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Distribution of glycosaminoglycans (Mucopolysaccharides) in the axual region of the developing chick… Kvist, Tage Nielson 1968

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THE DISTRIBUTION OF GLYCOSAMINOGLYCANS (MUCOPOLYSACCHARIDES) IN THE AXIAL REGION OF THE DEVELOPING CHICK EMBRYO by TAGE NIELSEN KVIST B. Sc., U n i v e r s i t y of B r i t i s h Columbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of ZOOLOGY We accept t h i s t hesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA DECEMBER, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f (j /&Ou The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date ^JLes^slfal, £j>} /?£<f ABSTRACT Environmental factors ( e x t r a c e l l u l a r macromolecules) possibly operating i n somite d i f f e r e n t i a t i o n were examined by using an in v i t r o system with myogenesis as the end point. I t was found that d i f f e r e n t i a t i o n depended on the time of removal of the somitic tissue from the host, i . e . between stages 17 and 26 (2% to 5 days of age), and the question was raised as to the r e l a t i o n of these observations and the appearance of glycosamino-glycans (mucopolysaccharides). A review of the l i t e r a t u r e revealed that no information was a v a i l a b l e on t h i s subject so that an examination of the time of appearance, d i s t r i b u t i o n , and nature of the glycosaminoglycans and neutral polysaccharides i n the a x i a l region (dermatome, myotome, scleratome, neural tube and notochord) of the developing chick embryo during early somite d i f f e r e n t i a t i o n was necessary. I t became apparent that both histochemical and biochemical analysis were required to i d e n t i f y , quantify, and l o c a l i z e the glycosaminoglycans since histochemical techniques alone l i m i t the inter p r e t a t i o n s possible because interference from proteins and glycoproteins could not be ruled out. Histochemical analysis indicated that there was very l i t t l e sulphated ani§nic glycosaminoglycan present i n the early embryonic stages examined. The cytoplasm of c e l l s i n a l l a x i a l areas contained strongly a c i d i c material, but e x t r a c e l l u l a r l y , sulphated anionic glycosaminoglycans were almost a l l confined to the notochordal sheath. The e x t r a c e l l u l a r matrix i n a l l areas contained weakly a c i d i c anionic glycosaminoglycans. With development, the weakly a c i d i c anionic glycosaminoglycans increased i n concentration i n most areas, but most noticeably i n the neural tube and scleratome. The concentration of sulphated anionic glycosaminoglycans also increased and they began to appear - i i -i n the e x t r a c e l l u l a r matrix i n a l l areas although never a t t a i n i n g the deep sta i n i n g i n t e n s i t y demonstrated by the weakly a c i d i c anionic glycosamino-glycans. By stage 25, however, the e x t r a c e l l u l a r matrix of the scleratome around the notochord was mostly sulphated anionic glycosaminoglycans. T e s t i c u l a r hyaluronidase digestion suggested that most of the stainable material was either hyaluronic a c i d or chondroitin 4- and/or 6-sulphate (chon-c r o i t i n sulphates A and/or C). A small!amount of strongly a c i d i c anionic glycosaminoglycan present i n the scleratome, neural tube and notochord was polysulphated. Biochemical analysis confirmed that the weakly a c i d i c anionic glycosaminoglycan was hyaluronic acid and that the sulphated anionic glycosaminoglycan was mainly chondroitin 4- and/or 6-sulphate (chondroitin sulphate A and/or C). Only trace amounts of dermatan sulphate (chondroitin sulphate B) were present. A small amount of heparin could be present since some glucosamine was present i n the sulphated f r a c t i o n s . This heparin could account for the polysulphated material observed with histochemical s t a i n i n g . On a q u a n t i t a t i v e b a s i s , the hyaluronic acid concentration (uronic acid/gm dry wt. of tissue) was at a peak between stages 21 to 25 and was greater than the chondroitin sulphate concentration up u n t i l stage 25. After that stage, the chondroitin sulphate concentration began to increase very r a p i d l y , concomitant with the formation of c a r t i l a g e around the ;noto-chord, and the hyaluronic acid concentration began to decline slowly. Thus, whereas the hyaluronic acid content was 2% times greater than the chondroitin.sulphate content i n stage 17 embryos, t h i s r a t i o was almost completely reversed by stage 28 due to the rapid increase i n chondroitin sulphate. Histochemical s t a i n i n g supported these f i n d i n g s . - i i i -It would seem that the increase in sulphated anionic glycosamino-glycans is directly related to cartilage formation while the high hyaluronic acid content present during stages 21 to 25, a time in development when myotube formation and scleratome c e l l aggregation and orientation are occurr-ing, may play some more general developmental role in somite differentiation. - i v -TABLE OF CONTENTS Page INTRODUCTION 1 MATERIAL AND METHODS 3 I Preparation for Histochemical Analysis 3 II Preparation for Glucosaminoglycan Extraction 7 II I Glycosaminoglycan Extraction 9 IV Separation of Extracted Glycosaminoglycans 11 V Analysis of the Glycosaminoglycans 13 RESULTS 16 Morphological Observations 16 Histochemical Analysis 16 Biochemical Analysis 40 I Ex t r a c t i o n and Separation of the Glycosamino-glycans 41 II Characterization of the Three Fractions 53 DISCUSSION 64 I S p e c i f i c i t y of Staining Techniques 64 II Histochemical L o c a l i z a t i o n of the Neutral Poly-saccharide and Glycosaminoglycans 69 II I Biochemical I d e n t i f i c a t i o n of the Anionic Glycosaminoglycans 74 IV Conclusions 77 SUMMARY 79 REFERENCES 82 - V -APPENDICES 89 A. Histochemical Techniques Employed 89 B. Uronic Acid Determinations 93 C. Hexosamine Reaction 94 D. Paper Chromatography 95 E. Stains Employed with Chromatography and Electrophoresis 97 - v i -LIST OF TABLES TABLE PAGE I A l c i a n blue/PAS Staining 20 II Methylation-saponification with A l c i a n blue/PAS 24, 25 I I I Aldehyde Fuchsin 29 IV A l c i a n blue - Aldehyde Fuchsin 32 V To l u i d i n e Blue Metachromasia 35 VI Azure A s t a i n i n g 36 VII A c r i d i n e Orange Fluorescence 39 VIII Properties of the Embryonic Tissues 42 IX Paper Electrophoresis of the Embryonic Extracts 45 X Column Chromatography of Embryonic Extracts 50 XI Uronic Acid Content of Each F r a c t i o n per Gram dry wt. of Tissue 51 XII Paper Electrophoresis of Isolated Fractions 55 XIII Characterization of F r a c t i o n 1 58 XIV Characterization of F r a c t i o n 2 59 v i i -LIST OF ILLUSTRATIONS FIGURE PAGE 1 to 5 Embryos from 3 to 5h days of age (stage 17 to 28) along with the excised t i s s u e from each 8 6 Flow sheet showing procedure followed i n separating the extracted glycosaminoglycans 15 7 Morphological appearance of a stage 17 embryo i n cross section 17 8 Morphological appearance of a stage 20 - 21 embryo in cross section 17 9 Morphological appearance of a stage 22 - 23 embryo i n cross section 18 10 Morphological appearance of a stage 26 embryo i n cross section 19 11 Longitudinal section through a stage 26 embryo 19 12 AB/PAS s t a i n i n g of stage 26 embryo section 22 13 PAS s t a i n i n g of a stage 26 embryo section 22 14 to 16 E f f e c t of acetylation-deacetylation on AB/PAS st a i n i n g 26 17 to 19 E f f e c t of methylation-demethylation on AB/PAS st a i n i n g 26 20 to 21 E f f e c t of t e s t i c u l a r hyaluronidase on AB/PAS st a i n i n g 28 22 to 24 E f f e c t of methylation-demethylation on AF st a i n i n g 31 25 E f f e c t of sulphation on AF s t a i n i n g 31 26 T o l u i d i n e blue metachromasia i n a stage 26 vertebrae centrum primordium 34 27 Azure A metachromasia (pH 4) 34 28 Paper electrophoresis of extract 44 29 Paper electrophoresis of extract 44 30 Column Chromatography of the Embryonic Extract from a stage 27 - 28 sample 47 v i i i 31 Paper electrophoresis of isolated fractions 48 32 Uronic acid/gm. dry wt. Embryonic tissue vs. Development 52 33 Paper electrophoresis of fraction 1 54 34 Paper electrophoresis of fraction 2 54 35 EtOH Fractionation of the calcium salts of fraction 2 from a stage 27 - 28 sample 57 36 Hexosamine paper chromatography of fraction 1 61 37 Hexosamine paper chromatography of fraction 2 61 38 to 41 Hexuronic acid paper chromatography 62 - 63 - i x -ACKNOWLEDGEMENT I wish to express appreciation to Dr. C. V. Finnegan for the guidance, support, and encouragement I have received throughout t h i s work. I am equally g r a t e f u l to Dr. R. H. Pearce for h i s support and for allowing me the f a c i l i t i e s of h i s laboratory for the biochemical work, and to B. J . Grimmer and J . M. Mathieson for th e i r assistance. I would also l i k e to thank Dr. A. B. Acton and Dr. P. Ford for th e i r advice and encouragement. This research was supported i n part by funds from the National Research Council of Canada. - 1 -INTRODUCTION The anionic glycosaminoglycans make up a group of polyanionic macromolecules to which hyaluronic acid, chondroitin 4-sulphate (chondroitin sulphate A), chondroitin 6-sulphate (chondroitin sulphate C), dermatan sulphate (chondroitin sulphate B), heparin, and keratosulphate belong. Chon-d r o i t i n , the non-sulphated analogue of chondroitin 4- and/or 6-sulphate, also belongs to t h i s group. A l l of these polyanionic macromolecules except chondroitin have been i s o l a t e d from connective tissue and characterized (Davidson and Meyer, 1955; Meyer, 1958; Hoffman et a l , 1957, 1960; Linker and Sampson, 1960; Bhavanandan and Meyer, 1966, 1967). The presence of chondroitin would seem to be indicated i n developing embryonic c a r t i l a g e (Thorp and Dorfman, 1963; Searls, 1965). The anionic glycosaminoglycans are widely d i s t r i b u t e d i n develop-ing tissues (Loewi and Meyer, 1958; Kaplan and Meyer, 1959; Mathews and Hinds, 1963). Both p h y s i o l o g i c a l and biochemical functions have been ascribed to these polyanions (Hoffman et a l , 1957; Loewi and Meyer, 1958; Rogers, 1961; Szabo and Roboz-Einstein, 1962). In the c a r t i l a g e matrix of 12 and 14 day old developing chick embryos, anionic glycosaminoglycans have been extracted and i d e n t i f i e d by biochemical assays (Searls, 1965; Meezan and Davidson, 1967), but very l i t t l e work has been done invo l v i n g precartilagenous t i s s u e s . The present study was designed to determine the d i s t r i b u t i o n and nature of the anionic glyco-saminoglycans present during early somite d i f f e r e n t i a t i o n when both chondro-genesis and myogenesis are i n i t i a t e d (2\ to 5\ days of age). To t h i s end, both histochemical and biochemical methods were employed. Franco-Browder et a l (1963) have examined chick somitic t i s s u e as early as stage 11 (40 hours - 2 -old) and found that a sulphated anionic glycosaminoglycan was present. By stage 23, several anionic glycosaminoglycans varying i n their degree of sulphation were present. None of these polyanions were i d e n t i f i e d other than by electrophoresis. - 3 -MATERIAL AND METHODS White Leghorn eggs incubated to the desired age at the Poultry Products B u i l d i n g (U.B.C.) were opened by breaking the crown (blunt end). The s h e l l membranes were peeled aside and the embryo removed onto a watch glass containing preheated Locke's so l u t i o n (Rugh, 1962). The extra-embryonic membranes were then removed and the embryos staged according to the scheme of Hamburger and Hamilton (1951). I. Preparation for Histochemical Analysis Stages ranging from 17 through 26 (56 - 120 hours incubation) were fixed for 48 hours i n 0.57. cfetylpyridinium bromide (CPB) i n 4% aqueous form-aldehyde (Williams and Jackson, 1956), rinsed one-half hour i n running tap water and dehydrated i n ethanol (EtOH). To f a c i l i t a t e subsequent embedding, block trimming, and sectioning, a l l embryos were stained 10 minutes with a 1% sol u t i o n of Eosin i n 90% EtOH. This treatment made the embryos r e a d i l y v i s i b l e i n p a r a f f i n . The embryos were cleared i n two changes of benzene (30 minutes each), impregnated f i r s t for one hour at 56° C with a 1:1 p a r a f f i n wax-benzene mix and then for one ad d i t i o n a l hour with a pure p a r a f f i n wax mixture (58:62), and subsequently embedded. Blocks were s e r i a l l y sectioned at 5 u and mounted on glass s l i d e s with albumin, heated at 44° C for 12 hours on a f l a t warming stage and stored at 4° C u n t i l stained. The ultimate ad-vantage of Eosin s t a i n i n g of the whole embryos l i e s i n the ease with which the s t a i n can be removed with 907. EtOH as the tiss u e sections are hydrated i n preparation for histochemical s t a i n i n g . Seven histochemical procedures were employed to detect glycosamino-- 4 -glycans (mucopolysaccharides). Periodic a c i d - S c h i f f (PAS) s t a i n i n g was employed to detect neutral polysaccharides (Scott and Clayton, 1953; Spicer, 1960). A combined A l c i a n blue/PAS (AB/PAS) s t a i n i n g sequence was used to d i f f e r e n t i a t e between neutral polysaccharides and anionic glycosaminoglycans (Spicer, 1960). Neutral polysaccharides are shown by red or magenta PAS s t a i n i n g , and anionic glycosaminoglycans by AB s t a i n i n g at pH 2.7. A purple s t a i n i n g r e a c t i o n indicated a mixture of the two types of polysaccharide. Gomori's Aldehyde Fuchsin (AF) as modified by Halmi was used to s t a i n for strongly a c i d i c sulphated glycosaminoglycans ( C u l l i n g , 1963). A combined AB-AF s t a i n i n g procedure was used to d i s t i n g u i s h between strongly and weakly a c i d i c glycosaminoglycans (Spicer and Meyer, 1960; Spicer, 1963; C u l l i n g , 1963). Weakly a c i d i c groups (carboxyl groups) having a strong a f f i n i t y for AB but not for AF w i l l s t a i n blue whereas strongly a c i d i c groups (sulphonate groups) having a strong a f f i n i t y for AF, but not for AB, w i l l s t a i n purple. Mixtures of weakly and strongly a c i d i c groups have an equal a f f i n i t y for both dyes and w i l l s t a i n v i o l e t . The c a t i o n i c thiazine dyes Toluidine blue (pH 4.5) (Vassar and C u l l i n g , 1959) and Azure A (pH 1.5 and 4) (Szirmai, 1963; Yamada, 1964; McConnachie and Ford, 1966) were both employed to d i f f e r e n t i a t e between strong-l y and weakly a c i d i c glycosaminoglycans. According to Szirmai (1963)- c a t i o n i c dye-binding i s of an e l e c t r o s t a t i c nature and w i l l therefore depend upon both the pH of the s t a i n i n g medium and the pK value of the anionic groups involved. Lowering the pH of the medium w i l l decrease the dissocia t i o n of carboxyl groups. This means that below pH 2, only sulphated groups are ionized and can bind dye while above pH 2 both sulphonate and carboxyl groups can bind dye. - 5 -Cationic thiazine dyes show increasing metachromasia depending on the degree of polymerization of dye molecules on glycosaminoglycans with free anionic groups (Sylven, 1954). The degree of dye polymerization which can be monomeric, dimeric or polymeric giving r i s e to orthochromasia (blue), beta metachromasia ( v i o l e t ) , or gamma metachromasia (red) r e s p e c t i v e l y (Michaelis and Granick, 1945; Michaelis, 1947), depends on both the surface charge density and on the a c i d i t y of the free anionic groups present on the glyco-saminoglycans (Sylven, 1954) . That i s , glycosaminoglycans with one anionic group per disaccharide unit w i l l show a higher degree of metachromasia i f that group i s a strongly a c i d i c sulphonate group rather than a weakly a c i d i c carboxyl group. Any purple metachromasia occurring with either T o l u i d i n e blue or Azure A st a i n i n g i s due to a mixture of orthochromasia and gamma metachromasia and i s not a true beta metachromasia (Schubert and Hamerman, 1956). Since only metachromasia due to strongly a c i d i c glycosaminoglycans can withstand dehydration (Michaelis, 1947; Sylven, 1954; Schubert and Hamerman, 1956) metachromatic a c t i v i t y was examined i n both water mounted and a i r dried t i s s u e sections. A c r i d i n e orange fluorescent s t a i n i n g was employed i n order to d i f f e r e n t i a t e between non-sulphated, mono-sulphated and poly-sulphated anionic glycosaminoglycans (Saunders, 1964). This method was based on the fi n d i n g that A c r i d i n e orange behaved l i k e a quaternary ammonium s a l t i n p r e c i p i t a t i n g anionic glycosaminoglycans. At neutral pH, Acridi n e orange could displace CPB from i o n i c carboxyl groups thereby s t a i n i n g only non-sulphated anionic glycosaminoglycans (t e s t s l i d e 1). At pH 3.2, both sulphonate and carboxyl groups could bind dye and th i s dye could then be d i f f e r e n t i a l l y removed from non-sulphated anionic glycosaminoglycans with 0.3 M NaCl ( t e s t s l i d e 2), and - 6 -from non-sulphated and mono-sulphated anionic glycosaminoglycans with 0.6 M