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The role of cyclic AMP and differentiation-inducing factor in stalk cell differentiation during the development… Sobolewski, Andre 1987

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THE ROLE OF CYCLIC AMP AND DIFFERENTIATION-INDUCING FACTOR IN STALK CELL DIFFERENTIATION DURING THE DEVELOPMENT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM. By ANDRE SOBOLEWSKI B.Sc. York University, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (B i ology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1987 © Andre Sobolewski, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) ABSTRACT The r o l e o f c y c l i c AMP a n d a d i f f e r e n t i a t i o n - i n d u e i n g f a c t o r ( D I F ) i n t h e d i f f e r e n t i a t i o n o f s t a l k c e l l s was i n v e s t i g a t e d i n t h e c e l l u l a r s l i m e mold D i c t y o s t e 1 i um  d i s c o ideum. I n t h i s o r g a n i s m , s t a r v a t i o n t r i g g e r s t h e a g g r e g a t i o n o f amoebae i n t o m u l t i c e l l u l a r masses w i t h i n w h i c h a s i m p l e , w e l l - r e g u l a t e d p a t t e r n o f p a r t i a l l y d i f f e r e n t i a t e d c e l l s i s f o r m e d and w h i c h u l t i m a t e l y f o r m f r u i t i n g b o d i e s c o m p r i s e d o f s p o r e a n d s t a l k c e l l s . I n a m o n o l a y e r s y s t e m a t low c e l l d e n s i t i e s , s t a l k c e l l f o r m a t i o n i s d e p e n d e n t on t h e p r e s e n c e o f b o t h c y c l i c AMP and D I F . B o t h f a c t o r s a c t w i t h i n a s h o r t t i m e o f e a c h o t h e r , i n d u c t i o n by c y c l i c AMP p r e c e d i n g i n d u c t i o n by D I F , b e g i n n i n g b e t w e e n 8 t o 10 h o u r s o f i n c u b a t i o n i n m o n o l a y e r s , and p r o g r e s s i v e l y c o m m i t t i n g an i n c r e a s i n g p r o p o r t i o n o f t h e c e l l s i n m o n o l a y e r t o f o r m s t a l k c e l l s . The r e l a t i v e e f f e c t i v e n e s s o f a n a l o g u e s o f c y c l i c AMP t o i n d u c e s t a l k c e l l f o r m a t i o n i n m o n o l a y e r s i n d i c a t e s t h a t t h e w e l l -c h a r a c t e r i z e d c e l l s u r f a c e c y c l i c AMP r e c e p t o r most p r o b a b l y m e d i a t e s t h e a c t i o n o f c y c l i c AMP. A l t h o u g h t h i s r e c e p t o r a p p e a r s e a r l y d u r i n g a g g r e g a t i o n , i t d o e s n o t become a c t i v a t e d u n t i l l a t e r d u r i n g d e v e l o p m e n t j j r i v i v o , p r o b a b l y b e c a u s e t h e c y c l i c AMP c o n c e n t r a t i o n s w i t h i n d e v e l o p i n g c e l l masses must b u i l d up t o l e v e l s h i g h e r t h a n t h o s e i n a g g r e g a t i o n s t r e a m s . The f i n d i n g t h a t c a f f e i n e i n h i b i t s s t a l k c e l l f o r m a t i o n i n low d e n s i t y m o n o l a y e r s and t h a t t h e . i i permeable analogue 8-Bromo-cyclic AMP can p a r t i a l l y reverse this i n h i b i t i o n suggests that a c t i v a t i o n of t h i s receptor leads to an increase in internal c y c l i c AMP levels as one of the steps in stalk c e l l d i f f e r e n t i a t i o n . The finding that the expression in low density monolayers of AP IV, a c e l l - t y p e non-specific isozyme of acid phosphatase, was c y c l i c AMP-dependent is consistent with the view that c y c l i c AMP induces non-specific postaggregative gene expression during development i n vivo. The findings that the expression of pre-stalk arid stalk c e l l s p e c i f i c antigens and of the pre-stalk c e l l s p e c i f i c isozyme AP 11 was DIF-dependent provide good evidence for the idea that both pre-stalk and stalk c e l l formation are induced by DIF. The fact that isolated pre-stalk c e l l s require DIF for stalk c e l l formation in low density monolayers further supports this idea. Whereas c e l l s independent of DIF for stalk c e l l formation in monolayers appear immediately a f t e r c y c l i c AMP-independent c e l l s during d i f f e r e n t i a t i o n in low density monolayers, DIF-independent c e l l s appear considerably later during development i n vivo. This evidence and the fact that developing c e l l masses contain elevated levels of D I F lead to the postulate that the factor(s) which triggers the formation of f r u i t i n g bodies also controls the pre-stalk to stalk c e l l conversion. i i i TABLE OF CONTENTS PAGES ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES v i i i ABBREVIATIONS xi ACKNOWLEDGEMENTS xi i i INTRODUCTION. 1 1. General features of D. disco ideum de ve 1 opment 1 2. The pre-stalk/pre-spore pattern in slugs 9 a. Morphological differences between pre-stalk and pre-spore c e l l s 10 b. Studies of biochemical markers for pre-stalk and pre-spore c e l l s 11 c. Regulation of the pre-stalk/pre-spore pattern in slugs 17 3. Factors regulating c e l l d i f f e r e n t i a t i o n : in v i tro studies 18 a. Early studies of the requirements for c e l l d i f f e r e n t i a t i o n 18 b. Investigations on the role of cycl ic AMP 19 c. Requirements for spore c e l l d i f f e r e n t i a t i o n 23 d. Requirements for stalk c e l l d i f f e r e n t i a t i o n 26 4. Aims of the thesis .27 MEDIA AND SOLUTIONS 28 MEDIA 28 BUFFER SOLUTIONS 29 i v METHODS . 35 1. Growth and j j i v i v o d i f f e r e n t i a t i o n 35 2. DIF p r e p a r a t i o n and e x t r a c t i o n . 37 3. D e t e r m i n a t i o n of DIF l e v e l s d u r i n g development j_n v i v o 38 4. S t a l k c e l l f o r m a t i o n i n monolayers 39 a. Low d e n s i t y monolayer a s s a y of DIF 39 b. F a c t o r removal and a d d i t i o n e x p e r i m e n t s i n low d e n s i t y monolayers 40 c. E x p e r i m e n t s to det e r m i n e e x p r e s s i o n of de v e l o p m e n t a l markers i n low d e n s i t y monolayers....... 40 d. High d e n s i t y monolayer e x p e r i m e n t s . . . . . . 41 5. D i s a g g r e g a t i o n e x p e r i m e n t s 42 6. I s o l a t i o n of p r e - s t a l k and p r e - s p o r e c e l l s on P e r c o l l g r a d i e n t s 43 7. P r o t e i n d e t e r m i n a t i o n 44 8. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and e l e c t r o b l o t t i n g on n i t r o c e l l u l o s e 45 9. Enzyme a s s a y s 46 a. U D P - g a l a c t o s e : m u c o p o l y s a c c h a r i d e t r a n s f e r a s e 46 b. A c i d phosphatase 47 10. Serum p r e p a r a t i o n 47 a. P r e p a r a t i o n of a r a b b i t immune serum a g a i n s t s t a l k c e l l e x t r a c t s 47 b. P r e p a r a t i o n of a r a b b i t immune serum a g a i n s t p r e - s t a l k c e l l e x t r a c t s 48 c. P r e p a r a t i o n of a mouse immune serum a g a i n s t s t a l k c e l l e x t r a c t s 49 d. A n t i b o d y p u r i f i c a t i o n 51 e. A n t i b o d y a d s o r p t i o n a g a i n s t c r o s s - r e a c t i v e a n t i g e n s 52 v 11. B i o t i n y l a t i o n protocol for reporter antibodies and enzymes 53 12. Reaction of n i t r o c e l l u l o s e blots with ant ibod ies 54 RESULTS 57 PART I CHARACTERIZATION OF STALK CELL FORMATION IN LOW DENSITY MONOLAYERS 57 A. Factor removal experiments 59 B. Factor addition experiments 66 C. Experiments with c y c l i c AMP analogues 69 D. Experiments with potential antagonists of c y c l i c AMP and DIF 73 i . Ammon ia 73 i i . Caf f e ine 78 SUMMARY OF PART I 84 PART II THE REQUIREMENTS OF CYCLIC AMP AND DIF FOR EXPRESSION OF PRE-STALK AND STALK CELL MARKERS 85 A. Acid phosphatase 85 B. Rabbit a n t i - s t a l k and anti-pre-stalk immune sera 94 C. Mouse an t i - s t a l k immune serum 100 SUMMARY OF PART II 103 PART III EXPERIMENTS WITH DISRUPTED DEVELOPING CELLS 106 A. Requirements of iri vivo developing c e l l s for stalk c e l l formation in monolayers 106 B. Studies of isolated pre-stalk and pre-spore c e l l s 109 SUMMARY OF PART III 116 DISCUSSION .117 REFERENCES 161 v i L I S T OF T A B L E S PAGES Table I The rela t i o n s h i p between the dependence of c y c l i c AMP and DIF for stalk c e l l formation in low density mono layers ... 63 Table II Table III Table IV Table V Determination of the minimum time required for the induction of stalk c e l l formation by DIF in low density mono layers 65 The r e l a t i v e a f f i n i t y of three c y c l i c AMP-binding proteins for c y c l i c AMP and c y c l i c AMP analogues 71 Effect of varying NH^Cl and DIF concentrations on stalk c e l l formation in low density monolayers 74 Requirements of pre-stalk and pre-spore c e l l s for stalk c e l l formation in low density monolayers I l l v i i L I S T OF F I G U R E S F igure 1 . F igure 2 . Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. F i gure 11. PAGES Aspects of the l i f e cycle of the c e l l u l a r slime mold disco ideum 3 DIF a c t i v i t y present in c e l l masses and lower pad solution during d i f f e r e n t i a t i o n on M i l l i p o r e f i l t e r s . . 5 8 The ef f e c t of DIF removal and c y c l i c AMP removal on stalk c e l l formation during d i f f e r e n t i a t i o n in low density monolayers 61 The effect of varying the time of c y c l i c AMP addition and i t s subsequent removal on stalk c e l l formation in low density monolayers 67 The e f f e c t of varying the time of DIF addition and i t s subsequent removal on stalk c e l l formation in low density monolayers 68 Comparison of the e f f e c t of various c y c l i c AMP analogues on stalk c e l l formation in low density monolayers The ef f e c t of c y c l i c AMP or DIF removal on stalk c e l l formation during d i f f e r e n t i a t i o n in low density monolayers at pH 72 7.5 76 NH^Cl The period of s e n s i t i v i t y to i n h i b i t i o n during d i f f e r e n t i a t i o n in low density monolayers, r e l a t i v e to the period of c y c l i c AMP requirement. The e f f e c t of caffeine in low density monolayers 77 79 The influence of calcium levels on caffeine i n h i b i t i o n of stalk c e l l formation in low density monolayers .80 The ef f e c t of 8-Bromo-cyclic AMP on the i n h i b i t i o n of stalk c e l l formation by caffeine in low density monolayers.... 82 v i i i Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. Figure 19. Figure 20. Figure 21 . Figure 22. Figure 23. Figure 24. The ef f e c t of caffeine on stalk c e l l induction by c y c l i c AMP and by DIF in low density monolayers 83 Acid phosphatase a c t i v i t y of slug c e l l s separated by density centrifugation on Percoll gradients 87 Acid phosphatase a c t i v i t y in c e l l s developing on Mi l l i p o r e f i l t e r s . 89 Acid phosphatase a c t i v i t y in extracts of c e l l s developing in monolayers at high c e l l densities 90 DIF-dependence of the expression of the pre-stalk s p e c i f i c acid phosphatase a c t i v i t y in low density mono layers .... 92 Acid phosphatase 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 in low density monolayers 93 S p e c i f i c i t y of the antigens detected by the adsorbed rabbit a n t i - s t a l k antibody preparation .96 Detection of a stalk antigen by an adsorbed rabbit a n t i - s t a l k antibody preparation during c e l l d i f f e r e n t i a t i o n in low density monolayers 97 Pre-stalk antigens detected in c e l l s developing in low density mono layers..99 Detection of developmentally regulated antigens by an adsorbed mouse ant i - s t a l k antibody preparation 101 Detection of developmentally regulated antigens by an adsorbed mouse an t i - s t a l k antibody preparation during c e l l d i f f e r e n t i a t i o n in low density monolayers 102 Expression of pre-stalk and stalk c e l l components during development in. vivo and in low density monolayers 105 The requirements of c e l l s developing i n v i vo for stalk c e l l formation in low density monolayers 107 i x Figure 25. Figure 26 Stalk c e l l formation by isolated pre-stalk and pre-spore c e l l s in response to increasing amounts of DIF 112 Kinetics of stalk induction by DIF for pre-stalk and pre-spore c e l l s in low dens i ty monolayers 11 4 Figure 27 Caffeine i n h i b i t i o n of stalk c e l l formation from isolated pre-stalk and pre-spore c e l l s in low density monolayers. . . 1 15 Figure 28 The requirements for c y c l i c in c e l l s developing in. vivo Summary of data AMP and DIF 1 17 Figure 29 C e l l d i f f e r e n t i a t i o n in D ictyoste1i um  disco ideum: a comparison between Schaap's model (1986) and a model derived from this thesis ..155 x ABBREVIATIONS AMP adenosine 5'-monophosphate BCIP 5'bromo-4'chloro-3'indoyl phosphate biotin-X-NHS biotiny1-€-aminocaproic acid N-hydroxysucc in i m ide BGG bovine gamma-globulin BHT 2,6-d i-tert-butylme thy1phenol c y c l i c AMP adenosine 3':5'-monophosphate cDNA complementary DNA DEAE diethylaminoethyl DIF di f f e r e n t i a t i o n - i n d u e i n g factor DTT d i t h i o t h r e i t o l EDTA ethylenediamine te t r a a c e t i c acid ELISA enzyme-linked immunosorbent assay FCA Freund's complete adjuvant HDMS high-density monolayer s a l t s IFA incomplete Freund's adjuvant IMP inosine 5'-monophosphate KK^ potassium phosphate (20 mM) buffer pH 7.0 LDMS low-density monolayer s a l t s LPS lower pad solution Mr apparent molecular weight MES 2-[N-morpholinolethanesulfonic acid mRNA messenger RNA Naphthol AS-MX 3-hydroxy-2-naphthoic acid 2,4-dime thy1-ani1ide x i NP-40 nonidet P-40 PBS phosphate buffered s a l t s PIF pre-spore inducing factor PMSF phenyl me t h y l s u l f o n y l f l u o r i d e SDS sodium dodecyl sulfate SPIF spore inducing factor TEMED N,N,N',N'-tetramethylethylenediamine Tr i s t r i(hydroxymethyl)am inome thane TBS t r i s buffered s a l t s TX-100 t r i t o n X-100 Tween 80 polyoxyethylene-sorb itan monooleate UDP ur i d ine-d iphosphate x i i ACKNOWLEDGEMENTS I h ave l e a r n e d a g r e a t d e a l f r o m many i n d i v i d u a l s d u r i n g t h i s w o r k , f r o m none so much as f r o m my s u p e r v i s o r Dr G e r r y Weeks. To a l l who h e l p e d me d u r i n g t h i s work I want t o e x p r e s s my most h e a r t f e l t t h a n k s . B o t h G e r r y and my p a r t n e r , J o a n n a Z i l s e l , have r e v i v e d my s p i r i t s w h i l e I was r e a d y t o g i v e up a n d t o them I a l s o want t o e x p r e s s my f u l l e s t g r a t i t u d e . L a s t l y , s p e c i a l t h a n k s go t o my s o n D a n i e l B i k o f o r e n a b l i n g me t o a p p r e c i a t e t h i s work i n i t s p r o p e r p e r s p e c t i v e . x i i i 1 INTRODUCTION A c e n t r a l t e n e t of d e v e l o p m e n t a l b i o l o g y i s t h a t the s h a p i n g of an organism and the d i s t r i b u t i o n of c e l l s i n i t s t i s s u e s are s p e c i f i e d by s i g n a l s such as d i f f u s i b l e morphogens or c e l l s u r f a c e m o l e c u l e s which are s p a t i a l l y and t e m p o r a l l y r e g u l a t e d . T h i s p r i n c i p l e was f i r s t e n u n c i a t e d n e a r l y 50 years ago by Spemann (1 9 3 8 ) , f o l l o w i n g h i s p i o n e e r i n g s t u d i e s of the i n d u c t i v e p r o p e r t i e s of mesodermal t i s s u e s d u r i n g the development of newt embryos. While t h e r e i s a l a r g e body of e v i d e n c e s u p p o r t i n g the concept of s p a t i a l l y r e g u l a t e d morphogens i n a v a r i e t y of d e v e l o p i n g o r g a n i s m s , o n l y a s m a l l number of morphogens have a c t u a l l y been i d e n t i f i e d . D i c t y o s t e l i um d i s c o ideum i s one of those organisms f o r which p r e s u m p t i v e morphogens have been c h a r a c t e r i zed. 1. G e n e r a l f e a t u r e s of D. Discoideum development The c e l l u l a r s l i m e mould D. d i s c o ideum was o r i g i n a l l y d i s c o v e r e d i n 1935 i n the d e c a y i n g l a y e r of f o r e s t s o i l s where i t feeds upon s o i l b a c t e r i a (Raper, 1935). I t belongs to the c l a s s Acras iae which c o m p r i s e s s e v e r a l s p e c i e s of amoebae a l l w i t h the a b i l i t y t o aggregate c h e m o t a c t i c a l 1 y to s p e c i f i c a t t r a c t a n t s (named a c r a s i n s ) i n t o m u l t i c e l l u l a r masses and to form f r u i t i n g b o d i e s (Bonner, 1967). 2 In the f i r s t major study of i t s development, Raper (1940) demonstrated that D. disco ideum amoebae d i f f e r e n t i a t e d into stalk and spore c e l l s in a highly organized pattern of development governed by a combination of external and internal signals. The developing c e l l aggregates exhibit two properties generally found in more complex organisms: a spe c i a l i z e d region which acts as an organizer, and the a b i l i t y to regenerate a normally proportioned f r u i t i n g body from any segment dissected from the c e l l mass (Raper, 1940). Thus, i t is hoped that insights into the development of this organism w i l l add to a general understanding of the fundamental questions in developmental biology. In the laboratory, the removal of the b a c t e r i a l food supply re s u l t s in cessation of growth and triggers entry into the d i s t i n c t program of development shown in figure 1. o When 10° washed amoebae are plated on a M i l l i p o r e f i l t e r over a pad saturated with a l i g h t l y buffered solution, this developmental process is completed in 24 hours with a high degree of synchrony (Sussman, 1966). 