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Alkaline phosphatase and embryogenesis in two urodele amphibian species O'Day , Danton H. 1969

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ALKALINE PHOSPHATASE AND EMBRYOGENESIS IN TWO URODELE AMPHIBIAN SPECIES by DANTON H. O'DAY B.Sc, The University of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE ' i n the Department of ZOOLOGY We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1969 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 r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I a g r e e 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 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 rposes may be g r a n t e d by the Head o f 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 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 not 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 . Department o f 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 - i -ABSTRACT The development of a l k a l i n e phosphatase (AP) has been studied i n two species of Urodele amphibian, Ambystoma  q r a c i l e and Taricha torosa. The enzyme i s present i n embryo homogenates at g a s t r u l a t i o n and increases immensely i n ac-t i v i t y as development proceeds to the free-swimming stages. The a c t i v i t y l e v e l i s a product of two isozymic forms that change q u a n t i t a t i v e l y . Using histochemical detection methods, i t was possible to co r r e l a t e the s p e c i f i c a c t i v i t y and elec-trophoretic data with h i s t o l o g i c a l AP development. Some func-t i o n of AP were rel a t e d to the available data. A c o r r e l a t i o n between substrate s p e c i f i c i t i e s and function i s proposed which may a s s i s t i n understanding the r o l e of AP i n the pro-cess of d i f f e r e n t i a t i o n . - i i -TABLE OF CONTENTS PAGE INTRODUCTION 1 MATERIALS AND METHODS 3 I. Preparation of Embryos 3 A. S p e c i f i c a c t i v i t y determinations 3 B. Electrophoresis 4 C. Histochemistry ~ 5 I I . S p e c i f i c A c t i v i t y Assays 5 A. Enzyme a c t i v i t y assays 6 B. E f f e c t of i n h i b i t o r s 7 C. Protein determinations 7 III . Gel Electrophoresis 8 A. Starch g e l electrophoresis 8 B. Disc electrophoresis 8 C. Gel sta i n i n g 8 IV. Histochemistry 9 RESULTS 10 I. S p e c i f i c A c t i v i t y 10 A. Optimal conditions for enzyme a c t i v i t y 10 1. pH optima 10 2. substrate optima 10 3. temperature optima 13 4. enzyme d i l u t i o n 13 5. time course . 15 6. e f f e c t of magnesium ions 15 B. E f f e c t of i n h i b i t o r s 15 C. Developmental changes i n s p e c i f i c 15 a c t i v i t y II. Gel Electrophoresis 23 A. Starch g e l electrophoresis 23 B. Disc gel electrophoresis 23 II I . Histochemistry 27 A. A. q r a c i l e 27 B. T. torosa 35 DISCUSSION 41 SUMMARY 50 BIBLIOGRAPHY 51 FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. LIST OF FIGURES The e f f e c t of pH on the a l k a l i n e phosphatase a c t i v i t y of extracts of free-swimming sala-mander larvae. The e f f e c t of substrate concentration on the rate of substrate hydrolysis by butanol extracts of free-swimming salamander larvae. PAGE 11 14 Some parameters of a l k a l i n e phosphatase a c t i v - 16 i t y . o f butanol extracts of A.,gracile embryos. E f f e c t of known i n h i b i t o r s of a l k a l i n e phos- 17 phatase on hydrolysis of PNPP by buffe r extracts of _T. torosa free-swimming larvae. Development of a l k a l i n e phosphatase s p e c i f i c 21 a c t i v i t y as revealed by the hydrolysis of PNPP. Development of a l k a l i n e phosphatase s p e c i f i c 22 a c t i v i t y as revealed by the hydrolysis of BGP. Change i n a c t i v i t y r a t i o (PNPP/BGP) during 25 development of A. g r a c i l e and T. torosa. E f f e c t of d i f f e r e n t extraction methods on 26 electrophoretic patterns of a l k a l i n e phospha-tases i n starch gels. Multimolecular forms of a l k a l i n e phosphatase 28 i n acrylamide g e l . .1 Diagrammatic representation of the developmen- 29 t a l 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 . - i v -TABLE I. LIST OF TABLES Comparison of s p e c i f i c a c t i v i t i e s with d i f f e r e n t extraction methods. PAGE 12 TABLE II. TABLE I I I . TABLE IV. TABLE V. TABLE VI. TABLE VII, TABLE VIII, TABLE IX. Comparison of crude homogenate a c t i v i t y 12 with supernatant a c t i v i t y . Hydrolysis of PNPP by butanol extracts of 19 A. g r a c i l e of d i f f e r e n t developmental stages. Hydrolysis of BGP by butanol extracts of 19 A. g r a c i l e of d i f f e r e n t developmental stages. Hydrolysis of PNPP by butanol extracts of 20 T. torosa of d i f f e r e n t developmental stages. Hydrolysis of BGP by butanol extracts of 20 T. torosa of d i f f e r e n t developmental stages. A c t i v i t y r a t i o s (PNPP/BGP) of butanol 24 extracts of A. g r a c i l e of d i f f e r e n t dev-elopmental stages. A c t i v i t y r a t i o s (PNPP/BGP) of butanol 24 extracts of T. torosa of d i f f e r e n t dev-elopmental stages. A table of the r e l a t i v e AP a c t i v i t y of 40 embryonic tissues at d i f f e r e n t stages of development. - V -LIST OF PLATES PAGE PLATE I. 36 A. Cross-section of st. 42, T. torosa frevealing s i t e s of AP a c t i v i t y i n various regions. B. Cross-section of st. 42, A. g r a c i l e showing a c t i v -i t y of the i n t e s t i n e . C. Cross-section of st. 42, T. t o r o s a { i n region of presumptive i n t e s t i n e . PLATE I I . 3 7 D. Cross-section of pronephric tubules of st. 32, A. g r a c i l e . E. Well developed pronephric tubules of s t . 42, A. g r a c i l e . F. Cross-section of the st. 38, A. g r a c i l e ^ showing a c t i v i t y of a x i a l mesenchyme. PLATE I I I . 38 G. A cross-section i n the region of the limb of s t . 42, T. torosa. H. Cross-section revealing a c t i v i t y of diencephalon and optic cup of s t . 3 5/36, T. torosa. I. A cross-section through the well developed eye of s t . 42, T. torosa. J . A c t i v i t y of the hypophysis of s t . 38, A. g r a c i l e . - v i -ACKNOWLEDGEMENT I am deeply g r a t e f u l fo Dr. CV. Finnegan for the guidance, encouragement, and support given to me through-out the course of thi s work. I would also l i k e to thank Dr. M. Gould (Somero) for teaching me the acrylamide gel technique and for much invaluable assistance and Dr. D. Francis for many stimulating discussions. Dr. Acton and Dr. Ford are thanked for t h e i r reading and c r i t i c i s m of the manuscript. This research was supported i n part by funds from the National Research Council of Canada. -1-INTRODUCTION It has been proposed that non-specific phosphatases are involved i n the basic biochemical mechanisms underlying and e s s e n t i a l f o r overt, or morphological, d i f f e r e n t i a t i o n (Moog, 1944 and 1952). Karzmer and Berg (1951), also, suggest that a d i f f e r e n t i a t i n g c e l l may have to pass through an "alkaline phosphatase-rich t r a n s i t i o n phase". Numerous studies have been concerned with the molecular d i f f e r e n t i a t i o n of a l k a l i n e phosphatases during normal development and have demonstrated changes i n the multimolecular forms of t h i s enzyme ( Moog et a l , 1965; Pfohl, 1965; Schneiderman, 1967; Solomon e_t a l , 1964). To date, no attempt has been made to correlate the multimolecular forms of a l k a l i n e phosphatase with i t s r o l e i n the processes of d i f f e r e n t i a t i o n . Recent papers s t i l l c i t e t h i s d i f f e r e n t i a t i o n function (Piatka and Gibley, 1967; Shah and Chakko, 1967) although the mechanism of action remains to be elucidated. As well, a l k a l i n e phosphatase has been implicated i n transport processes ( D a n i e l l i , 1952; Matthiessen, 1966; Tosteson et a_l, 1961), i n protein synthesis (Osawa, 1951; Vorbrodt, 19 58), i n mucopolysaccharide formation (Moog and Wenger, 1952; Matthiessen, 1966), i n bone formation (Matthies-sen, 1966; Stadtman, 1961; Schmidt and Laskowski, 1961) and, also, i n the control of DNA synthesis (Rubini et al,1964; Baserga, 1968). The present study involved an analysis of the development of a l k a l i n e phosphatase i n salamander embryos, a s i t u a t i o n that has been touched on (Karzmer and Berg, 1951; Krugelis, - 2 -1950; Lovtrup, 1953; and Osawa, 1951) but not adequately defined i n any one species. The use of histochemical detec-t i o n methods coupled with biochemical analyses, s p e c i f i c a c t i v i t y determinations and electrophoretic analysis, served here to define the ontogeny of the al k a l i n e phosphatases i n Ambystoma g r a c i l e and Taricha torosa embryos. -3-MATERIALS AND METHODS  I. Preparation of Embryos: Ambystoma g r a c i l e embryo masses were c o l l e c t e d i n early stages of development from natural pools i n the v i c i n i t y of Vancouver, B.C.. Thermos jugs containing Taricha torosa egg cluthces were received a i r express from Stanford and St. Marys, C a l i f o r n i a . Both species were allowed to develop at 8 - 1 . 