NaCl (test slide 3). Enzyme extractions were followed by both AB/PAS and Azure A stain-ing: malt diastase was used to remove glycogen (Culling, 1963); testicular hyaluronidase to remove hyaluronic acid, chondroitin 4-sulphate (chondroitin sulphate A), and chondroitin 6-sulphate (chondroitin sulphate C) (Culling, 1963; Shackleford and Bentley, 1964). Sulphation was carried out prior to AB/PAS, AF, and Azure A stain-ing to induce sulphate esters on hydroxyl and carboxyl groups carried on polysaccharides (Kramer and Windrum, 1954; Spicer, 1960). Methylation and methylation-demethylation blocking and unblocking reactions (Spicer, 1960) were used in conjunction with AB/PAS, AF, and Azure A staining. Methylation blocks anionic groups by forming methyl esters on carboxyl groups and hydrolyzing sulphate esters. Demethylation unblocks carboxyl groups only by hydrolyzing the methyl esters. The acetylation and acetylation-deacetylation blocking and unblock-ing sequence (Pearse, 1960; Yamada, 1963) was carried out in conjunction with AB/PAS staining. Acetylation blocks hydroxyl groups by forming acetyl esters and may hydrolyze alpha amino groups. Deacetylation unblocks the hydroxyl groups only by hydrolyzing the esters. All the preceding techniques are detailed in Appendix A. For comparative purposes, only the axial tissues (neural tissue and notochord) between the extremities of the anterior and the posterior limb buds along with their lateral tissues - scleratome, myotome, dermatome and epidermis - were examined. The only tissue examined ventral to the notochord was the limb bud. - 7 -I I . Preparation for Glycosaminoglycan Extraction The preparation of a l l embryos used for glycosaminoglycan extraction was done under s t e r i l e conditions. F i v e embryonic age groups were prepared. These were 2% - 3 day old embryos (stages 17 - 18), 3% day old embryos (stages 21 - 22), 4% day old embryos (stages 24 - 25), 5 day old embryos (stage 26), and 5% day old embryos (stages 27 - 28). (See Figures 1 to 5). Embryos that did not f i t into the stages given after each age group were discarded as were any embryos that showed signs of ab-normal development or abnormal s i z e . Embryos of the appropriate age groups were transferred through two changes of warm (37 - 38° C) Locke's s o l u t i o n , decapitated, and placed i n a p a r a f f i n operating d i s h . As many as 12 embryos were brought to t h i s stage before proceeding further. A l l dissections were performed under a di s s e c t i n g microscope with needles ground to a f i n e point. Excised tissue was removed with a pipette, rinsed i n Locke's sol u t i o n and either acetone dried or pre-pared for freeze-drying. Excised preparations consisted of the a x i a l tissues from the rhombencephalon back to the posterior edge of the hind limb bud along with t h e i r l a t e r a l tissues (scleratome, myotome, dermatome and epidermis) (Figures 1 to 5). A l l tissues ventral to the notochord, including the limb buds, were discarded. The t i s s u e was passed through 3 to 4 changes^of acetone for acetone drying and de f a t t i n g immediately after excision, treated for a further 48 hours with acetone at 4° C, a i r dried over calcium c h l o r i d e i n a dessicator at 4° C, and stored. Tissues prepared for freeze-drying were placed i n dry pre-weighed polyethylene caps (inverted hollow stoppers from Pioneer PP Jax FLA), the - 8 -Figures 1 to 5 - Embryos (all x 5.3) incubated for 3 days (stage 17 -18) (Fig. 1), 3h days (stage 21 - 22) (Fig. 2), 4% days (stage 24 - 25) (Fig. 3), 5 days (stage 26) (Fig. 4), and 5\ days.(stage 27 - 28) (Fig. 5) along with the excised tissue from each stage (x 5.3) except stage 17 - 18. Some of these stages are shown in cross section in Figures 7 to 10. - 9 -excess H2O quickly drawn o f f with a pipette and the tissues weighed on a Mettler B gram-atic balance. The caps were quick-frozen i n a t r i c h l o r o -ethylene - dry i c e bath (-80° to -85° C) for 15 - 20 minutes* wrapped i n t i n f o i l and stored i n dry i c e u n t i l l y o p h i l i z e d . Tissues were l y o p h i l i z e d (Lowry, 1953) i n a Fisher Freeze-dryer using l i q u i d nitrogen. The l y o p h i l i z a t i o n period ranged from 18 to 25 hours depending on the age of the t i s s u e . In order to avoid wetting the dried ti s s u e by condensation from the a i r , a 2 hour e q u i l i b r a t i o n period was required to allow the specimen chamber to come to room temperature before the vacuum was broken. The l y o p h i l i z e d t i s s u e was weighed quickly to determine the loss due to water, transferred to a screw cap glass tube and stored at -20° C over D r i e r i t e i n a sealed container. For freeze-drying, polyethylene caps were preferred over glass containers because the tiss u e did not adhere to the p l a s t i c a f t e r drying and could e a s i l y be transferred to a glass con-tainer and pooled without any tissue l o s s . The Locke's solutions i n which the tissue was dissected and rinsed were pooled, centrifuged to remove the discarded tissue (at 1000 X g) and f i l t e r e d through Whatman No. 1 f i l t e r paper. This s o l u t i o n was tested to see i f any glycosaminoglycan was l o s t during d i s s e c t i o n . I I I . Glycosaminoglycan Extraction The method of extracting anionic glycosaminoglycans ( a c i d i c MPS) from embryonic t i s s u e i s based, with some modification, on the technique of S c h i l l e r jet a l (1961) for the separation of a c i d i c MPS from rat ski n . Acetone dried and defatted tissue was used d i r e c t l y , but l y o p h i l i z e d t i s s u e had to be defatted p r i o r to enzyme dig e s t i o n . - 10 -The method of extracting l i p i d was that of Sweeny et a l (1963). Lyophilized tissue was treated with col d l i g h t petroleum (bp. 20 - 40° C) (20 ml/gm dry weight of t i s s u e ) , agitated 0.5 hours at 4° C on a Rotary Extractor and the ether removed by ce n t r i f u g a t i o n (20,000 rpm for 15 minutes o o at 4 C). Extraction was continued overnight with fresh ether at 4 C, the tis s u e rinsed once with l i g h t petroleum, dried at 4° C in vacuo over para-f f i n wax chips, and weighed to determine the loss due to l i p i d . Dried defatted tissue was soaked i n 0.1 M sodium acetate buffer, pH 5.5, containing 0.005 M disodium versenate (Fisher S c i . Co.). The volume of buffer used ranged from 10 to 20 ml per gram dry wt. of t i s s u e . After soaking for 6 to 8 hours, the suspension was made 0.005 M with respect to cysteine-HCl (Fisher S c i . Co.) and treated at 60° C i n a water bath. A 0.5% (w/v) suspension of papain (Worthington Biochem. Corp.) i n the same sodium acetate buffer, containing versenate and cysteine-HCl (both 0.005 M), was o activated at 60 C for 30 minutes, and then added to the tiss u e suspension to a f i n a l concentration of 2 mg/gm dry wt. of t i s s u e . The contents were mixed gently and digestion continued at 60° C for 24 hours. I f any tiss u e remained undigested after 24 hours, more activated papain was added and d i -gestion continued another 24 hours. The digest was dialyzed against cold running tap ^ 0 at 4° C another 24 hours. A 1% solu t i o n of tr y p s i n (2 X r e c r y s t a l l i z e d ; Worthington Biochemical Corp.) i n 10 N HC1 was then pre-pared and enough t r y p s i n s o l u t i o n added to the contents of the d i a l y s i s bag to give a f i n a l concentration of 2 mg trypsin/gm dry wt. of t i s s u e . D i a l y s i s was continued for 3 days at 37° C against a 0.1 M phosphate buffer, pH 7.8, with d a i l y changes of buffer to maintain optimal conditions for tr y p s i n digestion. The content of the d i a l y s i s bag was then measured i n a graduated c y l i n d e r , cooled i n an i c e bath and made 10% with respect to t r i c h l o r o a c e t i c - 11 -acid (TCA) by adding ice cold 100% TCA dropwise with continuous s t i r r i n g . A f t e r 24 hours at 4° C, the p r e c i p i t a t e that formed was removed by c e n t r i -fugation at 30,000 rpm for 30 minutes at 4° C. The clear s o l u t i o n was dialyzed 24 hours against d i s t i l l e d 1^0 at 4° C and evaporated to 1 or 2 ml on a Buchler Instrument. The glycosaminoglycans were p r e c i p i t a t e d from t h i s concentrated s o l u t i o n according to the method of Pearce and Mathieson (1967) by adding an equal volume of 1.0 H ac e t i c a c i d , c r y s t a l l i n e potassium ace-tate to 5% (w/v), and 2 volumes of absolute ethanol (EtOH) . The p r e c i p i t a t e that had formed after 24 hours at 4° C was c o l l e c t e d by c e n t r i f u g a t i o n at 2200 rpm for 20 minutes, washed once with 5 ml 75% EtOH, twice with absolute EtOH and dried i n vacuo at 37° C. A second p r e c i p i t a t e c o l l e c t e d 24 hours af t e r adding 2 volumes of EtOH to the supernatant was treated the same way. IV. Separation of Extracted Glycosaminoglycans A v a r i e t y of methods are a v a i l a b l e for the separation and further p u r i f i c a t i o n of i s o l a t e d anionic glycosaminoglycans. These procedures employ column chromatography of polyanions on anionic resins (Ringertz and Reichard, 1960; S c h i l l e r et al,1961; Pearce et a l , 1968) or they require complexing the polyanions with quaternary ammonium compounds (Scott, 1960) followed by thei r subsequent d i s s o c i a t i o n from i n e r t substrates ( S c h i l l e r et al, 1961; Antonopoulos et a l , 1961; Antonopoulos et a l , 1964). In a l l cases, increas-ing s a l t concentrations are used to elute the polyanions. Attempts at complexing the polysaccharide with catylpyridinium c h l o r i d e followed by s e l e c t i v e d i s s o c i a t i o n of these complexes from i n e r t c e l i t e ( S c h i l l e r et aJL, 1961) and Whatman C e l l u l o s e powder columns (Antono-poulos et al, 1964) were unsuccessful i n t h i s i n v e s t i g a t i o n . - 12 -The method adopted was that described by Pearce et a l , (1968). Glycosaminoglycans were dissolved i n 8 M urea and adsorbed onto a micro-column of AG 1x2 (CI ) r e s i n (from Calbiochem) packed with an 8 M urea s o l u t i o n . The s i z e of the columns and th e i r maximum adsorption c a p a c i t i e s are discussed by Pearce et a l (1968); s u f f i c e i t to say that maintaining the column load at or below 1 umole hexuronic acid/100 mg r e s i n insures that overloading does not occur. Urea was f i r s t used by Tomlinson and Tener (1962) to minimize non-electrostatic binding of protein and nucleotides to anionic resins and was included i n a l l eluents by Pearce _et al (1968)because i t seemed to have a si m i l a r e f f e c t and gave a better separation of glyco-saminoglycans . Loaded columns were washed with 8 M urea which was c o l l e c t e d and analyzed to detect any breakthrough of polysaccharide due to overloading and/or ,faulty r e s i n packing. Glycosaminoglycans were eluted from the column either by a continuous l i n e a r NaCl/8 M urea gradient (0 to 3 M CI ) and c o l l e c t e d by a f r a c t i o n c o l l e c t o r (LKB Radi Rac) i n 5 ml. samples from which corresponding CI determinations were made with a Technicon Autoanalyzer (Mabry et al, 1966) or by 50 ml batch elutions using stepwise increments of NaCl i n 8 M urea. E f f l u e n t s were placed i n c e l l u l o s e viscose tubing (A. H. Thomas Co.) and dialyzed 24 hours against cold running tap t^O and another 24 hours against d i s t i l l e d t^O at 4° C. The dialysates were then concentrated and the glycosaminoglycans p r e c i p i t a t e d , rinsed, and dried as described previously. To demonstrate the possible heterogeneity of f r a c t i o n 2 (mono-sulphates) the calcium s a l t s of t h i s f r a c t i o n were subjected to EtOH f r a c t i o n -ation (Meyer et a l , 1956; Loewi and Meyer, 1958). F r a c t i o n 2 was dissolved i n 5% calcium acetate i n 0.5 M ac e t i c acid (2 to 3 mg polysaccharide/ml) - 13 -and fractioned at room temperature by adding EtOH stepwise i n 5% increments (from 5 to 607.) at one-half hour i n t e r v a l s and c o l l e c t i n g each p r e c i p i t a t e that formed. This f r a c t i o n a t i o n i s based on the p r i n c i p l e that calcium s a l t s of dermatan sulphate (chondroitin sulphate B) p r e c i p i t a t e at an EtOH concen-t r a t i o n below 30% whereas chondroitin 4-sulphate and chondroitin 6-sulphate (chondroitin sulphates A and C respectively) p r e c i p i t a t e i n the 30 - 60% EtOH region. V. Analysis of the Glycosaminoglycans Uronic acids were determined by the carbazole-sulphuric acid method of Dische (1947) and by the modified carbazole-borosulphuric acid method of B i t t e r and Muir (1962) (Appendix B). The presence of borate speeds up the colour reaction (Gregory, 1960; Francois et a_l, 1962) and increases the colour y i e l d due to iduronic acid of dermatan sulphate from 41% to 83% of that of chondroitin 4- and 6-sulphates. A r a t i o of readings from the two carbazole te s t s w i l l therefore i n d i c a t e i f iduronic acid i s present. Hexosamine was determined by the Elson and Morgan (1933) reaction as modified by Boas (1953) and Pearson (1963) (Appendix C). A l l uronic acid and hexosamine analyses were done i n duplicate. Paper chromatography was done according to Fischer and Nebel (1955) for hexosamines and by a modified Anderson and Karamalla (1966) method for hexuronic acids (Appendix D). Reducing sugars were located by s i l v e r s t a i n i n g (Trevelyan et a_l, 1950) (Appendix E ) . Electrophoresis was done i n a Shandon tank on 3x22 cm Whatman No. 1 paper s t r i p s using a O.lMpyridine-formate buffer at pH 3 (Mathews and Hinds, 1963). A D.C. p o t e n t i a l of 20 v o l t s applied 14 to 18 hours produced adequate - 14 -migration. Anionic glycosaminoglycans were stained with a l c i a n blue as described by Foster and Pearce (1961) (Appendix E ) . The following standards were employed: sodium glucuronate mono-hydrate from Argo Chemical D i v i s i o n ; galactosamine and galactose from Fisher S c i e n t i f i c Company; iduronic a c i d , dermatan sulphate, chondroitin 4-sulphate, and heparin from Dr. R. H. Pearce, Department of Pathology, U n i v e r s i t y of B r i t i s h Columbia; and commercial hyaluronic acid and d-gluco-samine hydrochloride from N u t r i t i o n a l Biochemicals Corporation. A flow sheet (Figure 6) has been prepared to show the procedure followed i n separating and analyzing the extracted glycosaminoglycans. V - 15 -Figure 6 - Flow sheet showing the procedure followed in separating the extracted glycosaminoglycan (MPS). The uraoles MPS listed along the right margin indicate the approximate amount of MPS required to do the corresponding test shown for each fraction. Flow Sheet Tests Applied Extracted MPS Extracted MPS Sample applied to -Paper electrophoresis an AG (CI") column - B i t t e r & Muir Carb. test and eluted with increasing s a l t concentrations 0.65 M NaCl f r a c t i o n 1 F r a c t i o n 1 -Paper electrophoresis - B i t t e r & Muir Carb. test -Dische Carb. test -Paper chromatography of hexuronic acids -Hexosamine reaction and paper chromatography 1.8 M NaCl *. f r a c t i o n 2 F r a c t i o n 2 -Same as f r a c t i o n 1 3.0 M NaCl V  f r a c t i o n 3 F r a c t i o n 3 -Paper electrophoresis - B i t t e r & Muir carb. test - 16 -RESULTS  Morphological Observations Morphological changes occurring i n the dorsal embryonic region during the developmental period under i n v e s t i g a t i o n (stages 17 through 26) are shown i n Figures 7 to 11. Only major changes are pointed out i n the captions accompanying each figure since d e t a i l e d descriptions of morpholo-g i c a l changes are a v a i l a b l e i n the e x i s t i n g l i t e r a t u r e (Hamilton, 1952; Nelsen, 1953; Patten, 1958). The morphological changes shown i n Figures 7 to 10 occurred at the l e v e l of the anterior limb buds i n each stage. Observations made on embryo sections cut at i n t e r v a l s of 200 u from the anterior limb buds post-er iad, showed a gradual decrease i n development along the anterior-posterior axis i n each stage. However, though the extent of development decreases along the a n t e r i o r - p o s t e r i o r axis, the d i f f e r e n c e between the anterior and posterior region was no greater i n a s i n g l e embryo than that observed between comparable regions of embryos i n stages 17 - 19, and not as great as the d i f f e r e n c e observed between embryos i n the older stages. Histochemical Analysis Results of AB/PAS ( A l c i a n blue/Periodic a c i d - S c h i f f ) s t a i n i n g are shown i n Table I . PAS (Periodic a c i d - S c h i f f ) p o s i t i v e material was present i n the cytoplasm of some of the c e l l s i n a l l areas studied. In the spinal ganglia, dermatome, and limb bud p r e - c a r t i l a g e c e l l s , the s t a i n i n g i n t e n s i t y i n the c e l l cytoplasm was f a i n t and did not increase with developmental age. - 17 -Figure 7 - Morphological appearance of a stage 17 embryo i n cross section (x 215) stained with AF. The dermo-myotomic plate made up of the dermatome (D) and the myotome (M) s t i l l p e r s i s t