ann. Rheum. Dis. 21, 11- 22 (1962). DZIEWIATKOWSKI, D. D., and LEWIS, H. B., Glucuronic acid synthesis and the glycogen content of the liver of the rat. J. Bio!. Chem. 153, 49 - 52 (1944). ELLISON, M. L., AMBROSE, E. J., and EASTY, G. C., Chondrogenesis in chick embryo somites in vitro. J. Embryo!. expo Morph. 21,331- 340 (1969a). - - - Myogenesis in chick embryo somites in vitro. J. Embryo!. exp!. Morph. 21, 341- 346 (1969b). ELSDALE, T., and JONES, K., The independence and inter-dependence of cells in the amphibian embryos. Symp. Soc. expo Bio!.17, 257-272 (1963). GLEGG, R. E., CLERMONT, Y., and LEBLOND,C. P., The use of lead tetracetate benzidine o-dianizidine and film test technique, Stain Tech. 27, 277 - 301 (1952).
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GOEL, S. C., and JURAND, A., Electron microscopic studies on developing cartilage. II. Demonstration and distribution of glycogen in chick and mouse epiphyseal cartilage. J. Embryol. expo Morph. 28, 153-162 (1972). GOODRICH, E. S., Studies on the structure and development of vertebrates. London Constable 1930. GOULD, B. S., Ascorbic acid independent and Ascorbic acid dependent collagen forming mechanism. Ann. N. Y. Acad. Sci. 92, 168-174 (1961). - Collagen formation and fibrogenesis with special reference to the role of ascorbic acid. Int. Rev. Cytol. 15, 301- 361 (1963). GREEN, H., and GOLDBERG, B., Collagen synthesis of human fibroblast strains. Proc. SoC'. expt!. BioI. and Med. Il7, 258-261 (1964). GREEl', R., FISCHER, C. J., and MORSE, A., Histechemical demonstration of alkaline phosphatase in decalcified dental and osseous tissue. Scir nce 105, 666 (1947). GROBSTEIN, C., Autoradiography of interzene between tissues in inductive interaction. J. Exp. Zoo!. 142,203-213 (1959). - Mechanism of organogenetic tissue interactions. Nat. Can. Inst. Mongr. 26,279-282 (1967). HAY, L. D., The fine structure of blastema cells and differentiating cartilage cells in regenerations of limbs of Amblystoma larvae. J. Biophys, Biochern. Cyto!. 4,583-592 (1958). HOLTZER, H., Morphogenetic influence of tho spinal cord on the axial skeleton End museulat.urr-. Anat. Rec. 109, 113-114 (1951). Induction of chondrogenesis: a concept in quest of mechanism. In: Epithelia - Mesenchymal interactions. Ed. R. Fleischmajer and R. E. Billingham, PI'. 152-164. William & Wilkir.s ,Balti. more 1968. JEFFREY, J. J., and MARTIN, G. R., Collagen currents, 5, 114. Abst. 6th Int. Congo Biochem., New York 1964. KEECH, M. K., The formation of fibrils frem collagen solutione. IV. Effect of Mucepolysaccharides and Nucleic acids. An Electron Microscope study. J. Bicphys, Biochr m. Cyto!. 9, 192-209 (1961). KOBAYASHI, S., Acid Mucopolysaccharides in calcified tissues. Int. Rev. Cytol. G. H. Bourne and J. F. Danielli (Eds.). Acad. Press.,New York, 30, 257-371 (1971). LASH, J. W., Tissue interaction and specific metabolic responses: Chondrogenic induction and differont.iat.ion. In: cytodifferentiation and macromolecular synthesis. M. Locke (Ed.). Academic Press, New York, Pl'. 235- 260 (1963). HOLTZER, S., and HOLTZER, H., An Experimental analysis of the development of the spinal column. IV. Aspects of cartilage induction. Expt!. Cell Res. 13, 292 - 303 (1957). HOMMES, F. A., and ZILLIKEN, F., Induction of cell differentiation. In vitro induction of vertebral cartilage with a low molecular tissue component. Biochcm. Biophys. Acta 56, 313-319 (1962). LORCH,!. J., Demonstration of phosphatase in decalcified bone. Naturc 158, 269 (1946). MALINSKEY, J., Histochemistry of carbohydrates and lipids in intervertebral discs during phylogenetic development. Acta H istochem. 7, 107 - 124 (1959). MATUKAS, V. J., PANNER, B., and ORBISON, J. L., Studies on ultrastructural identification and dis-, tribution of protein polysaccharides in cartilage matrix. J. Cell Biol , 32, 365-377 (1967). MEDDA, J., and BOSE, A., A study of mitotic index in differentiating central nervous system of chick. Cytologia 34,1,36-44 (1969). Moss, M. L.,In: Calcification in Biological System. R. F. Sognnaes (Ed.); 1'.323. American Association Advanced Sciences Washington D. C. 1960. O'HARE, M. J., Differentiation of chick embryo sornites in chorioallantoic culture. J. Embryol. expo Morph. 27, 215-228 (1972a). Chondrogenesis in chick embryo somites grafted with adjacent and heterologous tissues. J. Embryo!. expo Morph , 27, 229-234 (1972b). Aspects of spinal cord induction of chondrogenesis in chick embryo somites. J. Embryol. expo Morph. 27, 235-243 (1972c).