3 > v v f v_ ^ f\ w 1 > v ' pr«*-oggr*90trv« aggregative poct-oggregativ* (tug culmination stag* stage stage 0 - 4 4 - 8 9 - 1 2 12—1 8 18 — 24 F i g . 1. As p e c t s of the l i f e c y c l e of the c e l l u l a r s l i m e mold D i c t y o s t e 1 i um d i sco ideum. The s t a g e s of development of the c e l l u l a r s l i m e mold D i c t y o s t e 1 i um d i sco ideum are d e p i c t e d i n t h i s f i g u r e , a l o n g w i t h the times ( i n hou r s ) a t which they o c c u r a f t e r the onset of s t a r v a t i o n i n s t r a i n V12 M2. S l u g s are made of two c e l l - t y p e s ( p r e - s t a l k and pr e - s p o r e c e l l s ) o r g a n i z e d i n t o a d i s t i n c t a n t e r i o r - p o s t e r i o r p a t t e r n f i r s t v i s i b l e a t 12 hour s . S t a l k and spore c e l l d i f f e r e n t i a t i o n t a k e s p l a c e d u r i n g the c u l m i n a t i o n stage (18 to 24 h o u r s ) . 4 With the onset of starvation, a few c e l l s spontaneously begin to secrete pulses of c y c l i c AMP, the D. d i sco ideum acrasin, a t t r a c t i n g other c e l l s and entraining them to synchronously pulse c y c l i c AMP (Described in Bonner, 1967; Loomis, 1975). Chemotactic c e l l s also express adhesive c e l l surface molecules and eventually c o l l e c t into t i g h t l y bound 5 c e l l masses comprising about IO'' amoebae. At this stage, the c e l l aggregates form tip s and eventually elongate into f i n g e r - l i k e structures (the finger stage). This structure f a l l s over onto the substratum and forms a migrating pseudoplasmodium or slug, which is comprised of d i s t i n c t l y d i f f e r e n t c e l l s in i t s anterior and posterior regions. After several hours of migration the slug condenses into a tipped mound that resembles the i n i t i a l tipped aggregate and the culmination stage begins. The formation of the stalk involves a general movement of the anterior c e l l s that has been aptly described as a reverse fountain process (Farnsworth, 1973). I n i t i a l l y , some c e l l s within the t i p region enlarge and become bounded by c e l l u l o s i c f i b r i l s which forms the upper part of a nascent stalk tube. As these c e l l s migrate down through the center of the c e l l mass, extending the stalk tube, the c e l l aggregate flattens into a c h a r a c t e r i s t i c shape (the Mexican hat stage). 5 When these c e l l s reach the substratum, they vacuolate and form outer walls which fuse with the bottom of the stalk tube, while c e l l s at the base of the aggregate also vacuolate and form a disk which anchors the stalk tube to the substratum. Thereafter, anterior c e l l s continue to migrate into the elongating stalk tube to form stalk c e l l s , while the remaining c e l l s are passively l i f t e d off the substratum and d i f f e r e n t i a t e into spore c e l l s . The r e s u l t i n g mature f r u i t i n g body comprises a sorus of dormant spores supported by a 1-2 mm c y l i n d r i c a l stalk encasing thick walled, vacuolated stalk c e l l s . External factors such as humidity, l i g h t and heat orient the migration of slugs, presumably d i r e c t i n g them to environments favorable to the dispersal of spores (Raper, 1940; Bonner §_t al_. , 1950). The phototactic properties of slugs are associated with the t i p (Francis, 1964), a speci a l i z e d region of the c e l l aggregate characterized by the presence of a small subset of morphologically d i s t i n c t c e l l s (Kopachik, 1982b). Slug migration can be abolished i f c e l l s are deposited over well buffered pads or greatly prolonged i f the pads are not buffered (Newell e_t al . , 1969b). Ammonia may dictate the choice of the morphogenetic pathway since i t has been shown that high levels in c e l l masses prolong slug migration, while its removal induces culmination (Schindler and Sussman, 1977). 6 G r a f t i n g a d d i t i o n a l t i p s onto a s l u g r e s u l t s in i t s fragmentation into s e v e r a l d i m i n u t i v e , autonomous slu g s which e v e n t u a l l y formed w e l l - p r o p o r t i o n e d f r u i t i n g bodies, suggesting that t i p s behave as ' o r g a n i z e r s ' (Raper, 1940; Rubin and Robertson, 1975). These ' o r g a n i z i n g ' p r o p e r t i e s of t i p s have g r a d u a l l y been d e t a i l e d over the years. Any segment d i s s e c t e d from a s l u g can regenerate a new t i p , c l e a r l y i n d i c a t i n g that a l l s l u g c e l l s have the p o t e n t i a l to form t i p s (Raper, 1940). When an impermeable b a r r i e r was i n s e r t e d into an aggregate whose t i p had been removed, the two segments thus formed generated new t i p s and continued to develop autonomously (Farnsworth, 1973). However, removal of the b a r r i e r w i t h i n 34 minutes of i n s e r t i o n r e s u l t e d in the formation of a s i n g l e t i p , i n d i c a t i n g that the formation of a new developmental a x i s r e q u i r e s at l e a s t 34 minutes. Moreover, each new developmental a x i s r e q u i r e d the continued presence of a t i p since removal of the b a r r i e r from an aggregate that had regenerated two t i p s , f o l l o w e d by the removal of one of the two t i p s always enabled the remaining one to assume c o n t r o l over the whole aggregate (Farnsworth, 1973). 7 Transplanting slug segments onto tipped or t i p l e s s slugs and measuring the a b i l i t y of these segments to regenerate tip s revealed the existence of a gradient of t i p in h i b i t o r emanating from tip s (Durston, 1976). If slug c e l l s are used in such transplantation experiments, only pre-stalk c e l l s can form tip s when injected into host slugs, indicating that t i p s arise from these eel Is (MacWi11iams, 1982). Since large c e l l aggregates often fragment into smaller c e l l masses, each under the control of a single t i p , the formation and maintenance of tip s through th i s i n h i b i t o r gradient probably serves to control the size of c e l l masses (Kopachik, 1982a; MacWilliams and David, 1985; Wang and Schaap, 1985). Mutants have been isolated which form proportionately more stalk or spore c e l l s in f r u i t i n g bodies than wild-type strains and which have correspondingly greater or smaller tips (MacWilliams, 1982). These mutants also have altered slug morphologies, and gr a f t i n g t i p s from mutant slugs onto t i p l e s s wild-types (or vice-versa) causes the recipient slug to assume the shape of the donor, suggesting that the shape of slugs is also controlled by tip s (MacWilliams, 1984). 8 The above e x p e r i m e n t s i n d i c a t e t h a t t h e m o r p h o g e n e t i c s e q u e n c e o f t h e d e v e l o p m e n t a l p r o g r a m and t h e s i z e o f d e v e l o p i n g c e l l masses a r e u n d e r t h e c o n t r o l o f t h e t i p r e g i o n . The f o l l o w i n g s e c t i o n w i l l p r e s e n t e v i d e n c e t h a t c e l l s w i t h i n d e v e l o p i n g c e l l masses a r e p a r t i a l l y d i f f e r e n t i a t e d a n d a r e l o c a l i z e d i n s p e c i f i c r e g i o n s . The f o r m a t i o n o f d i s t i n c t c e l l - t y p e s w i l l be shown t o a r i s e a t s p e c i f i c m o r p h o l o g i c a l s t a g e s o f d e v e l o p m e n t , b u t i t i s p r e s e n t l y u n c l e a r how t h e c o n t r o l o f m o r p h o g e n e s i s and o f c y t o d i f f e r e n t i a t i o n a r e i n t e r r e l a t e d . 