s . Other tissues indicated are: myocoel (m), notochord (N), neural crest (Nc) , neural tube (Nt) and scleratome (S). Note the e x t r a c e l l u l a r extensions between scleratome c e l l s and from the dermatome to the epidermis (arrow). Figure 8 - Morphological appearance of a stage 20 - 21 embryo in cross section (x 145) stained with AF. The myotome (M) has greatly increased i n s i z e i n r e l a t i o n to the dermatome (D) and has become d i s t i n c t from the scleratome (S) . The scleratome c e l l s have become oriented along the length of the myotome and are more c l o s e l y associated with the noto-chord (N) than i n stage 17. The marginal layer (mg) of the neural tube has formed and spinal ganglia (Sg) and nerves (Sn) are developing. - 18 -Figure 9 - Cross section through a stage 22 - 23 embryo (x 200) stained with AB-AF. The scleratome c e l l s (S).have become oriented and have f i l l e d the area around the notochord (N) and t h i c k e x t r a c e l l u l a r strands are associated with them (arrow) . Nerve f i b e r s now a r i s e from the mantle layer (mt) of the neural tube and pass into the now well formed spinal nerve (Sn). Other symbols used are: D = dermatome, M = myotome, Sg = spinal ganglia. - 19 -Figure 10 - Cross section through a stage 26 embryo stained with AF (x 132) . Clusters of scleratome c e l l aggregates (S) are c i r c u l a r l y oriented around the notochord (N) forming a vertebra centrum primordium. The dermatome (D) c e l l s are f i n e l y dispersed except for a few c e l l s along the myotome (arrow). The neural tube i s morphologically separable into a marginal layer (mg), mantle layer (mt), and ependymal layer (ed). Other symbols are: M = myotome, Nt = neural tube, Sg = sp i n a l ganglia, Sn = sp i n a l nerve. Figure 11 - Longitudinal section through a stage 26 embryo (x 132) stained with AB/PAS showing the cranio-caudal o r i e n t a t i o n of the myotomal c e l l s (M) and the intermyotomal l o c a t i o n of each centrum primordium (C). There was no d i r e c t connection between adjacent.myotomes. Other symbols used are: N = notochord, Sn = spinal nerve. Nt > Alcian blue/PAS Staining - TABLE I Area i n Cross Section 17 18 19 Stages 20-21 22-23 24-25 26 Notochordal sheath bl-H-++ bH-t-H- bl++++ bl-H-H- bl l 1 I I bll1 III b l l 1 1 1 I m++ m-H- m++ nrH- mft mH4 nri-H-Notochordal c e l l cytoplasm r-H- r+(r-H-f) r+(r-H-r) r+(r-r-H-) r+(r-H-r) r+(r+++) r+(r+++) " " surfaces bl-H- bl++ bl++ bl-H- bl+f bl-H- bl-H-Neural sheath bl+++ bl-J-H- bl-r-H- bl-HH- bl-f-H- bl+++ bl+-H-Neural tube c e l l cytoplasm r+ r+ r+ r+ r+(mf+) r+p+(nri-r) r+p+(nri4) " " surfaces bl-H- bl++ bl4+ bl-H- bl-r+(m++) bl-H-p+(m++) bl-H-p+(mH-) Spinal ganglia c e l l cytoplasm r+ r+ r+ r+ " " " surfaces bl++ bl-H- bl++ bl-H-Scleratome (a) Around notochord-cell cytoplasm r+ r+ r+ r++ r++ r++ r-H-- c e l l surfaces bl+ bl++ bl-H-f bl-H-r bl++-r bl-H-f-f bl-H-H-(b) Interjacent myotome & neural tissue - c e l l cytoplasm r+ r+ r+ r++ r++ r4+ r++ - c e l l surfaces &/or Matrix bl+ bl-H- bl-r-H- bl-H-H- bl-H-M- bl-H-H- bl-H-H-Myotome c e l l cytoplasm r+ r+ r+ r+ r+(r-r-r+) r+(r-H+) r+(r-r-H-) " " surfaces &/or matrix bl-H- bl+ bl+ bl+ bl+ bl+p+ bl-rp+ Dermatome c e l l cytoplasm r+ r+ r+ r+ r+ r+p+ r+p+ " " surfaces &/or matrix bl-H- bl-H- bl++ bl-H- bl-H- bl++p+ bl+p++ Limb bud pre-cartilage c e l l cytoplasm r+ r+ r+ r+ r+ Limb bud c e l l surfaces &/or matrix bl-H-f bl+++ bl+++ bl-H-H- bl-H-H-I Abbreviations used i n TABLES I through VII : b l - blue r = red - no stain present m = magenta v = v i o l e t + very faint colour p = purple MM very deep colour - 21 -In most of the myotomic c e l l s the i n t e n s i t y of PAS s t a i n i n g i n the cyto-plasm was also f a i n t , but from stage 22 on, some c e l l s stained a deep red. These deeply s t a i n i n g c e l l s were located i n areas of the myotome where a l c i a n o p h i l i c extensions projected i n from the adjacent scleratome ( F i g . 12). A few c e n t r a l l y located c e l l s i n the notochord also reacted strongly to PAS s t a i n i n g . In both the myotome and the notochord these deeply s t a i n i n g c e l l s are indicated i n Table I by placing the symbols representing them within brackets. The cytoplasm of the ependymal c e l l s making up the fl o o r of the ce n t r a l canal, along with the marginal layer immediately below i t , stained magenta with PAS whereas the other c e l l s i n the neural tube stained f a i n t l y red. This diff e r e n c e was apparent from stage 22 on when the regions of the neural tube had become morpologically d i s t i n c t . A r i n g of magenta also occurred along the inside of the notochordal sheath. Only within the cytoplasm of the scleratome c e l l s did the i n t e n s i t y of PAS st a i n i n g increase with embryonic age. The fibrous layer between the neural and notochordal sheaths also stained deeply red wherever these were separated by stage 26. E x t r a c e l l u l a r matrix i n a l l areas examined was a l c i a n o p h i l i c and stained p a r t i c u l a r l y deeply i n the notochordal and neural sheaths. In the notochord, neural tube, spinal ganglia, myotome and dermatome, the i n t e n s i t y of AB ( A l c i a n blue) s t a i n i n g remained unchanged with developmental age. In the neural tube, the c e l l s stained equally i n the early stages, but when the ependymal, mantle and marginal layers formed, only the ependymal layer main-tained the i n t e n s i t y of s t a i n i n g present i n the e a r l i e r stages. The other two layers became less intense i n s t a i n i n g capacity except i n the immediate area where nerve f i b e r s extended out to the spinal nerves from the mantle lay e r . The nerve f i b e r s i n the spinal nerves had an a f f i n i t y for AB equal - 22 -Figure 12 - Dark red s t a i n i n g c e l l s (arrows) demonstrable i n the myotome (M) and notochord (N) after AB/PAS s t a i n i n g . Stage 26 embryo (x 240). Other symbols used are defined i n Figure 10. Figure 13 - PAS st a i n i n g of a stage 26 embryo (x 150). Note that the scleratome (S) c e l l matrix stains red as well as the ce n t r a l notochordal c e l l s and sheath. Other symbols used are defined i n Figure 10. - 23 -to that of the marginal layer of the neural tube. The purple colour observed at c e l l surfaces was confined to the dermatome c e l l s condensed along the dorsal part of the myotome, to a few e x t r a c e l l u l a r extensions leading into the myotome from the dermatome, and to the c e l l processes i n the marginal layer of the neural tube. The increased a f f i n i t y for AB of the e x t r a c e l l u l a r matrix of the scleratome and limb buds during development was rela t e d to the aggregation and o r i e n t a t i o n of c e l l s r e s u l t i n g i n close apposition of c e l l surfaces and a greater deposition of c e l l matrix. The e x t r a c e l l u l a r matrix i n the scleratome was also PAS p o s i t i v e when the PAS sequence was used alone ( F i g . 13), but with the combined AB/PAS sequence t h i s p o s i t i v e material was blocked or masked by the AB (Spicer, 1960). Malt diastase reduced the i n t e n s i t y of PAS st a i n i n g i n the myotome and scleratome but PAS p o s i t i v e material was not diastase l a b i l e i n other t i s s u e s . Also, the deeply PAS p o s i t i v e c e l l s of the myotome were not reduced i n i n t e n s i t y by diastase treatment. A c e t y l a t i o n removed PAS p o s i t i v e material i n a l l areas and part-i a l l y removed AB s t a i n i n g a l s o . ( F i g s . 14, 15). Deacetylation restored PAS s t a i n i n g ( F i g . 15) and most of the a l c i a n o p h i l i a , except i n the s c l e r a -tome and myotome where both stains were only p a r t i a l l y restored. The magenta r i n g i n the notochordal sheath and the magenta i n the v e n t r a l neural tube was blocked on a c e t y l a t i o n and restored on deacetylation. Sulphation almost t o t a l l y blocked a l c i a n o p h i l i a i n a l l t i s s u e s . Only c e l l surfaces of the myotome and scleratome and the notochordal sheath stained f a i n t l y blue a f t e r sulphation. The e f f e c t s of the methylation-demethylation sequence on AB/PAS sta i n i n g are shown i n Table I I . Following raethylation, AB st a i n i n g was completely blocked on the notochordal, neural, g a n g l i a l , and dermatomal c e l l M e t h y l a t i o n - s a p o n i f i c a t i o n with A l c i a n blue/PAS - TABLE II Area i n Cross Methy la t ion Methyl ation-Demethylation Sect ion Stages 17 18 19 20 22 24 26 17 18 19 20 22 24 26 21 23 25 21 23 25 Notochordal sheath bl-H- bl++ bl-H- bl+ bl-H bl+ bl+ bl+++ bl-H-f bl+-H- bl-H-f bl-H-+ bl-H-f bl+++ m++ ra++ mH- iaH m+++ nri-H- nri-H- m++ m++ m H roH-r- nrt-H- nri-H- nrt-H-Notochordal (a) c e l l cytoplasm r+ - - r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ (r++) (r++) (r++) (r-H-) (r-H-) (r-H-) (r+++) <r-H+) (r+++) (r-H-t-) (r+H-) (r-W-) (b) c e l l surfaces - - - - - .7 bl+ bl+ bl+ bl+ bl+ bl+ bl+ Neural sheath r+ r+ r+ r+ r+ r+ r+ bl+++ bl-f-H- bl+++ bl+++ bl+++ bl-H-t- bl-r-H-Neural tube (a) c e l l cytoplasm r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ (m++)(m++)(m++) (m++) (mH-) (m++) (b) c e l l surfaces bl+ bl+ bl+ bl+ bl+ bl+ bl+ (m++) (m++) (#+) (m++) (m++) (m++) Sp ina l gangl ia (a) c e l l cytoplasm - - - - r+ r+ r+ r+ (b) c e l l surfaces - - bl+ bl+ bl+ bl+ Scleratome (1) Around notochord - r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ (a) c e l l cytoplasm (b) c e l l surfaces &/or matrix - - bl+ bl+ bl+ bl+ bl+ bl+ bl+ bl-H- bl-H- bl-f+t bl-H+ bl-H-(2) Interjacent myotome & Neural t i s sue (a) c e l l cytoplasm - r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ r+ (b) c e l l surfaces &/or matrix — bl+ bl+ bl+ bl+ bl+ bl+ bl+ bl+ bl+ bl-f-Hj bl-H+ bl-H-TABLE II (Cont'd.) Area i n Cross Section Methylation Methyl a t ion-D emethyla t i o n 17 18 19 Stages 20 22 21 23 24 25 26 17 18 19 Stages 20 21 22 23 24 25 26 Myotome (a) c e l l cytoplasm (b) c e l l surfaces &/or matrix - r+ r+ r+ r+ (r -H-bl+ r+ +) (r++ bl+ r+ +) (r-H-l-) bl+ r+ bl+ r+ bl+ r+ bl+ r+ bl+ r+ (r-H-4-) bl+:: r+ (r-t-H-) bl+p+ r+ (r-H-f) bl+p+ Dermatome (a) c e l l cytoplasm (b) c e l l surfaces &/or matrix r+ bl+ - - r+ - r+ - r+ bl -H-r+ bl++ r+ bl -H-T± bl -H-r+ bl++ r+p+ bl-H-p+ r+p+ bl-f+p+ Limb bud pre - c a r t i l a g e (a) c e l l cytoplasm (b) c e l l surfaces fit/or matrix r+ bl+ r+ bl+ r+ bl+ r+ bl+ r+ bit-r+ bl+ r+ bl+ r+ bl - H -r+ bl-H-r r+ bl -H-f - 26 -Figures 14. 15 and 16 - Unblocked co n t r o l (Figure 14) (x 128), a c e t y l -ated (Figure 15) (x 157) and deacetylated (Figure 16) (x 157) AB/PAS stained stage 24 -25 sections. The red and magenta colour i n the notochord (N) and the ventral neural tube (Nt) was removed on a c e t y l a t i o n and restored after deacetylation. AB s t a i n i n g was also p a r t i a l l y blocked and unblocked by t h i s treatment. Other symbols used are defined i n Figure 10. Figures 17, 18 and 19 - Unblocked (Figure 17), methylated (Figure 18), and demethylated (Figure 19) AB/PAS stained stage 26 sections,(x 100). AB. was almost com-p l e t e l y blocked following methylation whereas PAS p o s i t i v e material was more pronounced i n the notochordal and neural sheaths and along the myotomal-scleratomal junction. Demethyl-ation p a r t i a l l y restored AB s t a i n i n g . Symbols used are defined i n Figure 10. - 27 -surfaces and almost completely blocked on the myotomal and early scleratomal and limb bud p r e - c a r t i l a g e c e l l surfaces. The scleratome and limb bud pre-c a r t i l a g e c e l l matrix was f a i n t l y AB p o s i t i v e i n stages 22 to 26, as was the notochordal sheath ( F i g s . 17, 18). Demethylation p a r t i a l l y or f u l l y restored a l c i a n o p h i l i a to a l l tissues ( F i g s . 17, 19) (compare the demethylation data from Table II with that from Table I ) . The loss of a l c i a n o p h i l i a incurred a f t e r the methylation-saponification sequence was most pronounced in the notochordal sheath and i n the matrix of the scleratome and limb bud pre-c a r t i l a g e c e l l s . In these tissues c e l l s were aggregating i n preparation for c a r t i l a g e formation and could therefore presumably contain sulphated material. A s l i g h t loss of s t a i n i n g capacity occurred i n the notochordal, neural and g a n g l i a l t i s s u e s , but i n the myotome and dermatome a l c i a n o p h i l i a was f u l l y restored. PAS s t a i n i n g was not affected very much by methylation and sapon-f i f c a t i o n (Table I I ) , other than that the magenta of the notochordal sheath was more pronounced af t e r the treatment and the neural sheath stained red rather than blue. Spicer (I960) has also observed that methylation w i l l increase PAS s t a i n i n g previously masked by AB. The e f f e c t s of t e s t i c u l a r hyaluronidase i n s a l i n e on AB/PAS s t a i n -ing showed that a l c i a n o p h i l i a was l a b i l e to t h i s enzyme i n a l l tissues ( F i g s . 20, 21). This loss of a f f i n i t y for AB was most marked in the s c l e r a -tome c e l l matrix around the notochord. In the other areas, only a f a i n t s t a i n i n g reaction occurred compared to control sections treated with s a l i n e only. A summary of the r e s u l t s of using AF (Aldehyde Fuchsin) s t a i n i n g i s presented i n Table I I I and shows the general increase i n colour i n t e n s i t y with embryonic age. The cytoplasm of the myotome c e l l s stained weakly purple compared to the scleratome c e l l s . The neural t i s s u e c e l l cytoplasm - 28 -Figures 20 and 21 - E f f e c t of t e s t i c u l a r hyaluronidase on AB/PAS s t a i n -ing of a stage 24 - 25 embryo (x 145) . Hyaluronidase i n s a l i n e reduced AB st a i n i n g i n a l l tissues (Figure 21) when compared to a sa l i n e treated control (Figure 20). Symbols used are defined i n Figure 10. Aldehyde Fuchsin - TABLE I I I Area i n Cross Section 17 18 Stages 19 20-21 22-23 24-25 26 Notochordal sheath pi !" II pi H r p-H-H- p+-r-r+ pi 1 1 1 p-H-H- p-H-H-Notochordal c e l l cytoplasm P-H- P++ P-H- p++ p++ p-H- p++ " " surfaces p++ P++ p-H+ P+-H- p+H- p 4*1' "|- p+H-Neural sheath p - m - P+++ p+++ p+++ p+++ p+-H- p-H-r Neural tube c e l l cytoplasm p+ P+ P+ P+ P+ P+ P+ " " " surfaces p4+ p-f+ p++ P++ P++ P++ p++ Spinal ganglia c e l l cytoplasm p+ P+ P± p+ " " " surfaces p++ p++ p++ p++ Scleratome c e l l s around notochord (a) C e l l cytoplasm p4+ P++ P++ p++ p-H-f p+4-r pH~H* (b) C e l l surfaces &/or matrix P++ p+-H- P+-H- p+H- pl r H p-H-H- p+4-H-Scleratome c e l l s interjacent myotome & neural t i s s u e (a) C e l l cytoplasm P+ P+ P+ p++ P++ p++ P++ (b) C e l l surfaces &/or matrix p++ P++ p+f p-H-f p-HH- pt 1 1 Myotome c e l l cytoplasm - - • P± p+ P+ P± P± " " surfaces &/or matrix p-H- P+ P+ P++ p++ P++ p4-r-(P+++) (P+++) Dermatome c e l l cytoplasm P+ P+ p++ P++ P++ PH-" 11 surfaces &/or matrix P++ p++ p-H-f p++f p+H- p+++ p-H-4-Limb bud pr e - c a r t i l a g e (a) C e l l cytoplasm fH-f p++ P++ p-H- p-H-(b) C e l l surfaces &/or matrix p-H-r P+-H- p+++ p-H-r P++-H-- 30 -was likewise weakly purple. On the other hand, scleratome and limb bud c e l l s stained deeply purple. In a l l cases, c e l l surface coats were deeper purple than c e l l cytoplasm (see F i g s . 7, 8, 10), and t h i s a f f i n i t y for AF increased with developmental age as more c e l l matrix was deposited. Both the neural and notochordal sheaths stained intensely purple as did some c e l l surfaces i n the myotome of stage 19 to 21 embryos. Methylation com-p l e t e l y removed s t a i n i n g from a l l stages examined ( F i g s . 22, 23). I t was restored only very f a i n t l y i n the neural and notochordal sheaths, i n the limb buds, and i n the scleratome following demethylation ( F i g . 24). Sul-phation greatly increased AF st a i n i n g a c t i v i t y over that of control sections ( F i g . 