5 °C. i n a c o n t r o l l e d temperature r e f r i g e r a t o r u n t i l the desired stages of development were attained. For A. g r a c i l e (A.g.) staging was by the schema of Harrison for A. punctatum, as described i n Rugh (1962). T. torosa (T.t.) embryos were staged using the method of Twitty and Bodenstein (Rugh, 1962) fo r t h i s species. A f t e r stage 40, T. torosa was staged on the basis of limb development, using A. punctatum (Rugh, 1962) as a standard for comparison. Morphologically, the stages for the two species were almost i d e n t i c a l . A. S p e c i f i c a c t i v i t y determinations: Groups of f i f t e e n embryos from each of the major develop-mental periods were frozen i n small p l a s t i c capped test-tubes i n l i q u i d nitrogen and stored at -20 °C. u n t i l required. Fresh material was also used f o r comparative purposes. F i f -teen embryos were rou t i n e l y homogenized i n 1.0 ml. of borate b u f f e r (0.3 M, pH 8.6) i n a Bellco p l a s t i c homogenizer with t e f l o n pestle, maintained cold i n an ice-bath. To t h i s b r e i 1.5 volumes of n-butanol were added. This step was followed with further homogenization as the b r e i was allowed to a t t a i n room temperature (after Morton, 19 54). Subsequently, c e n t r i --4-fugation was c a r r i e d out i n an International r e f r i g e r a t e d centrifuge (model HR-1, Head 856) at 16,000 rpm. and 0-3 °C. for f i f t e e n minutes. The aqueous layer, l y i n g above the pre-c i p i t a t e d c e l l debris and below the l i g h t butanol and f a t t y layers, was removed and stored at -20°C. In c e r t a i n cases, the b r e i alone was stored for l a t e r determinations on a c t i v -i t y loss i n the p r e c i p i t a t e . I t was noted that frozen samp-les could be stored f o r months with l i t t l e detectable loss of a l k a l i n e phosphatase a c t i v i t y , as compared to fresh samp-le s . However, samples were usually used within one week. For comparative purposes an i d e n t i c a l b u f f e r extraction with-out butanol was done. For the developmental analysis of s p e c i f i c a l k a l i n e phosphatase a c t i v i t y , the extracts were prepared for each of the following periods of development: b l a s t u l a (T.t. stage 8), gastrula (A.g. s t . 12; T.t. s t . 13), neurula (A.g. s t . 16; T.t. s t . 16 and s t . 18), closed neural folds (T.t. s t . 21), t a i l bud (A.g. s t . 25; T.t. s t . 28), l a t e t a i l bud (A.g. s t . 31; T.t. s t . 32), C-flexure (A.g. s t . 33), S-flexure (A.g. s t . 35; T.t. s t . 36), and free-swimming larvae (A.g. st.38, s t . 41/42 and s t . 45/46; T.t. s t . 39, s t . 42/42+ and s t . 45/ 46. These extracts were then frozen and stored u n t i l a l l sta-ges were accumulated. Three separate extractions were done for each stage analyzed. B. Electrophoresis: Both butanol and b u f f e r extraction procedures were u t i l -ized for samples to be subjected to electrophoretic analysis. Here, 30 embryos were homogenized i n 0.2 ml. of buffer. I f -5-the number of embryos was increased the buffe r added was also increased proportionately. With butanol extracts i t was necessary to dialyze the samples against sucrose (20 % i n 0.3 M borate buffer) before e l e c t r o p h o r e s i s t t o remove the butanol. Routine d i a l y s i s of buffe r extracts was unnecessary since pre-liminary runs demonstrated i t did not q u a l i t a t i v e l y change the r e s u l t s . C. Histochemistry: Live embryos were fix e d overnight (approx. 12 hours) i n 80% ethanol and, a f t e r the fix e d embryos were taken to xylene through 95% and absolute ethanol, they were embedded i n a low melting point wax (Paraplast, M.P. 42-44°C.). The t o t a l time i n the wax was one hour. The p a r a f f i n blocks were stored at -20°C. , for the period, when the stages to be stud-ied were accumulated. The blocks were then trimmed and sec-tioned at 7-10 microns and the sections mounted on albumen-ized s l i d e s . II. S p e c i f i c A c t i v i t y Assays: In these experiments two d i f f e r e n t substrates were used for the analysis of a l k a l i n e phosphatase a c t i v i t y , sodium beta-glycerophosphate (BGP; N u t r i t i o n a l Biochemicals) and para-nitrophenyl phosphate (PNPP; Calbiochem). The s p e c i f i c a c t i v i t y for BGP was defined as micromoles of phosphate re-leased per minute per milligram of protein per m i l l i l i t e r . -3 For PNPP, the s p e c i f i c a c t i v i t y was defined as 1 X 10 mg. PNP released per min. per mg. protein per ml.. The s p e c i f i c a c t i v i t y values were then converted to percent of maximum a c t i v i t y for graphic representation. For comparison of -6-developmental changes, they were converted to the amount of substrate u t i l i z e d , expressed i n micromoles (^ iM), to allow a d i r e c t comparison of the hydrolysis of the two substrates by the embryo extracts. These data too were plotted as percent of maximum values. A. Enzyme a c t i v i t y assays: The standard reaction mixture , for each substrate, was modified from the method of Moog and Grey (1967) for beta-glycerophosphate and consisted of: 0.25 ml. of 0.25 M substrate (BGP or PNPP) 1.50 ml. of 0.10 M carbonate-bicarbonate buffer (pH 10.0) 0.2 5 ml. of 0.10 M MgCl 2 The concentrations of each of the reagents i n t h i s standard reaction mixture had been determined as optimal by prelim-inary experiments. Free-swimming larvae because of t h e i r abundance were used i n most cases for the determination of the optimal conditions and c h a r a c t e r i s t i c s of the a l k a l i n e phosphatase a c t i v i t y . The concentration of the substrate or MgC^/ the pH of the buff e r and temperature were varied i n accordance with the parameter under observation. For measure-ments of a c t i v i t y below pH 9.0, Tris/HCl b u f f e r (0.1 M) was used. To the reaction mixture, 0.2 ml. of sample was added, followed by rapid mixing on a Vortex Genie. The reaction was c a r r i e d out at room temperature and was " k i l l e d " with 0.8 ml. of 10% t r i c h l o r a c e t i c acid (TCA), a f t e r one hour un-less otherwise indicated. When BGP was used the released inorganic phosphate was measured by the method of Fiske and Subbarow (192 5) using a Technicon Autoanalyzer. Standards of known inorganic phosphate concentration (KFL^PO^) were run with each set of determinations. For PNPP the yellow colour of the released para-nitrophenol (PNP) was measured at 420 mp. i n a Spectronic 20 spectrophotometer. Since TCA destroys the colour, by pH depression, i t was redeveloped with 2.0 ml. 0.1 N NaOH (after Pfohl and Guidice, 1966). The amount of PNP released was determined from a standard curve prepared from a series of known concentrations of t h i s product (Calbiochem). Controls consisted of reaction mixtures to which TCA was added p r i o r to the addition of the sample (0-time con-t r o l ) and of substrate-free mixtures. B. E f f e c t of i n h i b i t o r s : The two amino acids, phenylalanine and tryptophan, and inorganic phosphate (Wf^PO^) were used, i n concentrations -3 -2 ranging from 1.25 X 10 to 1.5 X 10 Molar, to study the i n h i b i t i o n of l a r v a l phosphatase. C. Protein determination: Protein contents were determined by the technique of Lowry et al (1951) using the F o l i n Ciocalteau Phenol Reagent (Fischer) as described by Adams (1964). In t h i s case 0.2 ml. of sample rather than 0.4 ml. was used so as to make possible the d i r e c t c o r r e l a t i o n with the enzyme a c t i v i t y assays. No sample d i l u t i o n was necessary for these determinations. A standard curve was prepared with egg albumen treated the same as the experimental samples, ranging i n concentration from -8-_2 4.0 X 10 to 4.0 mg. of protein per ml.. Blanks f o r zero-ing the spectrophotometer consisted of a l l the reagents with water i n place of the embryo extracts. A Spectronic 20 spec-trophotometer was used i n a l l cases. I I I . Electrophoresis: A. Starch gel electrophoresis: An attempt to reveal multimolecular forms of a l k a l i n e phosphatase was made using the Tsyuki _et a_l (1962, 1963) modification of the