9 2• The p r e - s t a l k / p r e - s p o r e p a t t e r n of s l u g s Raper (1940) e s t a b l i s h e d f a t e maps f o r the c e l l s i n the m i g r a t i n g s l u g . He demonstrated t h a t c e l l s i n the a n t e r i o r q u a r t e r of s l u g s were d e s t i n e d to form the s t a l k c e l l s i n the t e r m i n a l f r u i t i n g body ( t h e y were termed p r e - s t a l k c e l l s ) w h i l e those i n the p o s t e r i o r p o r t i o n were d e s t i n e d to form the spore c e l l s ( t h e y were termed p r e - s p o r e c e l l s ) . T h i s s u g g e s t e d t h a t the c e l l p a t t e r n i n m i g r a t i n g s l u g s a n t i c i p a t e d t h a t of the t e r m i n a l f r u i t i n g body. However, the d e v e l o p m e n t a l f a t e of these p r e c u r s o r c e l l s was not f i x e d , i n t h a t fragments of s e c t i o n e d s l u g s c o u l d g e n e r a t e complete w e l l p r o p o r t i o n e d f r u i t i n g b o d i e s r e g a r d l e s s of whether they o r i g i n a t e d from the a n t e r i o r o r the p o s t e r i o r r e g i o n of s l u g s (Raper, 1940). The l a t t e r o b s e r v a t i o n a l s o i m p l i e d t h a t the p r o p o r t i o n s of s t a l k and spore c e l l s are t i g h l y r e g u l a t e d . A m i n o r i t y of c e l l s i n the extreme p o s t e r i o r r e g i o n of the s l u g which form the b a s a l d i s k i n the t e r m i n a l f r u i t i n g body were a l s o n o t e d (Raper, 1940) but these c e l l s have l a r g e l y been i g n o r e d i n subsequent r e s e a r c h . 10 a. Morphological differences between pre-stalk and pre-spore c e l l s Bonner o r i g i n a l l y demonstrated t h a t p r e - s t a l k c e l l s are d i s t i n c t l y l a r g e r than p r e - s p o r e c e l l s (Bonner, 1944; Bonner et a l • , 1955), and i t has been suggested t h a t t h i s d i f f e r e n c e a r i s e s from the s h r i n k a g e of p r e - s p o r e c e l l s by the r e l e a s e of water (Schaap ejt al . . , 1982). Bonner a l s o showed t h a t i f a s u s p e n s i o n of amoebae was s t a i n e d w i t h a v i t a l dye such as n e u t r a l r e d , o n l y the a n t e r i o r p r e - s t a l k zone remained s t r o n g l y s t a i n e d i n the m i g r a t i n g s l u g (Bonner, 1944, 1952). T h i s s t a i n i n g d i f f e r e n c e i s due to the r e t e n t i o n of a c i d i c a u t o p h a g i c v a c u o l e s i n p r e - s t a l k c e l l s (Yamamoto and T a k e u c h i , 1983) and i t has become a s t a n d a r d c r i t e r i o n to d i s t i n g u i s h p r e - s t a l k from p r e - s p o r e c e l l s . While p r e - s t a l k c e l l s are v i r t u a l l y i n d i s t i n g u i s h a b l e m o r p h o l o g i c a l l y from a g g r e g a t i n g c e l l s , p r e - s p o r e c e l l s p o s s e s s unique v e s i c l e s c o n t a i n i n g c h a r a c t e r i s t i c g r a n u l a r m a t e r i a l (Maeda and T a k e u c h i , 1969). These p r e - s p o r e v e s i c l e s appear to o r i g i n a t e from the G o l g i a p p a r a t u s (Oyama e t a l . , 1984). S t u d i e s which examined t h e i r f a t e d u r i n g development suggested t h a t these v e s i c l e s fuse w i t h plasma membranes d u r i n g spore f o r m a t i o n and t h a t t h e i r c o n t e n t s become i n c o r p o r a t e d i n t o the spore c e l l w a l l ( H o l h and Hamamoto, 1969). 11 b. Studies of biochemical markers for pre-spore and pre-s t a l k c e l l s The a p p e a r a n c e and s u b s e q u e n t d i s p o s i t i o n o f t h e p r e -s p o r e v a c u o l e s was c o n f i r m e d w i t h a s p o r e a n t i s e r u m d e v e l o p e d by i n j e c t i n g a r a b b i t w i t h s p o r e s f r o m t h e s p e c i e s D i c t y o s t e 1 i um mucoro i d e s a n d a d s o r b i n g t h e r e s u l t i n g immune serum w i t h v e g e t a t i v e c e l l e x t r a c t s ( T a k e u c h i , 1 9 6 3 ) . The a n t i g e n d e t e c t e d by t h i s s erum i n D. d i s c o ideum was l o c a l i z e d w i t h i n t h e p r e - s p o r e v e s i c l e s and l a t e r a p p e a r e d on t h e s u r f a c e o f t h e s p o r e c e l l s , f u r t h e r i n d i c a t i n g t h a t t h e m a t e r i a l i n c o r p o r a t e d i n t h e s p o r e c o a t o r i g i n a t e d f r o m t h e s e v e s i c l e s ( I k e d a a n d T a k e u c h i , 1971; T a k e u c h i , 1 9 7 2 ) . T h i s i s t h e s t r o n g e s t e v i d e n c e t h a t t h e p r e s u m p t i v e f a t e o f p r e - s p o r e c e l l s i s t o d i f f e r e n t i a t e i n t o s p o r e s . By c o n j u g a t i n g f l u o r e s c e i n o n t o t h e s p o r e a n t i s e r u m and m e a s u r i n g f l u o r e s c e n c e , T a k e u c h i a n d c o - w o r k e r s were a b l e t o f o l l o w t h e a p p e a r a n c e o f t h i s a n t i g e n d u r i n g d e v e l o p m e n t and t o d e t e r m i n e q u a n t i t a t i v e l y t h e p r o p o r t i o n o f c e l l s i n t h e p o p u l a t i o n e x p r e s s i n g i t . A few s t a i n e d c e l l s f i r s t a p p e a r e d d u r i n g t i p f o r m a t i o n a n d t h e i r numbers i n c r e a s e d r a p i d l y t o a maximum l e v e l o f 7 0 - 8 0 % o f t h e p o p u l a t i o n by t h e f i n g e r s t a g e a n d r e m a i n e d c o n s t a n t t h e r e a f t e r ( H a y a s h i a n d T a k e u c h i , 1976; Forman and G a r r o d , 1 9 7 7 a ) . C e l l s e x p r e s s i n g t h i s a n t i g e n f i r s t a p p e a r e d i n r a n d o m l y d i s t r i b u t e d c l u s t e r s w i t h i n a g g r e g a t e s a n d s u b s e q u e n t l y s o r t e d i n t o t h e p o s t e r i o r region at the finger stage (Takeuchi e_t al_., 1978). These data suggest that pre-spore c e l l d i f f e r e n t i a t i o n begins in tipped aggregates, and this is followed by c e l l sorting and regulation of c e l l proportions at the finger stage. Analysis of the developmental regulation of several pre-spore s p e c i f i c proteins has supported the view that d i f f e r e n t i a t i o n of pre-spore c e l l s is i n i t i a t e d at the tipped aggregate stage. The enzyme UDP-galactosy1: mucopolysaccharide transferase is s p e c i f i c for pre-spore c e l l s (Newell e_t al.. , 1969a) and although i t was o r i g i n a l l y reported to be f i r s t produced at the finger stage (Sussman and Osborn, 1964), subsequent investigations using more standard conditions for d i f f e r e n t i a t i o n consistently demonstrated that the enzyme f i r s t appeared during t i p formation (Sussman and Lovgren, 1965; Ashworth and Sussman, 1967). The three spore coat proteins SP60, SP70 and SP96 were f i r s t detected in tipped aggregates within the nascent pre-spore vesicles (Orlowski and Loomis, 1979; Devine e_t al . , 1982; Devine et al_. , 1983) and the mRNA for SP96 has been shown to precede the appearance of the protein by 1-2 hours (Dowds and Loomis, 1984; Morrissey e_t al.. , 1984). In study designed to characterize the early moments of pre-spore d i f f e r e n t i a t i o n , K r e f f t et al_. ( 1985) found that c e l l expressing a pre-spore s p e c i f i c surface antigen had s e l e c t i v e l y lost an antigen expressed in vegetative, aggregating and pre-stalk c e l l s . 13 At no time was there a sub-population of c e l l s expressing both antigens simultaneously. C e l l s expressing the pre-spore antigen were f i r s t detected in tipped aggregates, indicating that pre-spore c e l l s appear as a c l e a r l y d i s t i n c t cell-type at that stage of development. Most cloned cDNAs encoding mRNA sequences highly enriched in pre-spore c e l l s are f i r s t detected immediately before or during t i p formation (Mehdy ejb al.. , 1983; Barklis and Lodish, 1983; Chisholm et a l . , 1984). Clone D18 isolated by Chisholm et al_. was uncharacteristic in that i t was f i r s t expressed during aggregation and subsequently accumulated in pre-spore c e l l s . The above evidence generally supports that obtained with other pre-spore components, and indicates that overt pre-spore c e l l d i f f e r e n t i a t i o n begins shortly before or at the tipped aggregate stage. Although several enzyme a c t i v i t i e s such as the c y c l i c AMP dependent phosphodiesterase, 5* nucleotidase and trehalase are higher in pre-stalk c e l l s r e l a t i v e to pre-spore c e l l s (Reviewed by Rutherford ejb al.. , 1985), few components that are unique to pre-stalk have been described. One such component is an acid phosphatase isozyme which Oohata and Takeuchi (1977) detected in isolated pre-stalk c e l l s but which was absent from isolated pre-spore c e l l s . It was f i r s t detectable at the finger stage and remained expressed throughout development (Oohata, 1983). 