25) i n a l l areas. Results of a combined AB-AF st a i n i n g sequence are tabulated i n Table IV. The c e l l cytoplasm of a l l tissues examined stained purple. M a t e r i a l i n the neural and notochordal sheaths was a l c i a n o p h i l i c through a l l the stages although purple also appeared i n the neural and notochordal sheaths a f t e r stages 18 and 19 re s p e c t i v e l y . The purple s t a i n i n g material i n the neural sheath was, however, confined to the base of the sheath immediately overlying the notochord. A l l c e l l surfaces i n the neural tube and notochord stained v i o l e t . In the myotome and dermatome the surface coats also stained v i o l e t , but with increasing embryonic age, t h i s colour was gradually re-placed by purple. The c e l l matrix of the scleratome and limb buds was alciano-p h i l i c u n t i l stage 24 except for a b i t of v i o l e t i n the scleratome around the notochord. From stage 24 on, however, deep v i o l e t appeared i n the cl u s t e r s of scleratome c e l l aggregates that had enci r c l e d the notochord and a purple s t a i n i n g was beginning to appear i n the accumulating matrix of the limb bud p r e - c a r t i l a g e c e l l s . Areas stained with T o l u i d i n e blue at pH 4.5 and the r e l a t i v e - 31 -Figures 22, 23 and 24 - AF stained stage 24 - 25 sections (x 170) control (Figure 22), methylated (Figure 23), and demethylated (Figure 24). Methylation t o t a l l y blocked AF s t a i n i n g (Figure 23) and demethylation only restored f a i n t traces of the s t a i n (Figure 24). Symbols used are defined i n Figure 10. Figure 25 - E f f e c t of sulphation on AF s t a i n i n g of a stage 24 - 25 embryo (x 170) . Compare t h i s f i g u r e with Figure 22. Sulphation greatly increased the a f f i n i t y of the tissues for AF. Symbols used are defined i n Figure 10. A l c i a n blue - Aldehyde Fuchsin - TABLE IV Area i n Cross Section 17 18 Stages 19 20-21 22-23 24-25 26 Notochordal sheath bl+++ bl-H-f bl+++ bl-H-f bl-H- bl+++ bl4-H-P++ P++ P++ p++ Notochordal c e l l cytoplasm P+ P+ P+ P+ P+ P+ P+ " " surfaces v+p+ v+ v+ v+ v+ v+ v+ Neural sheath bl+++ bl-H-f bl-H-f bl-HH- bl+++ bl+++ p+++ base of sheath p++ P++ P++ P++ P++ Neural tube c e l l cytoplasm P+ P+ P+ P+ p+ P+ P++ " " surfaces v+ v+ v+ v+ v+ v+ v+bl+ Spinal ganglia cytoplasm P+ bl+p+ " " surfaces bl+ bl+ Scleratome c e l l s around notochord (a) C e l l cytoplasm j>++ p-H- P++ p+f p-Hh p++ P++ (b) C e l l surfaces &/or matrix bl-H- bl++v+ bl++ bl-H- bl++v+ V + + + V + + + Scleratome c e l l s interjacent myotome & neural t i s s u e (a) C e l l cytoplasm p++ p++ P 4+ p++ P++ P++ p+bl+ (b) C e l l surfaces &/or matrix bl-H- bl++ bl++ bl-H- bl+-H- bl+++ bH-H-Myotome c e l l cytoplasm P+ P+ P+ P+ p++ p++ P++ " " surfaces &/or matrix v+ v+bl+ v+ v+p+ v+p+ P++ Dermatome c e l l cytoplasm p-H- p-H- p++ p++ P++ p-H- P+ " " furfaces &/or matrix v-H-bl-H- v-H- p+++ p+++ p+++ V++P++ v++p++ Limb bud p r e - c a r t i l a g e (a) C e l l cytoplasm p++ p++ p-H- p++ p++ (b) C e l l surfaces &/or matrix bl-H-f- bl+++ bl-H-4- bl+++p+ bl-HH-p+ - 33 -i n t e n s i t y of the i metachromasia remaining after a i r drying and mounting are shown i n Table V. Scleratome and limb bud pr e - c a r t i l a g e c e l l s showed a steady increase i n free anionic groups with developmental age, as might be expected of c e l l s destined to form c a r t i l a g e . By stage 24, traces of strong gamma metachromasia (red) occurred immediately beneath the notochord i n the cl u s t e r s of scleratome c e l l aggregates making up the centra >. primordia of the vertebrae. By stage 26, t h i s strong metachromasia occurred around the notochord ( F i g . 26) and also i n the limb bud pr e - c a r t i l a g e c e l l matrix. The notochordal sheath i t s e l f , however, remained deeply purple through a l l the stages examined. The ventral neural sheath and notochordal c e l l s of older embryos stained only weakly purple. In the myotome, dermatome and neural t i s s u e , no metachromatic a c t i v i t y was present a f t e r a i r drying. Results of Azure A st a i n i n g at pH 4 and pH 1.5, observed i n d i s t -i l l e d water, are presented i n Table VI. As already pointed out, the purple colour i s due to a mixture of orthochromasia (blue) and gamma metachromasia (red). In Table VI, the presence of both colours i n most areas meant that some c e l l s were orthochromatic (blue) and others metachromatic (purple). In the notochordal sheath the ce n t r a l area stained orthochromatic with purple metachromasia at the inner edge. In the neural sheath, metachromasia occurred only i n the ven t r a l portion of the sheath immediately overlying the noto-chord. At pH 4, there was a steady increase i n orthochromasia with develop-mental age i n a l l tissues except the notochordal and myotomal c e l l s . Ortho-chromasia was most intense i n the notochordal and neural sheaths, epidermis, and spinal ganglia. In comparison, the neural c e l l s , scleratome c e l l s , and limb bud pr e - c a r t i l a g e c e l l s were moderately orthochromatic with the noto-chordal, dermatomal, and myotomal c e l l s l east orthochromatic. The greatest metachromatic response was e l i c i t e d i n the notochordal sheath and i n the - 34 -Figure 26 - T o l u i d i n e blue metachromasia i n a stage 26 centrum primordium of a vertebra (x 128). Symbols used are defined i n Figure 10. Figure 27 - A stage 24 - 25 water mount showing Azure A metachromasia at pH 4 i n the scleratome c e l l s surrounding the notochord (x 128). Symbols used are defined i n Figure 10. 3 T o l u i d i n e Blue Metachromasia - TABLE V Area i n Cross Section 17 18 19 Stages 20-21 22-23 24-25 26 Notochordal sheath P++ p-H- P++ p+++ P+++ P++++ P++++ Notochordal c e l l s - P± P± P± P+ P+ P+ Ventral Neural sheath P± P+ P+ P+ P+ P+ P+ Scleratome c e l l s (a) around notochord P± P+ P+ p++ p++ p++r+ p-H-r++ (b) interjacent myotome & neural tissue P± P± P± P+ P+ P+ P+ Limb bud pr e - c a r t i l a g e c e l l s p++ p-H- p++ p++r++ The purple (p) colour represents a mixture of orthochromasia and gamma metachromasia and i s not a true beta metachromasia. The red colour represents true gamma metachromasia. Azure A Staining - TABLE VI pH 4 pH 1.5 Area i n Stages Stages Cross Section 17 18 19 20 22 24 26 17 18 19 20 22 24 26 21 23 25 21 23 25 Notochordal sheath P+ p-W- P-H- p+++ p H—f- p-f-H- P+ P+ P+ p-H- P+± P+± P-H-bl-HH- bl+4-r bl-H-r bl-H-r bH-H-+ bl-H-H- bl++++ bl-H- bl-H- bl-H- bl-H-r bl-H-f bl-H-f bl+H-Notochordal c e l l s P+ P+ p-H- P++ p-H-f p-H- P++ P± P± P± P± P± P+ P+ bl-H- bl-H- bl-H- bl4+ bl-r+ bl++ bl-H- bl+ bl+ bl+ bl+ bl-r bl+ bl+ Ventral neural sheath P± P± P± p-H- p-H- p-H- P+± - - - P± P± P± P± bl+++ bl-H-r bl+++ bl-H-f bl+++ bl-H-f bl-H-H- bl+ bl+ bl+ bl-H- bl-H- bl-H- bl++ Neural c e l l s P+ P+ p-H- P+± P+± P+± P+± - - - P± P± P± P± bl-H- bl-r-H- bl-H-+ bl-t-H- bl-HH- bl-H-r bl+H- bl+ bl+ bl+ bl-r bl+ bl-r+ bl++ Spinal ganglia P+± P+± P+± P+± - - - -bl-H-r bl-H-f bl-H-H- bl-H-H- bl-H- bl-H- bl-H- bl+++ Scleratome c e l l s (a) around notochord P+ P++ p++ p++ p+++ p-H-f r+H- P+ P+ P+ p-H- p++ P-H- r-H-bl-H- bl-H- bl-H- bl-H- bl+++ bl-H-r bl-H-r bl+ bl+ bl+ bl-r bl-H- bl-H; bl-H-(b) interjacent myotome & neural tissue P+ P+± P+t p++ P++ p++ pH- - - P+ P+ p++ P+ P+ bl-H- bl-r++ b W + bl+++ bl-H+ bl-H-f- bl+++ bl+ bl+ bl-r bl-r bl-H- bl++ bl++ Myotome P± P± P+ P+ P+± P+± P+± bl-H- bl+ bl+ bl+ bl-H- bl-r bl-H- bl+ bl+ bl+ bl+ bl+ bl+ bl+ Dermatome P± P+ P++ P+± P+± P+± bl+f bl-H- bl-r+ bl-H- bl4+f bl-H- bl+++ bl+ bl+ bl+ bl+ bl+ bl+ bl -r Epidermis bl-H- bl-H-r bl-H- bl-H-f bl-H-r bl-H-r bl-H-H- bl+ bl+f bl-H- bl-H- bl-H- bl++ bl-H-Limb bud p+ p++ P++ r++ P+ P+ r-r bl-f-H- bl-f+f bl+H- bl-H-r bl-r+ bl-H- bl-H-- 37 -scleratome c e l l s around the notochord ( F i g . 27). By stage 26, centrum primordia were strongly metachromatic (red) as was the matrix i n the limb bud p r e - c a r t i l a g e area. In a l l other areas metachromatic a c t i v i t y was weak to moderate i n comparison. The epidermis was not metachromatic. I f sections were a i r dried, cleared i n x y l o l and mounted i n a manner sim i l a r to sections stained with To l u i d i n e blue, the metachromatic response was reduced almost to the point of e x t i n c t i o n i n a l l areas except the scleratome c e l l s around the notochord and the notochordal sheath. Even i n the scleratome, meta-chromasia was greatly reduced. Water mounted sections stained i n Azure A at pH 1.5 were much less r e a c t i v e than sections stained at pH 4. Ortho-chromasia was markedly reduced i n a l l tissues and almost t o t a l l y removed from the notochordal and myotomal c e l l s . Metachromatic a c t i v i t y was also reduced and confined almost e n t i r e l y to the notochordal sheath and c e l l s , scleratome and limb buds. Traces of metachromasia also occurred i n the neural t i s s u e and sheath of the older stages. I f sections were a i r dried and mounted, metachromatic a c t i v i t y was reduced to the immediate area around the notochord. Methylation-saponification t o t a l l y blocked a l l Azure A orthochrom-asia and metachromasia at pH 1.5. At pH 4, methylation blocked a l l st a i n i n g , but demethylation p a r t i a l l y restored orthochromasia to a l l areas. Meta-chromasia was only f a i n t l y restored after demethylation at pH 4 and then only i n the older stages. Following sulphation, orthochromasia with Azure A at pH 4 became so intense that metachromasia was almost completely o b l i t e r a t e d . Only a reddish-purple background heavily o v e r l a i d by blue could be discerned. De-creasing the s t a i n i n g period from 30 to 5 minutes did not decrease the intense orthochromasia. At pH 1.5, sulphation increased the i n t e n s i t y of - 38 -metachromasia whereas the orthochromasia remained the same or was s l i g h t l y reduced. At both pH values, metachromasia with Azure A was l a b i l e to t e s t i -cular hyaluronidase. Following enzyme treatment only traces of s t a i n remained i n the notochordal sheath and surrounding scleratome c e l l s . Ortho-chromatic a c t i v i t y was reduced to the greatest extent i n the notochordal, scleratomal, epidermal, and limb bud pr e - c a r t i l a g e c e l l s than i n any other t i s s u e s . These r e s u l t s were i n agreement with those obtained using AB/PAS following hyaluronidase d i g e s t i o n . Malt diastase had no apparent e f f e c t on Azure A s t a i n i n g at either pH. Results of Saunders Acridine orange fluorescence technique are tabulated i n Table VII. An orange-red fluorescence could be demonstrated i n a l l tissues examined i n test s l i d e 1. Fluorescence increased with embryonic age i n a l l areas although i n the notochordal sheath and epidermis i t was already intense at stage 17. The whole neural sheath fluoresced. Of the neural c e l l s , only the ependymal layer fluoresced i n the early stages but as the mantle layer developed, c e l l s located i n the area where nerve f i b e r s projected v e n t r a l l y into the spinal nerves also fluoresced. In the s c l e r a -tome, fluorescence interjacent to the myotome and neural ti s s u e was most intense along the myotome i n t e r f a c e while i n the rest of the do r s a l l y extend-ing scleratome c e l l s fluorescence was f a i n t . Myotome c e l l s of early embryonic stages were least r e a c t i v e . In test s l i d e 2, red fluorescence increased i n i n t e n s i t y with developmental age i n most areas where i t occurred. The notochordal sheath and epidermis again fluoresced most intensely. In the neural sheath fluorescence was confined to the ve n t r a l part overlying the notochord. Within the neural tube, only the v e n t r a l mantle c e l l s and the ependymal c e l l s making A c r i d i n e Orange Fluorescence - TABLE VII S l i d e 1 Sl i d e 2 Sl i d e 3 Area i n Stages Stages Stages Cross Section 17 18 19 20 21 22 24 23 25 26 17 18 19 20 21 22 23 24 25 26 17 18 19 20 21 22 23 24 25 26 Notochordal (a) Sheath 444 44-4 1' 4""i"'l'4' 1 1- t 1 r < r T ++ 4+ ++ +++ +++ 4+ + 4 + + + + + (b) C e l l s 44- 44- 4+ 44- +++ ++++ "fr" ^  1 1' 4 ++ ++ 44 44- 4+ 44 - + + + + + + Neural tube (a)Sheath 4-4 44- +++ 44-4 -f-H- 44-44 ++++ + + ++ 44 4+ 4+ 44- 4 4 + + 4 + 4 ( b ) C e l l s 44- 44 44- 44- 444- +++ 444- + + + + 4 4+ 44- + 4 - + 4 + + Spinal g a n g l i a l c e l l s 44- 444- 4444 44-H- + 4+ 44- 44- + 4 4 + Scleratome C e l l s (a) Around noto-chord 4 44- ++ 4+ ++ 4-4+ +++ + + + 4+ "h'\' 1" 1 \ 1" — + + 4 4 (b) Interjacent Myotome & Neural t i s s u e + 44- ++ 44- -H> 444 — + + + 4+ 44- 444- mm „ 4 4 4 Myotomal C e l l s + + + 4 44- 44- 44+ + Dermatomal C e l l s 44- 44- ++ ++ 44- 44- 1 1 1 T T T + + + + - + + - 4 4 + - 4 -Epidermis 44-4 +++ 4444 44-H- 44-4+ + + + +44 4++ +++ 444- 4 4 + + 4 + 4-- 40 -up the f l o o r of the c e n t r a l canal fluoresced. In the scleratome f l u o r -escence equalled that i n test s l i d e 1 i n the older stages but i n the younger embryos i t was not as intense and apparently absent i n stage 17. Fluorescence i n the dermatome was also markedly reduced compared to test s l i d e 1 and completely absent i n the myotome u n t i l stage 26. In test s l i d e 3, only a d i f f u s e red fluorescence could be discerned. What fluorescence there was appeared to be concentrated i n the notochordal sheath and c e l l s , s p i n a l ganglia, and epidermis. • In other areas, the fluorescence was very f a i n t i f not absent. Biochemical Analysis Preliminary examinations u t i l i z i n g 122 three and one-half day old (stage 21 - 22) and 11 f i v e and one-half day old (stage 27 - 28) acetone dried embryos indicated a uronic acid content of 0.72 and 0.48 umoles per sample r e s p e c t i v e l y . These r e s u l t s determined the number of embryos used i n the biochemical analysis (see F i g . 6) and also fixed the acetate buffer con-centration employed during the digestion process at 10 ml per gram dry weight of t i s s u e . Preliminary t e s t s , using l y o p h i l i z e d stage 27 - 28 preparations, also established that the high l i p i d content i n t e r f e r e d with the extraction procedure. D i r e c t papain digestion of l y o p h i l i z e d embryonic tissue resulted i n the formation of large f a t t y droplets which prevented complete tis s u e h y d r o l y s i s . Removal of t h i s l i p i d by shaking with two volumes of cold d i e t h y l ether or by pretreating the l y o p h i l i z e d t i s s u e with l i g h t petroleum removed enough l i p i d to allow complete tis s u e h y d r o l y s i s . A uniform extraction procedure was therefore adapted for a l l sub-- 41 -sequent age groups analyzed. Tissues were l y o p h i l i z e d and defatted with l i g h t petroleum rather than using acetone. I. E x traction and Separation of the Glycosaminoglycans The amount, weight, and other properties of the excised embryonic ti s s u e employed from each stage are tabulated i n Table VIII. The average dry weight of i n d i v i d u a l excised tissues increased from 0.21 mg i n stage 17 - 18 embryos to 2.74 mg i n stage 27 - 28 embryos. This increase i n weight was accompanied by a corresponding increase i n the s i z e of the excised ti s s u e with age (see F i g s . 1 to 5). The water content ranged from 90% to 91% of the wet weight and the l i p i d content from 7% to 11%, of the dry weight. The t o t a l uronic acid content of each extract, calculated per gram dry weight of t i s s u e , rose with developmental age from 7.62 umoles i n stage 17 - 18 to 16.40 umoles in stage 27 - 28, although between stage 21 - 22 and stage 24 - 25, no net increases appeared to occur. Independent analysis of two stage 21 - 22 and stage 26 batches of extract showed that the methods employed i n preparing the ti s s u e and i n extracting the glycosaminoglycan gave reproducible r e s u l t s . Microscopic examination of random samples showed that there had been no contamination by discarded embryonic t i s s u e . T o t a l glycosaminoglycan p r e c i p i t a t i o n occurred with the f i r s t EtOH treatment of the extraction medium i n a l l stages. A second p r e c i p i t a t e , harvested by subsequent addition of EtOH to the mother liquo r contained no glycosaminoglycan as determined by the B i t t e r and Muir carbazole test and by paper electrophoresis ( F i g . 29 bottom). To check for glycosaminoglycan loss incurred during t i s s u e prepar-ation, d i s s e c t i n g media from stages 17 - 18, 21 - 22 I, and 26 I were ana-lyzed. These media contained 0.41, 0.49, and 0.46 umoles of uronic acid Properties of the Embryonic Tissues - TABLE VIII The water content (7. H 2 O ) was determined from the loss i n weight r e s u l t i n g from l y o p h i l i z a t i o n , and ' the l i p i d content from the loss i n dry weight r e s u l t i n g from l i g h t petroleum treatment. The umoles uronic acid i n the t o t a l extract were calculated from duplicate B i t t e r and Muir reactions, read at 530 mi i n a Coleman J r . Spectrophotometer using a 19 mm round glass cuvette. Average T o t a l Uronic T o t a l T o t a l Dry Wt. Uronic Acid per Stage No. Emb. wet Dry per Emb. H 20 L i p i d a c i d gm dry Tissues Weight Weight Tissue Content Content Content Weight of ti s s u e of t i s s u e (7. of of extract of t i s s u e (mg) (mg) (mg) (%) dry wt.) (umoles) (umoles) 17-18 (2%-3 days old) 1972 4029.80 406.69 0.21 90 7 3.10 7.62 21-22 I (3% days old) 729 4453.28 436.42 0.60 90 9 4.62 10.59 21-22 I I Oh days old) 727 4280.21 425.00 0.58 90 11L 4.55 10.71 24-25 (4% days old) 521 5930.65 514.25 0.99 91 7 5.25 10.21 26 I (5 days old) 339 5024.55 442.45 1.30 91 9 5.25 11.85 26 II (5 days old) 327 4844.43 423.25 1.29 91 9 5.15 12.17 27-28 (5% days old) 292 ? 800.80 2.74 ? 