starch gel method of Smithies (1955, 19 59). Samples were run i n 12% gels i n the borate buffer system for 1% to 2 hours at 3°C. and 3 mA per g e l . Sample d i l u t i o n had no e f f e c t except to lower t o t a l a c t i v i t y . B. Disc electrophoresis: Disc electrophoresis i n 7% acrylamide gels as described by Ornstein (1962) and Davis (1962) was also used to reveal the a l k a l i n e phosphatase patterns of salamander embryo ex-t r a c t s . Samples were run i n a Canalco disc electrophoresis apparatus at 3 mA per gel f o r approximately 1 to V% hours depending on the rate of migration of the more electroneg-ative of the two embryonic pigment bands. The length of the run; corresponded c l o s e l y to the time required for a brom-phenol blue dye marker to migrate to the end of the g e l . When the most r a p i d l y migrating band reached the end of the running g e l the run was terminated. Sample volumes of 100 were layered d i r e c t l y on the surface of each stacking g e l . C. Gel sta i n i n g : Both starch and acrylamide gels were stained f o r l o c a l -i z a t i o n of a l k a l i n e phosphatase a c t i v i t y using sodium alpha--9-naphthyl phosphate (100 mg.) and Fast blue RR (50 mg.) i n carbonate-bicarbonate buffer (100 mis. of 0.1 M at pH 10), containing 1.0 ml. of 0.1 M MgC^. Staining was done at room temperature a f t e r which the gels were stored i n d i s -t i l l e d water (acrylamide gels) or i n Saran Wrap (starch g e l s ) , u n t i l photographed. The gels were photographed by trans-mitted l i g h t using Kodak Plus X Pan f i l m . The reddish-brown, azo-dye complex depicts s i t e s of a l k a l i n e phosphatase a c t i v i t y and i s stable i n d e f i n i t e l y , although darkening i n time, both at room temperature and i n the r e f r i g e r a t o r . IV. Histochemistry: Histochemical l o c a l i z a t i o n of a l k a l i n e phosphatase was by the Burstone (1962) Naphthol AS Phosphate Method, which i s s i m i l a r to that used i n the g e l staining procedure. Staining was performed at pH 9.0 because the s t a i n p r e c i p i -tates out at higher pHs and th i s region of pH i s s p e c i f i e d i n the method. On the basis of a developmental analysis of s p e c i f i c a l k a l i n e phosphatase a c t i v i t y c a r r i e d out i n t h i s laboratory, the use of t h i s pH seems legitimate, a l -though' not optimal (see Figure 1). After staining for one hour at 3 7 ° C , the sections were mounted i n Aquamount and covered with a c o v e r s l i p . Sections were viewed with a Wild microscope and a c t i v i t y values given i n a r b i t r a r y units (+ to ++++) based on a comparison with maximal gut a c t i v i t y of free-swimming larvae. Selected sections were photographed on a Zeiss Photomicroscope using Kodak High Speed Ektachrome (Type B). -10-RESULTS I . SPECIFIC ACTIVITY: A. Optimal condit ions for enzyme a c t i v i t y : 1. pH optima- Buffer extracts of free-swimming larvae (st. 40-46) demonstrated a s ing l e peak of a c t i v i t y w i t h i n the range of pH from 9.7 5-10.0 (Fig . 1. A , C ) . This he ld true for both T. torosa and A. g r a c i l e . Below pH 9.0 buf fer extracts showed a low l e v e l of a c t i v i t y which dropped o f f s lowly as the pH moved towards n e u t r a l i t y . Butanol extracts revealed (Fig . 1. B , D , E , and F) the same major a c t i v i t y peak, i n both species , as had the buf fer ex trac t s . No peak of ac-t i v i t y occurred below pH 9.0 i n butanol ex trac t s . Butanol ex trac t s , of both species , revealed a 6 - fo ld increase i n spe-c i f i c a c t i v i t y above that for buf fer ex trac t s . Morton (1954) has suggested that the increased s p e c i f i c a c t i v i t y i n butanol extracts i s due to increased enzyme re lease . However, the data i n Table I show that although butanol ex trac t ion pro-duces a modest increase i n absolute a c t i v i t y (column 3) , the major increase i n s p e c i f i c a c t i v i t y i s due to the much lower non-enzyme p r o t e i n i n butanol ex trac t s . This i n t e r p r e t a t i o n i s confirmed by comparing the amounts of AP a c t i v i t y l o s t to the p r e c i p i t a t e i n buf fer and butanol preparations ( c a l c u l -ated from data i n Table I I ) . The comparison of T. torosa butanol ex trac t a c t i v i t y , by volume (column 3, Table l ) , t o b r e i a c t i v i t y (Column 2, Table II) revealed that only about o n e - t h i r d (33.8%) of the t o t a l enzyme a c t i v i t y was l o s t i n the p r e c i p i t a t e d layer on c e n t r i f u g a t i o n . 2. Substrate optima- In A. g r a c i l e varying the substrate -11-FIGURE 1. The e f f e c t of pH on the a l k a l i n e phosphatase a c t i v -i t y of extracts of free-swimming larvae. A. Buffer extract of A. g r a c i l e (st. 42/44) embryos (substrate:PNPP). B. Butanol extract of A. g r a c i l e (st. 42/44) em-bryos (substrate:PNPP). C. Buffer extract of _T. torosa (st. 42/44) embryos (substrate:PNPP). D. Butanol extract of T. torosa (st. 42/44) em-bryos (substrate:PNPP). E. Butanol extract of A. g r a c i l e (st. 42/44) em-bryos (substrate: BGP). F. Butanol extract of T. torosa (st. 42/44) em-bryos (substrate: BGP). -12-TABLE I. Comparison of s p e c i f i c a c t i v i t i e s with d i f f e r -ent extraction methods ( A. g r a c i l e and _T. torosa stage 42 embryos; data represents the resu l t s of one set of experiments). TABLE II. Comparison of crude homogenate a c t i v i t y with supernatent a c t i v i t y ( _T_. torosa stage 42; buffer extraction). TABLE I COMPARISON OF SPECIFIC ACTIVITIES WITH DIFFERENT EXTRACTION METHODS SPECIES EXTRACTION METHOD A. g r a c i l e b u f f e r butanol T. torosa b u f f e r butanol -3 PNP X 10 RELEASED .23 5 .310 .399 .448 PROTEIN SPECIFIC ACTIVITY CONC. ACTIVITY INCREASE 1.30 0.29 1.10 0.21 0.181 1.05 0.362 2.13: 5.82 5.85 TABLE II SAMPLE Br e i Supernatant COMPARISON OF CRUDE HOMOGENATE ACTIVITY WITH SUPERNATANT ACTIVITY* PNP X 10~ 3 RELEASED .676 - .006 ,373 - .064 PROTEIN CONC. 2.4 - .20 .98 .19 SPECIFIC ACTIVITY .273 .380 Data represents means (- S.D.) of three experiments, -13-concentration of both PNPP and BGP gave maximal a c t i v i t y at _ 2 2.2 X 10 Molar (Figure 2). Some i n h i b i t i o n of enzyme a c t i v i t y occurred at higher substrate l e v e l s . This might be a r e s u l t of the free inorganic phosphate present i n the sub-strates. The high l e v e l of inorganic phosphate present i n PNPP gave very high zero-time control readings on the auto-analyzer and, thus, i t was more accurate to use the absorb-ance of the PNP as an index of enzyme a c t i v i t y rather than, the release of inorganic phosphate. Although T. torosa extracts have a higher s p e c i f i c a c t i v i t y at st. 42 (approx. 2 times that of g r a c i l e , Table I), the p l o t of enzyme ac t i v -i t y versus substrate concentration (for PNPP) appeared the same as that for A. g r a c i l e (Fig. 2, C). 3. Temperature optimum- The temperature optimum for A. g r a c i l e occurred around 37 °c . (Fig. 3, A). However, a c t i v -i t y i s s t i l l present at 5°C., at 10% of the maximum. The increase i n a c t i v i t y with temperature i s rather sharp a f t e r 30°C. as i s the decrease beyond 37^C.. Since the temper-ature at which maximum a c t i v i t y was present was so much higher than the temperatures these embryos normally encoun-ter, i t was decided to use room temperature (approx. 22°C.) for the temperature at which enzyme a c t i v i t y analysis would be done, though even t h i s i s a substantial increase over the usual temperature at which the embryos develop. 4. Enzyme d i l u t i o n - D i l u t i o n of embryo extracts of A. g r a c i l e revealed a l i n e a r increase i n a c t i v i t y with increas-ing enzyme concentration (Fig. 3, B). -14-FIGURE 2. The e f f e c t of substrate concentration on the rate of hydrolysis of substrate by butanol extracts of free-swimming salamander larvae: A. BGP hydrolysis by A. g r a c i l e (st. 42) extracts. B. PNPP hydrolysis by A. g r a c i l e (st. 42) extracts. C. BGP hydrolysis by T. torosa (st. 42) extracts. The enzyme a c t i v i t y units r e f e r to PNP X 10 re-leased per hour per ml. (for PNPP) and micromoles of inorganic phosphate released per hour per ml. •(for BGP) . -15-5'.. Time course- In extracts of A. g r a c i l e the rate of reaction i s l i n e a r with time for at lea s t two hours (Fig. 3, C). This was true f o r both A. g r a c i l e and T. torosa from gastrulae to free-swimming larvae. 