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Histochemistry of the developing alimentary tract of the Pacific big skate Raga binoculata Girard Evans, Robert E. 1974

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HISTOCHEMISTRY OF THE DEVELOPING ALIMENTARY TRACT OF THE PACIFIC BIG SKATE (RAJA BINOCULATA GIRARD). by ROBERT E. EVANS B.Sc. (1971) S i r George Wil l i a m s U n i v e r s i t y A Thesis Submitted i n P a r t i a l F u l f i l m e n t of the Requirements f o r the Degree Master of Science I n the Department of Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard of BRITI^I COLUMBIA March, 197^ 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 the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the 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 study. I f u r t h e r agree 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 purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of 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 not be a l l o w e d w ithout my w r i t t e n p e r m i s s i o n . Department o f Z o o Lag The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8 , Canada Date Wa^cA 21. iVf i ABSTRACT The h i s t o c h e m i c a l patterns of l e u c i n e naphthylamidase (LNAase) and a l k a l i n e phosphatase (AP) a c t i v i t y have been st u d i e d i n the developing alimentary t r a c t s of embryos (20 mm. - 200 mm.) of the skate Raja b i n o c u l a t a G i r a r d . The enzyme(s) LNAase was found only i n the s p i r a l v a l v e mucosae possessing elongated a d u l t - l i k e v i l l i . The a c t i v i t y l e v e l increases w i t h the growth of the embryo. The enzyme(s) i s probably not r e l a t e d to d i f f e r e n t i a t i v e events of the s p i r a l v a l ve but r a t h e r a m a n i f e s t a t i o n of a p a r t i c u l a r stage of f u n c t i o n a l d i f f e r e n t i a t i o n . Several p o s s i b l e p h y s i o l o g i c a l f u n c t i o n s of the enzyme(s) are discussed. The enzyme AP had a more general d i s t r i b u t i o n . I t was found i n many d i f f e r e n t i a t i n g regions of the d i g e s t i v e t r a c t . A c t i v i t y l e v e l s which were g e n e r a l l y high i n u n d i f f e r e n t i a t e d mesenchymal t i s s u e , decreased with f u r t h e r d i f f e r e n t i a t i o n i n some t i s s u e s . In other regions where AP remained i n the a d u l t , the enzyme accumulated w i t h advancing d i f f e r e n t i a t i o n . The s i g n i f i c a n c e of AP i n the v a r i o u s l o c a l i z a t i o n s i s discussed. AP isozyme patterns during alimentary t r a c t development were s t u d i e d u s i n g polyacrylamide g e l e l e c t r o p h o r e s i s . These data were c o r r e l a t e d w i t h the h i s t o c h e m i c a l l o c a l i z a t i o n of the enzyme a c t i v i t y . I t appears that new molecular forms of AP a r i s e during gut d i f f e r e n t i a t i o n . The r e l a t i o n s h i p between AP and d i f f e r e n t i a t i o n i s discussed and AP i s r e l a t e d to s p e c i f i c f u n c t i o n where a p p l i c a b l e . i i TABLE OF CONTENTS Page Abstract i L i s t of Tables i v L i s t of Plates v L i s t of Figures v i i i Acknowledgements i x INTRODUCTION 1 Leucine naphthylamidase 2 Alkaline Phosphatase ... 4 MATERIALS AND METHODS Experimental animals 3 Tissue 8 H i s t o l o g i c a l Technique ............... 9 Histochemleal Technique 9 ( i ) F i x a t i o n and embedding 9 ( i i ) Cryostat sections 10 ( i i i ) Gomori Calcium Cobalt Method .. 10 (iv) Naphthol AS-Phosphate HNF Method 10 (v) Leucine Naphthylamidase Method , 11 (vi) Controls 11 Disc Electrophoresis 11 ( i ) Tissue 11 ( i i ) Enzyme extraction 12 ( i i i ) Electrophoresis 13 (iv) Gel staining 13 * • • 111 Page RESULTS Histochemical Technique 14* Relative Alkaline Phosphatase Activity 15 Leucine Naphthylamidase Localization . 18 Alkaline Phosphatase Localization .... 21 Oesophagus 21 Cardiac stomach 23 Pyloric stomach 25 Spiral valve 28 Disc Electrophoresis 33 DISCUSSION Leucine Naphthylamidase ^0 Alkaline Phosphatase ^3 SUMMARY 56 REFERENCES 57 i v LIST OF TABLES TABLE I. A table of the r e l a t i v e AP a c t i v i t y of embryonic alimentary t r a c t tissues at d i f f e r e n t stages of development. TABLE I I . A table of the r e l a t i v e LNAase a c t i v i t y i n embryonic s p i r a l valves at d i f f e r e n t stages of development. Page. 17 19 LIST OF PLATES PLATE I. 1. Cross-seetion of s p i r a l valve from a 192 mm. embryo, revealing s i t e s of LNAase a c t i v i t y . PLATE I I . 2. Cross-section of oesophagus from a 73 mm. ©s&r embryo, showing AP a c t i v i t y using HNF. 3. Cross-section of oesophagus from an 80 mm. embryo, revealing AP a c t i v i t y . 4-. Cross-section of oesophagus from a 1^0 mm. embryo, i l l u s t r a t i n g response to AP substrate. 5. Cross-section of oesophagus from a 1?0 mm. embryo, i l l u s t r a t i n g AP l o c a l i z a t i o n . 6. Cross-section of oesophagus from a 192 mm. embryo, revealing AP response with HNF. PLATE I I I . 7. Cross-section of cardiac stomach from a kk mm. embryo showing AP a c t i v i t y . <p8. Cross-section of cardiac stomach from a 72 mm. embryo showing AP a c t i v i t y . 9. Gross-section of a portion of cardiac stomach from a 75 mm. embryo, revealing AP a c t i v i t y . 10. Cross-section of cardiac stomach from an 8k mm embryo showing s i t e s of AP a c t i v i t y . 11. Cross-section of cardiac stomach from a 112 mm embryo, showing s i t e s of AP a c t i v i t y . PLATE IV. 12. Cross-section of a portion of cardiac stomach from a 121 mm. embryo showing AP v i a HNF. 13. Cross-section of a portion of cardiac stomach from a 150 mm. embryo showing AP a c t i v i t y during cardiac gland development., Ik. Cross-seetion of a cardiac stomach f o l d from a 170 mm. embryo, manifesting s i t e s of AP. v i Page 15. Longitudinal-section of cardiac glands from a 170 mm. embryo showing AP localization. 26 16. Gross-section of cardiac glands from a 170 mm. embryo revealing sites of AP activity. 26 PLATE V. 17. Cross-section of the pyloric stomach from a 52 mm. embryo showing AP activity. 27 18. Cross-section of the pyloric stomach from a 75 mm. embryo manifesting AP activity. 27 19. Cross-section of the pyloric stomach from an 84 mm. embryo showing AP activity. 27 20. Cross-section of the pyloric stomach from a 112 mm. embryo showing AP activity with HNF Zr during epithelial folding. 27 21. Cross-section of the pyloric stomach from a 14-0 mm. embryo during epithelial folding. 27 PLATE VI. 22. Cross-section of the pyloric stomach from a 170 mm. embryo showing AP activity during pyloric gland development. 29 23. Cross-section of a pyloric stomach fold from a 180 mm. embryo showing AP localization. 29 24-. Cross-section of the pyloric stomach fold from the adult Ra.ja binoculata showing AP sites. 29 PLATE VII. 25. Cross-section of the spiral valve from a 44 mm. embryo showing AP localization. 30 26. Cross-section of the spiral valve from a 52 mm. embryo revealing sites of AP activity. 30 27. Cross-section of the spiral valve from a 72 mm. embryo showing AP localization. 30 28. Cross-section of the spiral valve from a 75 nun. embryo revealing sites of AP activity. 30 29. Cross-section of the spiral valve from a 95 mm. embryo manifesting AP response to substrate. 30 v i i Page PLATE VIII. 3 0 . Cross-section of spiral valve v i l l i from a 170 mm. embryo showing brush border AP activity. 32 3 1 . Cross-section of a spiral valve v i l l u s from a 170 mm. embryo i l l u s t r a t i n g brush border AP. 32 3 2 . Cross-section of spiral valve v i l l i from a 180 mm. embryo showing sites of AP activity. 32 33* Cross-section of a portion of a spiral valve v i l l u s from as adult Ra.ja binoculata. showingv*.. ; AP response to AP substrate i n the Golgi zone. 32 v i i i LIST OF FIGURES FIGURE U Adult (Group I) spiral valve alkaline phosphatases i n acrylamide gel. FIGURE 2. Diagrammatic representation of the adult (Group I) spiral valve alkaline phosphatases as pictured in FIGURE 1 . FIGURE 3. The developmental sequence of alkaline phosphatase differentiation from various embryonic alimentary tract regions. FIGURE k. Diagrammatic representation of the alkaline phosphatase differentiation i n various alimentary tract regions as pictured i n FIGURE 3. Page 3k 35 37 38 ACKNOWLEDGEMENT To my sup e r v i s o r , Dr. Peter Ford, I extend very s p e c i a l thanks f o r h i s i n t e r e s t , encouragement and as s i s t a n c e during the i n v e s t i g a t i o n and manuscript pr e p a r a t i o n . I am also g r a t e f u l to Dr. C V . Fin n i g a n and Dr. J.E. P h i l l i p s f o r reading the manuscript and o f f e r i n g h e l p f u l suggestions. S p e c i a l thanks are also extended to the captains and crews of the F i s h e r i e s Research v e s s e l s A.P. Knight and I n v e s t i g a t o r I I , f o r t h e i r work i n o b t a i n i n g specimens. The a s s i s t a n c e of Mr. W. Coward i n pro c u r i n g the F i s h e r i e s Research v e s s e l s was much appreciated. I am al s o g r a t e f u l to the Na t i o n a l Research C o u n c i l f o r t h e i r f i n a n c i a l support during part of the i n v e s t i g a t i o n . F i n a l l y , I would l i k e to express my a p p r e c i a t i o n to my wif e Linda, f o r her encouragement, and to my daughter Tara f o r her endearing charm. 1 INTRODUCTION The study of embryonic enzyme and isoenzyme patterns i s important because they may suggest where c r i t i c a l changes are occurring i n the esse n t i a l a c t i v i t i e s of organ and tissue development. I t i s apparent that c e l l u l a r a c t i v i t i e s are limited by t h e i r enzymatic composition. Therefore, the study of embryonic enzymology i s important, even i f i t i s done i n a simple descriptive manner (Moog, 1959). H i s t o^ j ' V v r « ; chemically, the amount of demonstrable enzyme may be in d i c a t i v e of the enzyme's contribution within the developing tissue (Moog, 1965). With few exceptions, the postulate of p a r a l l e l development of enzyme a c t i v i t y and enzyme function i s an accepted fact (Herrmann and Tootle, 196*0. In view of the orderly nature of embryonic l i f e , the embryo probably does not store a large reserve of enzymes (Boell, 1955). Therefore an increase i n enzyme a c t i v i t y may indicate an increase i n capacity of the enzyme system to carry out i t s metabolic function. We must however, be cautious i n in t e r p r e t i n g enzyme data since a r i s e i n enzyme l e v e l s may be due to d i f f e r e n t i a l growth of a c e l l type r i c h i n the enzyme rather than a r i s e i n enzyme l e v e l per c e l l . S i m i l a r l y the enzyme within the c e l l may function at submaximal le v e l s due to i t s involvement i n a metabolic pathway that l i m i t s i t s substrate supply. Also the enzyme and substrate may be s p a t i a l l y separated by some subcellular s t r u c t u r a l unit to which the enzyme may 2 be bound (Hermann and Tootle, 1964). There i s suggested a relationship between enzyme complement and cellular differentiation. Two groups of enzymes that we believed might be involved i n the cellular and morphologic differentiation of the alimentary tract were leucine naphthylamidase (LNAase) and non-specific alkaline phosphatase (AP). Therefore we studied the histochemical patterns of LNAase and AP localization, and AP isozymic patterns within the gastrointestinal tract of the skate Raja binoculata during development from 20 mm. to hatching (approx. 