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PEAItSE, A. G. E., Histochemistry - theoretical and applied, vol. 1, p. 336. J. & A. Churchill Ltd., London 1968. PETERKOFSKY, B., and UDENFRIEND, S., Enzymatic hydroxylation of protein in microsomal polypeptide leading to formation of collagen. Proc. Nat. Acad. Sci. U. S. 53,335-342 (1965). REYNOLDS, J. J., The effects of ascrobic on the growth of chick bone rudiments in chamically defined medium. Expt!. Cell Ress. 42,178-188 (1966). ROBERTSON, W. VON B., The biochemical role of ascorbic acid in connective tissue. Ann. N. Y., Acad. Sci. 92,159-167 (1961). ROMANOFF, A. L., The Avian Embryo. McMillan & Co., New York 1960. SOHNEIDEV, W., and STANDINGEV, H. J., Reduced Nicotinamide-adenine dinucleotide-dependent reduction of semi dehydro ascorbic acid. Biochem. Biophys. Acta 96, 157 -159 (1965). SCHIMIZU, Y., MCCANN, D. S., and KEECH, M. K., The effect of Ascorbic acid on human dermal fibroblasts in monolyaer tissue culture. J. Lab. Clin. Med. 65, 286-306 (1965). SENO, T., and BUYUKOZEV, T., and BUYUKOZEV, 1., Cartilage formation in somite grafts of chick blastoderm. Proc. Nat. Acad. Sci. U. S. 44, 1274-1284 (1958). STEINBERG,M. H., Ground substance, bone salts and cellular activity in bone formation and destruction. Am. J. Anat. 89,347-372 (1951). STOCKDALE, F., HOLTZER, H., and LASH, J., An experimental analysis of the development of the spinal column. VII. Response of the dissociated somite cells. Acta Embryol. Morph. Expt. 4, 40-46 (1961). STRUDEL,G., Induction of cartilage in vitro par I'extrait de tube nerveux et de chordate de l'embryon de pou let. Dev. Bio!. 4,47-86 (1962). - Autodifferentiation et induction de Cartilagca partit de me'senchyme somatique de poulet, cultive in vitro. J. Embryo!. expo Morph. 11,399-412 (1963). SYLVEN, B., The ground substance of connective tissue and cartilage in: "The Biochemistry and Physiology of Bone", Ed. G. H. Bourne. Academic Press, New York, pp. 53-80, 1959. THROP, F. K., and DORFMAN, A., Differentiation of connective tissues. in: Current Topics in Developmental Biology. Eds. A. Monroy and A. A. Moscona. Vol. 2, pp. 151-190. Academic Press, New York-London 1967. "VATTERSON, R. L., FOWLER, 1., and FOWLER, R. J., The role of the neural tube and notochord in development of the axial skeleton of chick. Am. J. Anat. 95, 337-383 (1954). WADDINGTON, C. H., The Epigenetics of Birds. Cambridge University Press, U. K. 1952. - Principles of Embryology. George Allen & Unwin U. K. 1956. WILLIAMS, J. L., The development of cervical vertebrae in chick under normal and experimental conditions. Am. J. Anat. 71,153-175 (1942). Address of the authors: e]c Dr. A. K. BOSE, Department of Zoology, Faculty of Science, University of Kalyani, Kalyani, Nadia, West Bengal, India.
Zoology Department, Kalyani University, Kalyani, Nadia, W. B.,India
A histochemical study on the differentiating vertebral column of chick By ARUN CHATTERJEE, LAKSHMI CHATTERJEE, SACHI NANDAN KUNDU and ASOKE BOSE (Received August 15, 1975)
Summary 1. The differentiation of the vertebral elements of the cervical region of chick has been studied from 4 to 23 day age group counted from the day of the commencement of incubation. 2. From the histochemical test.s it appears that chondrogenesis does not take place simultaneously in all the areas of the vertebra. 3. Histochemical changes usually follow histological differentiation. 4. The findings have been discussed in the light of inducing principles by the spinal cord.