6. E f f e c t of magnesium ions- In many cases magnesium ions are e s s e n t i a l for the a c t i v i t y of al k a l i n e phosphatase. No e f f e c t on enzyme a c t i v i t y was noted here when a range of concentrations of MgCl 2 from 9.0 X 10~ 2 to 9.0 X 10~ 4 Molar was used. B. E f f e c t of i n h i b i t o r s on phosphatase a c t i v i t y : Both tryptophan and phenylalanine, which are known i n -h i b i t o r s of other a l k a l i n e phosphatases (Fishman _et _al, 1963; Ghosh and Kotowitz, 1969; G r i f f i n and Cox, 1966), i n h i b i t the phosphatase a c t i v i t y present i n extracts of free-swimming larvae of A. g r a c i l e and T. torosa. The l e v e l of i n h i b i t i o n _3 (50% at 7.5 X 10 Molar) seems too low to be considered s i g n i f i c a n t (Fig. 4). Inorganic phosphate, too, i n h i b i t s the salamander a l k a l i n e phosphatase (Fig. 4) and i s less e f f i c i e n t i n t h i s respect than the amino acid i n h i b i t o r s (50% i n h i b i t i o n at about 1.25 X 10~ 2 Molar). C. Developmental changes i n s p e c i f i c a c t i v i t y : As shown i n Figures 5 and 6 the a l k a l i n e phosphatase a c t i v i t y of the salamander embryo extracts show a general over-a l l increase with development. This trend holds true for both species with both substrates. When PNPP was used as a substrate with A. g r a c i l e ex-tracts (Table III; Figure 5) the increase i n s p e c i f i c AP a c t i v i t y was gradual up to the appearance of a d e f i n i t e t a i l -16-FIGURE 3. Some parameters of a l k a l i n e phosphatase a c t i v i t y i n butanol extracts of A. g r a c i l e embryos: A. E f f e c t of temperature on PNPP hydrolysis by st. 42/44 extracts. B. E f f e c t of d i l u t i o n of enzyme extracts on ac t i v -i t y of st. 42/44 embryo extracts. C. Time course of PNPP hydrolysis by st. 3 5 embryo extracts. :(© and A represent r e s u l t s with d i f f e r e n t extracts). I O O T A 50i O O I O 2 0 3 0 4 0 5 0 T E M P E R A T U R E [ ° C ] I O O T B 50H O 1 1 1 O 0 5 I O D I L U T I O N F A C T O R I O O T C 50H O T 1 1 1 O 6 0 I 2 0 T I M E [min] -17-FIGURE 4. E f f e c t of known i n h i b i t o r s of a l k a l i n e phosphatase on PNPP hydrolysis by buffer extracts of T. torosa free-swimming larvae (st. 40): O Tyrosine @ Tryptophan. A Inorganic Phosphate ( K E U P O . ) . -18-bud (st. 25), which possessed a l e v e l of a c t i v i t y 5 times that of the gastrular l e v e l (st. 12), but became by stage 38 about 2 5 times the gastrular l e v e l . The increase was even more obvious within the next few stages of development, reaching ultimately (st. 45/46) about 150-fold the a c t i v i t y possessed by the gastrulae. Similar r e s u l t s were obtained when BGP was used as substrate (Table IV; Figure 6). How-ever, the increase between g.astrula and free-swimming stages reached a maximum of only 35-fold when BGP was used. The r e s u l t s with T. torosa extracts correlated very w e l l with those f o r A. g r a c i l e , although the former species usually possessed a greater a c t i v i t y i n the l a t e r stages of development. Comparing free-swimming a c t i v i t y (st. 42/42+) to gastrular s p e c i f i c AP a c t i v i t y there was a 60-fold i n -crease when PNPP was used and a 25-fold increase when BGP was used as substrate. Afte r stage 42/42+ a decrease i n s p e c i f i c a c t i v i t y was noted with both substrates. The foregoing discussion of r e s u l t s has been based on mean values calculated from three data points for each stage of development. Confidence l i m i t s (5%) were calculated, with the aid of a computer, and demonstrated large overlap below stages 35 (A.g.) and 36 (T.t.) but showed s i g n i f i c a n t d i f f e r -ences past these stages. This would indicate that between ga s t r u l a t i o n and the onset of autonomous muscle movement i n these salamander embryos no s i g n i f i c a n t difference i n enzyme a c t i v i t y e x i s t s . On the basis of work i n other Urodele species (Krugelis, 1950; Lovtrup, 1953), i n which s i m i l a r r e s u l t s were obtained, i t would seem that t h i s s i t u a t i o n -19-TABLE I I I . Hydrolysis of PNPP by butanol extracts of A. grac-i l e embryos of d i f f e r e n t developmental stages. TABLE IV. Hydrolysis of BGP by butanol extracts of A. g r a c i l e embryos of d i f f e r e n t developmental stages. TABLE III MEAN SUBSTRATE STAGE SPECIFIC ACTIVITY INCREASE % MAXIMUM USED (pM) 12 0.007 0. 600 0.051 0.002 16 0.009 0.720 0. 065 0.017 18 0.026 2.200 0.187 0.013 25 0.039 3.300 0.280 0.061 31 0.100 8.450 0.719 -0.026 33 0 o074 6.920 0. 530 0.022 35 0.096 8.120 0.690 0.092 38 0.188 15.90 1.350 0.730 41/42 0.918 73.90 6.600 0.362 45/46 1.180 100.0 8. 500 TABLE IV MEAN SUBSTRATE STAGE SPECIFIC ACTIVITY INCREASE % MAXIMUM USED OuM) 12 0.300 2.820 0.300 0.150 16 0.450 4.230 0.450 0.100 18 0.460 4.320 0.460 0.230 25 0.690 6.490 0.690 0.170 31 0.860 8.080 0.860 -0.190 33 0.670 5.490 0.670 0.210 35 0.880 8.260 0.880 0.270 38 1.150 10.80 1.150 5.460 41/42 6.180 63.94 6.810 3.840 45/46 10. 65 100.0 10.65 -20-TABLE V. Hydrolysis of PNPP by butanol extracts of T. torosa embryos of d i f f e r e n t developmental stages. TABLE VI. Hydrolysis of BGP by butanol extracts of T. torosa embryos of d i f f e r e n t developmental stages. TABLE V MEAN SUBSTRATE STAGE SPECIFIC ACTIVITY INCREASE % MAXIMUM USED (jiM) 13 0.035 -0.007 1.660 0.250 16 0.028 0.009 1.460 0.220 21 0.037 0.051 1.800 0.270 28 0.088 0.141 4.190 0.63 0 32 0.147 0.147 7.050 1.060 36 0.294 0. 502 14.20 2.120 39 0.796 1.295 38.00 5.720 42/42+ 2.091 -0.175 100.0 15.05 45/46 1.916 91.70 13.80 TABLE VI MEAN SUBSTRATE STAGE SPECIFIC ACTIVITY INCREASE % MAXIMUM USED (juM) 8 0.310 0.200 2.540 0.310 13 0. 510 -0.060 4.180 0.510 16 0.450 -0.060 3.690 0.450 21 0.390 -0.010 3.190 0.390 28 0.380 0.210 3.110 0.380 32 0.590 0.920 4.830 0. 590 36 1. 510 1.150 12.36 1.510 39 3.020 9.190 24.73 3.020 42/42+ 12.21 -0.640 100.0 12.21 45/46 11.57 94.75 11. 57 -21-FIGURE 5. Development of a l k a l i n e phosphatase s p e c i f i c a c t i -v i t y as revealed by the hydrolysis of PNPP. - 2 2 -FIGURE 6. Development of a l k a l i n e phosphatase s p e c i f i c a c t i -v i t y as revealed by the hydrolysis of the substrate BGP. I O O -A . G R A C I L E O T . T O R O S A >-> U < Z) x < 2 50H O 1-z LU u cc LU Q_ O ~ 1 — I O -o-— I — 2 0 3 0 4 0 S T A G E O F D E V E L O P M E N T -23-i s widespread i n developing salamanders. With A. g r a c i l e the r a t i o c alculations showed two peaks of high r a t i o (PNPP/BGP) corresponding to l a t e t a i l bud (st. 31) and early free-swimming larvae (st. 38)(Table VII; Figure 7). The r e s u l t s of the a c t i v i t y r a t i o s , when plotted as percent of maximum, were comparable between the two species, with the T. torosa values overlapping the A. g r a c i l e values continuously from g a s t r u l a t i o n to the free-swimming stage (Table VIII; Figure 7). II . GEL ELECTROPHORESIS: A. Starch gel electrophoresis: The r e s u l t s with Tsyuki's (1962, 1963) microstarch gel electrophoretic system were discouraging. It was necessary to use butanol extracts to obtain migration. However only a dense band streaking out approximately one-half inch from the o r i g i n occurred (Figure 8). Some sub-banding was evident within the streak, but was not reproducible. An analysis of the general protein patterns (stained with Amido Black 10B) revealed many c l e a r l y delineated bands and commercial intes-t i n a l phosphatase (ly o p h i l i z e d , bovine; Calbiochem) gave a s i n g l e band of a c t i v i t y . Therefore, the f a i l u r e to obtain banding of AP l i e s with the embryo extracts rather than the starch gel technique. B. Acrylamide gel electrophoresis: Clear r e s u l t s were obtained with acrylamide gel e l e c t r o -phoresis. It was found that butanol extracts, a f t e r d i a l y s i s , gave the same electrophoretic pattern as buffer extracts of any one stage. This i s shown i n Figure 9. Migration was -24-TABLE VII. A c t i v i t y r a t i o s (PNPP/BGP) of butanol extracts of A. g r a c i l e from d i f f e r e n t developmental stages. TABLE VIII. A c t i v i t y r a t i o s (PNPP/BGP) of butanol extracts of T. torosa from d i f f e r e n t developmental stages. TABLE VII STAGE ACTIVITY RATIO (PNPP/BGP) % MAXIMUM OF RATIO 12 0.170 14.50 16 0.140 11.95 18 0.410 35.05 25 0.420 35.90 31 0.840 71.80 33 0.790 67. 50 35 0.780 66.70 38 1.170 100.0 41/42 0.970 82.80 45/46 0.800 68.30 TABLE VIII STAGE ACTIVITY RATIO (PNPP/BGP) % MAXIMUM OF RATIO 13 0.490 26.05 16 0.490 26.05 21 0.690 36.70 28 1.650 87.80 32 1.800 95. 