14 Another component recognized by a monoclonal antibody was detectable in c e l l s of the anterior region of slugs (Tasaka ejt al_. , 1983). C e l l s stained with this antibody were f i r s t detectable early during aggregation and they increased to a maximum of 80% of the c e l l population p r i o r to t i p formation. However, by the finger stage the number of stained c e l l s had decreased to about 20% of the population and this antigen remained expressed in pre-stalk and stalk c e l l s (Tasaka et al.. , 1983). Three c e l l surface proteins recognized by di f f e r e n t antibody preparations have comparable patterns of expression, being f i r s t detected in vegetative or early aggregating c e l l s and subsequently becoming r e s t r i c t e d to pre-stalk c e l l s (Krefft e_t a_L., 1985; Barclay and Smith, 1986; Chadwick, 1986). These data suggest that some components of pre-stalk c e l l are expressed early in development but are subsequently lost in pre-spore c e l l s . Using cDNA probes, a number of pre-stalk mRNAs have been detected which f a l l mainly into two classes: early mRNAs which are f i r s t synthesized during aggregation and late mRNAs which are f i r s t synthesized at t i p formation (Mehdy e_t al.., 1983; Barklis and Lodish, 1983; Chisholm et a l . , 1984). Other mRNA sequences such as the acti n 4 mRNA are present in vegetative and aggregating c e l l s , and are also later detectable only in pre-stalk c e l l s (Tsang e_t a I ., 1982). 15 The above data again indicate that pre-stalk c e l l s share several constituents with aggregating c e l l s and that they might arise during aggregation. However, i t has not been c l e a r l y established that the expression of these early pre-stalk mRNAs is r e s t r i c t e d to a small subset of aggregating c e l l s or whether they accumulate in a l l c e l l s p r ior to any overt d i f f e r e n t i a t i o n and then become s e l e c t i v e l y degraded in pre-spore c e l l s . Several studies have u t i l i z e d two-dimensional gel electrophoresis to characterize the timing of the synthesis of individual proteins during d i f f e r e n t i a t i o n (Alton and Brenner, 1979; Morrissey et al_. , 1981; Ratner and Borth, 1983; Morrissey et al_., 1984; Carde 11 i e_t al_., 1985). In a l l these studies, there were very few pre-stalk or pre-spore s p e c i f i c proteins exclusively r e s t r i c t e d to the slug stage (less than 1% of the total number detected). Morrissey et a l . (1984) found that 16 out of 17 pre-spore proteins began accumulating during or s l i g h t l y a f t e r t i p formation and that most of these were present in mature spores. S i m i l a r l y , ten of the twelve pre-spore proteins i d e n t i f i e d by Ratner and Borth (1983) were undetectable or synthesized at very low levels before t i p formation, but they were synthesized at a high rate by the f i r s t finger stage and subsequently appeared in mature spores. 16 Five pre-stalk s p e c i f i c proteins and three proteins enriched in pre-stalk c e l l s were detected during aggregation, while only one or two appeared later at t i p formation (Borth and Ratner, 1983; Morrissey, 1984). None of these pre-stalk proteins were detectable in stalk c e l l s . In fact, every study concluded that most stalk c e l l proteins were synthesized during culmination when stalk c e l l d i f f e r e n t i a t i o n takes place (Borth and Ratner, 1983; Morrissey, 1984; Kopach i k e_t al_., 1985). Several conclusions can be drawn from these studies. Most pre-spore s p e c i f i c proteins are f i r s t detected at t i p formation while pre-spore mRNAs are detected at or s l i g h t l y before t i p formation, suggesting that pre-spore c e l l induction occurs at or s l i g h t l y before this stage. Many of these components continue to be synthesized during culmination and accumulate in mature spores. In contrast, many pre-stalk s p e c i f i c components are f i r s t synthesized during aggregation but are not synthesized during culmination and are not detected in stalk c e l l s . The synthesis of stalk c e l l components is r e s t r i c t e d predominantly to the period of stalk c e l l formation during the culmination stage. Thus, while pre-spore, spore and stalk c e l l s and their constituents appear at c l e a r l y defined times, the time at which pre-stalk c e l l s appear s t i l l remains poorly defined and a controversial subject (Gomer e_t al . , 1986; Williams et al.. , 1987). c. Regulation of the pre-stalk/pre-spore pattern in slugs The f i n a l stalk:spore r a t i o can be altered in response to changes in temperature during development up to the culmination stage (Farnsworth, 1975). Moreover, i t has been found that the proportions of pre-stalk and pre-spore c e l l s are e s s e n t i a l l y invariant in slugs that d i f f e r in size by over three orders of magnitude (Wi11iams et a l . , 1981), suggesting that the a b i l i t y to regulate proportions is a property of pre-stalk and pre-spore c e l l s . Tasaka and Takeuchi (1983) demonstrated that prolonged migration of slugs resulted in the loss of the pre-spore c e l l s located next to the substratum but that the o r i g i n a l proportion of pre-spore c e l l s was restored within 1-2 hours of the induction of culmination, suggesting that regulation of the pre-stalk/pre-spore r a t i o can also occur shortly a f t e r the induction of culmination. The existence of mutants which exhibit a normal pre-stalk/pre-spore pattern but which form either only stalks or only spores as t h e i r terminal structures supports this view (Morrissey et al.. , 1981). S i g n i f i c a n t l y , pre-spore c e l l s were intermixed with pre-stalk c e l l s during regulation in early culminates, indicating that the c e l l proportioning is not a position dependent process (Tasaka and Takeuchi, 1983). 18 3. Factors regulating c e l l d i f f e r e n t i a t i o n ; In v i t r o studies One might expect an organism as simple as Dictyostelium  d i sco ideum to have a r e l a t i v e l y small number of elements c o n t r o l l i n g the establishment and regulation of the pre-stalk /pre-spore pattern during development. Several in. v i tro systems have enabled investigators to identi f y some of the factors c o n t r o l l i n g development and these findings, along with supportive evidence from studies of developmentally deranged mutants, w i l l be summarized below. a. Early studies of the requ irements for c e l l d i f f e r e n t i a t i o n Investigations c a r r i e d out in the early 70's in Sussman's lab indicated that disruption of developing aggregates arrested c e l l d i f f e r e n t i a t i o n and disrupted the accumulation of developmentally regulated enzymes (Summarized in Sussman and Newell, 1972). However, when disaggregated c e l l s were replated on M i l l i p o r e f i l t e r s at their o r i g i n a l density they promptly resumed development and resynthesized the regulated enzymes. These studies and others (Gregg, 1971; Takeuchi and Sakai, 1971; Okamoto and Takeuchi, 1976) suggested that close c e l l - c e l l interactions such as c e l l contacts might be necessary for development to proceed. 19 A requirement for c y c l i c AMP for c e l l d i f f e r e n t i a t i o n as well as for the aggregation process was suggested by Bonner's observation that stalk c e l l formation could be induced by p l a t i n g starved, unaggregated amoebae on non-nutrient agar containing 1 mM c y c l i c AMP (Bonner, 1970). The induced c e l l s were judged to be stalk c e l l s by t h e i r very prominent vacuoles and thick c e l l walls staining p o s i t i v e l y with a c e l l u l o s e - s p e c i f i c indicator. Ce l l d i f f e r e n t i a t i o n was s t r i c t l y dependent on c y c l i c AMP and was found to respond to c e l l density. These two studies suggested that both soluble factors and c e l l - c e l l contact might control de ve1opment. b. Investigations on the role of c y c l i c AMP The role of c y c l i c AMP in a c t i v a t i n g gene expression during aggregation has been well documented (Reviewed by Williams §_t al ., 1986) and w i l l not be presented here. Bonner's o r i g i n a l report that c y c l i c AMP induces stalk c e l l formation at high c e l l density was l a t e r confirmed and extended by Town et al_. ( 1976) who presented evidence that the dependence on c e l l density resulted from the requirement for an additional low molecular weight factor. In the presence of this factor and c y c l i c AMP, c e l l s at low densities r e a d i l y formed stalk c e l l s , suggesting that c e l l contact was not essential for stalk c e l l d i f f e r e n t i a t i o n . 