References The chondrification of different vertebral elements in birds has not been properly studied (ROMANOFF 1960). The process, according to GOODRICH (1930), does not occur simultaneously in all the parts of the spinal column. Moreover, the maturation of different chondrogenic foci (arcualia) may occur at different periods of epigenesis {O'HARE 1972a). Along with histogenesis, there is also a biochemical differentiation in the cartilage (AMPRINO 1955a, b, MALINSKY 1959). Among many other substances that are involved with cartilage differentiation, chondroitin sulphate has been found to be an essential component (CABRINI 1961, KOBAYASHI 1971). The occurrence of this substance in a vertebral zone, may indicate chondrification of the tissue of that region. At the same time, the biogenesis of chondroitin sulphate depends on the availability of glycogen as the starting material. After its intracellular synthesis (DzIEWIATKOWSKI and LEWIS 1944), the mucopolysaccharide molecule is released in the extracellular matrix where it helps in the precipitation of collagen fibres (GOULD 1961, KEECH 1961). In this step of collagen precipitation, the role of ascorbic acid has been shown to be of great importance (GOULD 1961, 1963, REYNOLDS 1966). The extracellular matrix thus becomes much organised. The significance of extracellular matrix in tissue differentiation has already been explained by different authors (ELSDALE and JONES 1963; GROBSTEIN 1959, 1967, O'HARE 1972a, b). With a view to these facts, a histological and histochemical study on differentiation of cervical vertebra of chick has been undertaken. The findings may reveal the relationship between the chemical and tissue differentiation of the various components of a cervical vertebra at its various phases of epignesis.
A histochemical study on the differentiating vertebral column
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Material and Methods The differentiating cervical vertebrae of chick embryo of Rhode Island Red breed at 4 to 20 days of incubation as also those of 2-uay old chicks are the materials of observations. The embryos at specific age groups were dissocted out and the cervical vertebrae were fixed in suitable fixatives for histological and histochemical studies.
Histological observation Tissues fixed in BOUIN'S fluid and embedded in paraffin after the routine process of dehydration, were sectioned at 8 p.m thickness. The sections were stained in Haematoxylin and Eosin.
Detection Chondroitin sulphate For detection of chondroitin sulphate, the tissues were fixed in ZENKER'S fluid and metachromatic reactions with 0.1 % Toluidine blue in paraffin embedded tissue sections were taken into consideration. As it is generally agreed (STEINBERG 1951, SYLVEN 1959, KOBAYASHI 1971) that sites of metachromasia may contain some other substances apart from chondroitin sulphate, a control series of slides treated with hyaluronidase (L. Light & Co., U. K.) was also run. The enzyme is expected to remove chondroitin sulphate A and C (CABRINI 1961, PEARSE 1968). The slides after being deparaffinised and hydrated, were treated for 60 min with citrate buffer at pH = 4.5 to remove trace of calcium that may interfere the staining reaction with toluidine blue (LORCH 1946, GREEN et al , 1947). They were brought to pH = 7.0 and stained with Toluidine blue. Further, as there has been much debate as to the nature of the metachromatic substances visible after staining with Toluidine blue, sections were studied in water and then their alcohol fastness was studied by dehydrating quickly in grades of butyl alcohol (PEARSE 1968). However, after dehydration very little change in colour intensity or spectral shift was noticed by visual comparison.
Detection of Glycogen Glycogen was studied cytochemically in the sections of BODIN'S fixed tissue by PAS method with and without a prior treatment with 2% diastase solution (GLEGG et al. 1952).
Detection of Ascorbic acid Tissues were fixed and impregnated with 5 % silver nitrate. The paraffin sections at 8 pm thickness were developed in thiosulphate after the routine process (PEARSE 1968). Each observation was based on tissues of 10 different embryos.
Observations In 4-day embryos, the cells in the middle of the centrum show vacuolations. The mesenchyme round the perichordal tube are elongated, compact and parallel in distribution; but they are spherical and loose in the intervertebral region. The reaction for glycogen is intense in the membranes of mesenchyme cells and in the perichordal tube but it is less in the cells of the intervertebral area. The reactions for ascorbic acid are very feeble. There is no display of metachromasia which may indicate that the cartilage matrix has not yet been established in the 4-day embryos. The differentiation of the matrix as evidenced by the manifestation of metachromasia is seen in the middle of the centrum of 6.day embryos. In this period of development, the glycogen becomes increased particularly in the membrane of mesoderm cells. In the centrum, the peripheral cells display more reactions for glycogen and
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and A.