7 13.13 16.40 - 43 -p o s i t i v e material r e s p e c t i v e l y . This amounted to 127. of the t o t a l uronic acid p r e c i p i t a t e d i n stage 17 - 18, 10% i n stage 21 - 22 I, and 8% i n stage 26 I . However, a brownish-red colour reaction i n d i c a t i v e of true sugars (Dische, 1947) rather than hexuronic acids was observed suggesting that these figures grossly overestimated the actual glycosaminoglycan l o s s . For one thing, carbohydrates l i k e glucose dissolved from the embryonic tis s u e i n the d i s s e c t i n g medium would contribute to the colour reaction (Gurin and Hood, 1939; Seibert and Atno, 1946; Holzman et a l , 1947) as would proteins at low hexuronic acid concentrations (Balazs, et a l , 1965). For another thing, discarded embryonic t i s s u e would also contribute to the soluble uronic acid p o s i t i v e pool i n the d i s s e c t i n g medium. To as c e r t a i n what the components of each extract might be, 10 u l samples containing approximately 0.03 umoles uronic acid each were subjected to paper electrophoresis. Two main components were resolved i n each age group. In stage 17 - 18 to stage 26 ( F i g . 28), the distance migrated by the slower moving components (Table IX) gave r a t i o s similar to commercial hyaluronic acid (0.60) when divided by the distance migrated by chondroitin 4-sulphate. These r a t i o s provided a means of comparing the mobility of each spot r e l a t i v e to the standard reference chondroitin 4-sulphate. The faster moving components i n these same stages gave r a t i o s ranging from 0.85 to 0.95 suggesting the possible presence of a heterogeneous population of mono-sulphated glycosaminoglycans. In stage 27 - 28 ( F i g . 29), the slower moving components also had a mobility s i m i l a r to that of hyaluronic acid, t h e i r r a t i o s being 0.72 and 0.70 r e s p e c t i v e l y . The faster moving component gave a r a t i o of 0.98 i n d i c a t i n g a mobility s i m i l a r to that of the chondroitin sulphates. No heparin-like component was demonstrable i n any of the stages. Unfortunately, r a t i o s from d i f f e r e n t runs were not comparable since v a r i a t i o n s - 44 -Figure 28 - Paper electrophoresis (x 0.45) of stage 17 - 18 to stage 26 extracts. Two main components l o c a l i z e d by A l c i a n blue s t a i n i n g were present i n each stage. The distance migrated by each spot was recorded i n Table IX. A l l samples were run together for 16% hours at 20 v o l t s . Figure 29 - Paper electrophoresis (x 0.45) of a stage 27 - 28 extract showing two components l o c a l i z e d by A l c i a n blue s t a i n i n g . The distance migrated by each spot was recorded i n Table IX. A second p r e c i p i t a t e harvested by a second EtOH t r e a t -ment of the extraction medium contained no glycosamino-glycan (bottom of p i c t u r e ) , A l l samples were run together for 16% hours at 20 v o l t s . Dernatan Sulphata Chondroitin-4-Sulphata St 17-18 to t a l extract St 21-22 II t o t a l extract St 24-25 to t a l extract St 26 I to t a l extract Hyaluronic acid T • St 27-28 second EtOH ext. after ppt. MPS - 45 -Paper Electrophoresis of the Embryonic Extracts - TABLE IX The distance migrated by each spot was calculated according to Foster and Pearce (1961) as the furthest, distance of the s t a i n from the point of a p p l i c a t i o n l e s s one-half the width of the spot. The l a t t e r measure corrected for d i f f u s i o n during the run. The mobility of each sample and standard was compared to that of chondroitin-4-sulphate by d i v i d i n g the distance migrated by each into the distance migrated by chondroitin-4-sulphate. RUN I (Figure 28) RUN II (Figure 29) Distance migrated (cm) M o b i l i t y Distance migrated (cm) M o b i l i t y Chondroitin -4-sulphate 9.4 1.00 8.3 1.00 Dermatan Sulphate 8.1 0.86 7.7 0.93 Heparin - - 10.8 1.30 Hyaluronic Acid 5.6 0.60 5.8 0.70 St. 17-18 8.0 5.6 0.85 0.60 St. 21-22 II 8.5 5.8 0.90 0.62 St. 24-25 8.9 5.8 0.95 0.62 St. 26 I 8.9 5.7 0.95 0.61 St. 27-28 8.2 6.0 0.98 0.72 - 46 -i n the voltage applied (Hartman, 1947) and the length of time i t i s applied (Smoluchowski, 1921), a f f e c t the distances migrated by charged p a r t i c l e s i n an e l e c t r i c f i e l d to a d i f f e r e n t extent, depending on the net charge of the p a r t i c l e . For that reason, standard references were included with every run. To determine the f e a s i b i l i t y of separating and i s o l a t i n g the two components present i n each extract, a stage 27 - 28 sample containing 5.25 umoles uronic acid was applied to an AG 1x2 (CI ) column and eluted using a continuous NaCl gradient ( F i g . 30). Four d i s t i n c t peaks were resolved. Chloride analysis established that the f i r s t peak ( f r a c t i o n 1) and the second peak ( f r a c t i o n 2) could be eluted from the column i n succession with 0.65 and 1.8 M NaCl (indicated by arrows i n F i g . 30). The t h i r d and fourth peaks together ( f r a c t i o n 3) could subsequently be eluted from the column with 3.0 M NaCl. The recovery i n each f r a c t i o n from the sample was recorded i n Table X, stage 27 - 28 ( c ) . Paper electrophoresis of each f r a c t i o n ( F i g . 31) showed that the slower moving hyaluronate-like component was located in the 0.65 M NaCl eff l u e n t ( f r a c t i o n 1) and the faster moving chondroitin sulphate-like com-ponent i n the 1.8 M NaCl ef f l u e n t ( f r a c t i o n 2). No anionic glycosaminoglycan-l i k e substance could be demonstrated i n the 3.0 M NaCl ef f l u e n t ( f r a c t i o n 3), even a f t e r a ten f o l d increase i n the concentration of the applied sample. Having established that components in the extracts could be separated by column chromatography, the remaining samples were applied to columns and separated into three f r a c t i o n s with stepwise 0.65, 1.8, and 3.0 M NaCl batch e l u t i o n s . No s i g n i f i c a n t breakthrough of sample occurred i n any of the columns since the uronic acid detectable i n the bed volumes was less than 1% of the t o t a l sample applied (Table X). T o t a l recovery i n the three f r a c t -- 47 -Figure 30 - Continuous NaCl gradient e l u t i o n of a stage 27 - 28 sample from an AG 1x2 (CI") column showing the main com-ponents present and the s a l t concentrations required to separate them (indicated by arrows). The umoles uronic acid were calculated for each f r a c t i o n from the average of duplicate B i t t e r and Muir carbazole reactions '. The amount of sample applied and the recovery i n each f r a c t i o n was recorded i n Table X, stage 27 - 28 ( c ) • Column Chromatography of the Embryonic Extract from a stage 27-28 sample 25 50 75 100 125 150 175 200 Volume (ml) (5 ml fractions) k» - 48 -Figure 31 - Paper electrophoresis (x 0.45) of f r a c t i o n s i s o l a t e d by column chromatography. The 0.65 M NaCl e f f l u e n t ( f r a c t i o n 1) contained a component with a mobility s i m i l a r to hyaluronic acid, and the 1.8 M NaCl ef f l u e n t ( f r a c t i o n 2) a component with a mobility intermittent between that of hyaluronic acid and chondroitin 4-sulphate. No A l c i a n blue p o s i t i v e s t a i n i n g material was present in the 3.0 M NaCl ef f l u e n t ( f r a c t i o n 3). The distance migrated by each spot was recorded i n Table XII. A l l samples were run together for 14% hours at 20 v o l t s . Hyaluronic acid Heparin Chondro i t in-4-Sulphate St 27-28 0.6S M NaCl/8 H urea St 27-28 1.8 M NaCl/urea St 27-28 3.0 H NaCl/urea - 49 -ions ranged from 76% to 95%. As expected, the main bulk of eluted material was l o c a l i z e d i n fr a c t i o n s 1 and 2 (Table X). Very l i t t l e material was i s o l a t e d i n f r a c t i o n 3. The content of f r a c t i o n 2 increased from 28% to 667. of the t o t a l amount of recovered material while that of f r a c t i o n 1 decreased from 69% to 28%. In comparison, the content of f r a c t i o n 3 remained low i n a l l stages examined, reaching a maximum of only 6% of the t o t a l recovered material by stage 27 -28. Two independent analyses of the stage 27 - 28 extract (samples a and b), and of both stage 26 preparations showed that batch e l u t i o n from columns using increasing s a l t concentrations gave reproducible r e s u l t s . The t h i r d stage 27 - 28 sample analyzed (sample c) was derived from continuous NaCl gradient e l u t i o n ( F i g . 30). For comparative purposes, the uronic acid content per gram dry weight of embryonic t i s s u e was calculated (Table XI) and plotted ( F i g . 32) for each f r a c t i o n . The uronic acid content of both f r a c t i o n s 1 and 2 increased from stage 17 - 18 to stage 21 - 22 and then l e v e l l e d o f f u n t i l stage 24 - 25. Although f r a c t i o n 2 did increase i n content at a rate faster than f r a c t i o n 1 throughout these stages, i t remained lower i n content than f r a c t i o n 1. Ratios of the two f r a c t i o n s showed that by stage 24 - 25 the content of f r a c t i o n 2 had increased from 0.40 times to 0.72 times that of f r a c t i o n 1. I t was evident that the content of f r a c t i o n 1 had reached a peak between stages 21 - 22 and 24 - 25 and was beginning to decrease again a f t e r stage 24 - 25. Not u n t i l t h i s decrease had commenced did the content of f r a c t i o n 2 begin to increase very r a p i d l y . Ratios of the two f r a c t i o n s showed that f r a c t i o n 2 had already exceeded f r a c t i o n 1 i n content by stage 26, and was 2.42 (average of three r a t i o s ) times higher than f r a c t i o n 1 by stage 27 - 28. The content of f r a c t i o n 3 was i n s i g n i f i c a n t compared to the Column Chromatography of Embryonic Extracts - TABLE X The umoles of extract applied to each column and the amount eluted i n each f r a c t i o n was calculated from uronic a c i d analyses. Duplicate B i t t e r and Muir carbazole t e s t s were performed on a l l e f f l u e n t s . Both breakthrough and t o t a l recovery were expressed as a percentage of the applied sample. A l l samples were fractioned by stepwise batch NaCl e l u t i o n except s t . 27 - 28 (c) which was eluted by a continuous NaCl gradient. Stage umoles applied Bed V o l . (umoles) Break-through (%) Content fract.1 (umoles) Content fract.2 (umoles) Content fr a c t . 3 (umoles) Re-covery (%) D i s t r i b u t i o n of Eluted M a t e r i a l fract.1 % fract.2 % fract.3 % 17-18 2.84 0.02 0.8 1.50 0.60 0.06 76 69 28 3 21-22 II 4.41 0.02 0.3 2.05 1.38 0.03 79 59 40 1 24-25 5.01 0.04 0.8 2.48 1.78 0.08 87 57 41 2 26 I 5.01 0.01 0.2 1.78 2.02 0.11 78 45 52 3 26 I I 4.92 0.02 0.5 1.73 2.15 0.11 81 43 54 3 27-28 (a) 5.25 0.03 0.6 1.33 3.21 0.28 92 28 66 6 (b); 1.68 0.01 0.6 0.47 1.02 0.10 95 30 64 6 (c) 5.25 0.02 0.4 1.23 3.28 0.31 92 26 68 6 - 51 -Uronic Acid Content of Each F r a c t i o n per Gram dry wt. of Tissue - TABLE XI  In each f r a c t i o n , the uronic acid content per gram dry weight of t i s s u e was calculated from the t o t a l uronic acid content per gram dry weight of t i s s u e from each stage (recorded i n Table VIII) a f t e r the recovery i n each f r a c t i o n had been determined (Table X). umoles uronic a ./gm dry wt. Ratio of f r a c t . 2 Stage f r a c t . 1 f r a c t . 2 f r a c t . 3 T o t a l f r a c t . 1 17-18 4.02 1.61 0.16 5.79 0.40 21-22 I I 4.98 3.35 0.07 8.40 0.67 24-25 5.05 3.63 0.16 8.84 0.72 26 I 4.21 4.78 0.26 9.25 1.14 26 II 4.28 5.32 0.27 9.87 1.24 27-28 (a) 4.15 10.03 0.88 15.06 2.41 (b) 4.59 9.95 0.98 15.52 2.17 (c) 3.84 10.25 0.97 15.06 2.67 - 52 -Figure 32 - Graph showing the increase i n each f r a c t i o n of the uronic acid content per gram dry weight of t i s s u e . The t o t a l uronic acid content per gram dry weight of tissue was plotted both before and af t e r separation into f r a c t i o n s to show the difference due to t h i s treatment. Where the average value of several readings were plotted, the deviation i s shown by v e r t i c a l bars. Uronic acid/gm dry wt. Embryonic tis s u e vs. Development T o t a l before (st.17-18) (at.21-22) (st.24-25) (st.26) (st.27-28) Developmental age ( i n days) » - 53 -other two f r a c t i o n s although i t did increase to almost 1 umole uronic acid per gram dry weight of tissue by stage 27 - 28. The t o t a l uronic acid content per gram dry weight of t i s s u e before and after column chromatography (recorded in Tables VIII and XI respectively) was also plotted i n F i g . 32. In a l l stages the t o t a l was less a f t e r column chromatography since recoveries were not 100%. However, whereas the t o t a l uronic acid content per gram dry weight before column chromatography did not increase between stages 21 - 22 and 24 - 25, the t o t a l uronic acid content a f t e r column chromatography d e f i n i t e l y d id. Whether the uronic acid p o s i t i v e material not recovered i n the three f r a c t i o n s i n each stage was due to impurities retained by the column, or due to anionic glycosaminoglycans which would have come o f f the column at a s a l t concentration higher than 3.0 M NaCl was not determined. I I . Characterization of the Three Fractions Fractions obtained by column chromatography were subjected to paper electrophoresis. One main spot was observed in each f r a c t i o n . In f r a c t i o n 1, components from each stage ( F i g s . 31 and 33) had m o b i l i t i e s similar to, or s l i g h t l y faster than that of commercial hyaluronic acid (Table X I I ) . The e l i p t i c a l shapes of these spots, p a r t i c u l a r l y noticeable i n stages 21 - 22 and 24 - 25, suggested that part of these components could be either degraded and therefore faster moving (Foster and Pearce, 1961) or a l t e r n a t i v e l y p a r t l y sulphated. The material observed remaining at the point of a p p l i c a t i o n i s apparently a common find i n g (Foster and Pearce, 1961) and could be minimized by c e n t r i f u g a t i o n of the sample sol u t i o n before a p p l i c -a t i o n . In f r a c t i o n 2, components from each stage ( F i g s . 31 and 34) had - 54 -Figure 33 - Paper electrophoresis (x 0.45) of f r a c t i o n 1 samples from d i f f e r e n t stages, i s o l a t e d by column chromatography. The distance migrated by each spot was recorded i n Table XII. Note that some streaking towards chondroitin sulphate did occur i n these spots. The spots were l o c a l i z e d with A l c i a n blue s t a i n i n g . A l l samples were run together for 15 hours at 20 v o l t s . Figure 34 - Paper electrophoresis (x 0.45) of f r a c t i o n 2 samples from d i f f e r e n t stages i s o l a t e d by column chromatography. Spots were l o c a l i z e d with A l c i a n blue st a i n i n g , and the distance migrated by each spot recorded i n Table XII. There was some t r a i l i n g towards the hyaluronic acid component i n a l l the samples, but no d e f i n i t e spot occurred. A l l samples were run together for 16% hours at 20 v o l t s . 33 Hyaluronic acid St 17-18 fraction 1 St 21-22 II fraction 1 St 24-25 fraction 1 St 26 I fraction 1 St 26 II fraction 1 Chondro i tin-4-S u l p h a t e Hyaluronic acid Dermatan Sulphate St 17-18 frac t i o n 2 St 21-22 II frac t i o n 2 St 24-25 fr a c t i o n 2 34 St 261 frac t i o n 2 ii Chondro i t i n - 4-Sulphate m m m - 55 -Paper Electrophoresis of Isolated Fractions - TABLE XII Distance migrated by each spot and the mobility r e l a t i v e to chondroitin-4-sulphate was explained i n Table IX. RUN I (Figure 31) RUN (Figui II e 33) RUN I I I (Figure 34) Sample )istance i i grated (cm) M o b i l i t y Distance migrated (cm) M o b i l i t y Distance migrated (cm) M o b i l i t y Chondroitin -4-sulphate 7.5 1.00 7.6 1.00 8.4 1.00 Dermatan Sulphate - - - 7.4 0.88 Heparin 8.3 1.11 - - - -Hyaluronic Acid 5.0 0.67 4.7 0.62 4.9 0.58 St.17-18 4.6 0.61 7.5 0.89 St.21-22 I I 5.4 0.71 7.9 0.94 St.24-25 5.2 0.68 7.4 0.88 St.26 I 4.6 0.61 7.4 0.88 St.26 I I 4.7 0.62 St.27-28 0.65 M NaCl 5.1 0.68 1.8 M NaCl 6.7 0.89 - 56 -m o b i l i t i e s s i m i l a r to that of dermatan sulphate. Some t r a i l i n g did occur with most of the spots, but no hyaluronate-like component occurred. Neither f r a c t i o n 1 nor 2 contained any heparin-like component and, as was the case with the stage 27- 28 sample ( F i g . 31), no anionic glycosaminoglycan-like material could be demonstrated i n the t h i r d f r a c t i o n of any age group. Further f r a c t i o n a t i o n of f r a c t i o n 2, by p r e c i p i t a t i n g the calcium s a l t s of a stage 27 - 28 sample with EtOH, indicated that only one component was present. P r e c i p i t a t i o n of t h i s component i n the 30 - 457« EtOH region ( F i g . 