20 A mutant was isolated which could form both spore and stalk c e l l s when plated on agar in the presence of c y c l i c AMP, suggesting that c y c l i c AMP might also be required for spore c e l l d i f f e r e n t i a t i o n (Town e_t al.. , 1976). Although wild-type c e l l s did not d i f f e r e n t i a t e into spore c e l l s on agar, they formed pre-spore vacuoles in high density monolayers in the presence of c y c l i c AMP. These results suggested that c y c l i c AMP was essential for both stalk and spore c e l l d i f f e r e n t i a t i o n and was therefore pathway indifferent (Kay et a l . , 1978). Town and Gross (1978) also demonstrated that the presence of 1 mM c y c l i c AMP induced the expression of two postaggregative enzymes in c e l l suspensions shaken at high speed to prevent the formation of c e l l contacts. They proposed that c y c l i c AMP was required for postaggregative gene expression during normal development. It was subsequently demonstrated that the expression of pre-stalk c e l l s p e c i f i c mRNAs was induced by c y c l i c AMP in shaken suspensions (Mehdy e_t al.. , 1983), but expression of some pre-spore c e l l s p e c i f i c mRNAs required the formation of c e l l contacts, and i t was argued that c e l l - c e l l contacts were a necessary requirement for the expression of pre-spore components (Barklis and Lodish, 1983; Mehdy ejt al.. , 1983; Chisholm e_t al_. , 1984). 21 Chisholm et al_. ( 1984) described a pre-stalk c e l l mRNA which was expressed in shaken suspension in the absence of c y c l i c AMP and a pre-spore c e l l mRNA which only required c y c l i c AMP. They suggested that there were three requirements for expression of cell-type s p e c i f i c genes: class I pre-stalk mRNAs were induced by starvation and were expressed during aggregation, class II pre-stalk mRNAs and class I pre-spore mRNAs required c y c l i c AMP and were expressed late in aggregation and class II pre-spore mRNAs, which they argued represented the majority of pre-spore mRNAs, were induced by s p e c i f i c c e l l contacts in c e l l aggregates. While the above studies did not suggest that c y c l i c AMP was responsible for cel l - t y p e s p e c i f i c d i f f e r e n t i a t i o n they c l e a r l y confirmed a requirement for c y c l i c AMP for postaggregative gene expression. A c o r r e l a t i o n was observed between the induction of aggregation stage components by exogenous c y c l i c AMP and an increased i n t r a c e l l u l a r c y c l i c AMP concentration (Sampson et a l . , 1978) suggesting that exogenous c y c l i c AMP might activate gene expression by elevating c e l l u l a r c y c l i c AMP pools. Moreover, i t has been demonstrated that during normal development the total ( i n t r a c e l l u l a r and e x t r a c e l l u l a r ) levels of c y c l i c AMP r i s e with the formation of c e l l aggregates (Pahl ic and Rutherford, 1979; Merkle e_t al . , 1984). Although there is as yet no direc t evidence to support this hypothesis, developmentally regulated internal 22 c y c l i c AMP receptors such as c y c l i c AMP-dependent protein kinases (Sampson, 1977; L e i c h t l i n g et al_. , 1982; Rutherford et al . , 1982; de Gunzburg and Vernon, 1982) and other i n t r a c e l l u l a r c y c l i c AMP-binding proteins (Tsang and Tasaka, 1986) have been described that could mediate the eff e c t s of elevated internal c y c l i c AMP l e v e l s . Studies from Lodish's lab have also suggested a role for c y c l i c AMP in the s t a b i l i z a t i o n of postaggregative mRNAs (Reviewed by Lodish e_t al_., 1982). Messenger RNAs in both growing and developing c e l l s were found to have h a l f - l i v e s of 4 hours (Margolskee §_t al.. , 1980; Mang iarot t i e_t al . , 1981) but disruption of c e l l aggregates s p e c i f i c a l l y reduced the h a l f - l i f e of postaggregative mRNAs to 30 minutes and led to a rapid loss of this mRNA in c e l l s maintained in suspension as single c e l l s (Landfear and Lodish, 1980; Chung et al . , 1981). However, the mRNA levels could be maintained by adding c y c l i c AMP to the suspension. These results suggested that c y c l i c AMP was necessary for both induction and maintenance of many developmentally regulated mRNAs (Barklis and Lodish, 1983; Mehdy §_t al.. , 1983; Chisholm e_t a l . , 1984). 23 c. Requirements for spore c e l l d i f f e r e n t i a t i o n It has been argued that c e l l - c e l l contacts are necessary for spore c e l l d i f f e r e n t i a t i o n since wild-type c e l l s have not been observed to form spore c e l l s without the maintenance of c e l l contacts (Sussman, 1982; Wilkinson e_t a l . , 1985). Spore c e l l s are formed when washed amoebae or disaggregated slug c e l l s are maintained in suspension in slowly rotating r o l l e r tubes (Gerisch, 1968; Sternfeld and Bonner, 1977; Take uch i et al_. , 1977; Forman and Garrod, 1977b). Pre-stalk and pre-spore c e l l s i n i t i a l l y appear uniformly d i s t r i b u t e d within the large amorphous aggregates formed under these conditions and they subsequently sort themselves into separate regions where they eventually d i f f e r e n t i a t e into stalk and spore c e l l s (Sternfeld and Bonner, 1977; Tasaka and Takeuchi, 1979, 1981). In addition to ce11-ce11 contacts, some reports have indicated a requirement for c y c l i c AMP and a heat-stable, low molecular weight component present in conditioned medium (ie medium that previously contained c e l l s ) for spore c e l l d i f f e r e n t i a t i o n (Wilcox and Sussman, 1978; Sternfeld and David, 1979; Ishida, 1982; Wilkinson et al.. , 1985). Ammonia enhances spore c e l l formation although its presence is not absolutely essential (Wilcox and Sussman, 1978; Sternfeld and David, 1979; Gross e_t a l . , 1983; Weeks, 1984). 24 Kay (1982) and Weeks (1984) reported that pre-spore c e l l formation could occur in monolayers without c e l l contact in the presence of both c y c l i c AMP and conditioned medium, and Mehdy and F i r t e l (1985) reported that expression of a pre-spore mRNA did not require c e l l contact so long as c y c l i c AMP and conditioned medium factor(s) were present. Okamoto (1981) o r i g i n a l l y described a shaken suspension system in which pre-spore c e l l formation required c y c l i c AMP and the prior formation of c e l l aggregates, but a later report implied that formation of c e l l contacts per se was not required (Okamoto, 1985). Most recently, Kumagai and Okamoto (1986) characterized a low molecular weight factor present in conditioned media which, together with c y c l i c AMP, was necessary for pre-spore c e l l formation in low density monolayer. Taken together, these studies indicate that c y c l i c AMP and a low molecular weight factor in conditioned media are necessary for the formation of pre-spore c e l l s , and they do not support the claim that c e l l contacts are essential for this process (Lodish e_t al . , 1982; Chisholm et al_., 1984). The requirements for pre-spore to spore conversion and the possible role of c e l l contacts in this process remain unclear. A study by Wilkinson et al_. ( 1985) showed that spore coat proteins that are expressed late in development required a low molecular weight factor and the maintenance of c e l l contact for expression in. v i tro, indicating perhaps 25 that the terminal step in spore d i f f e r e n t i a t i o n requires both c e l l contacts and a soluble factor. However, mutants with defective contact s i t e A, a c e l l surface molecule considered to mediate c e l l - c e l l contacts in c e l l aggregates, were able to form spores during development (Murray et al_. , 1985). Another mutant with a thermosensitive defect in a second adhesive system expressed l a t e r in development forms spore and stalk c e l l s as well as the parental s t r a i n (Wilcox and Sussman, 1981) and some sporogenous mutants form spore c e l l s in monolayers in the absence of c e l l contact (Kay, 1982; Weeks, 1984). Thus, although there is circumstantial evidence implicating c e l l - c e l l contact in the process of spore formation, dir e c t evidence supporting this idea is s t i l l wanting. 