B OSE
ascorbic acid than the central cells. The neural arch differentiates with a sign of chondrification. The chondroblast s, in the middle of the neural arch , are loosely arranged and they are sur rounded by cells oriented parallel with the nerve tube. The base of t he arch shows deep localizations for ascorbic acid while the peripher al part show s less reaction. In thc intervertebral area, there is a wedge shaped intrusion of some cells arranged irregularly with less reaction for glycogen . Th e ventral portion of the spinal cord shows a deep react ion for ascorbic acid than that in other areas. In 8-day embryos, as more mat rix begins t o appea r in t he cent ru m and in the n eural arch, t he concentrat ions of both glycogen and ascorbic acid become increased . The mesen chyme in the intervert ebral area is m ore compact and basophilic. The neural arch shows less metachr omasia than the cent rum. Th e react ions become en hanced in the cells of the middle of the arch of the IO-day embryos. In this phase of development, the localizations for glycogen in the cent rum, the neural arch and in the intervertebral area become decreased. The lev el of ascorbic acid remains more or less same like that of the 8-day embryos and it is observed particularly in relation of the chondroblasts in the ventral portion of the spinal cord. When examin ed at 12-day of development, the matrix in the centrum is found t o be well esta blished with in creased metachromasia but less glycoge n . The formation of the neural arch is complet e. I n the centrum, the rea cti ons for ascorbic acid becomes decreased. Som e reactions a re, however, obser ved in t he periphery of the centrum and that of the neural arch . In the ventral part of the spinal cord, the localiz ations for ascorbic acid become remarkably decreased . The cent rum of I4-day embry os displays more or less uniform metachromasia an d glyc ogen localizations at the two ends but they are ve ry feeble in the middle part. The chondroblasts are few in t he centrum. The ap ex of the neural arch sho ws some metachromasia and glycoge n localization but t he base shows less metachromatic property. The distribution of asco rbic acid shows a similar patt ern a nd it is distributed mainly with the chondroblasts . The spina l cord shows some localizati on for ascorbic acid. There is an overall decrease in the metachromatic property and glycog en localizations in the vertebral area of 16-day embryos. Some of the cells of the centrum and of the neural arch display some metachromatia but those within the lacunae and in the periphery of the centrum are elongat ed and nonmetachromatic. The apex of the neural arch with the neural spine do es not show any metachr omasia. There is a slight ris e in the ascorbic acid concentration in the centrum and in the spinal cord but its level is very high in the cho ndroblasts of the neural a rch . Ther e is a further diminution of metachromasia in the vert ebral area of IS-day em bryos except in the ba se of the neural arch whi ch displays a metachromatic stain . Both glycogen and ascorbic acid show a decline in t hei r localizations in the neural arch a nd in the cent ru m . I n the spina l cord also ther e is a decrease in conce nt ration of ascorbic acid. In 20-day embryos and in 2-day old chick t he centrum do es n ot sho w any metachromasia but cells with deep Toloudine blue st ain are observ ed . The lacunae are without any colouration. The neural arch sh ows a simil ar reaction. Ther e is practi cally no reaction for
A histochemical study on the differentiating vertebral column
103
glycogen. The cell membranes which so long showed some stain practically present a negative reaction. The distribution pattern of ascorbic acid in 20-day embryos is very much similar with that of IS-day ones with a slight increase in concentration of the substance in the peripheral portion of the neural arch. In 2-day chick, there is a depletion of ascorbic acid concentration in different portions of the vertebral elements.