50 36 1.400 74.50 39 1.880 100.0 42/42+ 1.230 65.40 45/46 1.190 63.20 -25-FIGURE 7. Change i n a c t i v i t y r a t i o (PNPP/BGP) during dev-elopment of A. g r a c i l e and T. torosa. -26-FIGURE 8. E f f e c t of d i f f e r e n t extraction methods on elec-trophoretic patterns of a l k a l i n e phosphatases i n starch gels, ( a l l are A. g r a c i l e embryo extracts; 3 5 and 42 r e f e r to stages of development; c stands for co n t r o l ) . + — — i "~ 42 C 42 C42 BUTANOL EXTRACT + • 35 C 42 C 35 ~ BUFFER EXTRACT -27-slower with butanol extracts than with buffer extracts, which may r e f l e c t the lower protein content of the butanol extracts. As a r e s u l t , buffer extracts were used routinely. The maximum number of a l k a l i n e phosphatase bands was two. These components migrated very close together making i t d i f f i c u l t to quantify the i n d i v i d u a l bands. Microdensito-meter tracings of the stained gels have been made and such tracings reveal the slower component of the zymogram as a shoulder of the faster, more dense band. That the a c t i v i t y of the bands increased, and markedly with development, was evident. The same re s u l t s were obtained f o r both species and a summary of the development of the enzyme patterns i s shown i n Figure 10. Designating the more electronegative band as band 2 and the more e l e c t r o p o s i t i v e as band 1 the develop-mental trend can be described as an increase i n band 2 a c t i v -i t y and a decrease of band 1. By stage 40 the a c t i v i t y i s so great that separation of the bands v i s u a l l y i s d i f f i c u l t . I t was impossible to detect any subtle changes i n the band a c t i v i t i e s for a comparison of t h e i r r e l a t i v e a c t i v i t y , and increased running time, up to 2% hours, did not aid e l e c t r o -phoretic band separation. I I I . HISTOCHEMISTRY: A. A. g r a c i l e : The f i r s t histochemically detectable a l k a l i n e phosphatase a c t i v i t y occurred during t a i l bud stages (st. 27) and was l o c a l i z e d i n the yet u n d i f f e r e n t i a t i e d endoderm, two regions of ectoderm (the stomodeal and proctodeal regions) and i n the forebrain (composed at t h i s time of the telencephalon and diencephalon). The general a c t i v i t y of the gut and -28-FIGURE 9. Migration of a l k a l i n e phosphatases i n acrylamide gel (10/12, 22/24, 40 r e f e r to develop-mental stages; 10/12 buffe r and 10/12 butanol compare r e s u l t s with d i f f e r e n t extraction methods. 50 JJLI. of extract were applied to each g e l . IO/12 22/24 30 34 4 0 DEVELOPMENTAL PATTERN T . T IO/I2 IO/I2 B U F F E R B U T A N O L -29-FIGURE 10. Diagrammatic representation of 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 and a comparison of the extraction procedures as pictured i n Figure 9. (symbols as for Fig. 9). j A A T A T T IQfl2 22J2A 3 0 3 4 4 0 IQfl2 IC/12 BUFFER BUTANOL D E V E L O P M E N T A L P A T T E R N -3 0-b r a i n regions was low, whereas that of the developing stora-odeum and proctodeum regions was higher but r e s t r i c t e d , only occurring i n a few sections. As the embryos continued to develop into the la t e t a i l bud stage (st. 30/31), a l k a l i n e phosphatase a c t i v i t y became evident i n other areas. The l i v e r diverticulum, which f i r s t appeared as a simple ventral extension of the foregut into the yolk, had a r e l a t i v e l y high l e v e l of a c t i v i t y i n i t i a l l y , with the major amount of a c t i v i t y disposed d o r s a l l y towards the foregut. The pronephric tubules, which at t h i s time have just become morphologically d i s t i n c t , had l i t t l e a c t i v i t y . Although l i t t l e d i f f e r e n t i a t i o n of the heart had occurred there was a detectable l e v e l of a c t i v i t y and the head and branchial mesenchymes showed low le v e l s of AP a c t i v i t y as we l l . The only other change by stage 3 0/31 was a s l i g h t increase i n the stomodeal a c t i v i t y . As development proceeds, evidenced externally by an elongation of the embryo and an increase i n the siz e of tlhe g i l l buds, some new tissues are added to the ensemble of a l k a l i n e phosphatase producers. By stage 33, the spinal cord possessed a detectable l e v e l of the enzyme whereas the optic cup and stalk had a higher l e v e l of AP a c t i v i t y . I t was not u n t i l t h i s time that AP enzyme d i f f e r e n t i a t i o n of the optic stalk and cup became obvious, yet the i n i t i a l morphological d i f f e r e n t i a t i o n had started some time during m i d - t a i l bud. The mid-brain region (mesencephalon),too, had a low l e v e l of enzyme a c t i v i t y . Concomitant with the appearance of these new enzyme sources, c e r t a i n other tissues had been increasing t h e i r l e v e l s of a c t i v i t y . The AP a c t i v i t y of both ectodermal s i t e s had increased, as had the anterior-most region of the pharynx. The regions of the gut as well as the l i v e r diverticulum (transverse duodenum) had not undergone any detectable change. The pronephric tubules (Plate II, D), now with a c e n t r a l lumen, had a high l e v e l of a c t i v i t y disposed towards the lumen. S p e c i f i c tissue l e v e l l o c a l i z a t i o n of enzyme a c t i v i t y had been evident only i n the pronephric tubules. The heart at t h i s time was s t i l l only i n i t s early stages of d i f f e r e n t i a t i o n but did show increased a c t i v i t y . By the time the embryos had the capacity for f u l l S-flexure (st. 36), a l l of the neurectodermal components demonstrated a l k a l i n e phosphatase a c t i v i t y histochemically. This a c t i v i t y was highest i n the diencephalon with more inten-se l o c a l i z a t i o n s where the optic stalks associated. Although the telencephalon possessed a very low l e v e l of AP a c t i v i t y the remaining regions of the brain, as well as the spinal cord, had a general, s l i g h t l y higher l e v e l of a c t i v i t y and the optic cup had i t s maximal amount of ac t i v i t y ; (see T. torosa, Plate III, H.). Although a c t i v i t y i n the ectodermal deriv-atives became reduced (especially i n the proctodeum) the hypo-physis, now morphologically evident, demonstrated a f a i r l y high l e v e l of a c t i v i t y where i t associated with the dieceph-alon. The amount of a c t i v i t y i n the ventral prosencephalon seemed to be higher than other regions as w e l l . While the endoderm had not improved as an a l k a l i n e phosphatase con-t r i b u t o r the yolk a c t i v i t y began to show an increase. The heart, with an increase i n a c t i v i t y as well as i n i t s -32-morphological development, now revealed that the major ac-t i v i t y was associated with the endocardium though other r e s t r i c t e d areas of l i g h t staining did occur within the heart. The head mesenchyme at thi s stage generally demon-strated a low l e v e l of a c t i v i t y that was more intense where i t associated with the optic cup and stalk. The branchial mesenchyme AP a c t i v i t y was very intense. With the appear-ance of the limb bud, a low l e v e l of a c t i v i t y was associated with the dense mesenchyme of the limb. In the region of the future a x i a l skeleton the a x i a l mesenchyme also demon-strated a low l e v e l of a c t i v i t y . A l l the tissues, other than endodermal or yolk, that had not previously reached t h e i r maximal l e v e l attained i t by stage 38. However, the stomodeal a c t i v i t y was reduced and the proctodeal a c t i v i t y t o t a l l y l o s t as these openings became morphologically complete. The hypophysis, whose association with the infundibulum was now more intimate, showed a very high l e v e l of a c t i v i t y (Plate III, J ) . In general the enzyme a c t i v i t i e s i n the diencephalon and telen-cephalon had increased to a f a i r l y high l e v e l while increas-es i n the mesencephalon, rhombencephalon and spinal cord were not so marked. S p e c i f i c a l l y the telencephalon had a general, o v e r a l l staining whereas the diencephalon a c t i v i t y was located i n the region of the optic s t a l k s . The s t a i n -ing of the optic stalk was a l l - i n c l u s i v e but the cup a c t i v i t y was l o c a l i z e d i n the c e l l s around the lumen. The AP a c t i v i t y of the mesencephalon , rhombencephalon, and spinal cord was more intense i n the region of the white matter and was d i s --33-posed more v e n t r a l l y than d o r s a l l y . The foregut enzyme a c t i v i t y was at a f a i r l y high l e v e l yet the midgut a c t i v i t y was s l i g h t and