26 d. Requ irements for s t a l k c e l l d i f f e r e n t i a t i o n Using the wild-type isolate V12 M2, which formed stalk c e l l s more e f f i c i e n t l y than the more commonly used wild-type s t r a i n NC-4, Town e_t al... ( 1976) demonstrated that stalk c e l l formation in an .in. v i tro monolayer system only required c y c l i c AMP and a low molecular weight factor that has since been termed DIF ( D i f f e r e n t i a t i o n Inducing Factor). DIF has been characterized as a non-polar l i p i d i c molecule based on it s s o l u b i l i t y in organic solvents and i t s chromatographic properties (Gross e_t al.. , 1981; Kay et al.. , 1983). DIF begins to accumulate at the tipped aggregate stage of development with approximately the same k i n e t i c s as the pre-spore s p e c i f i c enzyme UDP-galactosyl:mucopolysaccharide transferase (Brookman et al_. , 1982). It was proposed that DIF was the pre-stalk c e l l inducer (Gross e_t al.. , 1981), an idea strengthened by the i s o l a t i o n of a mutant that was unable to produce DIF and required the addition of exogenous DIF to form pre-stalk and stalk c e l l s (Kopachik et. al.. , 1983). Since DIF also inhibited formation of pre-spore c e l l s and ammonia promoted spore formation and inhibited stalk c e l l formation in monolayers, Gross et. al_. (1981) proposed that DIF acted as the activ a t o r and ammonia the i n h i b i t o r of pre-stalk c e l l formation in an a c t i v a t o r - i n h i b i t o r model of pattern regulation of the kind proposed by Gierer and Meinhardt ( 1972).-However, the requirements for stalk c e l l formation during culmination have remained undefined. 27 4. Aims of the t h e s i s Although the data on the DIF requiring mutant were consistent with the idea that DIF was the inducer of pre-stalk formation, there were no data from wild-type c e l l s to confirm t h i s hypothesis, and the p o s s i b i l i t y that DIF might mediate the conversion of pre-stalk to stalk c e l l could not be. ruled out. One of the goals of t h i s thesis has been to. es t a b l i s h whether pre-stalk formation or pre-stalk to stalk c e l l conversion is the DIF-dependent step, using low density monolayers as the experimental system. To t h i s end, the requirements for c y c l i c AMP and DIF have been characterized for stalk c e l l formation in monolayers. Subsequently, attempts were made to relate the process of d i f f e r e n t i a t i o n in low density monolayers to jn vivo d i f f e r e n t i a t i o n . To achieve these goals, components unique to pre-stalk and stalk c e l l s that could be detected in low density monolayers were i d e n t i f i e d and the requirements for t h e i r expression were assessed. 28 MEDIA AND S O L U T I O N S MEDIA 1. SM agar Nutrient medium for vegetative c e l l growth Dextrose 10 g Bac t e r i o l o g i c a l peptone 10 g Yeast extract 1.0 g MgSOA.7H20 1.0 g KH2POz, 1.0 g Na2HPOA.7H20 1.5 g Agar 20 g Water 1.0 l i t e r 2. Non-nutrient agar Medium for c e l l d i f f e r e n t i a t i o n NaCl 30.0 mg KCI 37.5 mg CaCl 2 15.0 mg Agar 20 g Water 1.0 l i t e r 29 B U F F E R S O L U T I O N S 1. Bonner's s a l t s Final Concentration NaCl 0.60 g 1 0 mM KCI 0 . 7 5 g 1 0 mM CaCl2 0.30 g 2 . 0 mM Water 1 . 0 l i t e r This solution was s t e r i l i z e d by autoclaving. 2. Phosphate buffered saline (PBS) NaCl 8.00 g KCI 0 . 2 0 g K H 2 P O 4 0 . 2 0 g N a 2 H P 0 4 . 7 H 2 0 2.17 g Water 1 . 0 l i t e r This gave a solution containing about 150 mM NaCl, 2 . 7 mM KCI and 1 0 mM phosphate at pH 6.0. When indicated, NaN3 was included at 0 . 2 % (w/v), and Tween 80 at 0.05% (v/v). 3. Tris-Buffered Salts (TBS) Tr i s 6.1 g 50 mM NaCl 8.8 g 150 mM Water 1.0 l i t e r The pH of thi s solution was adjusted to 7 . 0 with 12 M HCI. 30 4. Potassium phosphate buffer (KK 2 buffer) K2HPOA 0.45 g KH2POA 2.4 g Water 1.0 l i t e r This gave a solution containing 20 mM phosphate at pH 6.0 and i t was s t e r i l i z e d by autoclaving. 5. Lower pad solution (LPS) Final Concentrat i on KCI 1 .5 g 20 mM MgCl 2.6H 20 0.50 g 2.5 mM Streptomycin sulfate 0.50 g 500 pg/ml Phosphate buffer 50 mM, pH 6.5 1.0 l i t e r This solution was s t e r i l i z e d and stored at -20°C. 6. Low Density Monolayer Salts (LDMS) MES 0.50 M, pH 6.2 1.0 ml Streptomycin su l f a t e , 10 mg/ml 1.0 ml Bonner's s a l t s 100 ml When indicated, 0.50 M T r i s - C l , pH 7.5 was substituted for 0.50 M MES, pH 6.2. 5 mM 100 pg/ml 7. High Density Monolayer Salts (HDMS) NaCl 2.0 M KCI 2.0 M CaCl 2 0.10 M Prepared by adding 1.0 ml of a concentrated s t e r i l e stock solution. MES 0.50 M, pH 6.2 2.0 ml Streptomycin sulfate, 10 mg/ml 2.0 ml Tetracycline, 7.5 mg/ml 200 pi 20 mM 20 mM 1 .0 mM 10 mM 200 pg/ml 15 pg/ml S t e r i l e water, to bring the f i n a l volume to 100 ml 8. S o l u t i o n s f o r SDS-polyacrvlamide g e l  e l e c t r o p h o r e s i s (a) 30% A c r y l a m i d e / b i s - a c r y l a m i d e stock s o l u t i o n Acrylamide 29.2 g B i s - a c r y l a m i d e 0.80 g Water, to b r i n g the f i n a l volume to 100 ml. The s o l u t i o n was f i l t e r e d through g l a s s wool and s t o r e d at 4°C. (b) Running g e l F i n a l C o n c e n t r a t i o n A c r y l a m i d e / b i s - a c r y l a m i d e 0.90 ml 6. 8% SDS, 10% (w/v) 40 p i 0. 10% T r i s - C l 1.5 M, pH 8.8 1. 1 ml 0. 42 M TEMED 15 p i 0. 38% Ammonium p e r s u l f a t e , 10% <w/v) 15 p i 0. 038% Water 1.9 ml S t a c k i n g g e l Ac r y l a m i d e / b i s - a c r y l a m i d e 0.33 ml 3. 0% T r i s - C l 0.50 M, pH 6.8 0.46 ml 69 mM SDS, 10% (w/v) 33 p i 0. 10% TEMED 2 p i 0. 06% Ammonium p e r s u l f a t e , 10% (w/v) 5 Ml 0. 015% Water 2.5 ml 32 (d) Running b u f f e r F i n a l C o n c e n t r a t i o n G l y c i n e 2.16 g 192 mM T r i s 0.45 g 25 mM SDS, 10% (w/v) 0.15 g 0.10% Water 150 ml (e) Sample b u f f e r T r i s - C l 0.50 M, pH 6.8 1.0 ml 0.10 M fl-mercaptoethanol 100 p i 2.0% SDS, 10% (w/v) 1.0 ml 2.0% G l y c e r o l 2.0 ml 40% Water, to b r i n g the f i n a l volume up t o 5.0 ml. 9 • S o l u t i o n s f o r n o n - d e n a t u r i n g p o l y a c r y l a m i d e g e l s <a> 12% A c r y l a m i d e / b i s - a c r y l a m l d e s t o c k s o l u t i o n A c r y l a m i d e 6.0 g 12% B i s - a c r y l a m i d e 0.16 g 0.32% Water, t o b r i n g the f i n a l volume t o 50 ml. T h i s s o l u t i o n was f i l t e r e d t h r o u g h g l a s s wool and s t o r e d a t 4°C. (b) Running g e l 3% A c r y l a m i d e A c r y l a m i d e / b i s - a c r y l a m i d e 0.63 ml 3.0% I m i d a z o l e 0.50 M, pH 6.8 0.50 ml 0.10 M Ammonium p e r s u l f a t e , 10% (w/v) 15 p i 0.060% TEMED 1 p i 0.04% Water 1.36 ml 33 6% A c r y l a m i d e F i n a l C o n c e n t r a t i o n A c r y l a m i d e / b i s - a c r y l a m i d e 1.25 ml 6.0% I m i d a z o l e 0.50 M, pH 7.8 0.50 ml 0.10 M Ammonium p e r s u l f a t e , 10% (w/v) 15 p i 0.060% TEMED 2 p i 0.08% Water 0.73 ml T h i s g e l c o n s i s t s of a l i n e a r g r a d i e n t from 3-6% a c r y l a m i d e , u s i n g the above s o l u t i o n s . <c) Running b u f f e r I m i d a z o l e 0.80 g 59 mM Sodium a c e t a t e 0.80 g 43 mM A c e t i c a c i d ( g l a c i a l ) 0.35 ml to b r i n g pH t o 7.0 Water, t o b r i n g the f i n a l volume t o 200 ml. 10. S o l u t i o n s f o r enzyme a s s a y s (a) T r a n s f e r a s e s t o c k s o l u t i o n T r i c i n e , pH 7.5 250 mM KC1 70 mM M g C l 2 9 mM UDP-galactose 0.1 mM UDP-C6- 3H]-galactose 60 pCi/ml (b) I m i d a z o l e b u f f e r I m i d a z o l e , pH 6.8 0.10 M NP-40 0.5% G l y c e r o l 15% Tra c e s of Bromophenol Bl u e 34 (c) Stain for acid phosphatase a c t i v i t y i . C itrate buffer at 0.10 M, pH 4.8 C i t r i c acid (monohydrate) 1.56 g Tri-sodium c i t r a t e 3.67 g Water, to bring the f i n a l volume up to 200 ml. i i . Dye-substrate solution Final Concentrat i on Naphthol AS-MX phosphate 20 mg 0.05% Fast Garnet GBC s a l t 40 mg 0.10% The dyes were dissolved in 40 ml 0.1 M c i t r a t e buffer, pH 4.8 with rapid s t i r r i n g and f i l t e r e d through a 0.45 pm M i l l i p o r e f i l t e r . 11. E l e c t r o b l o t t i n q buffer Glycine 43.2 g 192 mM T r i s 9.0 g 25 mM SDS 3.0 g 0.10% Methanol 600 ml 20% Water was added to bring the f i n a l volume to 3.0 l i t e r s , and the solution was kept at 4°C. 12. Coomassie Blue solution Reagent for determination of protein concentration. i . 100 mg Coomassie B r i l l i a n t Blue G-250 was dissolved in 50 ml 95% ethanol. i i . To this solution, 100 ml 85% phosphoric acid was added with vigorous s t i r r i n g . i i i . Water was added slowly to bring the f i n a l volume to 1.0 l i t e r . 