Discussion The essential outcome of this study is that in chick chondrification of a cervical vertebra does not take place simultaneously throughout its area and the process is intimately associated with histochemical changes of the structure. As evident from metachromatic reactions, chondrification, starts from the centrum. Though the mechanism and causal factors involved in the process are with much controversy (HOLTZER 1951, 1968, WATTERSON et aJ. 1954, AVERY et aJ. 1956, SENO and BUYUKOZER 1958, STOCKDALE et aJ. 1961, LASH et aJ. 1962, LASH 1963, STRUDEL 1962, 1963, ELLISON et aJ. 1969a, b, KOBAYASHI 1971, O'HARE 1972a, b) as regards the synthesis of macromolecules connected with chondrification, it is generally accepted that a limited period of association between spinal cord and somite is necessary for induction of cartilage (LASH et aJ. 1957, KABAYASHI 1971, O'HARE 1972a). This inducing process must have some effects over the surrounding mesenchyme tissue. It is evident from the present study that on the 4th day of development, the cells of the centrum and notochord are basophilic and non metachromatic. On thc 6th day, metachromasia begins to appear in the middle region of the centrum, in the basal cells of the neural arch and also in the somites, This is indicative of the synthesis of chondroitin sulpahte in these areas. There upon the accumulation of metachromatic substance goes on increasing upto 14 daya of incubation. The appearance of chondroitin sulphate in the centrum on the 6th day of development deserves an explanation. It is known that the central nerVOUB system in birds becomes differentiated at 48 h of incubation (WADDINGTON 1952, 1956, ROMANOFF 1960). The spinal cord at this stage of development and with high mitotic index (MEDDAand BOSE 1969) appears to start a "general cartilage promoting activity" (O'HARE 1972c) that results in the establiehment of basiventral cartilage on the 6th day (WILLIAMB 1942, ROMANOFF 1960) along with the appearance of metachromasia in the vertebral area as evidenced in the present study. This fits well with the observations of LASH (1963) that synt.hesis of mucopolysaccharide in a cartilage starts precisely on the fourth day of its induction. As the spinal cord mainte.ins the mitotic index more than I % level upto lO-day of incubation (MEDDA and BOBE 1969) metachromatic substance increases in the vertebral zone upto 14 day of incubation. The occurrence of chondroitin sulphate in the subsequent phases of development in relation to the neural arch may, however, be related to the "somite specif'ic activity" of the spinal cord (MOBS 1960, KOBAYASHI 1971, O'HARE 1972a). On the other hand, the localization of glycogen shows a somewhat different picture. It is observed that though metaohromaaia is not demonstrated in 4-day embryos, glycogen becomes concentrated in different cell typos particularly in the membrane area. Moreover, the concentration of glycogen becomes increased prior to the increase of chondroitin sulphate. This may be explained as due to the contribution of this carbohydrate in the formation of amino sugar which is an essential component of the mucopolysaccharide (MALINSKY 1959, ROMANOFF 1960, CABRINI 1961, THROP and DORFMAN 1967). ThB concentration of glycogen becomes decreased after 12 days of incubation when localization of chondroitin sulphate also becomes less demonstrable indicating the commencement of ossification. This gradual diminution of chondroitin sulphate may be correlated with the activity of alkaline phosphatase associated with the formation of bones in the vertebral area that starts after 10 days of incubation (BOSE 1960) and in the mighbourhood of 12 days of incubation (ROMANOFF 1960). The significance of the identical picture in the distribution of glycogen in the present study and alkaline phosphatase as recorded previously in the vertebral elements of chick embryos at dif-
104
A. CHATTERJEE, L. CHATTERJEE, S. N. KUNDU and A. BOSE
ferent stages of development (BOSE 1960) is possibly due to utilization of glucose in the formation or ossification of chondrocytes by the break down of either glucose-B-phosphate or phosphopyruvate due to the activity of alkaline phosphatase. It may be presumed that the liberated hydrogen phosphate might diffuse to the matrix to form calcium phosphate. Thus, though the role of glycogen in chondrogenesis has led to controversial findings (HAY 1958, ANDERSON 1964, MATUKAS et a!. 1967, DAVIES et a!. 1962, GOEL and JURAND 1972), the simultaneous occurrence of glycogen and chondroitin sulphate in the differentiating vertebral elements deserves attention. At the same time, the localization of ascorbic acid in differentiating vertebral column shows a pattern which is very much similar to that of the chondroitin sulphate and it is always found to be associated with the chondroblasts. Whenever, the matrix appears to be laid down, the ascorbic acid is found to occur in that area. As the chondroblasts gradually recede from the middle portion of the centrum, the localization of ascorbic acid also becomes decreased in the area. Same is the condition with other portion of the vertebral body undergoing differentiation. This may indicate that in the synthesis of extracellular matrix and its deposition, ascorbic acid may play some role to maintain the normal polysaccharide/collagen ratio (GOULD 1961, 1963, ROBERTSON 1961, SCHNEIDER and STAUDINGER 1965, JEFFREY and MARTIN 1964, REYNOLDS 1966) possibly by stimulating collagen synthesis as also by inhibiting abnormal hydration of chondroblasta in the vertebral area (GREEN and GOLDBERGER 1964, SCHIMIZU et a!. 1965, PETERKOFSKY and UDENFRIEND 1965, REYNOLDS 1966). The importance of extracellular matrix material synthesised in association with spinal cord and somite in enhancing chondrogenesis has been stressed previously (O'HARE 1972b). In the deposition of extracellular matrix and in the process of chondrogenesis (GROBSTEIN 1959, COHEN and HAY 1971), the spinal cord, thus, may have some role.
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