35) strongly suggested that i t was chondroitin 4- and/or 6-sulphate (Meyer et al., 1956; Loewi and Meyer, 1958) rather than dermatan sulphate as indicated by electrophoresis. Any calcium s a l t s of a dermatan sulphate-l i k e substance should have p r e c i p i t a t e d i n the 18 - 25% EtOH region. Results of B i t t e r and Muir carbazole t e s t s , Dische carbazole t e s t s , and hexosamine tests on samples from each age group were tabulated i n Table XIII and Table XIV. In f r a c t i o n 1 from each stage (Table X I I I ) , the r e s u l t s of the Dische and B i t t e r & Muir carbazole tests gave hexuronic acid r a t i o s very close to one, i n d i c a t i n g that iduronic acid was not a contaminent. The Dische and hexosamine tests also gave r a t i o s close to one, i n d i c a t i n g an equimolar d i s t r i b u t i o n of hexuronic acid to hexosamine. In f r a c t i o n 2 from each stage (Table XIV), Dische over B i t t e r and Muir carbazole tests also gave r a t i o s close to one, i n d i c a t i n g the absence of iduronic a c i d . Dische over hexosamine tests gave r a t i o s even closer to unity than in f r a c t i o n 1, again i n d i c a t i n g an equimolar hexuronic acid'.hexosamine content. The small amount of material recovered i n f r a c t i o n 3 l i m i t e d ana-l y s i s of t h i s f r a c t i o n to B i t t e r and Muir carbazole tests and paper e l e c t r o -phoresis. Results of these tests have already been recorded. Paper chromatography i d e n t i f i e d the hexosamine of f r a c t i o n 1 from - 57 -Figure 35 - Fractionation of the calcium salts of a fraction 2 stage 27 - 28 sample by EtOH precipitation. A l l uronic acid tests were done in duplicate. Only one component precipitated in the 30 - 45% EtOH region. EtOH Fractionation of the Calcium S a l t s of F r a c t i o n 2 from a stage 27 - 28 sample - 58 -Characterization of F r a c t i o n I - TABLE XIII Uronic a. Uronic a. Ratio Ratio determined determined Hexosamine of of Stage by the B+M by Dische's test Dische Dische Carb. test Carb. test B+M Hexosam. (umoles) (umoles) (umoles) 17-18 1.50 1.58 1.28 1.05 1.23 21-22 I I 2.05 2.18 1.90 1.08 1.15 24-25 2.48 2.32 2.08 0.96 1.12 26 I 1.78 1.88 1.73 1.09 1.09 26 II 1.73 1.72 1.63 1.00 1.06 27-28 1.50 1.32 1.15 0.88 1.15 (a+c) - 59 -Characterization of F r a c t i o n 2 - TABLE XIV Uronic a. Uronic a. Ratio Ratio determined determined Hexosamine of of Stage by the B+M by Dische's test Dische Dische Carb. test Carb. test B+M Hexosam. (umoles) (umoles) (umoles) 17-18 0.60 0.61 0.59 1.01 1.03 21-22 I I 1.38 1.47 1.31 1.06 1.12 24-25 1.78 1.82 1.89 1.02 0.97 26 I 2.02 2.10 2.14 1.04 0.98 26 II 2.15 2.08 2.15 0.97 0.97 27-28 (a) 0.76 0.76 0.70 1.00 1.09 (b) 1.02 1.02 - 1.00 -(c) 0.77 0.78 - 1.01 -- 60 -each stage as glucosamine ( F i g . 36), and the main hexosamine of f r a c t i o n 2 ( F i g . 37) as galactosamine with a small amount of glucosamine present a l s o . In neither f r a c t i o n was any galactose present, i n d i c a t i n g that keratosulphate could not be a s i g n i f i c a n t contaminant. Paper chromatograms of the hexuronic acids from a l l f r a c t i o n s ( F i g s . 38 to 41) revealed that the acid i n a l l cases was glucuronic a c i d . Iduronic acid could only be detected i n trace amounts i n f r a c t i o n 2 of each stage. The two spots observed for most of the standards and samples on the chromatograms were the r e s u l t of an acid-lactone e q u i l i b r i a . Ammonium hydroxide had been added to the samples p r i o r to subjecting them to paper chromatography i n order to convert the lactones to free acid, but apparently t h i s was not successful i n many cases. A major disadvantage i n using s i l v e r n i t r a t e to locate hexuronic acids was that a l l reducing components present stained, r e s u l t i n g i n the appearance of many u n i d e n t i f i a b l e spots. I t was i n t e r e s t i n g to note that the reducing components i n f r a c t i o n 1 near the o r i g i n of the chromatogram were simi l a r i n character to those present i n the commercial hyaluronic acid stan-dard ( F i g . 39) while i n f r a c t i o n 2 they were similar i n character to those present i n chondroitin 4-sulphate ( F i g . 41). In neither f r a c t i o n did the reducing components resemble those present i n dermatan sulphate. Unfortunately, not enough glycosaminoglycan was recovered i n stage 17 - 18 to permit the detection of hexuronic acid by paper chromato-graphy of f r a c t i o n 2. An attempt to chromatograph f r a c t i o n 1 of t h i s same stage was also unsuccessful ( F i g . 38) due to interference from an abnormally high s a l t content. - 61 -Figure 36 - Hexosamine paper chromatogram (x 0.55) of fraction 1 samples from a l l stages examined. Spots were located with silver nitrate staining. Only glucosamine was present in the samples. The galactosamine standard was the spot closest to the origin and galactose the spot farthest from the origin. Figure 37 - Hexosamine paper chromatogram (x 0.55) of fraction 2 samples from a l l the stages. The spots were localized with silver nitrate. Galactosamine was the main hexosamine present in a l l the stages with a small amount of glucosamine also present. No galactose was present in any fraction. Galactosamine Glucosamine Galactose St 27-28 frac t i o n 1 St 26 II frac t i o n 1 St 26 I fr a c t i o n 1 St 24-25 frac t i o n 1 St 21-22 II frac t i o n 1 St 17-18 fr a c t i o n 1 Glucosamine 36 37 St 21-22 I fr a c t i o n 2 St 27-28 fr a c t i o n 2 St 26 II f r a c t i o n 2 Galactosamine Glucosamine Galactose St 26 I fr a c t i o n 2 St 24-25 fr a c t i o n 2 St 21-22 frac t i o n 2 St 17-18 fraction 2 • - 62 -Figure 38 - Hexuronic acid paper chromatogram (x 0.55) of f r a c t i o n 1 from stages 17 - 18, 21 - 22 I I , and 26 I. The spots were l o c a l i z e d with s i l v e r n i t r a t e s t a i n i n g . The two spots seen for most of the samples and standards were due to e q u i l i b r i a between the free acid and i t s lactone. The e f f e c t of too much s a l t on the chromatogram was shown by f r a c t i o n 1 of stage 17 - 18. Figure 39 - Hexuronic acid paper chromatogram (x 0.55) of f r a c t i o n 1 from stage 24 - 25 and 26 I I , and of f r a c t i o n 2 from stage 26 I I . Staining was the same as i n Figure 38. Note the c h a r a c t e r i s t i c pattern of the reducing compounds near the o r i g i n . Those of f r a c t i o n 1 are si m i l a r i n pattern to those found i n commercial hyaluronic acid, and that of f r a c t i o n 2 similar to those i n chondroitin 4-sulphate. Na glucuronate St 26 I fra c t i o n 1 St 21-22 II fr a c t i o n 1 St 17-18 fra c t i o n 1 Hyaluronic acid Dermatan Sulphate Chondroitin-4-Sulphate T Na glucuronate 38 Na glucuronate st 2611 fraction 2 St 2611 fraction 1 St 24-25 fraction 1 Hyaluronic acid Dermatan Sulphate Chondroi tin-4-Sulphate 39 - 63 -Figure 40 - Hexuronic acid chromatogram (x 0.55) of both f r a c t i o n s from the stage 27 - 28 embryo. D e t a i l s are the same as those given i n Figure 38. Figure 41 - Hexuronic acid chromatogram (x 0.55) of f r a c t i o n 2 from stages 21 - 22 I I , 24 - 25, and stage 26 I . D e t a i l s are the same as those given i n Figure 38. Note that the st a i n i n g pattern of the reducing compounds near the o r i g i n s i n a l l the samples i s similar i n pattern to that found i n chondroitin 4-sulphate. Na glucuronate Dermatan Sulphate Chondroitin-4-Sulphate Hyaluronic acid St 27-28 frac t i o n 2 St 27-28 fraction 1 Unhydrol. Na glucuronate Na glucuronate 40 Iduronic acid St 26 I frac t i o n 2 St 24-25 fraction 2 St 21-22 II frac t i o n 2 Chondroitin-4-Sulphate Dermatan Sulphate Hyaluronic acid Na glucuronate - 64 -DISCUSSION I. S p e c i f i c i t y of Staining Techniques S p e c i f i c i t y of s t a i n i n g techniques i s a problem pertinent to any histochemical study. In t h i s i n v e s t i g a t i o n several stains were used to demonstrate anionic glycosaminoglycans: AB ( A l c i a n Blue) of the AB/PAS (AB/ Periodic a c i d - S c h i f f ) procedure, AF (Aldehyde Fuchsin), a combined AB-AF procedure, Azure A, Toluidine blue, and Saunders fluorescent Acridine orange method. Agreement between the stains was generally good. Only the AF and AB-AF reactions, the only two which involve Aldehyde Fuchsin, gave r e s u l t s inconsistent with the d i s t r i b u t i o n pattern of anionic glycosamino-glycans demonstrable with the other s t a i n s . In a l l tissues sulphated anionic glycosaminoglycans were demonstrable using Azure A and Acri d i n e orange but the AB-AF sequence completely f a i l e d to indicate the presence of such strongly a c i d i c anionic glycosaminoglycans i n the spinal ganglia and scleratome i n t e r -jacent to the myotome and neural ti s s u e and did not indicate the presence of sulphated material i n the neural sheath, notochordal sheath, and surrounding scleratome c e l l s u n t i l stage 20, or i n the limb buds u n t i l stage 24. Nor did AB-AF st a i n i n g indicate the presence of non-sulphated anionic glycosamino-glycans i n the scleratome and myotome of stage 26 embryos although such weakly a c i d i c anionic glycosaminoglycans were d e f i n i t e l y indicated i n these tissues with Acri d i n e orange fluorescence. AF s t a i n i n g showed a substantial amount of sulphated material i n the myotome and dermatome whereas Azure A and Acridine orange showed very l i t t l e sulphated anionic glycosaminoglycan present. AF had a very high a f f i n i t y for the strongly a c i d i c sulphonate groups of anionic glycosaminoglycans as indicated by the complete blocking of - 65 -AF s t a i n i n g i n a l l tissues following methylation and the f a i n t r estoration of s t a i n i n g to only the neural sheath, notochordal sheath and surrounding scleratome c e l l s and the limb buds after demethylation, coupled with the observed increase i n s t a i n i n g a c t i v i t y following sulphation. However, contrary to the findings of many other investigators (Scott and Clayton, 1953; Spicer and Meyer, 1960; Spicer, 1962; Conklin, 1953), AF was not s p e c i f i c for sulphonate groups since carboxyl groups unblocked by demethyl-ati o n stained, though f a i n t l y . This would support the work of Ortman et a l , (1966) showing that s t a i n i n g solutions of AF are unstable and give r i s e to intermediate compounds which react with carboxyl groups. The a f f i n i t y of AB for carboxyl groups of anionic glycosamino-glycans was shown by the blocking of a l c i a n o p h i l i a by methylation and i t s r e s t o r a t i o n following demethylation. The p a r t i a l resistance of AB s t a i n i n g to methylation i n the notochordal sheath and surrounding scleratome c e l l s , and the limb bud p r e c a r t i l a g e c e l l s from stage 22 on and the subsequent f a i l u r e of demethylation to restore t h i s a l c i a n o p h i l i a i n these areas i s , according to Spicer (I960) and Spicer and Duvenci (1964), c h a r a c t e r i s t i c of c e r t a i n sulphated anionic glycosaminoglycans found in cartilagenous t i s s u e . That AB was s t a i n i n g sulphated glycosaminoglycans was supported by the f a i l u r e of sulphation to completely block a l c i a n o p h i l i a i n the areas r e s i s t a n t to methylation. These r e s u l t s are i n agreement with the work of Spicer (1960, 1963) and Mowry (1963) that AB i s more s p e c i f i c for carboxyl r a d i c a l s present on the glycosaminoglycans than for sulphonate groups. Since the greatest a f f i n i t y of AB and AF i s for carboxyl and sulphonate r a d i c a l s r e s p e c t i v e l y , there i s no apparent explanation for the observed discrepancy i n s t a i n i n g between AB-AF and the other s t a i n s . AB was used f i r s t i n the sequence and may i n some way prevent the access of - 66 -sulphonate groups to AF. The AF s t a i n i n g s o l u t i o n i s very acid (pH 1.5) and i t could be that proteins were i n t e r f e r i n g with AF by competing with the a v a i l a b l e anionic s i t e s . Schubert and Hamerman (1956), Spicer (1962), and Szirmai (1963) found that decreasing the pH ionizes polycations which may react with the anionic groups of the glycosaminoglycans. Protein may also i n t e r f e r e with Azure A s t a i n i n g . S i a l i c a c i d , shown by Kraemer (1966) to be a regular constituent of c e l l surfaces of mammalian c e l l l i n e s grown i n t i s s u e c u l t u r e , w i l l a f f e c t s t a i n i n g by contributing carboxyl groups (Spicer and Duvenci, 1964; Q u i n t a r e l l i and Dellovo, 1965) and could, i f present in chick embryonic ti s s u e s , account for some of the s t a i n i n g v a r i a b i l i t y observed. Neuraminidase applied i n conjunction with AB/PAS s t a i n i n g did not have any e f f e c t . However, i n several tissues containing s i a l i c acid the enzymatic a c t i v i t y i s apparently not shown ( Q u i n t a r e l l i and Dellovo, 1965). A c e t y l a t i o n and deacetylation, designed to block hydroxyl and amino groups and then to restore hydroxyl groups only, p a r t i a l l y removed and only p a r t i a l l y restored a l c i a n o p h i l i a i n a l l tissues of a l l stages. This implied that hydroxyl groups as well as amino groups can influence AB s t a i n -ing. Yamada (1963, 1964) observed similar r e s u l t s using known sulphated anionic glycosaminoglycans. The f a i l u r e of deacetylation to restore alciano-p h i l i a completely may, as suggested by Yamada (1964), be due to the e f f e c t of the a l k a l i i n the deacetylating s o l u t i o n on the a c i d i c groups of the anionic glycosaminoglycans. I t i s possible that both the a c e t y l a t i o n -deacetylation and the methylation-demethylation sequences could cause some loss of stainable material due to the extreme chemical treatments employed. A small loss i s indicated by the f a i l u r e of s a p o n i f i c a t i o n to restore s t a i n -ing completely i n a l l the tissues following these blocking and unblocking - 67 -procedures. A p p l i c a t i o n of the metachromatic reaction at c o n t r o l l e d pH and with hyaluronidase permitted i d e n t i f i c a t i o n of sulphated anionic glyco-saminoglycans. However, non-sulphated anionic glycosaminoglycans were d i f f i c u l t to l o c a l i z e since sulphated anionic glycosaminoglycans occurred i n the same areas. Although t e s t i c u l a r hyaluronidase was of no help i n d i f f e r e n t i a t i n g between sulphated and non-sulphated anionic glycosamino-glycans where both of these stained at the same time (pH 4), t h i s enzyme did indicate the presence of hyaluronic acid, chondroitin, chondroitin 4-sulphate or chondroitin 6-sulphate for a l l of which i t i s s p e c i f i c (Meyer et a l , 1958, 1960; Curran, 1961; Walker, 1961) when Azure A s t a i n i n g at both pH 1.5 and 4 were compared. The complete blocking of Azure A a c t i v i t y following the methyla-tion-demethylation sequence indicated that a l l metachromasia and ortho-chromasia at pH 1.5 was due to sulphated anionic glycosaminoglycans. At pH 4 some metachromasia was restored i n stage 24 to 26 embryos and most of the orthochromasia i n a l l areas of a l l the stages following methylation-demethylation. Therefore, a small amount of non-sulphated anionic glyco-saminoglycan did partake i n the metachromatic r e a c t i o n . Most weakly a c i d i c anionic glycosaminoglycans were orthochromatic. Meyer (1955r 1956) and Schubert and Hamerman (1956) claim that hyaluronic acid can give a meta-chromatic reaction i f i t i s in a highly polymerized state, or highly concen-trated i n any given area. In general though, hyaluronic acid has been found to s t a i n orthochromatically only with thiazine c a t i o n i c dyes (Michaelis and Granick, 1945; Sylven and Malmgren, 1952; Sylven, 1954; Walton and Ricke t t s , 1954). The fact that sulphation increased the orthochromatic reaction of - 68 -Azure A at pH 4 to the point of exclusion of metachromasia i s not s u r p r i s i n g considering that metachromasia depends upon oriented anionic groups (Barka and Anderson, 1963). Conklin (1963) and Spicer (1963) found sulphation to have a s i m i l a r suppressing e f f e c t on metachromasia of sulphated anionic glycosaminoglycans. The e f f e c t of sulphation at pH 1.5 cannot be explained. Agreement between Toluidine blue and Azure A metachromasia at pH 1.5 showed that a i r drying did remove a l l metachromasia not due to strongly a c i d i c sulphonate groups. This i s consistent with the work of Michaelis (1947), Sylven (1954), and Schubert and Hamerman (1956), showing that only strong chromotropes were r e s i s t a n t to dehydration. PAS p o s i t i v e material which was not l a b i l e to diastase and was blocked and unblocked by the acetylation-deacetylation sequence was assumed to be neutral polysaccharide. Anionic glycosaminoglycans can t h e o r e t i c a l l y react with PAS and have been shown to do so under c e r t a i n conditions (Zugibe, 1962; Meyer, 1966). However anionic glycosaminoglycans have not been found to react with PAS to any appreciable extent using the conventional PAS procedure (Conklin, 1963; Zugibe, 1963; Yamada, 1964). Any PAS p o s i t i v e l i p i d material should, according to Barka and Anderson (1963) be r e s i s t a n t to a c e t y l a t i o n and therefore separable from neutral polysaccharide s t a i n i n g . The complete blocking of PAS s t a i n i n g following sulphation and the increase i n PAS s t a i n i n g following methylation would be expected since both procedures a f f e c t the a v a i l a b i l i t y of hydroxyl groups required for aldehyde formation. I t i s obvious that there are l i m i t s to the degree of i d e n t i f i c a -t i o n of glycosaminoglycans using histochemical techniques. Of the methods used, only the PAS reaction i d e n t i f i e d neutral polysaccharides. L o c a l i z a t i o n of non-sulphated anionic glycosaminoglycans was achieved on the basis of - 69 -r e l a t i v e a c i d i t y , blocking and unblocking