200 mm.). The skate Raja binoculata Girard was selected for this study because previous reports of this nature have been concerned with teleost fishes and higher vertebrates, and nothing i s known oftthe more specialized and primitive elasmobranchs. The microscopic anatomy of the alimentary tract of the adult skate was found to be similar to that described by M.X. Sullivan (1907) for the elasmobranchs Mustellus canis and Garcharias l i t t o r a l i s . The embryonic development was also similar to that of the Elasmobranchii as described by W. Balfour (1881), F.M. Balfour (1878) and specifically for Raja batis, (Beard, 1890). Leucine Naphthylaimdase Enzymes that act upon the substrate leucyl-^-naphthylamide (LNA) were formerly referred to as leucine aminopeptidase (LAP). Pew studies of LNA s p l i t t i n g enzymes 3 during embryonic development and p a r t i c u l a r l y i n the embryonic g a s t r o - i n t e s t i n a l t r a c t have been c a r r i e d out. Baxter-Grillo, ( 1 9 7 0 ) , observed an increase i n LAP from 8 t h - l 6 t h day i n chick when rapid c e l l u l a r degeneration and p r o l i f e r a t i o n at the morphological d i f f e r e n t i a t i o n s of the stomach-complex and in t e s t i n e take place. Changes i n the isozyme pattern of LAP from i n t e s t i n e s of developing human foetusesrwas observed by Pataryas and Christodoulou, ( 1 9 7 0 ) . In 1 9 6 3 , conclusive evidence was offered to show that LAP was not s i g n i f i c a n t l y responsible f o r the hydrolysis of aminoacyl naphthylamidase by tissue sections (Patterson et a l . , 1963; Hanson et al., 1 9 6 3 ) . In a series of papers Sylven showed that at lea s t s i x d i f f e r e n t groups of enzymes were capable of hydrolyzing the chromogenie substrate LNA (Sylven and Bois, 1962; Sylven and Bois, 1963; Sylven and Snellman, 1964; Sylven and Snellman, 1968; Sylven and Bois-Svensson, 1 9 6 4 ) . Therefore we w i l l r e f e r to the enzymatic response to LNA substrate (hydrolysis of the CN bond) as LNAase (Pearse, 1 9 7 2 ) . The LNA s p l i t t i n g LAP enzymes are exopeptidases and require a free «*-amino group (Chayen et al,, 1 9 6 9 ) . Hydrolysis however i s not r e s t r i c t e d to l e u c y l compounds. The rate of hydrolysis depends on the nature of the side chain of the N-terminal residue (Smith and Spackman, 1955)* In t h i s work, LNAase i s operationally defined as an enzyme a c t i v i t y that hydrolyzes L-leucyl-^-naphthylamide to li b e r a t e naphthylamine. The product of the reaction i s 1* trapped with a diazonium s a l t to form an insoluble azo dye. This histochemical approach demonstrates only the enzyme s i t e s available to substrate i n t e r a c t i o n . Therefore i n i n  vivo conditions these enzymes probably were active and therefore able to s p l i t the CN bond of the LNA (Sylven and Bois, 1963). Although an attempt i s made to discuss the b i o l o g i c a l implications of t h i s group of LNA hydrolyzing enzymes i n the s p i r a l valve and i t s v i r t u a l absence i n other t r a c t areas, we are i n h i b i t e d by an ignorance of the natural substrate(s). Alkaline Phosphatase The phosphatases can be divided into s p e c i f i c and non-specific groups. In t h i s report we w i l l be concerned with non-specific phosphatase and i n p a r t i c u l a r a l k a l i n e phosphatase (E.C.3131) as opposed to acid phosphatase. These are r e a d i l y distinguished by t h e i r pH optima (alkaline pH 9.5, and ac i d pH 5)« Alkaline phosphatase i s a phosphomonoesterase which hydrolyzes a v a r i e t y of organic phosphates. I t i s assumed that only the phosphoric ac i d group of the substrates i s e s s e n t i a l f o r the reaction since there i s a lack of s p e c i f i c i t y toward the alcohol r a d i c a l attached to the phosphoryl group. Therefore i t was suggested that the most important i n vivo a c t i v i t y of the phosphatases i s concerned with the transfer of phosphate from one aleohol to another (Dixon and Webb, 196k; Pearse, 1968). Alkaline phosphatase i s probably not involved i n phosphate ester synthesis since Stadtman (1961) found that the reactant concentrations had 5 to be extremely high f o r synthesis to occur. Although there i s no evidence that phosphatase a c t i v i t y i s a manifestation of phosphate transfer rather than hydrolysis of phosphate esters, histochemical data become more s i g n i f i c a n t i f wesassume the former. However, caution i s necessary i n that the histochemical approach may give us an i n d i c a t i o n of p o t e n t i a l i n vivo enzyme a c t i v i t y and not . the actual i n vivo a c t i v i t y (Pearse, 1968). No s p e c i f i c function can be a t t r i b u t e d to AP i n embryos and adults, and many speculations have been proposed. AP has been associated with d i f f e r e n t i a t i o n (Moog, 1952), morphogenesis (Piatka and Gibley, 1967)» and organogenesis (Brachet, 19^6). Phosphatases have also been linked to secretion ( D a n i e l l i , 1952), bone formation (Lorch, 19^9b), protein and nucleic acid metabolism (McWhinnie and Saunders, 1966; Ikeda, 1959), c e l l adhesion (Millington and Brown, 19&7), and transduction of energy f o r movement (Clark, 1961). More s p e c i f i c a l l y , i n t e s t i n a l mucosa AP has been implicated with digestion (Holt and M i l l e r , 1961j M i l l e r and Crane, 1961; Rothstein et. al., 1953)* absorption and transport across the plasma membrane (Bonneville and Weinstock, 197©)» u t i l i z a t i o n of L-amino acids ( Watanabe and Fishman, 1963)» and i n providing inorganic phosphate fo r metabolic processes (Narayanan et' alw 1972; Schmidt and Laskowski, 1961). When the histochemical data was completed we studied, through disc electrophoresis, the isozymic pattern of alimentary t r a c t AP during development. Polyacrylamide gel 6 electrophoresis on AP extracts was done to observe i f ' indeed the electrophoretic patterns of AP change q u a l i t a t i v e l y during d i f f e r e n t i a t i o n of the alimentary t r a c t . AP isozyme patterns have been observed to a l t e r during morphogenesis and c e l l u l a r d i f f e r e n t i a t i o n i n other species (Soloman et al., 196*4-; Beckman and Johnson, 1964bj E t z l e r and Moog, 1968bj Moog, Vire and Grey, 1966; Moog, 1965; Pataryas and Christodoulou, 1970). The isozymic pattern of a tissue may r e f l e c t the state of d i f f e r e n t i a t i o n of i t s c e l l s . For example, the appearance of a new phosphatase isozyme coincides with the d i f f e r e n t i a t i o n of the m i c r o v i l l i of c e l l s at the v i l l i t i p s into the long, narrow form c h a r a c t e r i s t i c of the mature animal (Overton, 1965). This may r e f l e c t gene a l t e r a t i o n during development or s t r u c t u r a l changes which may a l t e r enzyme binding and/or a c t i v i t y . Since each tissue has i t s own c h a r a c t e r i s t i c isozyme pattern f o r a p a r t i c u l a r group of enzymes with s i m i l a r a c t i v i t y , a s p e c i f i c p h ysiological r o l e f o r each isozyme i s implied (Markert and Apella, 1961). Using the s p i r a l valves from four d i f f e r e n t adult elasmobranchs, the r e p r o d u c i b i l i t y of the electrophoretic techniques were tested. Then data was c o l l e c t e d from several developmental periods and from each i n d i v i d u a l gastro-i n t e s t i n a l t r a c t organ where tissue quantities permitted. The electrophoretic data obtained was correlated with the histochemical r e s u l t s . A discussion includes the possible functional 7 signi f i c a n c e s of the enzymes LNAase and AP at t h e i r various histochemical l o c a l i z a t i o n s during p a r t i c u l a r stages of development. Comparison i s often made with other species. The possible relevance of the changing isozyme patterns i s discussed. 8 MATERIAL AND METHODS Experimental Animals The animal selected for t h i s i n v e s t i g a t i o n was the embryo of the P a c i f i c b i g skate (Ra.ja binoculata Girard). The egg cases of t h i s oviparous elasmobranch were c o l l e c t e d by drag from the S t r a i t of Georgia o f f Comox on Vancouver Island. Seventy egg cases containing an average of four embryos per egg case were c o l l e c t e d at i n t e r v a l s between September, 1971 and August, 1973• A l l embryos were measured to the nearest millimeter from the t i p of the rostrum to the end of the t a i l . Only embryos that measured 20 millimeters or greater i n t o t a l length were used. I t should be mentioned that the actual embryo length, or r o s t r a l - c l o a c a l length, i s less than h a l f the rostral-caudal length. The smaller the embryo, the greater i s the proportion of t a i l i n the r o s t r a l -caudal length (Ford, 1971 )• Live specimens, within t h e i r protective egg cases, were maintained f o r several weeks i n sea water aquaria with a water temperature of approximately 10°C. Other embryos were either f i x e d or were frozen over dry i c e immediately a f t e r capture. For the larger embryos (greater than 100 mm.), the alimentary t r a c t was dissected out p r i o r to f i x a t i o n or freezing. A few adult animals were also used f o r comparative purposes. Tissue A l l embryos that were less than 100 mm. i n t o t a l body length were l e f t i n t a c t and used as such. Although observations were made on the whole alimentary t r a c t , only 9 the main organs w i l l be considered. The larger embryos had t h e i r alimentary t r a c t s excised and then divided into four regions; oesophagus, cardiac stomach, p y l o r i c stomach, s;and s p i r a l valve. The mid-region of each organ was chosen for study. This proved to be a s a t i s f a c t o r y method of gaining an o v e r a l l picture of enzyme a c t i v i t y i n the t r a c t since several preparations that were made from tissue at the extremities of the organs were histochemically consistant with the c e n t r a l l y located areas. The duodenal and r e c t a l areas of the s p i r a l valve did show morphological differences and should be considered separately. H i s t o l o g i c a l Technique Following f i x a t i o n i n Bouin's f l u i d f o r a minimum of 24- hours, whole embryos or portions of t h e i r alimentary t r a c t s were dehydrated i n ethanol, cleared i n terpineol and embedded i n wax (Fisher Tissuemat) i n the usual manner. S e r i a l transverse sections of the smaller embryos were cut at 8 microns. For the larger embryos where only a portion of the oesophagus, cardiac