the l i v e r diverticulum showed a decline i n enzyme a c t i v i t y . The well developed pronephric tubules of these embryos possessed a b r i l l i a n t s c a r l e t staining i n d i -cating very high lev e l s of enzyme a c t i v i t y . This kidney a c t i v i t y demonstrated the precise l o c a l i z a t i o n afforded by the method, as the staining i n the tubule c e l l s was l o c a l -ized at the surface towards the lumen and no d i f f u s i o n was observed. The heart at t h i s time had a f a i r l y high l e v e l of a c t i v i t y and the l o c a l i z a t i o n s remained as previously desc-ribed. The a c t i v i t y of the head mesenchyme had increased markedly as had that of the a x i a l mesenchyme, which includ-ed regions around the notochord, spi n a l cord and dorsal aorta.. (Plate II, P) . Yolk staining remained about the same. In the free-swimming larvae (st. 42 )a reduced number of tissues possessing a l k a l i n e phosphatase a c t i v i t y were seen, and most of these demonstrated a decline i n a c t i v i t y . The only active ectodermal deri v a t i v e was the hypophysis which had l o s t a noticeable amount. In the brain, the telen-cephalon and diencephalon possessed very low le v e l s of phosphatase a c t i v i t y , which was associated with the white matter (see T. torosa, Plate III, I), and the remaining regions of the b r a i n did not demonstrate the presence of any enzyme a c t i v i t y . The a c t i v i t y of the optic cup had dropped to almost nothing, whereas, i n contrast, the optic stalk was s t i l l f a i r l y intense. The eye by t h i s time has undergone extensive d i f f e r e n t i a t i o n and the s p e c i f i c l o c a l -i z a t i o n s were i n two regions; one was i n the region between the nerve f i b e r layer and r e t i n a l neuroblasts and the other was i n the region between the inner and outer nuclear layers of r e t i n a l neuroblasts (see _T. torosa, Plate III, I ) . In contrast, the optic stalk (nerve) was stained throughout (see T.torosa, Plate III, I ) . The endoderm, too, had under-gone a great deal of h i s t o l o g i c a l d i f f e r e n t i a t i o n . The pharynx had no demonstrable, a c t i v i t y . The fore-gut remained as previously described, while v e n t r a l l y and p o s t e r i o r l y i n the transverse duodenum, a decrease i n a c t i v i t y had occurred Where the transverse duodenum associated with the r e s t of the i n t e s t i n e the a c t i v i t y increased again and became very intense posterior to the transverse duodenum (Plate I, B). The o r i g i n a l gut c a v i t y (the archenteron) was no longer v i s i b l e and the region where i t had been present no longer demonstrated AP a c t i v i t y . Posteriorly, where the i n t e s t i n e was present as simply a s p l i t i n the yolk material, the ac-t i v i t y bordering the lumen was just as intense as that of the more anterior, c l e a r l y formed, gut regions and the gener-a l yolk s t a i n i n g i n t h i s region was also very intense (see T. torosa, Plate I, C). The pronephric tubules possessed an intense though s l i g h t l y lower a c t i v i t y than the stage 38 embryos while the heart a c t i v i t y remained the same (for tubule a c t i v i t y see T. torosa, Plate II, E). Both the a x i a l and head mesenchyme had decreased to a just detectable l e v e l . The limb a c t i v i t y , seemingly, had increased, and proximal to the embryo a f a i r l e v e l of a c t i v i t y was present i n the c e n t r a l limb mesenchyme. Moving d i s t a l l y i n the limb -3 5-an increase i n a c t i v i t y was followed by a decrease,although a c t i v i t y was s t i l l present as f a r d i s t a l as the d i g i t s . Within the limb was a central dense region, which seemed to be c a r t i l a g e , around which the staining was most intense but l a t e r a l to t h i s c e n t r a l region the a c t i v i t y dropped (see T. torosa, Plate III, G). Another region where only l i g h t staining occurred was the somite t i p . At t h i s stage, a staining of the mesenchyme associated with the f i n was at a higher l e v e l than previously. B. T. torosa: The a l k a l i n e phosphatase histochemical picture i s basic-a l l y the same for T. torosa, so that the A. g r a c i l e descrip-tion, i n general, should s u f f i c e for the C a l i f o r n i a species as w e l l . However, s l i g h t differences, a l l associated with ectoderm or ectodermal derivatives, were noted. The T. torosa embryos possessed no staining at any stage i n the stomodeum, proctodeum or hypophysis. Local-i z a t i o n s of AP did occur i n the o t i c placode, which was not seen i n the A. g r a c i l e embryos, and was present i n torosa i n only low l e v e l s at two stages (St. 36 and 38). The em-bryo epithelium possessed no detectable a c t i v i t y u n t i l stage 42, when there appeared a general low l e v e l of staining with more intense l o c a l i z a t i o n s i n the epithelium l y i n g adjacent to the eye. F i n a l l y , the AP staining at any one stage, i n a l l responding torosa tissues, was more intense than the counterpart staining i n A. g r a c i l e embryos though the tempor-a l changes, as w e l l as the l o c a l i z a t i o n s and r e l a t i v e changes PLATE I Cross-section of s t . 42 _T. torosa embryo r e v e a l l i n g s i t e s of AP a c t i v i t y i n the pronephric tubules (p), stomach (s.) , transverse duodenum (td), limb bud (lb) and spin a l cord (sp) . (X 34) . Cross-section of St. 42 A. g r a c i l e free-swimming larva show-ing intense l o c a l i z a t i o n towards the i n t e s t i n a l lumen (1). (X160). Cross-section of st. 42 T. torosa i n region of the presum-ptive i n t e s t i n e (pi) where a c t i v i t y i s intense. A c t i v i t y i n the spin a l cord and yolk (y) i s also noticeable here. (X 55). PLATE II Cross-section of pronephric tubules (p) of st. 32 A. gra-c i l e embryo showing l o c a l i z a t i o n towards the lumen. (X 100). Well developed pronephric tubules (p) of st. 42 T. torosa i n cross-section, showing l o c a l i z a t i o n towards lumen. A c t i v i t y i n the adjacent mesenchyme (m) i s also evident. (X 110). Cross-section of st. 38 A. g r a c i l e showing a c t i v i t y of a x i a l mesenchyme surrounding spinal cord (sp), notochord (n), and dorsal aorta (da). (X 100). -38-PLATE III G. A cross-section i n the limb region (anterior) of s t . 42 T. torosa showing l o c a l i z a t i o n i n c e ntral dense region (c) (presumed to be c a r t i l a g e ) and i n surrounding mesen-chyme (m). (X 100). H. Cross-section revealing a c t i v i t y of diencephalon (d) region of b r a i n and optic cup (oc) of an early T. torosa embryo (st. 35/36) . (X 45) . I. A cross-section through the well developed eye of s t . 42 T. torosa showing the l o c a l i z a t i o n i n the optic stalk (os) and i n two layers within the eye (shown by arrows; describ-ed i n r e s u l t s ) ; a c t i v i t y i n the diencephalon (d) and head mesenchyme (hm) i s also evident. (X 100). J . A c t i v i t y of the hypophysis of s t . 38 A. g r a c i l e embryo as revealed by cross-section. (X 100). -39-were the same. The histochemical r e s u l t s are summarized i n Table IX. -40-TABLE IX. A table of the r e l a t i v e AP a c t i v i t y (as compared to maximal gut a c t i v i t y of s t . 