of s p e c i f i c acid groups and resistance to s a l t extraction at d i f f e r e n t m o l a r i t i e s . Acridine orange fluorescence s t a i n i n g permitted further i d e n t i f i c a t i o n of the sulphated anionic glycosaminoglycans into mono-and poly-sulphated groups. Hyaluron-idase l a b i l i t y showed that some of the non-sulphated anionic glycosaminoglycan was hyaluronic acid (or chondroitin) and most of the sulphated anionic glycosaminoglycan chondroitin 4-and/or 6-sulphate. None of the stains employed were completely s p e c i f i c for glyco-saminoglycans. The p o s s i b i l i t y of protein and glycoprotein ( s i a l i c acid) interference could not be ruled out. These factors l i m i t the interpretations possible and also make quantitative histochemical determination impossible. I I . Histochemical L o c a l i z a t i o n of the Neutral Polysaccharide and  Glycosaminoglycans Examination of the histochemical r e s u l t s i n the d i f f e r e n t develop-mental stages shows a progression i n the appearance of anionic glycosamino-glycans . Neutral polysaccharides were present i n small amounts i n the cytoplasm of c e l l s i n c e l l tissues examined at a l l age groups. With the exception of a few notochordal, myotomal, and ependymal c e l l s , only the scleratome c e l l s oriented along the length of the myotome and e n c i r c l i n g the notochord showed an increasing cytoplasmic neutral polysaccharide' content with developmental age. This increase was c o i n c i d e n t a l with deposition of e x t r a c e l l u l a r matrix by the c e l l s . A few c e n t r a l l y located notochordal c e l l s had acquired a high neutral polysaccharide content as early as stage 18. Myotomal c e l l s , c l o s e l y associated with strands containing non-sulphated - 70 -anionic glycosaminoglycan material which are being deposited close to the scleratome i n t e r f a c e from stage 22 on, and the ependymal c e l l s making up the f l o o r p late of the neural tube, a l l showed a s i m i l a r l y high neutral poly-saccharide content. In the ependymal c e l l s , however, t h i s high neutral polysaccharide content was d i f f e r e n t i n character from that i n the notochord and myotome since i t stained magenta rather than red with the PAS reaction and appeared to be associated with a correspondingly high amount of magenta coloured neutral polysaccharide located i n the marginal layer immediately v e n t r a l to i t . The i n d i c a t i o n i n these cases was that c e l l s with a high neutral polysaccharide content were a c t i v e l y engaged i n e x t r a c e l l u l a r matrix formation. C e l l cytoplasm of t i s s u e i n a l l stages examined contained sulphated material (with the possible exception of the myotome c e l l s ) i n addition to neutral polysaccharides. This i s consistent with work showing that anionic glycosaminoglycan precursors (neutral polysaccharides) are formed i n t r a -c e l l u l a r l y and may be sulphated p r i o r to being extruded into the i n t e r s t i t i a l matrix (Mancini et a l , 1961; Franco-Browder et aJL, 1963; Mathews and Hinds, 1963) . E x t r a c e l l u l a r neutral polysaccharides were l o c a l i z e d i n the matrix of scleratome c e l l s aggregated around the notochord and along the length of the myotome as well as i n the neural and notochordal sheaths and interposed fibrous material where these two were separated. These r e s u l t s indicate that neutral polysaccharides, c h a r a c t e r i s t i c components of cartilagenous matrix (Spicer and Duvenci, 1964; Q u i n t a r e l l i and Dellovo, 1966) are present i n pre-cartilagenous embryonic t i s s u e . Glycogen was detected only within the scleratomal and myotomal c e l l s from stage 22 on. - 71 -Unlike the neutral polysaccharides, the anionic glycosamino-glycans were l o c a l i z e d e x t r a c e l l u l a r l y i n the i n t e r s t i t i a l ground substance. Histochemical s t a i n i n g showed that non-sulphated anionic glyco-saminoglycans were already present at c e l l surfaces of a l l tissues by stage 17. These weakly a c i d i c anionic glycosaminoglycans were most apparent i n the notochordal and neural sheaths and i n the epidermis where they remained concentrated throughout the developmental stages examined. With progressive development, the non-sulphated anionic glyco-saminoglycan content increased i n the e x t r a c e l l u l a r matrix of the scleratome and neural ti s s u e , but i n the myotome and dermatome, which appeared to contain the least amount of glycosaminoglycan, only small increases occurred. In the dermatome, myotome and scleratome, weakly a c i d i c anionic glycosamino-glycans appeared to be associated with very t h i n c e l l u l a r processes j o i n i n g the c e l l s . The observed increase i n i n t e r s t i t i a l glycosaminoglycans i n the scleratome and limb buds was concomitant with c e l l aggregation i n preparation for c a r t i l a g e formation. E x t r a c e l l u l a r extensions between the epidermis and dermatome i n stage 17 and 18 embryos where the dermo-myotomic plate s t i l l p ersisted also contained non-sulphated anionic glycosaminoglycans. These extensions were very si m i l a r to the " e x t r a c e l l u l a r material" described by Moscona (1960) and Steinberg (1963), and l o c a l i z e d i n skate neurulae by McConnachie and Ford (1966) . The structure of the " e x t r a c e l l u l a r material" i s a basement-membrane-like unit with e x t r a c e l l u l a r extensions to adjacent t i s s u e layers ( F i g . 7). Similar extensions occur between the notochordal sheath and scleratome c e l l s . This " e x t r a c e l l u l a r material" could function i n tissue adhesion. In the neural ti s s u e and ganglia, ventral neural sheath, dermatome, - 72 -myotome, and scleratome condensed along the myotome in t e r f a c e , Azure A metachromasia at pH 4 showed that some of the non-sulphated glycosamino-glycans were highly polymerized. Hyaluronidase l a b i l i t y of a large proportion of the non-sulphated anionic glycosaminoglycan, both metachromatic and orthochromatic, indicated that i t was hyaluronic acid (or chondroitin). The highly hydrated chemical structure of hyaluronic acid would make i t very viscous (Schubert and Hamerman, 1956) and possibly endow a s t r u c t u r a l function to the anionic glycosaminoglycan by providing r i g i d i t y to the ground substance as well as a ph y s i o l o g i c a l function of c o n t r o l l i n g molecular d i f f u s i o n (Rogers, 1961) . Sulphated anionic glycosaminoglycans f i r s t appeared i n the noto-chordal sheath and then spread to the ventral neural sheath, notochordal c e l l s , and surrounding scleratome c e l l s as development progressed. U n t i l stage 19 or 20, there was l i t t l e sulphated anionic glycosaminoglycan present i n any tiss u e except the notochordal sheath. The content of the epidermis then rose r a p i d l y . The dermatome contained very l i t t l e strongly a c i d i c anionic glycosaminoglycan and the myotome was completely void of any u n t i l stage 26, although AF s t a i n i n g did in d i c a t e the presence of anionic glyco-saminoglycans i n the myotome at stages 19 and 20 e s p e c i a l l y . Only the notochordal sheath and c e l l s , the ventral neural sheath, the scleratome and the limb bud c e l l s gave a metachromatic response with Toluidine blue and Azure A (pH 1.5) i n d i c a t i n g that these tissues contained highly polymerized sulphated glycosaminoglycans. Azure A dehydration further indicated that the notochordal sheath and surrounding scleratome contained the strongest a c i d i c anionic glycosaminoglycans. Hyaluronidase digestion showed that most of the st a i n i n g material was chondroitin 4- and/or 6-sulphate. - 73 -With developmental age, the greatest sulphated anionic glyco-saminoglycan increase occurred i n the scleratome and limb bud regions destined to form c a r t i l a g e . The close apposition of the scleratome c e l l s oriented around the notochord and along the length of the myotome from stage 20 on enhanced the formation* of a uniform sulphated anionic glycosaminoglycan matrix. There was no apparent morphological difference between the s c l e r a -tomal c e l l s surrounded by anionic glycosaminoglycan matrix and those which were not. By stage 26 however, c l u s t e r s of scleratomal c e l l aggregates had formed centra primordia around the notochord and the c e l l s i n these c l u s t e r s began to take on the appearance of chondroblasts. Strong gamma metachromasia i n both the scleratome and limb bud p r e - c a r t i l a g e matrix at stage 26 denoted the presence of highly polymerized anionic glycosamino-glycans. Thus, the main function of the sulphated anionic glycosaminoglycans appears to be s t r u c t u r a l . The presence of anionic glycosaminoglycans i n neural ti s s u e may suggest a p h y s i o l o g i c a l or biochemical function. Morphologically, d i f f e r e n t -i a t i o n of the neural tube into a marginal, mantle, and ependymal layer was accompanied by a decrease i n s t a i n i n g capacity to a l l areas except the ependymal layer and the v e n t r a l horns of the mantle l a y e r . Non-sulphated anionic glycosaminoglycans were most concentrated i n the ependymal layer and spinal ganglia. Some weakly a c i d i c anionic glycosaminoglycans were present i n the ventral horn of the mantle layer i n close association with the nerve f i b e r s . T h i s , i n addition to the presence of small amounts of non-sulphated anionic glycosaminoglycans i n the marginal layer and spinal nerves, might in d i c a t e , as suggested by Szabo and Roboz-Einstein (1962) that weakly a c i d i c anionic glycosaminoglycans (hyaluronic acid) function i n myelin construction. The presence of sulphated anionic glycosaminoglycans i n the mantle layer also - 74 -might function i n some aspect of neuron development. Poly-sulphated anionic glycosaminoglycan concentrations were too low to permit p o s i t i v e i d e n t i f i c a t i o n i n most tissues studied. I t appeared as i f the notochordal sheath and c e l l s , s p i n a l ganglia, and epidermis could contain trace amounts of highly a c i d i c anionic glycosaminoglycans. No function could be ascribed to t h i s glycosaminoglycan. I l l . Biochemical I d e n t i f i c a t i o n of the Anionic Glycosaminoglycans Two main glycosaminoglycan f r a c t i o n s - one non-sulphated and the other mono-sulphated - were i s o l a t e d from stage 17 to 28 embryos. A t h i r d f r a c t i o n i s o l a t e d from each stage, containing only trace amounts of uronic acid p o s i t i v e material, may have been an a r t i f a c t peculiar to column e l u t i o n since electrophoresis indicated that no AB p o s i t i v e anionic glyco-saminoglycan was present. Analysis based on anion column chromatography, electrophoretic mobility, hexuronic acid and hexosamine determinations and chromatography, and EtOH f r a c t i o n a t i o n of the calcium s a l t s , i d e n t i f i e d the content of the non-sulphated f r a c t i o n as hyaluronic a c i d and that of the sulphated f r a c t i o n as mainly chondroitin 4- and/or 6-sulphate, confirming the histochemical r e s u l t s observed using hyaluronidase. The presence of a small amount of glucosamine i n the chondroitin sulphate f r a c t i o n could be the r e s u l t of heparin contamination since a small shoulder did occur on the t r a i l i n g portion of the chondroitin sulphate e l u t i o n peak ( F i g . 30), and since histochemical s t a i n i n g with a c r i d i n e orange indicated the presence of a poly-sulphated glycosaminoglycan. That the contaminant was hyaluronic acid could not be ruled out since no component with an electrophoretic mobility - 75 -si m i l a r to heparin was observed and since separation of the two fract i o n s was not complete. The r e l a t i v e l y low electrophoretic mobility of the i s o l a t e d chondroitin sulphates i n comparison to chondroitin 4-sulphate suggested the presence of a glycosaminoglycan low i n sulphate content (Franco-Browder et a l , 1963; B i t t e r and Muir, 1966). The r e l a t i v e l y weak metachromatic response (purple colour) observed i n the tissues supported the presence of such a low sulphate containing anionic glycosaminoglycan. I s o l a t i o n of a low sulphate-containing chondroitin sulphate from 14 day old chick embryonic c a r t i l a g e , which had an electrophoretic mobility slower than chondroitin 4-sulphate and which was capable of accepting sulphate has been achieved by Meezan and Davidson (1967) . Chondroitin, a sulphate free or low sulphate containing analogue of chondroitin 4- and/or 6-sulphate, has also been i d e n t i f i e d i n 12 and 13 day old chick embryonic c a r t i l a g e (Thorp and Dorfman, 1963; Searls, 1965). I t appears, therefore that low sulphate containing chondroitin sulphates are c h a r a c t e r i s t i c of young developing cartilagenous tissues where matrix i s being a c t i v e l y synthesized. The f a i l u r e of galactose to appear on hexosamine chromatograms was construed to mean that keratosulphate was not a contaminant i n any of the i s o l a t e d f r a c t i o n s . This agrees with the work of Kaplan and Meyer (1959) and Lash et al (1960) showing that keratosulphate i s not present i n young embryonic c a r t i l a g e . Although dermatan sulphate was shown to be absent from a l l stages examined using the Dische:Bitter and Muir hexuronic a c i d r a t i o s and EtOH f r a c t i o n a t i o n of the calcium s a l t s of the sulphated anionic glycosaminoglycans, trace amounts were indicated by the presence of iduronic acid on the hexuronic acid chromatograms. Dermatan sulphate has not previously been found i n early - 76 -chick embryo development (Fr anco-Browder et a l , 1963; Searls, 1965). Possibly the sample sizes employed by these investigators were not large enough to detect such a small concentration of dermatan sulphate. Preliminary electron microscopy of developing chick scleratome matrix (Low, 1967) has shown that microfibers present i n the e x t r a c e l l u l a r matrix i n 2% to 5% day old embryos do not give r i s e to unit collagen during the f i r s t week of development. Since Searls (1965) could not demonstrate s i g n i f i c a n t amounts of dermatan sulphate i n chick c a r t i l a g e u n t i l the twelfth day of development, i t i s possible that an association between dermatan sulphate and the appearance of coarse collagen f i b e r s i n connective tissue does ex i s t (Hoffman et a l , 1957; Loewi and Meyer, 1958). Since collagen may be required to form stable c a r t i l a g e (Gross et J l , 1960) the implication i s that chondroitin 4- and/or 6-sulphate i s deposited i n the i n t e r s t i t i a l t i s s u e before synthesis of dermatan sulphate stimulated collagen fi b e r formation and hence stable c a r t i l a g e . The rapid increase i n chondroitin 4- and/or 6-sulphate concentr-ation (uronic acid/gm dry wt. of tissue) with developmental age was related to the formation of c a r t i l a g e i n the scleratome as shown by histochemical s t a i n i n g . After stage 25 when the chondroitin sulphate concentration exceeded the concentration of hyaluronic a c i d , strong gamma metachromasia, i n d i c a t i v e of a high concentration of sulphated anionic glycosaminoglycans, appeared i n the scleratome. The high hyaluronic acid concentration present during stages 21 to 25, a time i n development when myotube formation (Holtzer et a l , 1957) and scleratome c e l l aggregation and o r i e n t a t i o n are occurring, may play some r o l e i n somite d i f f e r e n t i a t i o n , as already outlined. The drop i n hyaluronic acid concentration a f t e r stage 25, which i s not obvious with histochemical techniques, was probably masked by the high l e v e l of chondroitin - 77 -sulphate present at that time i n development. IV. Conclusions Both the histochemical and the biochemical analysis i d e n t i f i e d the major anionic glycosaminoglycans present as hyaluronic acid and chondroitin 4- and/or 6-sulphate. The presence of chondroitin could not be p o s i t i v e l y ruled out by either histochemical or biochemical means since i t , l i k e hyaluronic a c i d , i s l a b i l e to t e s t i c u l a r hyaluronidase and since i t has a repeating unit similar to chondroitin 4 and/or 6-sulphate. Chon-d r o i t i n could account for the slower moving chondroitin sulphate observed on electrophoresis but sulphate determinations would be required to make a p o s i t i v e i d e n t i f i c a t i o n . Although the presence of polysulphated anionic glycosaminoglycan was indicated by Acridi n e orange fluorescent s t a i n i n g i t could not be i d e n t i f i e d biochemically. The presence of a small amount of glucosamine to the chondroitin sulphate f r a c t i o n could be caused by heparin, but contamination by hyaluronic acid could not be ruled out. F i n a l l y , dermatan sulphate, which i s not l a b i l e to t e s t i c u l a r hyaluronidase, could not be l o c a l i z e d histochemically since i t could not be distinguished from any sulphomucins that might be present and st a i n i n g , but, based on the chemical properties of iduronic acid as compared to glucuronic a c i d , trace amounts of dermatan sulphate were i d e n t i f i e d by biochemical a n a l y s i s . Lash et ail (1960) have reported that the macromolecules charact-e r i s t i c of c a r t i l a g e appear only when morphologically distinguishable matrix i s formed. The r e s u l t s of t h i s i n v e s t i g a t i o n agree with that f i n d i n g since the chondroitin sulphate content was found to increase concomitant with the formation of c a r t i l a g e matrix. The presence of glycosaminoglycan precursors - 78 -inside c e l l s at a l l stages and the i d e n t i f i c a t i o n of chondroitin 4- and/or 6-sulphate i n early d i f f e r e n t i a t i n g somites would imply that chemical d i f f e r e n t i a t i o n of the c e l l s precede th e i r morphological d i f f e r e n t i a t i o n , p a r t i c u l a r l y since chondroblasts could not be i d e n t i f i e d morphologically u n t i l stage 26, when c a r t i l a g e matrix formation was already well advanced. - 79 -SUMMARY 1. The a x i a l regions (dermatome, myotome, scleratome, neural tube and notochord) of developing chick embryos, ranging from stage 17 through 28 (2% to 5% days of age), were analyzed, both histochemically and biochemically, to determine the d i s t r i b u t i o n and nature of the glyco-saminoglycans present during early somite d i f f e r e n t i a t i o n . 