stomach, p y l o r i c stomach, or s p i r a l valve was u t i l i z e d , at l e a s t three s l i d e preparations of each were made. These were also cut at 8 microns. The s l i d e preparations were then stained with Heidenhain's Iron Hematoxylin and counterstained with Eosin (Galigher and Kozloff, 1964). Histochemical Technique ( i ) F i x a t i o n and embedding Live whole embryos or small pieces of gut were f i x e d f o r 2k hours at k°C. i n absolute acetone. After dehydration i n ethanol the tissues were double embedded (Pearse, 1968). The p a r a f f i n wax embedding was done at 56°C. f o r one hour. Transverse sections were cut at 8 microns and tested f o r a l k a l i n e phosphatase, ( i i ) Gryostat sections To avoid f i x a t i o n and f o r comparative purposes, fresh cryostat sections were prepared. Live tissues were frozen by quenching i n an isopentane-dry i c e mixture and embedded i n Cryoform (IEC). Sections were cut at 16 microns on an International-Harris cryostat, Model CT (IEC), mounted on coverglasses, and a i r dried f o r ten minutes. Alternate sections were tested histochemically f o r a l k a l i n e phosphatase and leucine naphthylamidase l o c a l i z a t i o n , ( i i i ) Gomori Calcium Cobalt Method Histochemical l o c a l i z a t i o n of non-specific a l k a l i n e phosphatase was c a r r i e d out by the Gomori Calcium Cobalt method (Pearse, 1968) on acetone f i x e d tissue. This technique produces sharper pictures with f i n e r granulation than most other a l k a l i n e phosphatase methods. Incubation time with Na-/J-glycerophosphate as substrate was considered maximal at one hour. Actual incubation times f o r minimal a c t i v i t y were much l e s s . (iv) Naphthol AS-Phosphate Hexazotized New Fuchsin (HNF) For comparative purposes the Naphthol AS-Phosphate HNF (Triamino-tritolylmethane chloride) method f o r a l k a l i n e phosphatase (Stutte, 1967) was employed on both acetone f i x e d and on cryostat sections. With naphthol AS-MX phosphate as 11 substrate (Pearse, 1968), incubation time once again was usually much less than one hour to obtain visi b l e reaction. In some instances with this procedure and similarly for the Gomori method, incubation times were reduced to less than one minute. This was particularly true of the larger embryos, (v) Leucine Naphthylamidase Method Cryostat tissue sections that were employed for leucine naphthylamidase (LNAase) localization with L-leucyl-^-naphthylamide (LNA) as substrate and Past Blue B as diazonium salt (Chayen et al., 19^9) were incubated for a maximum of two hours. Fresh substrate was substituted every fifteen minutes. In most cases visible reaction was obtained within the f i r s t fifteen minutes, (vi) Controls Control slides were prepared for most of the substrate treated sections. For the Gomori Calcium Cobalt Technique water was substituted for substrate. In the Naphthol AS-Phosphate HNF method only the substrate was omitted from the reaction mixture. Similarly, L-leucyl-£-naphthylamide was omitted from the leucine naphthylamidase reactions as a control. Disc Electrophoresis (i) Tissue Due to a limited supply of tissue during the electrophoretic studies, only the following groups of embryos were examined. Group I consisted of the spiral valves from several adults of the elasmobranchs Ra.ja binoculata, Ra.ja 12 ghina, Ra.ia k i n c a i d i i . and Squalus acanthi as. Groups II and III consisted of the complete alimentary t r a c t s from embryos o f R a - i a binoculata ranging i n size from 4o-6l mm. and 78-91 mm. respectively. Extracts from embryos i n the size range 138-15^ mm., Group IV, were made from s p i r a l valves, oesophagus, and combined cardiac and pylorie stomachs. F i n a l l y , Group V embryos of 187-20^ mm. included separate extracts from each gut organ. ( i i ) Enzyme extraction A modification of the n-butanol extraction procedure of Ahmed and King ( i 9 6 0 ) was u t i l i z e d f o r samples to be subjected to electrophoretic analysis. One gram of b l o t dried» f i n e l y cut up tissue was added to l i m l . of d i s t i l l e d water i n a pyrex (2 ml.) tissue grinder. This mixture was homogenized f o r two minutes i n an i c e bath. While homogenizing, 2 ml. of n-butanol were added inO. 5 ml. aliquots over the next 8 minutes. (Solution volumes were altered accordingly when smaller weights of tissue were used.) This emulsion was s t i r r e d vigorously f o r 25 minutes at room temperature. I t was then warmed f o r 5 minutes at 3 5°C while being s t i r r e d . The extract was then centrifuged f o r 15 minutes at 2000 rpm. The aqueous zone, l y i n g above the heavy p r e c i p i t a t e and below the butanol and l i g h t p a r t i c l e layers, was removed by suction. This extract was then subjected to freeze drying i n order to concentrate the enzyme. Water extracts without butanol were found to have mueh less active enzyme than the n-butanol extracted samples. The increase i n enzymatic a c t i v i t y a f t e r n'-butanol extraction probably indicates l i b e r a t i o n of 13 l i p o p r o t e i n bound enzyme, ( i i i ) Electrophoresis Disc electrophoresis i n 7*5% acrylamide gels, with a T r i s - g l y c i n e upper buffer and Tris-HGl lower buffer, as described i n the Polyanalyst (Buchler Instruments) i n s t r u c t i o n manual, was used to reveal the a l k a l i n e phosphatase patterns of the tissues. Samples were run i n a Polyanalyst disc electrophoresis apparatus (Buchler Instrument Go.) at 3 milliamps per gel f o r approximately one hour and f i f t e e n minutes. The length of the run corresponded c l o s e l y to the time required f o r a bromophenol blue dye marker to migrate to within 5 millimeters of the end of the g e l . Sample density was increased with sucrose. Sample volumes of approximately 75 m i c r o l i t e r s were layered with a syringe on the surface of a 3% acrylamide stacking gel (Ornstein, 1964; Davis, 1964) . Three runs were done from each sample, (iv) Gel staining The gels were stained f o r AP l o c a l i z a t i o n using the HNF histochemical procedure of Stutte (1967)* as outlined by Pearse (1968), page 716. The reaction was c a r r i e d out i n a 0.1M. Tris-HCl buffer at pH 9 .2 at room temperature f o r one hour. AP enzyme bands were coloured red. The use of t h i s procedure allowed f o r comparison with the histochemically demonstrable enzymes since the same method with the same substrate (Naphthol AS-MX phosphate) was employed. The gels were then stored at room temperature i n a mixture of methanol, water and acetic a c i d ( 5 » 5 s l ) u n t i l photographed. 1 4 RESULTS Histochemical Technique Gomori Calcium Cobalt Method The black p r e c i p i t a t e of cobalt sulphide formed i n the tissue at s i t e s of AP a c t i v i t y gave excellent c l a r i t y and contrast for observational and photographic purposes. Nuclear st a i n i n g was a consistant observation i n a l l areas that showed a response to substrate. The bulk of experimental work on t h i s aspect of the Gomori method suggest that nuclear staining i s an a r t e f a c t . Calcium phosphate, an intermediate i n the reaction, i s p r e f e r e n t i a l l y absorbed by nuclei (Novikoff et al., 1 9 5 2 ) . The azo dye techniques as well as electron microscopic methods for AP (Hugon and Borgers, 1 9 6 6 ) show no evidence of nuclear reactions. S i m i l a r l y no AP a c t i v i t y was observed i n i s o l a t e d nuclei of mouse (Yokoyama et a l . . 1 9 5 0 ) and rat l i v e r c e l l s (Novikoff et al . . 1 9 5 0 ) . Therefore nuclear staining i n skate tissue was presumably due to artefact and was not considered to be s i g n i f i c a n t . Naphthol AS-Phosphate HNF Method The HNF method of Stutte on acetone f i x e d tissue was also observed to give consistant and cl e a r r e s u l t s . Although the degree of response was s i m i l a r to the Gomori technique, the red azo dye did not produce the contrast that was necessary for black and white photographs. However, acetone f i x e d tissue did not give a nuclear response and a l l the regions that reacted were in- accord with those observed i n the 15 Gomori procedure. The c r y o s t a t s e c t i o n s t h a t were attempted proved to give the greatest response to substrate although d i f f u s i o n a r t e f a c t was considerable. Leucine Naphthylamidase Method The l e u c i n e naphthylamidase method wi t h c r y o s t a t s e c t i o n s gave a p a r t i c u l a t e response w i t h some d i f f u s e s t a i n as compared to the f i n e g r a n u l a t i o n of the AP r e a c t i o n products. The c r y o s t a t s e c t i o n s however d i d not y i e l d sharp l o c a l i z a t i o n and made i n t e r p r e t a t i o n d i f f i c u l t . S l i d e preparations were kept only two days since the r e a c t i o n products were very unstable. R e l a t i v e A l k a l i n e Phosphatase A c t i v i t y  General I n a c t i v i t y Tissue s e c t i o n s of the alimentary t r a c t of the 20 mm. embryo were devoid of AP a c t i v i t y (Table I ) . The l i m i t of s e n s i t i v i t y o f the AP technique of Gomori was approximately 25 micromoles (about 1 Bodansky u n i t ) per gram of r e a c t i n g t i s s u e i f the time of i n c u b a t i o n was one hour (Gomori, 1950c). Therefore i t was p o s s i b l e that undetectable t r a c e amounts of enzyme were present i n the embryonic t i s s u e . Endoderm A summary of r e s u l t s presented i n Table I i l l u s t r a t e s t hat d e f i n i t e patterns of enzyme a c t i v i t y e x i s t e d w i t h i n the four organs s t u d i e d and i n some cases were s i m i l a r i n a l l the organs s t u d i e d . The endodermal component f o r example, which e v e n t u a l l y formed the e p i t h e l i u m o f the mucosa was c o n s i s t a n t l y AP negative i n the oesophagus, ca r d i a c stomach, 16 and p y l o r i c stomach. In the s p i r a l valve however, the epidermal c e l l s were negative up to the point when the brush borders began to d i f f e r e n t i a t e . The brush border d i f f e r e n t i a t i o n had concomitant AP response as reported for other species including frog, guinea pig, f o e t a l mouse, and chick embryo (Brown and Millington, 1968). Golgi Apparatus According to Bourne (1943)# a Golgi response i s a f a m i l i a r phenomenon of the i n t e s t i n a l epithelium. Throughout t h i s study no enzyme reaction products were observed i n the Golgi zone of the gut e p i t h e l i a l c e l l s of skate embryos, i n contrast to other species (Deane and Dempsey, 19^5; Emmel, 1945; Hugon, 1970; Hugon and Borgers, 1968; Novikoff et al.. 1952). The AP reaction product was c l e a r l y observable i n the Golgi zone of the adult skate s p i r a l valve epithelium. I t i s not uncommon however that no response was observable i n the Golgi region of the embryos since none was seen i n embryos and adult steelhead trout (Prakash, 1961). S i m i l a r l y no reaction was observed i n goblet c e l l s of the oesophagus or s p i r a l valve throughout t h i s study. Mesoderm The mesodermal components of each organ displayed some consistant features. For example, the mesenchyme of a l l the organs studied acquired AP a c t i v i t y at an early point i n development. Mesenchyme c e l l processes were very reactive. As t h i s tissue reverted to the connective tissues of the lamina propria and/or submucosa, the f i b r o b l a s t and macrophage cytoplasmic c e l l processes continued to show Table of r e l a t i v e AP a c t i v i t y based on a maximal a c t i v i t y i n the adult brush border (BB) a f t e r one hour incubation i n substrate. *- observations based on the HNF r e s u l t s , a l l other r e s u l t s based on the Gomori method. o- represents a response i n the outer l o n g i t u d i n a l muscle layer of the muscularis. A l l other references to the muscularis represent both c i r c u l a r and l o n g i t u d i n a l muscle layers. RELATIVE ACTIVITY TISSUE OESOPHAGUS Epithelium Mesenchyme Submucosa Muscularis Serosa Leydig Organ CARDIAC STOMACH Epithelium Mesenchyme Lamina propria Submucosa Muscularis Serosa PYLORIC STOMACH Epithelium Mesenchyme Lamina propria Submucosa Muscularis Serosa SPIRAL VALVE Epithelium (BB) . Mesenchyme Lamina propria Muscularis mucosa Submucosa Muscularis Serosa EMBRYO LENGTH IN MILLIMETERS 20* 30 44 52 61* 72 80 84 95 112* 121* 140 150 170 180* 190 Adult + +--. + ++ + + ++ ++ ++ ++ ++ ++ ++ +++ ++ ++ ++ + - - - - -++ ++ + +- +- + +-+ + - - + -- + -' - - +++ +++ +++ +++ ++•;•• +++ +++ ++ +++ +++ +++ +++ ++ + +- +- - - -.