42 larvae) of embryonic tissues at d i f f e r e n t stages of develop-ment. (data refers to both species, A.g. and T.t., ex-cept where indicated). TISSUE RELATIVE ACTIVITY STAGE STAGE STAGE STAGE STAGE STAGE 27/28 3 0/3 2 33/34 36 38 42 ECTODERM: aStomodeum +- + ++- +- — — Proctodeum + + ++- ++- +- -^Hypophysis - - . - ++ +++ ++-•^Epithelium - - - - • - +-Otic placode — - — +- +- — NEURECTODERM: Telencephalon +- +- +- + ++ + Diencephalon +- +- + ++ ++ +-Mesencephalon - - + ++ ++- +-Rhombencephalon - - - + + +-Spinal cord - - - - +- +-Optic cup - - + ++- ++ +-Optic s t a l k — — + ++- ++ ++ ENDODERM: Archenteron-foregut +- +- +- +- + ++-midgut +- + + +- - -hindgut +- +- +- - - - -l i v e r d i v e r t . - + ++- ++ ++ + Intestine — — + ++- +++ ++++ MESODERM: Pronephros - + ++ +++ ++++ +++ Heart - +- + ++ + + Mesenchyme-head - +- + ++ +++- ++-branchial - + + ++- +++- + limb - - - + ++ +++-a x i a l - - - +- ++- ++-f i n — — — — +- + YOLK: +- + + ++- ++ +++ a) only, detected i n A. g r a c i l e b) only detected i n T. torosa -41-DISCUSSION It has been stated (Moog, 1967) that an analysis of the s p e c i f i c enzyme a c t i v i t y of whole embryo or tissue homogenates requires an associated analysis of the enzyme a c t i v i t y at the histochemical l e v e l . I t i s evident i n the present i n v e s t i -gation that, although the general trend of s p e c i f i c AP a c t i v -i t y of homogenates of developing salamanders i s towards a higher l e v e l as development proceeds, t h i s does not indicate a general tissue increase but, instead, i s the cumulative expression of a multitude of r e g u l a r l y increasing and decreas-ing enzyme le v e l s within i n d i v i d u a l tissues. This i s espec-i a l l y evident i n these salamander embryos a f t e r stage 38, when the t o t a l number of tissues contributing to the measur-ed AP a c t i v i t y begins to drop r a p i d l y while the actual homo-genate a c t i v i t y increases prodigiously. The major source of the increased homogenate a c t i v i t y i s the d i f f e r e n t i a t i n g endoderm (gut). Lovtrup (1953) suggested, since the i n t e s t i n a l mucosa i s one of the r i c h e s t sources of AP, wherein i t may play a r o l e i n transport ( D a n i e l l i , 1952; Matthiessen, 1966; Tos-teson _e_t a_l, 1961) and f a t absorption (Warnock, 1968), that the increase or "break" noted at t h i s time i n s p e c i f i c a c t i v i t y of Urodele embryo homogenates (A. punctatum, Lov-trup, 1953; A. mexicanum, Krugelis, 19 51) was due to endo-dermal d i f f e r e n t i a t i o n . More recently Harris (1967), i n a study of the morphogenesis of the stomach and i n t e s t i n e of A. maculatum (A. punctatum), revealed that about the time of the described AP increase, the gut begins i t s d i f f e r e n t i a t i o n , -42-but, no histochemical data were made available. The present work shows that the assumption of Lovtrup (1953) was indeed correct, and further reveals that the enzyme a c t i v i t y , as expected, i s low i n the stomach but becomes very intense as one progresses v e n t r a l l y and p o s t e r i o r l y past the transverse duodenum ( l i v e r diverticulum) and into the i n t e s t i n e of free-swimming larvae. However important the gut increase may be, the contribution of other tissues should not be ignored since c e r t a i n of them show t h e i r greatest a c t i v i t y at t h i s time. The best example of t h i s i s found i n the limb components but l o c a l i z a t i o n i n the yolk was also intense i n d i c a t i n g , most l i k e l y , the increased u t i l i z a t i o n of t h i s material by the free-swimming embryos. T here i s evidence that AP i s involved i n the mobilization of these yolk components that serve as a source of raw materials and free-energy for embry-onic development (Williams, 1967). The A. g r a c i l e gut dev-elops more slowly than that of A. maculatum, probably as a r e s u l t of i t s higher yolk content which could undoubtedly retard t h i s morphogenesis. This species v a r i a t i o n i n embryo-nal yolk content may explain the s l i g h t l y e a r l i e r sharp i n -crease (after s t . 36) and the f i n a l a c t i v i t y decrease.in the T. torosa s p e c i f i c a c t i v i t y curves. The torosa embryos possess less yolk than g r a c i l e and presumably gut d i f f e r e n -t i a t i o n would be completed e a r l i e r than i n A. g r a c i l e embryos, an idea which was supported by observation of the histochemi-c a l sections. Further, the data could be considered to sup-port the theory of non-archenteric gut formation proposed by -43-Balinsky (1947) and c l a r i f i e d by Harris (1967)t since the presumptive i n t e s t i n e passage through the yolk was sharply demarcated by intense staining (indicating a high l e v e l of AP a c t i v i t y ) , a c h a r a c t e r i s t i c shared by both the d i f f e r e n -t i a t i n g and the mature gut, but not by the archenteron. Piatka and Gibley (1967), using the Gomori technique, have studied the histochemical l o c a l i z a t i o n of a l k a l i n e phosphatase of the developing pronephros of the frog and Osawa (1952) described s i m i l a r observations i n two other amphibian species, the Urodele Hynobius tokyoensis and the Anuran Rhacophorus s c h e l e g l e i i . The present r e s u l t s c o r r e l -ate w e l l with both of those observations of pronephric AP development. That the enzyme i s concentrated at the luminal border of the tubules i n f e r s a r o l e i n the reabsorption and possible secretory function of the proximal tubules (Piatka and Gibley, 1967; D a n i e l l i , 1952). S p e c i f i c l o c a l i z a t i o n s of high phosphatase a c t i v i t y i n the limb mesenchyme during c a r t i l a g e deposition, as seen i n the present study, had been described previously i n A. punctatum by Karzmer and Berg (1951), both during normal ontogeny and during regeneration. The enzyme can be implicated i n the process of c a l c i f i c a t i o n (Karzmer and Berg, 1951; Matthiessen, 1966; Stadtman, 1961; Schmidt, 1961; Shah and Chakko, 1967) i n which i t seems to act by the production of high l e v e l s of phosphate ions f a c i l -i t a t i n g the formation of calcium phosphate. This chondro-genic function would also explain the high lev e l s of phos-phatase present i n the head mesenchyme, where the cranium w i l l form and i n the a x i a l mesenchyme where the a x i a l -44-skeleton w i l l form. Moog and Wenger (1952) discovered h i s -tochemically that mucopolysaccharide i s generally present i n large amounts where high l e v e l s of AP are detected and the association of AP a c t i v i t y with c a r t i l a g e i n regions of future bone formation has i n t e r e s t i n g connotations i n view of the presence of mucopolysaccharide i n t h i s connective ti s s u e . Later work (Moog and Grey, 1966) revealed that mucopolysaccharide forms an i n t e g r a l part of the duodenal AP molecules and may play a s i g n i f i c a n t role, v i a t h i s moiety, i n morphogenesis of the v i l l i . This association might also explain the i n a b i l i t y of McWhinnie and Saunders (1966) to obtain a s o l u t i o n of phosphatase of bone, even with butanol, i f i t i s assumed that the carbohydrate constituent makes an undissociable bonding with the limb bone or c a r t i l a g e . The presence of AP i n the epidermis seems to vary with the spec-ies used. Shah and Chakko (1967) f e l t that where i t i s present i t functions i n the formation of fibrous proteins and i n the passage of metabolites across the c e l l membrane. Of importance to t h i s study i s the hypothesis that t h i s enzyme plays a r o l e i n the processes of d i f f e r e n t i a t i o n (Moog, 1944, 1952; Karzmer and Berg, 1951). The hypothesis i s a product of evidence, at once p l e n t i f u l and circumstan-t i a l , that AP i s i n v a r i a b l y associated with tissues during the early stages of t h e i r d i f f e r e n t i a t i o n , but decreases i n amount or i s l o s t e n t i r e l y on morphological or functional d i f f e r e n t i a t i o n of these tissues. This idea would appear to have relevance i n explaining the increase and subsequent de-crease i n l e v e l s of phosphatases i n the hypophysis, the -45-various regions of the brain, the transverse duodenum, the heart, and the areas of the limb not involved i n chondrogen-e s i s . I t also could explain the precocious AP increase i n tissues where i t plays a functional r o l e i n the mature t i s -sue, such as transport i n the kidney (Piatka and Gibley, 1967; Fortak _et a_l, 1962). I t i s evident, i n the present inv e s t i g a t i o n , that AP does not show an increase and subse-quent decrease i n a l l tissues. In c e r t a i n tissues, notably, the ectodermal sense placodes, AP shows no increasing a c t i v -i t y p r i o r to morphological d i f f e r e n t i a t i o n . Indeed, many of these tissues possess no histochemically detectable amounts of AP so that the suggestion of Moog (1944 and 1952) should be modified. AP seems to be involved i n c e r t a i n biochemical events which precede, and might be e s s e n t i a l for, the d i f f e r -e n t i a t i o n process i n some but not a l l tissues. The cautious-ness of such a statement i s necessitated by the absence of any experimental data on i n h i b i t i o n of these enzymes i n dev-eloping systems. I t seems from the present work, that such data may not be immediately forthcoming, considering the nature and quantity of the i n h i b i t o r s necessary for exten-sive i n h i b i t o n of AP a c t i v i t y . In view of the postulated r o l e for AP i n development, taken with the increasing i n t e r e s t i n multimolecular forms of enzymes, i t . i s i n t e r e s t i n g that no attempt has been made to determine i f the "differentiation-phosphatases" are