2. Neutral polysaccharides were l o c a l i z e d histochemically within the cytoplasm of c e l l s i n a l l the a x i a l tissues and t h e i r content was seen to increase with developmental age i n c e l l s engaged i n e x t r a c e l l u l a r matrix deposition. E x t r a c e l l u l a r l y , neutral polysaccharides (PAS posit i v e ) appeared i n i n t e r s t i t i a l ground substance destined to form c a r t i l a g e matrix and to some extent i n the marginal layer of the neural tube. 3. Histochemical analysis indicated that the matrix between c e l l s of a l l tissues i n the a x i a l region contained weakly a c i d i c anionic glycosamino-glycans. These were i n i t i a l l y most concentrated i n the notochordal and neural sheaths and i n the epidermis, but with progressive development they increased i n concentration i n most t i s s u e s , p a r t i c u l a r l y i n the neural tube and scleratome. Digestion with t e s t i c u l a r hyaluronidase suggested that most of the stainable matrix was hyaluronic a c i d . 4. Histochemical analysis also indicated that strongly a c i d i c anionic glyco-saminoglycans were concentrated i n the notochordal sheath i n stage 17 embryos. From stage 19 - 20 on, these sulphated anionic glycosamino-glycans increased, i n the ve n t r a l neural sheath as well as the notochordal, epidermal, and scleratomal c e l l matrix. By stage 26 the scleratomal c e l l matrix around the notochord was mostly sulphated anionic glycosamino-- 80 -glycan. The myotomal matrix was completely devoid of any sulphated anionic glycosaminoglycan u n t i l stage 26, although sulphated material was present i n the c e l l cytoplasm of a l l the tissues throughout the stages examined. T e s t i c u l a r hyaluronidase l a b i l i t y indicated that most of the stainable material was chondroitin 4- and/or 6-sulphate. 5. Biochemical analysis confirmed that the weakly a c i d i c glycosaminoglycan from each age group was hyaluronic acid and that the strongly a c i d i c \ glycosaminoglycan was mainly chondroitin 4- and/or 6-sulphate. Only trace amounts of dermatan sulphate were present. Keratosulphate was not found i n any of the age groups examined. 6. A small amount of polysulphated anionic glycosaminoglycan l o c a l i z e d histochemically i n the notochordal sheath and c e l l s , spinal ganglia and epidermis could not be p o s i t i v e l y i d e n t i f i e d biochemically. I t i s possible that t h i s glycosaminoglycan i s heparin. 7. On a quantitative basis, the hyaluronic acid concentration (uronic acid/gm. dry wt. of tissue) was at a peak between stages 21 to 25. The hyaluronic acid concentration was greater than the chondroitin sulphate concentration u n t i l stage 25. After that stage, the chondroitin sulphate concentration began to increase very r a p i d l y , an increase which was concomitant with the formation of c a r t i l a g e around the notochord. The hyaluronic acid concentration began to decline slowly a f t e r stage 25 so that whereas the hyaluronic content was 2\ times greater than the chondroitin sulphate content i n stage 17 embryos, t h i s r a t i o was almost completely reversed by stage 28 due to the rapid increase i n chondroitin sulphate. 8. In chondrogenesis, chemical d i f f e r e n t i a t i o n of the c e l l s preceded t h e i r morphological d i f f e r e n t i a t i o n since the anionic glycosaminoglycans - 81 -c h a r a c t e r i s t i c of c a r t i l a g e were present i n the c e l l matrix before any morphological c e l l types could be i d e n t i f i e d . - 82 -REFERENCES Anderson, D.M.W., and Karamalla, K.A. (1966). Studies on uronic acid mat-e r i a l s : Part XII. The composition of Acacia Gum exudates. J . Chem. Soc. (C); 762 - 764 Antonopoulos, C.A., Borelius, E., G a r d e l l , S., Hamnstrom, B., and Scott, J.E. (1961). 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Quantitative approaches i n the histochemistry of muco-polysaccharides. J . Histochem. Cytochem. 11; 2 4 - 3 4 Thorp, F.K., and Dorfman, A. (1963). The occurrence of i n t r a c e l l u l a r chon-d r o i t i n s u l f a t e . J . C e l l . B i o l . 18; 13 - 17 Tomlinson, R.V., and Tener, G.M. (1962). The use of urea to eliminate the secondary binding forces i n ion exchange chromatography of polynucleotides. J . Am. Chem. Soc. 84; 2644 Trevelyan, W.E., Procter, D.P., and Harrison, J.S. (1950). Detection of sugars on paper chromatograms. Nat. 166; 444 - 445 Vassar, P.S., and C u l l i n g , C.F.A. (1959). F i b r o s i s of the breast. Arch. Pathol. 67; 128 - 133 Walker, P.G. (1961). The enzymatic degradation of mucopolysaccharides. Biochem. Soc. Symp. No. 20, pp 109 - 125, Cambridge Univ. Press, London Walton, K.W., and Ricket t s , C R. (1954). Investigations of the histochemical basis of metachromasia. B r i t i s h J . E x p t l . Pathol. 35; 227 - 240 Williams, G. and Jackson, D.S. (1956). Two organic f i x a t i v e s for acid muco-polysaccharides. Stain Tech. _31; 189 - 191 Yamada, K. (1963). Staining of sulphated polysaccharides by means of A l c i a n blue. Nat. 198; 799 - 800 Yamada, K. (1964). The reaction of sulfated polysaccharides to several histochemical t e s t s . J . Histochem. Cytochem. 12; 327 - 332 Zugibe, F.T. (1963). Mucopolysaccharides of the a r t e r i a l w a l l . J . Histochem. Cytochem. U ; 35 - 39 - 89 -APPENDIX A  Histochemical Techniques Employed 1. Staining a. PAS (Spicer, 1960) Oxidize 10 minutes i n 17, aqueous periodic acid, wash i n d i s t i l l e d H2O and treat 30 minutes with S c h i f f reagent. Rinse i n three changes of sulphite (0.57o i n 0.05 N CHI), wash, dehydrate and mount. b. AB/PAS (Spicer. 1960) Stain 30 minutes i n 17. A l c i a n blue (Hartman-Leddon Co.) i n 37o acetic a c i d , r i n s e 10 minutes i n running ^ 0 , follow with Periodic acid S c h i f f treatment as described i n part (a), dehydrate and mount. c. Gomori's Aldehyde Fuchsin as modified by Halmi ( C u l l i n g , 1963) Sections were brought to 707* EtOH, stained 10 minutes i n a fresh aldehyde fuchsin s o l u t i o n (0.57o basic fuchsin i n 607, EtOH ripened 24 hours with 1 ml paraldehyde and 1.5 ml concentrated H C 1 per 100 ml s t a i n i n g s o l u t i o n ) , rinsed i n 707> EtOH, dehydrated and mounted. d. Combined AB-AF (Spicer and Meyer. 1960; C u l l i n g , 1963) Stain i n AB as described i n part (b) , bring to 707o EtOH and s t a i n with AF as described i n part ( c ) . Dehydrate and mount. e. T o l u i d i n e blue (Vassar and C u l l i n g . 1959) Stain for 10 seconds i n 0.257» Tol u i d i n e blue (G. T. Gurr) i n Michaelis's Veronal acetate-hydrochloric acid buffer at pH 4.5. Rinse i n d i s t i l l e d H 2 O , a i r dry and mount i n permount. - 90 -f• Azure A (Szirmai, 1963; Yamada, 1964; McConnachie and Ford, 1966) Stain for 30 minutes i n 0.27. Azure A (Fisher S c i e n t i f i c Co.) in d i s t i l l e d H 20 with the pH adjusted to 4.0 or 1.5 with HC1. Sections were examined either i n d i s t i l l e d H^ O or after a i r drying and mounting, g. Acrid i n e Orange (Saunders, 1964) Treat 3 p a r a l l e l s l i d e s i n 1% CPC for 5 minutes, wash 10 minutes i n running H^O, and treat with 0.1% ribonuclease i n d i s t i l l e d H 20 at 45° C for 2 hours. Treat s l i d e 1 i n CPC another 5 minutes, wash i n running H^0 for 10 minutes and s t a i n 3 minutes i n neutral aqueous 0.17. Acridine orange (Fisher S c i e n t i f i c Co.). Stain s l i d e 2 i n 0.1% Acridi n e orange i n 0.01 M acetic acid (pH 3.2) for 5 minutes, wash i n running H2O for 10 minutes and d i f f e r e n t i a t e i n 0.3 M NaCl i n 0.01 M acetic acid for 20 minutes. Treat s l i d e 3 as s l i d e 2, but d i f f e r e n t i a t e i n 0.6 M NaCl i n 0.01 M ac e t i c acid for 20 minutes. Wash a l l s l i d e s i n running 1^0, a i r dry, and mount i n Flourmount (E. Gurr). Red fluorescence indicates the presence of hyaluronic acid in s l i d e 1, chondroitin sulphates and heparin i n s l i d e 2, and heparin i n s l i d e 3. Enzyme Treatment a. Diastase ( C u l l i n g . 1963) Digest sections i n 0.17. commercial malt diastase ( N u t r i t i o n a l Biochemicals Corporation) i n d i s t i l l e d H2O for 30 minutes at 37° C to - 91 -remove glycogen. Incubation prolonged for 3 or more hours may remove a l l carbohydrates as well as oligosaccharides. Control s l i d e s were incubated i n d i s t i l l e d H 20 at 37° C. b. T e s t i c u l a r hyaluronidase ( C u l l i n g , 1963) Incubate sections for 3 hours at 37° C i n 0.1% t e s t i c u l a r hyaluronidase ( N u t r i t i o n a l Biochemicals Corporation) i n 0.85% sa l i n e and wash i n d i s t i l l e d H2O. Control s l i d e s were treated at 37° C i n 0.85% s a l i n e only. A c i d i c MPS s t a i n i n g i n the controls but not in the enzyme treated s l i d e s may be either hyaluronic acid, chondroitin 4-sulphate (chondroitin sulphate A) or chondroitin 6-sulphate (chondroitin sulphate C). 3. S e l e c t i v e blocking and unblocking a. Sulphation (Kramer and Windrum, 1954; Spicer, 1960) Dehydrated sections were treated i n sealed containers for 20 minutes at room temperature i n a concentrated H 2S04-glacial acetic acid s o l u t i o n mixed i n the r a t i o of 1:3 (v/v/), rinsed i n absolute EtOH, hydrated and stained. b. Methylation-demethylation (Spicer, 1960) Dehydrated sections were treated 4 hours at 60° C i n 0.1 N HC1 in methanol, rinsed i n absolute EtOH, hydrated and stained. To saponify, methylated sections were brought to 70% EtOH and treated with 1% KOH i n 70% EtOH for 30 minutes at room temperature. c. Acetylation-deacetylation (Pearse. 1960; Yamada. 1963) Dehydrated sections were acetylated i n 40% a c e t i c anhydride o i n anhydrous pyridine for 6 hours at 60 C, hydrated and stained. Acetylated sections were deacetylated i n a 70% EtOH:H20:NH^0H - 92 -mixture (7:1:2 by volumes) for 24 hours at 37° C, rinsed i n 707. EtOH and stained. - 93 -APPENDIX B Uronic Acid Determinations Hexuronic acids and hexuronides heated i n concentrated sulphuric acid (Fisher S c i e n t i f i c Co., Reagent grade) with or without borate and treated with carbazole (Eastman Organic Chemicals), develop a stable red colour. 1• Dische (1947) Carbazole-sulphuric acid method To 1 ml c h i l l e d sample add 6 ml i c e cold concentrated H2SO4, mix and heat 20 minutes on a b o i l i n g H2O bath. Cool i n i c e H2O for 10 minutes, add 0.2 ml 0.1% carbazole i n 95% EtOH, mix and stand at room temperature for 2 hours. Read o p t i c a l d e n s i t i e s at 530 mu. The hexuronic acid content of the unknown was calculated from a standard curve obtained using sodium glucuronate monohydrate (0.05 to umoles). 2. B i t t e r and Muir (1962) Carbazole-borosulphuric acid methdd To 1 ml c h i l l e d sample add 6 ml i c e cold 0.025 M borate i n con-centrated H2SO4, mix and heat on a b o i l i n g H2O bath for 10 minutes. Cool i n i c e H2O and add 0.2 ml 0.125% Carbazole i n absolute EtOH, mix and heat 15 minutes on a b o i l i n g ^ 0 bath. Cool 10 minutes i n i c e ^ 0 and read the o p t i c a l d e n s i t i e s at 530 mu.. The hexuronic acid content was calculated as i n the Dische method. - 94 -APPENDIX C Hexosamine Reaction Hexosamines treated with acetylacetone i n mild a l k a l i and then with E h r l i c h ' s reagent develop a c h a r a c t e r i s t i c colour. 1. Method (Elson and Morgan. 1933; Boas. 1953; and Pearson. 1963) Acetylacetone so l u t i o n - mix 1 ml pentan-2, 4-dione (Eastman Organic Chemicals) with 50 ml 2 M carbonate buffer at pH 10. E h r l i c h ' s Reagent - Dissolve 13.4 gms p-dimethylaminobenzaldehyde (Matheson, Coleman and B e l l ) in 250 ml concentrated HC1 and add 250 ml absolute EtOH. D i l u t e t h i s with 4 parts absolute EtOH just p r i o r to use. To 1 ml samples and d-glucosamine hydrochloride standards (0.05 to 2 umoles) are added concentrated HC1 to 0.4 N. The tubes are evacuated, sealed and hydrolyzed 16 hours on a b o i l i n g ^ 0 bath. The seal i s broken and the hydrolyzates dried i n vacuo over NaOH. Hydrolyzates are redissolved i n d i s t i l l e d ^ 0 , 1 ml samples mixed with 1 ml acetylacetone s o l u t i o n and heated for 45 minutes at 90° C i n sealed tubes. Unhydrolyzed standards are treated s i m i l a r l y . To each tube, 5 ml E h r l i c h ' s reagent are added, mixed and l e f t at room temperature for 90 minutes. O p t i c a l densities are read at 530 mu and the hexosamine content calculated from a standard curve prepared from the hydrolyzed standards. The non-hydrolyzed standards are used to check the destruction incurred during h y d r o l y s i s . - 95 -APPENDIX D Paper Chromatography Hydrolyzates of glycosaminoglycans are spotted on f i l t e r paper and placed i n an atmosphere saturated with a stationary solvent phase. A second, descending solvent phase i r r i g a t e s the paper and separates the hydro-lyzate components on the basis of s o l u b i l i t y . 1. Hexosamine Chromatography (Fischer and Nebel, 1955) Stationary Phase - pyridine:ethyl acetate: H2O (11:40:6 by volumes) Mobile Phase - pyrid i n e : e t h y l acetate:acetic a c i d i ^ O (5:5:1:3) About 0.06 - 0 . 1 umoles glycosaminoglycan i s hydrolyzed and dried as described i n Appendix C. The hydrolyzate i s redissolved i n 10 u l d i s t i l l e d H2O and spotted on 46 x 24 cm Whatman No. 1 paper sheets. 0 . 1 umoles of glucosamine, galactosamine and galactose standards are also spotted. The paper i s allowed to e q u i l i b r a t e with the stationary phase (saturated tank atmosphere) for 2 hours before the mobile phase i s added. 18 to 24 hour runs gave good separations. Reducing sugars are located by s i l v e r s t a i n i n g (Appendix E). 2. Hexuronic acid Chromatography (Anderson and Karamalla. 1966) Stationary and Mobile Phases - ethyl a c e t a t e : g l a c i a l acetic acid: formic a c i d ^ O (18:3:1:4 by volumes) 0.4 to 0.5 umoles, glycosaminoglycan i n 1 N HC1 were hydrolyzed on a b o i l i n g H2O bath for 4 hours and dried in vacuo over NaOH. The hydrolyzates were redissolved i n 5 ul d i s t i l l e d H2O, made - 96 -1 N with NH.OH to convert the lactones to their free hexuronic acids, 4 and spotted on Whatman No. 1 paper. Sodium glucuronate monohydrate, iduronic acid, hyaluronic acid, dermatan sulphate and chondroitin 4-sulphate standards were treated similarly. Chromatographs were then run as in part 1, and the spots located with silver staining (Appendix E). - 97 -APPENDIX E Stains Employed with Chromatography and Electrophoresis S i l v e r Stain for Chromatograp'hy (Trevelyan et a l . 1950) S i l v e r N i t r a t e Solution - Dissolve 1.8 gms Ag(N0^)2 *-n 5 ml d i s t i l l e d r^O and add 300 ml acetone. I f a white p r e c i p i t a t e forms, add H 2 O dropwise u n t i l i t d i s s o l v e s . Sodium Ethylate - Add 1.5 ml saturated NaOH to 300 ml absolute EtOH. The chromatograph was passed quickly through the s i l v e r n i t r a t e s o l u t i o n and a i r d r i e d . The spots were then made v i s i b l e by passing the paper through sodium ethylate, and d i f f e r e n t i a t e d i n 57o Na thiosulphate. A l c i a n Blue 8GS for Paper Electrophoresis (Foster and Pearce, 1961) Buffered EtOH - To 0.1 M c i t r i c acid adjusted to pH 3 with KOH i s added an equal volume of absolute EtOH. Paper electrophoresis s t r i p s were stained for 4 hours at room temperature i n 1% A l c i a n blue i n 957, EtOH d i l u t e d with 9 parts buffered EtOH. The s t r i p s were d i f f e r e n t i a t e d i n three changes of buffered EtOH at 60° C, blo t t e d on f i l t e r paper and a i r d r i e d . 

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