+- +- +-° *° - - -++ ++ +- + + ++ — +- — - +++ +++ +++,+++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ .+- ++ +n - - + + + ++ ++° +° - - - -++ ++ + +- +- +- + - - + -- - +- +- + + + + ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +- ++ ++ ++ ++ + + + + + +- ++ +++ +++ ++ + - — ++ ++ + - — - ++ 1 - f — +-- - +- +- - — - - — - ' +- -18 response to substrate i n most cases. This was s i m i l a r i n d i f f e r e n t i a t i n g smooth muscle tissue of the alimentary t r a c t . The AP d i f f e r e n t i a t i o n i n the smooth muscle layers of the muscularis was i n t e r e s t i n g . The reactive mesenchyme tissue became more compact at some point i n each organ (approx. 52 mm. i n oesophagus and 72 mm. i n the other gut areas). The AP response persisted as the conversion from mesenchyme to smooth muscle c e l l s took place. Then when the smooth muscle c e l l s appeared to be morphologically d i f f e r e n t i a t e d the AP r e a c t i v i t y was l o s t (Table I ) . The lamina propria of the cardiac stomach, p y l o r i c stomach and s p i r a l valve was always active f o r AP. This observation was eonsistant with that i n steelhead trout (Prakash, 1 9 6 l ) . Leucine Naphthylamidase L o c a l i z a t i o n The only tissue that gave a response to the LNA substrate was the s p i r a l valve. Reactivity was f i r s t observed i n the 112 mm. embryo whereas none was observed i n the 97 mm. embryo even a f t e r two hours incubation time (Table I I ) . A l l s p i r a l valves i n embryos larger than 112 mm. were p o s i t i v e . When LNAase a c t i v i t y was observed i t was always manifested a f t e r only 15 minutes incubation and more l i g h t l y so a f t e r 5 minutes. The brush border and terminal web region appeared to be the most strongly p o s i t i v e area. However, only the lower portion of each v i l l u s extending to the crypts of Lieberkuhn showed a response. E p i t h e l i a l c e l i s i n the crypts gave a weaker response. The mucosa of the uppermost region of each v i l l u s 19 Table I I . Table of r e l a t i v e LNAase a c t i v i t y i n s p i r a l valve based on a maximal a c t i v i t y i n the adult brush border a f t e r two hours incubation i n substrate. Note: A l l other organs studied as well as the s p i r a l valves from embryos of 97 mm. and less were LNAase negative. TISSUE SPIRAL VALVE  Mucosa-Brush Border E p i t h e l i u m Lamina p r o p r i a Muscularis mucosa Submucosa Muscularis v i l l u s t i p v i l l u s base c r y p t o f Lieberkuhn v i l l u s t i p v i l l u s base c r y p t o f Leiberkuhn v i l l u s t i p v i l l u s base c r y p t o f Lieberkuhn v i l l u s t i p v i l l u s base c r y p t o f Lieberkuhn Serosa RELATIVE ACTIVITY EMBRYONIC LENGTH IN MILLIMETERS 97 112 121 140 150 170 180 . 192 Adul-++ +++ +++ +++ ++++ ++++ ++++ ++++ + + ++ ++ ++ +++ +++ +++ +- +- +- + + + + +- +- +- +- + + + + +- +- +- +- +- •f- + +- +- +- +- +- +- +- •f 26 PLATE I. 0 1. Cross-section of s p i r a l valve from a 192 mm. embryo. Cryostat: Leucine Naphthylamidase, 15 minutes incubation. Sites of LNAase a c t i v i t y appear black. 200X 20a 21 was non-reactive. The extremely p a r t i c u l a t e p r e c i p i t a t e was also observable i n the lamina propria to a l e s s e r extent. Muscularis mucosa, submucosa and serosa were non-reactive. Response to the LNAase substrate seemed to increase with the s i z e of the embryo (PLATE,I, 1 ) . Alkaline Phosphatase L o c a l i z a t i o n  Oesophagus The AP a c t i v i t y i n the oesophageal region i s the lowest of any area i n the g a s t r o - i n t e s t i n a l t r a c t . The oesophagus of the embryonic skate showed no histochemically demonstrable AP enzyme a c t i v i t y u n t i l the embryo had attained a t o t a l length of 52 mm. Enzyme l o c a l i z a t i o n was r e s t r i c t e d to the region of the developing muscularis and serosa (PLATE I I , 2 and 3 ) . The enzyme a c t i v i t y was l i m i t e d to smooth muscle c e l l s and endothelial c e l l s of c a p i l l a r i e s between groups of mesenchyme c e l l s . The a c t i v i t y was i n i t i a l l y quite low but increases somewhat i n larger embryos. When the muscularis had reached morphological maturity i n embryos larger than 14-0 mm., the enzyme reaction i n the smooth muscle c e l l s was l o s t . AP a c t i v i t y became evident i n another region of the oesophagus i n embryos of 112 mm. or greater (PLATE I I , 4). The submucosa which had now d i f f e r e n t i a t e d below the oesophageal epithelium, since the lamina propria was r e s t r i c t e d to the basement membrane, contained a broad band of weak^enzymevactivity i n the areolar connective tissue. This band of enzyme active tissue increased i n response to 22 PLATE I I . 2. Cross-section of oesophagus from a 73 mm. embryo showing AP a c t i v i t y i n the developing muscularis, at the arrow. HNF- 1 hour incubation. 500X 3. Cross-section of oesophagus from an 80 mm. embryo revealing AP a c t i v i t y i n the developing muscularis. (arrow) Gomori- 1 hour incubation. 200X 4. Cross-section of oesophagus from a IkO mm. embryo i l l u s t r a t i n g AP a c t i v i t y i n developing muscularis and submucosa (arrows). Gomori -1 hour incubation. 200X 5. Cross-section of oesophagus from a 170 mm. embryo showing AP a c t i v i t y i n submucosa (arrow). Gomori- 30 minutes incubation. 350X 6. Cross-section of oesophagus from a 192 mm. embryo showing submucosal AP a c t i v i t y (arrow). Cryostat HNF- 1 hour incubation. 200X Legend- e- epithelium, Lo- Leydig organ, L- lumen, S- submucosa. 22a 23 substrate and was condensed to a more narrow region as e p i t h e l i a l f o l d i n g took place and the embryos increased i n size (PLATE I I , 5 ) . This small enzyme r i c h region of the submucosa was found io p e r s i s t i n already hatched skates (PLATE I I , 6 ) . As previously mentioned the epithelium was always non-reactive. The serosa showed variable p o s i t i v e a c t i v i t y (Table I ) . The Leydig organs which began to d i f f e r e n t i a t e at about 6 l mm., also manifested spotted r e a c t i v i t y . The lymphocytes and granulocytes of t h i s organ remained negative. Cardiac Stomach Histochemically demonstrable AP a c t i v i t y was very evident i n the mesenchyme tissue surrounding the epithelium of the cardiac stomach of the 37 mm. embryo (PLATE I I I , 7 ) . Enzyme l o c a l i z a t i o n was maintained i n t h i s region f o r a considerable period during development (PLATE I I I , 8 and 9)« I t was unchanged i n the cardiac stomach of 84 mm. embryos where the d i f f e r e n t i a t i n g muscularis maintained i t s response to substrate (PLATE I I I , 1 0 ) . From t h i s point on, a decrease i n t o t a l area of enzyme a c t i v i t y was observed. The enzyme gradually assumed position i n the c e l l s of the presumptive lamina propria where the cardiac glands were to develop, as well as i n the muscularis (PLATE I I I , 11 and PLATE IV, 1 2 ) . A broad mesenchymal region destined to be the submucosa l o s t i t s enzymatic response. E p i t h e l i a l f o l d i n g and gland development were apparent by the 121 mm. stage and AP was extremely reactive to substrate PLATE I I I . 7. Gross-section of cardiac stomach from a 44 mm. embryo showing mesenchyme l o c a l i z e d AP a c t i v i t y (arrow). Gomori- 1 hour incubation. 500X 8. Cross-section of cardiac stomach from a 72 mm. embryo, i l l u s t r a t i n g mesenchyme l o c a l i z e d AP a c t i v i t y (arrow). Gomori- 1 hour incubation. 200X 9. Cross-section of a portion of cardiac stomach from a 75 mm. embryo revealing mesenchymal AP a c t i v i t y . Gomori- 1 hour incubation. 500X 10. Cross-section of cardiac stomach from an 84 mm. embryo revealing AP a c t i v i t y i n mesenchymal and developing muscularis regions. Gomori- 1 hour incubation. 200X 11. Gross-sectiom© ;f ©ar&iac,stamaeh:;.£r©m:M112^mm. embryo showing AP a c t i v i t y i n developing lamina propria and muscularis regions (arrows). Cryostat HNF- 1 Hour incubation. 200X Legend- e- epithelium, M- mesenchyme, L- lumen, Ms- muscularis. 24a 2 5 in this zone. The muscularis on the other hand was approaching morphological maturity and by 14-0 mm. stage had lost i t s enzyme activity i n the inner circular layer (PLATE IV, 13). The outer longitudinal layer maintained i t s weak positive response u n t i l this too had disappeared i n the 1?0 mm. embryo. As the long tubular glands of the cardiac stomach differentiated, the gland cells gave no response to histo-chemical substrate (PLATE IV, 14). A moderate reaction was s t i l l obtained i n the connective tissue?, capillaries, and lymphatics of the lamina propria between the glands as was observed i n both longitudinal and transverse gland sections of the 170 mm. embryo (PLATE IV, 15 and 1 6 ) . Therefore i t appeared that the cardiac glands of this species were differentiated in an AP rich milieu which persisted around the glands i n the adult. The serosa showed more consistant response to substrate than that surrounding the oesophagus. Pyloric Stomach The pyloric stomach as a distinct organ or region of the gut was f i r s t seen i n the 37 nan. stage. Unlike the cardiac stomach of the same embryo this tissue was non-reactive to the histochemical methods employed for AP. However, the mesenchyme surrounding the pyloric stomach epithelium of the 44 mm. stage embryo was very rich in enzyme activity (PLATE V, 17). This area as well as i t s enzyme response remained largely unaltered through a considerable period of embryonic growth as shown by increased body length (PLATE V, 18 and 19). A considerable amount of expansion PLATE IV. 12. Cross-section of a portion of cardiac stomach from a 121 mm. embryo showing AP a c t i v i t y i n the presumptive lamina propria during e p i t h e l i a l f o l d i n g processes (arrow). Cryostat HNF- 1 hour incubation. 