unique forms of AP, or whether t h e i r importance l i e s i n a type of dose-response mechanism as postulated by Karzmer and Berg (1952) and c a l l e d a "phosphatase-rich t r a n s i t i o n phase". -46-The a c t i v i t y r a t i o data revealed, for both species, two peaks of PNPP-preference or high r a t i o . I t should be under-stood that, as used i n t h i s context, "PNPP-prefering" and "BGP-prefering" enzymes are r e l a t i v e terms which serve only to indicate changes i n substrate preference, since the enzymes have not been p u r i f i e d . The f i r s t peak occurred during the period of f i r s t autonomous muscle movements and the second occurred j u s t p r i o r to the free-swimming stage. Up to about stage 31 (A.g.) or 32 (T.t.), very l i t t l e h i s t o l o g i c a l d i f f e r -e n t i a t i o n i s obvious, the neurectoderm of both species shows some d i f f e r e n t i a t i o n of the various major b r a i n regions and most of these possess a low l e v e l of AP a c t i v i t y . This observation suggests that most phosphatases present at t h i s time are those involved i n the biochemical processes of d i f f e r e n t i a t i o n and thus, these enzymes can now be considered as PNPP-prefering enzymes. After stages 31 and 32, when a decrease i n r a t i o i s noted, the pronephric tubules are i n -volved i n t h e i r histogenesis which has associated with i t an enzymic d i f f e r e n t i a t i o n of AP revealed as an increase i n a c t i v i t y . Since the tubules are histochemically the most reactive tissues at t h i s stage and are r e l a t i v e l y extensive, they are most l i k e l y the source of the AP enzymes with the BGP-preference. By stages 35 (A.g.) and 36 (T.t.) numerous tissues are contributing to the l e v e l of AP a c t i v i t y . Since most of these w i l l show decreasing a c t i v i t y a f t e r stage 38 (A.g.) or 39 (T.t.), i n association with increasing d i f f e r -e n t i a t i o n , we assume, as did Moog (1944 and 19 52), that these enzymes are involved i n the d i f f e r e n t i a t i o n process. -47-Th e r a t i o s observed for t h i s period indicate these are PNPP-prefering enzymes. It has been observed that, a f t e r stages 38 and 39, the d i f f e r e n t i a t i o n of the alimentary t r a c t with i t s high AP l e v e l s becomes the dominant enzyme contributor. The r a t i o decrease noted at that time then must be a r e s u l t of t h i s d i f f e r e n t i a t i o n and further indicates that the "mature" or. d i f f e r e n t i a t e d enzymes are BGP-prefering enzymes. I t i s evident from the foregoing that, on the basis of the observations made i n t h i s investigation, the AP enzymes a f f i l i a t e d with the d i f f e r e n t i a t i o n process may be PNPP-pre-f e r i n g enzymes while those functioning i n the gut and kidney are considered to have a BGP-preference. This view i s sup-ported by Moog1s (1966) work on the AP of the mouse duodenum, i n which the APs of t h i s region have a BGP-preference (as compared to PNPP) at b i r t h . No other quantitative work with both substrates which would further c l a r i f y t h i s idea has come to our attention. Since the acrylamide gel electropherograms revealed two molecular forms of a l k a l i n e phosphatase that were pre-sent throughout development, i t i s cl e a r that the r o l e of AP i n d i f f e r e n t i a t i o n i s not a function of new AP molecular species. Coupling these data with the s p e c i f i c a c t i v i t y data i t i s evident that a simple developmental sequence i n which band 1 decreases as band 2 increases i s not v a l i d . What i s more l i k e l y i s that the r a t i o s of band 1 to 2 do change, but that such a change i s masked by the proximity of the bands to each other. Thus the a c t i v i t y l e v e l , as well as the r a t i o change, may be important i n the d i f f e r e n t i a t i o n -48-process. The l i t e r a t u r e on multimolecular forms of AP indicates that a general theme for the formation of new molecular forms, regardless of species or tissue source of the enzyme, ex i s t s . New molecular forms of a l k a l i n e phosphatase that appear during development can almost i n v a r i a b l y be considered as products of modification of other AP species. Moog (1966) has indicated that the duodenal AP i s synthesized i n a moder-at e l y active (mice) or in a c t i v e (chick) form which at s p e c i f i c times i n l a t e r development become activated by, i n theory, the removal of a s p e c i f i c i n h i b i t o r which then allows the conversion of one molecular form to two other more active forms. In Drosophila (Schneiderman, 1967; Schneiderman _et a l , 1966), the development from larvae to pupae has assoc-ia t e d with i t a change i n the types of AP molecules present i n t o t a l homogenates which involves, i n one case, the con-version of one molecular form to another. In t h i s instance however, the conversion involves a t a i l o r i n g of skin phos-phatases by t r y p s i n or " t r y p s i n - l i k e enzymes" a f t e r the entrapment of skin c e l l s i n the yellow body of mature larvae. Schlesinger and Anderson (1968) report that induction of AP i n cultures of E. c o l i , by lowering of the external inorgan-i c phosphate concentration, produces a high l e v e l of AP per c e l l which was due to the conversion of a slow migrating (in acrylamide gel) molecular component to a fa s t e r migra-ti n g form. Although no experimental evidence on th i s point i s a v a i l a b l e for the salamander species studied i t seems l i k e l y that AP development may include the formation of -49-multimolecular forms which, i n many instances, would occur by a modification of enzyme species previously present or, more simply, by molecular conversion reactions or molecular t a i l o r i n g . Since i n the known cases these conversions i n -volve the formation of a fas t e r migrating form i n starch or acrylamide gel electrophoresis (Schneiderman, 1967; Schles-inger and Anderson, 19 6 8 ) / i t follows that.the salamander s i t u a t i o n observed i n the present i n v e s t i g a t i o n could i n -volve the formation i n i t i a l l y of band one (1) which i s then converted to band two (2). The rate of the conversion reaction then might determine the r e l a t i v e amounts of the two enzymes (the r a t i o determining factor) and thereby play a r o l e i n d i f f e r e n t i a t i o n . -50-SUMMARY The ontogeny of a l k a l i n e phosphatase (AP) i n developing embryos of two species of Urodele amphibian, A. g r a c i l e and _T. torosa, has been studied using biochemical assays ( s p e c i f i c a c t i v i t y determinations, gel electrophoresis) and histochemical methods. AP i s present i n embryo homogenates from g a s t r u l a t i o n to free-swimming larvae, over which time i t increases 150-fold ( with PNPP) or 35-fold (with BGP) i n A. g r a c i l e and 60-fold (with PNPP) or 25-fold (with BGP) i n T. tor-osa. P l o t t i n g the s p e c i f i c a c t i v i t y data as a r a t i o of a c t i v i t y on the two d i f f e r e n t substrates (PNPP/BGP), two peaks of high r a t i o are seen during t h i s period of devel-opment. The AP l e v e l i n the homogenates was found, by acrylamide g e l electrophoresis, to be a product of two molecular forms. The h i s t o l o g i c a l development of AP was related to the biochemical data and to proposed functions of the enzyme. I t was evident that the d i f f e r e n t i a t i o n function of AP does not seem to be a product of new molecular forms. A c o r r e l a t i o n between substrate s p e c i f i c i t i e s and func-t i o n was proposed which may allow a closer scrutiny of the r o l e of AP i n the process of d i f f e r e n t i a t i o n . -51-REFERENCES Adams, E., 1962. The ontogeny of isozymes of l a c t i c dehy-drogenase i n two amphibian species. M.Sc. Thesis, Univ. of B.C. Library. Baserga, R., 1968. Biochemistry of the c e l l cycle: a review. C e l l and Tissue Kin. JL: 167-191. Berwick, L. and D.R. Coman, 1962. Some chemical factors i n c e l l u l a r adhesion and s t i c k i n e s s . Cancer Res. 22:982-986. Burstone, M.S., 1962. Enzyme Histochemistry- and i t s applic-ation i n the study of neoplasms. Academic Press, N.Y.. D a n i e l l i , J.F., 1952. St r u c t u r a l factors i n c e l l permeabil-i t y and secretion. Symp. Soc. Exp. B i o l . J5:l-15. Davis, B.J., 1964. Disc electrophoresis. II. Method and ap p l i c a t i o n to human serum proteins. Ann. N.Y. Acad. S c i . 121:404-427. 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