200X 13. Cross-section of a portion of cardiac stomach from a 150 mm. embryo showing AP a c t i v i t y i n presumptive lamina propria and submucosal regions during gland formation (arrows). Gomori-1 hour incubation. 500X 14. Cross-section of a cardiac stomach f o l d from a 170 mm. embryo showing lamina propria and serosal AP a c t i v i t y (arrows). Gomori- 20 minutes incubation. 275X 15. Longitudinal- section of cardiac glands from a 170 mm. embryo showing lamina propria AP a c t i v i t y (arrows). Gomori- 10 minutes incubation. 500X 16. Cross-section of cardiac glands from a 170 mm. embryo showing lamina propria AP a c t i v i t y (arrow). Gomori- 10 minutes incubation. 500X Legend- e- epithelium, L- lumen, S- submucosa. 2 6 a PLATE V 17. Cross-section of the pyloric stomach from a 5 2 mm. embryo showing mesenchymal A P activity (arrow). Gomori- 1 hour incubation. 1 2 5 0 X 18. Cross-section of the pyloric stomach from a 75 mm. embryo showing mesenchymal A P activity (arrow). Gomori- 1 hour incubation. 5 0 0 X 19. Cross-section of the pyloric stomach from an 84 mm. embryo revealing mesenchymal A P activity. Gomori- 1 hour incubation. 1 2 5 0 X 2 0 . Cross-section of the pyloric stomach from a 1 1 2 mm. embryo showing A P activity in the presumptive lamina propria and submucosa during epithelial folding processes (arrows). Cryostat HNF- 15 minutes incubation. 2 0 0 X 2 1 . Cross-section of the pyloric stomach from a 140 mm. embryo showing A P activity i n the developing muscularis, submucosa and lamina propria during epithelial folding (arrows). Gomori- 1 hour incubation. 3 0 0 X Legend- e- epithelium, M- mesenchyme, L- lumen 2?a 28 of this organ occurred between the 84 mm. stage and the 112 mm. stage (PLATE V, 2 0 ) . The epithelial surface area increased several fold and was accompanied by folding or rugae formation. The AP reactive areas assumed more or less the same pattern as observed in the cardiac stomach. The muscularis appeared to have lost i t s enzyme activity i n the inner circular layer by the 140 mm. stage (PLATE V, 2 1 ) , whereas the outer longitudinal layer retained i t s activity up to the 170 mm. stage. The tissue adjacent to the epithelium possessed strong enzymatic response (Table I), the major activity was in the presumptive lamina propria and much less in the submucosa (PLATE VI, 2 2 ) . The described reaction remained the same as the embryo enlarged. Pyloric «glaihd^development was observed to occur in an enzyme rich environment (PLATE VI, 23)» as seen at the 170 mm. stage. When the glands were mature, the enzyme was localized to the lamina propria, and the gland ce l l s were unreactive (PLATE VI, 24). The serosa as in the cardiac stomach showed a variable pattern of reactivity. Spiral Valve Although in the 20 mm. embryo there had already commenced the process of evagination ,of the endoderm tube to form the spiral valve, no histochemically demonstrable AP was observed. At the 30 mm. stage however the mesodermal components surrounding the epithelium showed light reaction. This reaction was quite strong at the 44 mm. stage (PLATE VII» 2 5 ) , and f e l l off considerably as the embryo enlarged further PLATE VI. 22. Cross-section of the p y l o r i c stomach from a 170 mm. embryo showing lamina propria and submucosal AP a c t i v i t y during gland development (arrow). Gomori- 1 hour incubation. 240X 2 3 . Cross-section of a p y l o r i c stomach f o l d of a 180 mm. embryo showing lamina propria AP l o c a l i z a t i o n (arrow). Gomori- 10 minutes incubation. 240X 24. Cross-section of the p y l o r i c stomach f o l d i n the adult Ra.ja binoculata i l l u s t r a t i n g lamina propria enzyme (arrow). Gomori- 10 minutes incubation. 36OX Legend- e- epithelium, S- submucosa, L- lumen. 2 9 a 30 PLATE VII. 25. Cross-section of the s p i r a l valve from a 44 mm. embryo showing mesenchymal AP l o c a l i z a t i o n {(arrow). Gomori- 1 hour incubation. 200X 26. Cross-section of the s p i r a l valve from a 52 mm. embryo revealing mesenchymal AP l o c a l i z a t i o n (arrow). Gomori- 1 hour incubation. 200X 27. Cross-section of the s p i r a l valve from a 72 mm. embryo i l l u s t r a t i n g brush border and d i f f e r e n t i a t i n g connective tissue AP a c t i v i t y (arrows). Gomori- 1 hour incubation. 200X 28. Cross-section of the s p i r a l valve from a 75 mm. embryo revealing connective tissue AP a c t i v i t y (arrow). Gomori- 1 hour incubation. 200X 29. Cross-section of the s p i r a l valve from a 95 mm. embryo showing brush border and connective tissue AP a c t i v i t y just p r i o r to v i l l i formation (arrows). Gomori- 1 hour incubation. 200X Legend- e- epithelium, M - mesenchyme. 3 0 a 31 (PLATE VII, 2 6 ) . Brush border AP at the free end of the columnar e p i t h e l i a l c e l l s f i r s t became observable at the 51 mm. stage. I t appears, i n agreement with Moog (1962), that development of the s t r i a t e d border and the appearance of phosphatase are i n fact synchronous and apparently inseparable events. As the s p i r a l valve enlarged and the mesodermal tissue d i f f e r e n t i a t e d into three d i s t i n c t zones, two of connective tissue and one of smooth muscle c e l l s , enzyme a c t i v i t y persisted (PLATE VII, 2? and 28). The muscularis, unlike the three previous gut components did not appear to have any active enzyme. The developing muscularis mucosa mentioned above showed the strongest a c t i v i t y followed by the connective tissue of the lamina propria on either side of i t (PLATE VII, 2 9 ) . The i n t e s t i n a l v i l l i commenced morphological d i f f e r e n t i a t i o n by the 97 mm. stage. This was accompanied by considerable a c t i v i t y i n the muscularis mucosa region, as well as i n the areolar connective tissue of the lamina propria. The brush border showed a tremendous increase i n response to substrate (Table I ) . I t reached a peak at 112 mm. and remained at t h i s high l e v e l throughout the remainder of gut development (PLATE VIII, 30, 31 and 3 2 ) . The lamina propria increased i n enzyme a c t i v i t y as the embryo reached maturity. As the v i l l i elongated to assume t h e i r f i n a l form, i t was observed that c e l l s were sloughed o f f at t h e i r t i p s . I t was conjectured that enzyme i n t h i s zone had been denatured and therefore no PLATE VIII. 30. Gross-section of the s p i r a l valve v i l l i from a 170 mm. embryo showing brush border AP a c t i v i t y (arrow). Gomori- JO seconds incubation. 200X 31. Cross-section of a s p i r a l valve v i l l u s from a 170 mm. embryo showing brush border AP a c t i v i t y (arrow). Gomori- 1 minute incubation. 50GX 32. Cross-section of s p i r a l valve v i l l i from a 180 mm. embryo showing brush border and lamina propria AP a c t i v i t y (arrows). Gomori -1 hour incubation. 200X 33. Gross-section of a portion of a s p i r a l valve v i l l u s from an adult Ra.ja binoculata showing AP response to substrate i n the Golgi zone (arrows). Gomori- 1 hour incubation. 1250X Legend- e- epithelium, L- lumen, l p - lamina propria. 32a 33 a c t i v i t y was observed (Hugon and Borgers, 1968) . The brush borders of the remaining portion of the v i l l u s extending to the crypts were strongly reactive. The Golgi zone of the adult s p i r a l valve e p i t h e l i a was reactive to substrate as opposed to a negative response i n the embryos (PLATE VIII, 33)• Disc Electrophoresis The electrophoretic r e s u l t s showed three major zones of electrophoretic enzyme a c t i v i t y based on charge and molecular weight heterogeneity. Electrophoretic comparison of various adult (Group I) elasmobranch s p i r a l valve AP revealed two major zones of enzyme a c t i v i t y (FIGURE 1 and 2 ) . The two zones are based upon r e l a t i v e electrophoretic m o b i l i t i e s , zone 3 having the greatest anodal mobility. The three skates studied had two common zones of AP a c t i v i t y , zones 1 and 3 . I t appears that zone 1 of the s p i r a l valve of the skate Ra.ja binoculata i s divided into two components whereas t h i s i s not r e a d i l y apparent i n the other skates. The Squalus acanthias samples, as compared to the skates, were lacking zone 1 . Our studies on the histochemistry of the adult s p i r a l valve of Ra.ja binoculata indicate that two l o c a l i z a t i o n s of AP were present. I t i s possible that the brush border AP represents one band of AP a c t i v i t y and that the lamina propria enzyme represents the other. This i s a simple approach however and any one or both zones of AP a c t i v i t y may be present i n the two histochemieal l o c a l i z a t i o n s . These 34 FIGURE 1 . Migration of s p i r a l valve a l k a l i n e phosphatases i n acrylamide gel from a v a r i e t y of adult elasmobranchs. (Group I) Squalus acanthias Ra.ja b i n o c u l a t a GURE 2. Diagrammatic r e p r e s e n t a t i o n o f v a r i o u s a d u l t (Group I ) s p i r a l v a l v e a l k a l i n e phosphatases as p i c t u r e d i n FIGURE 1 . Squalus a c a n t h i a s I I 1 Ra.ja k i n c a i d i i I I T - CO N M I I • » Ra.ja r h i n a Ra.ja b i n o c u l a t a 36 electrophoretic r e s u l t s proved to be r e a d i l y reproducible and so further studies were c a r r i e d out. In the studies of Group II embryos, only one electrophoretic zone of AP was discovered, zone 2 (FIGURE 3 and 4). When we consider the histochemical l o c a l i z a t i o n of the enzyme at t h i s time, i t becomes probable that the enzyme i s derived mainly from d i f f e r e n t i a t i n g mesenchymal tissue i n a l l the gut organs. Brush border AP was present during the l a t e r stages of t h i s Group but did not contribute s i g n i f i c a n t l y to the extractable enzyme. Group III whole t r a c t extracts containing zone 1 and zone 2 bands had AP derived from d i f f e r e n t i a t i n g muscularis of the oesophagus, mesenchymal tissue of the stomach regions and the lamina propria and brush border regions of the s p i r a l valve (FIGURE 3 and 4). The new zone of AP, zone 1, probably represents a new isozyme i n one or several of the areas aforementioned. In the embryonic ontogeny of the s p i r a l valve (FIGURE 3 and 4), zone 1 f i r s t became apparent i n the whole t r a c t extracts of Group III and was present i n the s p i r a l valve from t h i s point u n t i l maturity. This zone as we saw previously appeared i n the adult s p i r a l valve. Group IV s p i r a l valves l o s t zone 2 a c t i v i t y and were found to contain enzyme i n zone 1 and zone 3« The AP i s believed to be derived mainly from the lamina propria and brush border. This isozyme pattern was found to be the same i n Group V and Group I skate s p i r a l valves where histochemical l o c a l i z a t i o n was 37 FIGURE 3 . The developmental sequence of a l k a l i n e phosphatase d i f f e r e n t i a t i o n from various embryonic alimentary t r a c t regions. 37a Group I I 4o -6l mm. embryos Whole t r a c t e x t r a c t s Group I I I 78-91 mm. embryos Whole t r a c t e x t r a c t s Oesophagus Oesophagus • Group IV 138-154 mm. embryos Cardiac and P y l o r i c Stomachs Group V 187-204 mm. embryos Cardiac Stomach P y l o r i c Stomach 1 S p i r a l v a l v e GURE 4 Diagrammatic representation of the a l k a l i n e phosphatase d i f f e r e n t i a t i o n i n various alimentary t r a c t regions as pictured i n FIGURE 3 . 38 , Group II '40-61-- mm. embryos Whole tract extracts Q Group III 78-91 mm. embryo Whole tract extracts Oesophagus Oesophagus Group IV 138-154 mm. embryos Cardiac £ Pyloric Stomachs Z 1 Z 2 Z 3 Group V 187-204 mm. embryos Cardiac Pyloric Stomach Stomach Spiral valve y Spiral valve y 39 s i m i l a r . Therefore i t appears that the adult isozyme complement of the s p i r a l valve i s present at a r e l a t i v e l y early stage i n development (Group IV) and that one component, zone 1, appears e a r l i e r than the other, zone 3. Of the Group IV samples only the oesophagus retained the banding pattern of Group III (FIGURE 3 and 4). This was presumably due to the l a t e morphological d i f f e r e n t i a t i o n of the mesenchymal c e l l s within the submucosal and muscularis regions which account f o r the AP l o c a l i z a t i o n . Once again i t i s uncertain whether one or both AP bands are derived from one or both histochemical l o c a l i z a t i o n s . A l l the other Group IV extracts had no zone 2 a c t i v i t y . Therefore zone 2 appears to be a zone present only i n the early d i f f e r e n t i a t i n g stages of gut development. The oesophagus of Group IV, whose AP a c t i v i t y i s derived histochemically from the submucosa, manifested banding i n zones 1 and 3 with a loss of zone 2 a c t i v i t y . A combination of cardiac and p y l o r i c stomach tissue from Group IV produced zone 1 and zone 3 banding. The major complement of enzyme from the stomach regions at t h i s stage was from the lamina propria, with minor amounts from the submucosa and muscularis. The cardiac stomach of Group V, whose AP a c t i v i t y was present i n the lamina propria, showed zone 1 and zone 3 a c t i v i t y with zone 1" having two bands. The p y l o r i c stomach of Group Ifcfwhose a c t i v i t y i s also derived mostly from the lamina propria, produced only zone 1. I t i s possible that the p y l o r i c stomach of Group IV also gave only zone 1 banding, the other a c t i v i t y being derived from the cardiac stomach. DISCUSSION Leucine Naphthylamidase (LNAase) The histochemical r e s u l t s obtained f o r the enzyme(s) LNAase, although mostly negative, were indeed i n t e r e s t i n g . I t was s u r p r i s i n g that the s p i r a l valve was the only organ of the g a s t r o - i n t e s t i n a l t r a c t to show a response to substrate, since the oesophagus and stomach of guinea pig and r a t respectively are known to contain LAP (LNAase) a c t i v i t y (Nachlas et al . , 1957a). Previous data suggest that i n any single tissue there are present simultaneously, several d i f f e r e n t enzymes capable of s p l i t t i n g the chromogenic substrate LNA between pH 6 .5-7 .1 (Sylven and Bois, 1962; Nachlas et al. 1962? Smith, i 9 6 0 ) . This would suggest therefore that i n the skate embryo g a s t r o - i n t e s t i n a l t r a c t , these enzymes were lacking or were present below the concentration ''threshold" necessary f o r a chromogenic response. However, t h i s "threshold" has been found to be quite low (Sylvan and Bois, 1963) . Absence of enzyme(s) may be due to a metabolite-controlled repression of t h e i r formation. Therefore i t i s reasonable that there were i n s u f f i c i e n t or no enzyme s i t e s available f o r substrate i n t e r a c t i o n i n contrast to the s p i r a l valve. I n t e s t i n a l LNAase a c t i v i t y i s a common phenomenon i n both adult animals and embryos (Pennttila and Gripenberg, 1969; McCabe and Chayen, 1965; Holt and M i l l e r , 1962; Sylven and Bois, 1962; Rosenholtz and Wattenberg, 1 9 6 l ; Burstone and Folk, 1956). Since no LNAase a c t i v i t y was present i n the 41 s p i r a l valve of the 97 mm. embryo and intense reaction obtained i n the 112 mm. stage, a study of the respective stages of development seemed appropriate. I t was c l e a r from the h i s t o l o g i c a l preparations of the 97 mm. stage that the epithelium was beginning to show signs of f o l d i n g . Between t h i s stage and the 112 mm. stage the folds had undergone considerable elongation and assumed an a d u l t - l i k e v i l l u s form. During t h i s process the LNAase enzyme(s) became apparent. V o l l r a t h (1969) s i m i l a r l y observed that the i n i t i a l appearance and growth of the i n t e s t i n a l v i l l i go hand i n hand with increases i n LAP a c t i v i t y i n r a t . Whether there was a gradual increase or a sudden elevation i n enzyme l e v e l ( s ) , i n our ease, i s unknown. I t i s also unclear whether new enzyme(s) were synthesized at t h i s time or whether ina c t i v e precursor molecules became activated. I t i s not the opinion of the author that LNAase i s d i r e c t l y involved with the process of v i l l u s formation. I t i s more l i k e l y that as the v i l l i assume the adult c h a r a c t e r i s t i c s , LNAase enzyme(s) become operational i n some capacity other than d i f f e r e n t i a t i o n . The l o c a l i z a t i o n of LNA s p l i t t i n g enzyme(s) i n skate s p i r a l valve brush borders and lamina propria was s i m i l a r to that observed i n dog jejunum (Nachlas et al.., 1957a), chicken in t e s t i n e (Michael and Hodges, 1973)» and r a t i n t e s t i n e (McCabe and Chayen, 1965). In the v i l l i t i p s i t i s possible that LNAase enzyme(s) were denatured due to c e l l death and sloughing o f f i n t h i s zone. Since the reaction product was observed as both pa r t i c u l a t e and diffuse s t a i n one might 4 2 question the methodology. However, the LNA hydrolyzing enzymes were found both i n soluble and p a r t i c u l a t e f r a c t i o n s of tissue homogenates (Patterson e_t a l . , 1963). The p a r t i c u l a t e : histochemical reaction product has been referred toaas lysosomal (Chayen et al?> 1969; P e n t t i l a and Gripenberg, 1969; Sylven and Bois, 1962). Baxter-Grillo, (1970), has associated chick embryo lysosomal LAP (LNAase) with c e l l death i n the i n t e s t i n e . I t was suggested that the enzyme(s) may act on proteins produced during c e l l death and proteins which enter the i n t e s t i n a l c e l l i n i t s absorptive phase. LAP (LNAase) l o c a l i z a t i o n i n brush borders also l e d Holt and M i l l e r , ( I 9 6 I ) , to the conclusion of a protein digestion function. Although the LNAase enzyme(s) hydrolyze a v a r i e t y of substrates which possess a free amino group, the natural substrates are unknown (Shnitka, i 9 6 0 ; Sylven and Bois, 1964; Smith and Spackman, 1955)* The role of these enzymes i n connection with c e l l growth, c e l l u l a r injury, and c e l l death remains a mystery. Most enzymes of the g a s t r o - i n t e s t i n a l mucosa are active i n secretion, absorption, active transport, and energy producing mechanisms. LNAase l o c a l i z a t i o n i n brush border would support a secretory or absorptive function. Protein digestion i s an obvious speculation on the action of the enzyme(s) i n skate and other animals. The significance of p a r t i c u l a t e and diffuse enzyme i n one tissue as well as evidence f o r d i f f e r e n t isozymic forms (Pataryas and Christodoulou, 1970; Beckman et .al., 1966a), would suggest more than one function. The enzyme a c t i v i t y does not appear 43 to be related to the d i f f e r e n t i a t i v e events of the gastro-i n t e s t i n a l t r a c t of the skate Ra.ja binoculata. However the appearance of the enzyme(s) i n v i l l i e p i t h e l i a l c e l l s may be an i n d i c a t o r of a c e r t a i n stage of c e l l d i f f e r e n t i a t i o n there. Non-Specific Alkaline Phosphatase This study and previous reports indicate that AP i s c l o s e l y r e l a t e d to d i f f e r e n t i a t i o n at the morphological and c e l l u l a r l e v e l (Moog, 1944; Moog, 1946; Karczmar and Berg, 1951). D i f f e r e n t i a t i o n i s often characterized at the -vs.**?.**! chemical l e v e l by the appearance of, an increase i n , or the disappearance of an enzyme a c t i v i t y that i s proper to, or unnecessary to a c e l l type or tissue (Moog, 1952; E t z l e r and Moog, I966). However, the appearance of an enzyme does not mean that i t i s the i n i t i a t o r of the d i f f e r e n t i a t i o n , f o r enzymes can be tools but scarcely true c a u s i t i v e agents i n morphogenesis (Holter, 1949). The ultimate control of d i f f e r e n t i a t i o n and morphogenesis may reside i n gene ac t i v a t i o n and repression (Schjeide and DeVellis, 1970). AP has been l o c a l i z e d i n the adult i n t e s t i n a l brush borders of a l l species of vertebrates studied (mammals, birds, r e p t i l e s , amphibians, and f i s h ) , as well as i n some invertebrates. In fact, the i n t e s t i n a l mucosa has a higher a l k a l i n e phosphatase content than any other t i s s u e . In embryos on the other hand, i t i s quite common that no brush border AP i s present i n i n t e s t i n a l e p i t h e l i a l c e l l s during t h e i r early development. The teleost f i s h , Oryzias l a t i p e s shows brush 44 border l o c a l i z a t i o n only during the l a t e r stages of embryonic l i f e (Ikeda, 1959). In steelhead trout, brush borders and AP aris e concomitantly i n the l a t e prehatching stage (Prakash, 1961). This has also been found i n f o e t a l mouse inte s t i n e (Hugon and Borgers, 1969), chick embryonic duodenum ( P e n t t i l a and Gripenberg, 1969), and f o e t a l r at small i n t e s t i n e (Nordstrom gt a l . , 1968). Our observations i n skate s p i r a l valve confirms these observations. Small i n t e s t i n e m i c r o v i l l i have a unit membrane covering a fibrous core which extends into the a p i c a l cytoplasm of the e p i t h e l i a l c e l l s . AP has been l o c a l i z e d u l t r a s t r u c t u r a l l y i n the core of the m i c r o v i l l i and on the membranes (Mayahara ej; al., 1967). D i f f e r e n t i a t i o n of the e p i t h e l i a l c e l l s appears to have a decisive e f f e c t on the development of AP since i t has been observed that AP does not accumulate i n i n t e s t i n a l epithelium allowed to spread as sheets of squamous tissue i n culture. I t was also observed i n the presence of adrenocorticoids which are known to increase AP a c t i v i t y i n t h i s tissue. In organ culture AP developed normally (Moog, 1965). The reports suggest that as the brush border develops, AP i s b u i l t into i t and that when the brush border i s completed and a l l the AP-accomodating l o c i are occupied, the process of enzyme formation ceases (Moog, 1956). Moog (1950 and 1951), has observed that brush border AP i n chick reaches a peak i n a c t i v i t y just before hatching and that there i s a prenatal and postnatal r i s e i n brush border AP i n mouse. In Rana temporaria i t was reported that both i n t e s t i n a l brush borders and AP developed only when feeding ^5 had commenced (Brown and Millington, 1968). The enzyme pattern of the e p i t h e l i a l c e l l s indicates that these c e l l s mature just before or at the onset of the digestive function, both morphologically and enzymatically. The skate i s not an exception since AP was present during yolk absorption at an early stage and increased i n a c t i v i t y during c e l l d i f f e r e n t i a t i o n . Therefore enzyme development may be a functional expression of gradual morphologic development of the brush border regions. The observation i n Raja binoculata that the embryonic mesenchyme c e l l s were AP po s i t i v e during some part of t h e i r development would lend support to Karczmar's (1951) generalization that the d i f f e r e n t i a t i n g c e l l s pass through a t r a n s i t i o n phase represented i n phosphatase r i c h mesenchyme. Our acrylamide gel data shows only one isozyme at the early stages of alimentary t r a c t development, which appears to be common to a l l the mesenchymal areas. This suggests a common and general function f o r the enzyme a c t i v i t y . Ikeda (1959) noted i n h i s studies of Oryzias l a t i p e s that the mesenchyme c e l l s surrounding the alimentary t r a c t were r i c h i n AP. Si m i l a r l y , Prakash ( 196l ) and Moog (1944) observed the same phenomenon i n early prehatching stage trout and chick embryo respectively. I t i s possible that during t h i s period of mesenchymal c e l l d i f f e r e n t i a t i o n the AP r i c h phase represents a period of increased i n t r a c e l l u l a r phosphate transfer. This would suggest that high energy u t i l i z i n g processes are i n operation. AP l o c a l i z e d i n the lamina propria i s a common observation (Gomori, 1 9 4 l a ; Moog, 1944; Prakash, 1961; Moog, 1962; Malegalada, 46 et a l . , 1971). Therefore our r e s u l t s i n Ra.ja b i n o c u l a t a were not at a l l s u r p r i s i n g . I t was c l e a r that the mesenchymal AP was maintained i n a s i m i l a r or d i f f e r e n t , but no l e s s a c t i v e form, i n the d i f f e r e n t i a t i n g lamina p r o p r i a . The f i b r o b l a s t c e l l s of t h i s t i s s u e were AP p o s i t i v e and i t has been speculated t h a t AP may f u n c t i o n i n f i b r o g e n e s i s (Schmidt and Weary, 1962). I n o l d e r embryos of skate, the lamina p r o p r i a AP enzyme was present throughout stomach e p i t h e l i a l f o l d i n g processes and c a r d i a c and p y l o r i c gland development, suggesting t h a t the enzyme may be i n v o l v e d i n the d i f f e r e n t i a t i o n which occurs at t h i s stage even though gland c e l l s were AP negative. Whether AP i s an e s s e n t i a l f a c t o r during gland d i f f e r e n t i a t i o n i s unknown. Enzyme i n h i b i t o r s t u d i e s would be r e l e v a n t here, however a p r e c i s e i n h i b i t o r substance has yet to be found. The AP of the submucosa i n Ra.ja b i n o c u l a t a was v a r i a b l e throughout the d i g e s t i v e t r a c t . Only i n the oesophagus where the connective t i s s u e of the mucosa i s r e s t r i c t e d to a basement membrane d i d the submucosa show a d e f i n i t e p a t t e r n of p o s i t i v e AP a c t i v i t y . The submucosa of a l l the organs stu d i e d , w i t h the exception of the oesophagus, q u i c k l y l o s t AP r e a c t i v i t y as they d i f f e r e n t i a t e d from mesenchyme. Since t h i s and other areas show decrease i n AP a c t i v i t y w i t h i n c r e a s i n g d i f f e r e n t i a t i o n , i t seems reasonable to suggest that t h i s enzyme i s i n v o l v e d i n the process of d i f f e r e n t i a t i o n . I t would seem, w i t h the l o s s o f the more r i g i d lamina p r o p r i a i n the oesophagus, that there would also be no AP a c t i v i t y . However, our observations show tha t 47 AP appears i n the loose meshlike layer of f i b r o e l a s t i c tissue of the submucosa i n oesophagus of older embryos and adults. This i s an example of the accumulation of AP with morphological d i f f e r e n t i a t i o n . I t i s possible therefore that the oesophageal submucosa has assumed the ro l e , at l e a s t with respect to AP, Vr,u.): that the lamina propria serves i n the other areas of the t r a c t . This i s assuming of course, from AP l o c a l i z a t i o n , that i t serves a s i m i l a r physiological function i n these tissues. This function once again i s obscure. The outer part of the digestive t r a c t mucosa, the muscularis mucosa was d i s t i n c t only i n the s p i r a l valve. In the other organs i t was present only as scattered smooth muscle c e l l s which made AP l o c a l i z a t i o n impractical. AP l o c a l i z a t i o n i n the s p i r a l valve during embryonic development was spotty. What significance t h i s enzyme would have i n the muscularis i s not known. I t i s possible that the d i f f e r e n t i a t i o n of these c e l l s requires a turning on and o f f of AP synthesizing systems or a c t i v a t i o n of an inactive precursor during c e r t a i n c r u c i a l stages. The muscularis (externa) of the skate alimentary t r a c t maintained the AP a c t i v i t y that was present i n the mesenchyme tissue that i t d i f f e r e n t i a t e d from. After further embryonic growth and muscular d i f f e r e n t i a t i o n , AP a c t i v i t y disappeared. In the s p i r a l valve the muscularis completed d i f f e r e n t i a t i o n e a r l i e r than i n the other gut regions, probably due to i t s early function i n digestion. AP a c t i v i t y was observed to disappear from the muscularis i n t h i s organ e a r l i e r than i n the other organs studied. Therefore the r e s u l t s give us the 48 impression that when AP disappears from d i f f e r e n t i a t i n g muscle tissue, the muscle becomes functionally, mature. Low l e v e l s of AP are c h a r a c t e r i s t i c of d i f f e r e n t i a t e d muscle (McWhinnie and Saunders, 1966; Moog, 1944; Bourne, 1954a; Schmidt and Weary, 1962). This i s made more apparent when we consider that the s p i r a l valve functions i n yolk absorption at an early embryonic stage and AP also disappears r e l a t i v e l y early from i t s muscle tissue. Oesophagus, cardiac stomach and p y l o r i c stomach do not function i n digestion u n t i l a f t e r hatching and AP becomes inactive i n t h e i r muscularis l a t e r i n embryonic development. Therefore i t appears that muscle d i f f e r e n t i a t i o n and maturity are associated with decreasing AP a c t i v i t y . Our observations indicate that AP i s associated with the r i s e to functional maturation of the smooth muscle of the muscularis. I t has been speculated that AP may disappear with the onset of muscular contraction (Boell, 1955)* Our observations i n Ra.ja binoculata that the e p i t h e l i a l c e l l s were AP negative i n oesophagus, cardiac stomach, and p y l o r i c stomach are generally i n agreement with the findings of other researchers. Prakash (1961) fo r example observed no AP a c t i v i t y i n the endodermal c e l l s of l a t e prehatching stage steelhead trout embryos. Early prehatching stage trout were found to have low endodermal cytoplasmic AP a c t i v i t y . The columnar c e l l s of the mucosa l i n i n g the oesophagus and g a s t r i c rugae i n cardiac an&ypyloric stomach regions of adult trout were devoid of AP a c t i v i t y (Weinreb and B i l s t a d , 1955). Moog, (1944), observed that i n 8 day o l d chick embryos the whole 4-9 epithelium of the alimentary t r a c t was AP negative. The r e s u l t s suggest that AP was not synthesized i n the embryonic or adult e p i t h e l i a l c e l l s of oesophagus, cardiac stomach or py l o r i c stomach of skate and that i t played no s i g n i f i c a n t r o l e i n t h e i r d i f f e r e n t i a t i o n . The enzyme probably has no s i g n i f i c a n t physiological function i n the adult c e l l s . I t i s possible that an AP r i c h period occurred i n the endoderm c e l l s p r i o r to the 20 mm. stage i f such a t r a n s i t i o n phase i s re q u i s i t e f o r c e l l d i f f e r e n t i a t i o n as postulated by Karczmar (1951). The serosal layer of the alimentary t r a c t i n Ra.ja  binoculata showed poorly developed patterns of AP a c t i v i t y . This has been found i n other species (Bourne, 19^4; P e n t t i l a and Gripenberg, 19^9; Ikeda, 1959). The physiological s i g n i f i c a n c e of the enzyme i n t h i s tissue i s unknown and i t s low and variable histochemical response i s reminiscent of a c t i v i t y observed i n endothelial c e l l s . S i m i l a r l y , the Leydig organs showe'd almost no reaction to histochemical substrate. Since they remained on the whole AP negative, one can only assume that AP was not important i n c e l l d i f f e r e n t i a t i o n and organ maturity within t h i s lymphoid structure. Therefore AP seems to play a ro l e i n the d i f f e r e n t i a t i v e events of some but not a l l tissues i n the alimentary t r a c t . Due to the v a r i a b i l i t y of b i o l o g i c a l systems i t i s reasonable that more than one d i f f e r e n t i a t i v e system exists within the digestive t r a c t of skates and other animals. The l o c a l i z a t i o n of AP d i r e c t l y beneath the gut epithelium 50 during development would favour the assumption that the enzyme i s i n d i r e c t l y associated with the secretory processes of the e p i t h e l i a l c e l l s . There i s no evidence f o r t h i s and no mechanisms have been proposed. The oesophagus and stomach regions of skate do not appear to p a r t i c i p a t e i n the digestive process u n t i l a f t e r hatching since the yolk passes v i a the i n t e r n a l yolk sac d i r e c t l y into the s p i r a l valve at the duodenum (TeWinkel, 1941 and 1943). I f we assume then that these areas are not a c t i v e l y secreting, then a more general function f o r AP would be favoured. Patterns of AP accumulation suggest t h e i r importance at the onset of the ph y s i o l o g i c a l processes of the e p i t h e l i a l v i l l u s c e l l . In the early t h i r t i e s , several workers believed brush border AP served i n the transport of glucose and f a t t y acids through the i n t e s t i n a l wall (Moog, 1946). That i t functions i n absorption and transport across the plasma membrane i s a l o g i c a l speculation due to i t s l o c a l i z a t i o n , high l e v e l s of a c t i v i t y there, and i t s high l e v e l of a c t i v i t y preceding function (Moog, 1962? Bonneville and Weinstock, 1970; Moog and Glazier, 1972;). Furthermore, there i s a deficiency of brush border AP i n patients with malabsorption syndrome (Shnitka, i960). Johnson and Kugler (1953) suggested that AP functioned i n glucose absorption v i a a phosphorylation a f t e r entry into the c e l l , and a dephosphorylation upon e x i t into the bloodstream at the base of the e p i t h e l i a l c e l l s . Crane and Krane (1956) however, have ruled out AP i n the transport of glucose and presently i n t e r e s t centres upon i t s association 51 with f a t absorption (Glickman et al., 1970; Linscheer et a l M 1971) . I t has been shown that AP i s subject to quantitative vari a t i o n s r e l a t e d to d i e t . A high protein or f a t diet shows more AP a c t i v i t y than a high carbohydrate diet (Tuba and Dickie, 1 9 5 5 ) . I t appears that the increase i n i n t e s t i n a l AP i s a r e l a t i v e l y slow process that proceeds during digestion and absorption (Jackson, 1 9 5 2 ) . S i m i l a r l y , V o l l r a t h (1969) indicated that the changes i n enzyme patterns a f t e r b i r t h i n rat i n t e s t i n e were probably under the influence of a food intake factor. I t i s possible then that the u t i l i z a t i o n of yolk i n Raja binoculata influences to some extent the brush border enzyme pattern either q u a l i t a t i v e l y , q u a n t i t a t i v e l y or both. In view of the past and present data i t would be j u s t i f i a b l e to say that since the skate embryo brush border AP developmental pattern i s reminiscent of chick and mouse, that i t serves a s i m i l a r physiological function. This precise function s t i l l eludes us. An u l t r a s t r u e t u r a l study would be necessary to determine whether minute quantities of AP are present i n the Golgi apparatus of embryonic s p i r a l valve e p i t h e l i a l c e l l s i n skate. However, the fact that we observed Golgi l o c a l i z e d AP i n the adult ska$e and not i n the embryos might also r e l a t e d to the state of d i f f e r e n t i a t i o n of the s p i r a l valve e p i t h e l i a l c e l l s or of t h e i r Golgi apparatus. I t was shown i n mouse that on the 1 6 t h day of neonatal l i f e the duodenal e p i t h e l i a l c e l l s assumed the adult form and f o r the f i r s t time AP appeared i n the Golgi cisternae (Hugon, 1 9 7 0 ) . 52 I t has been suggested t h a t the G o l g i apparatus, endoplasmic r e t i c u l u m , and lysosomes, o r the "GERL" complex o f N o v i k o f f , p l a y s a s i g n i f i c a n t r o l e i n the e l a b o r a t i o n and t r a n s p o r t o f AP and o t h e r h y d r o l y t i c enzymes (Hugon and Borgers, 1968). P r z e l e c k a et a l . , (1962), on the o t h e r hand, has shown a c o r r e l a t i o n o f G o l g i l o c a l i z e d AP a c t i v i t y with the presence o f i n t r a e p i t h e l i a l l i p i d . G o l g i AP t h e r e f o r e seems to be connected w i t h p h o s p h o l i p i d b i o s y n t h e s i s . Shnitka, ( i960) , mentions t h a t G o l g i AP may be a s s o c i a t e d w i t h the d e p h o s p h o r y l a t i o n o f absorbed l i p i d s . Whatever process the G o l g i AP i s i n v o l v e d w i t h i s perhaps unnecessary i n skate embryonic s p i r a l v a l v e e p i t h e l i a l c e l l s . T h i s process, i f e s s e n t i a l , may be accomplished through an a l t e r n a t i v e r o u t e a t l e a s t u n t i l the a d u l t stage. Isozymes as we know are m u l t i p l e m o l e c u l a r forms o f enzymes seen a f t e r s e p a r a t i o n procedures such as e l e c t r o p h o r e s i s . Two isozymes i n the s p i r a l v a l v e were found to be common to t h r e e s p e c i e s o f skate. Whitmore and Goldberg (1972), found t h a t f o u r d i f f e r e n t s p e c i e s o f t r o u t had c l o s e l y r e l a t e d i n t e s t i n a l isozyme p a t t e r n s . T h i s i s s i g n i f i c a n t i n t h a t isozyme p a t t e r n s from s i m i l a r t i s s u e s may be p h y l o g e n e t i c a l l y r e l a t e d . Not a l l evidence supports the e x i s t a n e e o f more than one AP per t i s s u e where s e v e r a l isozymes oc c u r . Although t h r e e isozyme zones were found i n human i n t e s t i n e , the enzyme p r o p e r t i e s i n the g e l zones were found so s i m i l a r t h a t the c o n c l u s i o n reached was t h a t one isozyme was d e r i v e d from another f o r example by a process o f d e g r a d a t i o n d u r i n g 53 c e l l processes or enzyme extraction (Moog, 1965; Warnock, 1966). Therefore the hypothesis of one enzyme per tissue was proposed. On the other hand, electrophoretic patterns of AP have "been found to change both q u a n t i t a t i v e l y and q u a l i t a t i v e l y during morphogenesis and c e l l u l a r d i f f e r e n t i a t i o n . These patterns are believed to change with the varying metabolic requirements of the d i f f e r e n t i a t i n g c e l l s . Therefore c e l l u l a r d i f f e r e n t i a t i o n and AP enzyme complement al t e r a t i o n s appear to be related (Soloman et a l . , 1964). As our r e s u l t s show, isozyme patterns do change q u a l i t a t i v e l y during tissue and organ development i n the digestive t r a c t of the skate Ra.ja binoculata. This i s i n contrast to the findings i n two urodele amphibians where d i f f e r e n t i a t i o n was found to be associated with varying quantities of two molecular forms of AP and not with changes i n the isozyme pattern (O'Day, 1969). Recent research on AP isozymes indicates that multiple molecular forms are derived from one another during development and are i n most cases, not newly synthesized molecular forms (Moog, 1966; Schneidermanj 196?; Schneiderman et al., 1966} Schlesinger and Anderson, 1968). I f t h i s general phenomenon applies i n skates, then the two zones of.AP:that are found i n the digestive t r a c t of prehatching skates, are derived from the one isozyme that was present i n mesenchymal tissue during the early stages of gut development. The oesophagus was found to r e t a i n the o r i g i n a l AP isozyme longer than the other gut regions. I t also takes the 54 oesophagus l o n g e r to d i f f e r e n t i a t e t o t h e a d u l t form. T h e r e f o r e i t appears t h a t the r a t e o f c o n v e r s i o n o f t h e o r i g i n a l AP isozyme t o t h e o t h e r forms may somehow be a f a c t o r r e l a t e d to t h e r a t e o f d i f f e r e n t i a t i o n . The s p i r a l v a l v e AP r e a c h e s th e h i g h l e v e l s o f t h e a d u l t s t a t e e a r l y i n development and AP i s a l s o shown to be q u a l i t a t i v e l y d i f f e r e n t i a t e d a t an e a r l y s t a g e i f o u r g e l d a t a i s an i n d i c a t i o n . T h e r e f o r e each a l i m e n t a r y t r a c t o r g a n d e v e l o p s i t s isozyme p a t t e r n s a t i t s own pace and t h i s appears to be r e l a t e d to i t s r a t e o f d i f f e r e n t i a t i o n . The d i f f e r e n t i a l growth o f AP i n s k a t e and o t h e r embryos p r o b a b l y r e f l e c t s m e t a b o l i c a d a p t a t i o n t o t h e c h a n g i n g r e q u i r e m e n t s d u r i n g t h e i r development (Moog, 1959)* The appearance o f AP i n a t i s s u e f o r t h e f i r s t t i m e o r an i n c r e a s e i n i t s r e s p o n s e to h i s t o c h e m i c a l s u b s t r a t e may be under th e c o n t r o l o f one o r s e v e r a l r e g u l a t i n g p r o c e s s e s . F o r example, t h e r e may be a more a c t i v e enzyme s y n t h e s i z i n g system p r e s e n t a t some s t a g e s (Moog, 1950). C o r t i c a l hormones a r e known to v a r y AP l e v e l s by a c t i n g upon whole d i f f e r e n t i a t i n g systems (Moog, I 9 6 2 ) . We must a l s o c o n s i d e r t h a t a change i n h i s t o c h e m i c a l r e s p o n s e may be due t o an a l t e r a t i o n i n t h e enzyme m o l e c u l e s t h e m s e l v e s , a c o n f o r m a t i o n a l change f o r i n s t a n c e , r a t h e r t h a n an i n c r e a s e i n m o l e c u l e number. B l o c k i n g o r i n h i b i t i n g p r o t e i n m o l e c u l e s may be p r e s e n t d u r i n g low o r i n a c t i v e AP p e r i o d s t h a t i n h i b i t enzyme a c t i v i t y o r t h e i r c o n v e r s i o n t o a more a c t i v e f orm (Moog, 1964). Then we must c o n s i d e r t h e p o s s i b i l i t y t h a t s u b s t r a t e a v a i l a b i l i t y may d i r e c t t h e a c t u a l enzyme f o r m a t i o n ( B o e l l , 1955). 5 5 One speculation isathat s t r u c t u r a l development l i m i t s or promotes growth of or regulates the a c t i v i t y of the enzyme (Moog, 1 9 6 5 ) . This i s consistant with AP association with d i f f e r e n t i a t i o n . Ultimately we must be aware of t r a n s c r i p t i o n a l and t r a n s l a t i o n a l controls as well as i n t r a c e l l u l a r regulatory c i r c u i t s that could be operational at any or a l l stages of f o e t a l development (Schjeidi and DeVellis, 1 9 7 0 ) . However the change i n isozyme patterns i s performed, i t c e r t a i n l y appears that the early AP isozyme of skate alimentary t r a c t i s a function of the d i f f e r e n t i a t i v e events of the gut and that the AP isozymes of older embryos and adults are consequent on d i f f e r e n t i a t i o n . 56 SUMMARY 1 . The histochemical patterns of leucine naphthylamidase (LNAase) and a l k a l i n e phosphatase (AP) ontogeny during the development of the alimentary t r a c t of the skate Ra.ja binoculata have been studied. 2. S i m i l a r l y the ontogenic pattern of AP isozymes i n the developing alimentary t r a c t have been studied by polyacrylamide gel electrophoresis. 3. LNAase a c t i v i t y was l o c a l i z e d only i n the s p i r a l valve mucosa of developing Raja binoculata embryos. The enzyme probably plays no r o l e i n c e l l or morphologic d i f f e r e n t i a t i o n , however other proposed functions are discussed. 4 . AP enzyme patterns show that AP a c t i v i t y occurs i n various tissues of the alimentary t r a c t independently of other regions and i n variable quantity. 5. Several instances of circumstantial evidence r e l a t e AP a c t i v i t y to d i f f e r e n t i a t i v e events of the alimentary t r a c t . 6. The AP of embryonic and adult digestive t r a c t tissue homogenates was found to be the product of at l e a s t three molecular forms. Two s p i r a l valve isozymes were found to be common to three species of skate. 7. The histochemical development of AP was r e l a t e d to the isozyme data and to proposed functions of the enzyme. 8. The d i f f e r e n t i a t i v e function of AP seems to be a product of new molecular forms. 9. » The early AP isozymes are a function of d i f f e r e n t i a t i o n , the prehatching isozymes consequent on differentiation.' 57 REFERENCES Ahmed, Z. and E.J. King ( i 9 6 0 ) . P u r i f i c a t i o n of placental a l k a l i n e phosphatase. 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