35 METHODS 1 • Growth and in v i v o d i f f e r e n t i a t i o n D i c t y o s t e 1 i u m d i sco ideum s t r a i n V12 M2 was used throughout t h i s s t u d y . To produce a c o n f l u e n t lawn of v e g e t a t i v e D. d i sco ideum c e l l s , a s t a n d a r d loop of amoebae was suspended in a f r e s h c u l t u r e of E n t e r o b a c t e r aerogenes and 0.1 ml and 0.3 ml a l i q u o t s were p l a t e d in P e t r i d i s h e s c o n t a i n i n g SM n u t r i e n t agar (see Media and S o l u t i o n s ) and i n c u b a t e d at 2 2 ° C for a p p r o x i m a t e l y 48 h o u r s . To i n i t i a t e in. v i v o development , amoebae were washed free of b a c t e r i a by r e p e a t e d l y c e n t r i f u g i n g f o r 5 minutes at 700g and resuspended in potass ium phosphate b u f f e r ( K K 2 b u f f e r ) , u n t i l s u p e m a t a n t s were c l e a r . Washed c e l l s were resuspended in K K 2 b u f f e r , counted and p l a t e d at 1 0 ° c e l l s on 4 cm M i l l i p o r e f i l t e r s (0 .45 pm pore s i z e ) suppor ted by f i l t e r pads s a t u r a t e d w i t h KK 2 b u f f e r . In some e x p e r i m e n t s , lower pad s o l u t i o n (LPS) i n s t e a d of K K 2 was used wi thout any a p p r e c i a b l e e f f e c t on deve lopment . To o b t a i n m i g r a t i n g s l u g s f o r the p u r i f i c a t i o n of p r e - s t a l k and p r e - s p o r e c e l l s on P e r c o l l g r a d i e n t s , a p p r o x i m a t e l y a 2x10 washed c e l l s were p l a t e d in P e t r i d i s h e s c o n t a i n i n g 36 n o n - n u t r i e n t a g a r ( s e e M e d i a a n d S o l u t i o n s ) a n d a l l o w e d t o d i f f e r e n t i a t e f o r 16-18 h o u r s a t 22°C. To o b t a i n l a r g e numbers o f f r u i t i n g b o d i e s f o r p r e p a r a t i o n o f s p o r e and s t a l k c e l l e x t r a c t s , 12 s t a n d a r d l o o p s o f amoebae were m i x e d w i t h 6 ml o f f r e s h l y g r o w n E n t e r o b a c t e r  a e r o g e n e s a n d p l a t e d i n s t e r i l i z e d s t a i n l e s s s t e e l t r a y s ( e a c h e q u i v a l e n t t o 19 P e t r i d i s h e s ) c o n t a i n i n g SM n u t r i e n t a g a r and i n c u b a t e d a t 22°C f o r 5 t o 7 d a y s . I n a l l t h e e x p e r i m e n t s d e s c r i b e d i n t h i s t h e s i s , d e v e l o p m e n t p r o c e e d e d a s f o l l o w : v i s i b l e a g g r e g a t i o n b e g a n b e t w e e n 4 and 6 h o u r s a n d e n d e d w i t h t h e f o r m a t i o n o f l o o s e mounds a t 8 h o u r s . T i p s e m e r g e d on t h e s e a g g r e g a t e s a t 9-10 h o u r s , and t h e y e l o n g a t e d t o f o r m f i n g e r s f r o m 10 t o 12 h o u r s . F i n g e r s ( a l s o c a l l e d s t a n d i n g s l u g s ) f e l l o n t o t h e s u b s t r a t u m a n d m i g r a t e d a s s l u g s u n t i l a b o u t 18 t o 20 h o u r s . A t t h a t t i m e , t h e y e n t e r e d t h e c u l m i n a t i o n s t a g e and f o r m e d f r u i t i n g b o d i e s , w h i c h were c o m p l e t e d by 24 h o u r s . 37 2• DIF preparation and extraction S o l u t i o n s c o n t a i n i n g DIF a c t i v i t y were prepared e s s e n t i a l l y as d e s c r i b e d by Town and S t a n f o r d (Town and S t a n f o r d , 1979). q 1-2x10^ washed V12 M2 c e l l s were d e p o s i t e d between 2 s t e r i l i z e d c e l l o p h a n e sheets ( o b t a i n e d from B r i t i s h Cellophane) supported by a 28 x 28 cm p l a s t i c screen and maintained in c o n t a c t with 600 ml 50% Bonner's s a l t s in a s t e r i l i z e d 36 x 36 x 3 cm p l a s t i c t r a y covered with aluminum f o i l . S e v e r a l t r a y s thus prepared were rocked g e n t l y at 22°C. A f t e r 3 hours, t h i s s o l u t i o n was d i s c a r d e d and t r a y s were r e p l e n i s h e d with 600 ml 50% Bonner's s a l t s c o n t a i n i n g 1 mM c y c l i c AMP and 100 ug/ml streptomycin s u l f a t e . A f t e r a f u r t h e r 36 to 48 hours i n c u b a t i o n , t h i s s o l u t i o n was c o l l e c t e d and c o n c e n t r a t e d in. vacuo at 48 °C to approximately 100 ml before e x t r a c t i n g the DIF a c t i v i t y . The aqueous s o l u t i o n was thoroughly mixed with an equal volume of heptane and c e n t r i f u g e d at lOOOg f o r 10 minutes to completely separate the two phases. The o r g a n i c phase was recovered, the aqueous phase was r e - e x t r a c t e d with heptane and the o r g a n i c phases were pooled. In e a r l y experiments, petroleum ether was used i n s t e a d of heptane. Both s o l v e n t s e x t r a c t e d DIF e q u a l l y , but heptane was p r e f e r r e d because i t d i d not evaporate a f t e r prolonged storage at -20°C or -70°C. 38 P o o l e d o r g a n i c e x t r a c t s were e v a p o r a t e d under N 2 a t 48°C t o about 100 p i , t w i c e r e suspended w i t h 1 ml e t h a n o l , and r e -c o n c e n t r a t e d as above. The f i n a l volume was a d j u s t e d t o between 0.5 and 2.0 ml e t h a n o l , such t h a t a l i n e a r dose-response was o b t a i n e d when 0.5 t o 5 p i samples were a s s a y e d . B u t y l a t e d h y d r o x y t o l u e n e (BHT) was added t o the e x t r a c t s b e f o r e e v a p o r a t i o n t o g i v e a f i n a l c o n c e n t r a t i o n o f 1 mg/ml i n the e t h a n o l i c s o l u t i o n . P o o l e d heptane e x t r a c t s g r e a t e r than 25 ml were e v a p o r a t e d In vacuo a t 48°C t o about 8 ml p r i o r t o c o n c e n t r a t i o n under N 2 . A l l aqueous s o l u t i o n s w i t h DIF a c t i v i t y were c o n t a i n e d i n v e s s e l s made of p o l y e t h y l e n e or s i l a n i z e d g l a s s w a r e t o p r e v e n t l o s s e s caused by a d s o r p t i o n . 3. D e t e r m i n a t i o n of DIF l e v e l s d u r i n g development In v i v o To d e t e r m i n e DIF l e v e l s d u r i n g development i n v i v o , 10° washed v e g e t a t i v e c e l l s were p l a t e d on M i l l i p o r e f i l t e r s s u p p o r t e d by 3.5 cm ( i n n e r d i a m e t e r ) p l e x i g l a s s r i n g s , and kept i n c o n t a c t w i t h 6.7 ml lower pad s o l u t i o n ( L P S ) . F i l t e r s c a r r y i n g d e v e l o p i n g c e l l s were b l o t t e d d r y on paper t o w e l s , i n s e r t e d i n s i l a n i z e d t e s t tubes and e x t r a c t e d t w i c e by m i x i n g v i g o r o u s l y i n 5 ml heptane. LPS c o n t a i n i n g c e l l u l a r e x u d ates was e x t r a c t e d w i t h heptane as d e s c r i b e d above. DIF a c t i v i t y was d e t e r m i n e d w i t h the s t a n d a r d low d e n s i t y monolayer a s s a y d e s c r i b e d below. 39 4. S t a l k c e l l formation i n monolayers (a) Low d e n s i t y monolayer assay of DIF •a 9 Washed V12 M2 c e l l s were p l a t e d at 10° c e l l s / c m ^ in 65 mm Nunc or F a l c o n t i s s u e c u l t u r e d i s h e s c o n t a i n i n g 2 ml LDMS (Low d e n s i t y monolayer s o l u t i o n , see Media and S o l u t i o n s ) with 1 mM c y c l i c AMP and 1 to 5 p i of an e t h a n o l i c s o l u t i o n of DIF. C o n t r o l experiments showed that 5 p i ethanol d i d not a f f e c t c e l l d i f f e r e n t i a t i o n . E a r l i e r experiments i n c l u d e d 5 pg/ml BHT in the above assay mix, and i t s subsequent omission had no n o t i c e a b l e e f f e c t s . S t a l k c e l l formation was assessed by s c o r i n g at l e a s t 200 c e l l s by phase c o n t r a s t microscopy. Reproducible q u a n t i t a t i o n of s t a l k c e l l s was achieved by c o u n t i n g as p o s i t i v e only those c e l l s that were vacuolated to 50% of t h e i r volume. A l l measurements were performed in d u p l i c a t e or t r i p l i c a t e and were g e n e r a l l y w i t h i n 20% of each other. The dose-response curve f o r DIF i n d u c t i o n of s t a l k c e l l s was l i n e a r up to l e v e l s inducing about 50% s t a l k c e l l formation. Each sample was assayed at s e v e r a l l e v e l s of DIF and a c t i v i t y was determined from the l i n e a r p o r t i o n of the curve. One u n i t of DIF was d e f i n e d as the amount which induces 1% s t a l k c e l l formation in t h i s standard assay (Brookman e_t al_. , 1982). 40 (b) F a c t o r removal and a d d i t i o n e x p e r i m e n t s i n low d e n s i t y monolayers In f a c t o r removal e x p e r i m e n t s , c e l l s were p l a t e d i n monolayers i n LDMS c o n t a i n i n g the i n d i c a t e d amount of c y c l i c AMP and DIF. S u p e r n a t a n t s were removed a t the i n d i c a t e d t i m e s and c e l l s were g e n t l y r i n s e d t h r e e t i m e s w i t h Bonner's s a l t s . LDMS l a c k i n g e i t h e r c y c l i c AMP o r DIF was r e p l e n i s h e d . I n f a c t o r a d d i t i o n e x p e r i m e n t s , 0.5 ml from a monolayer s u p e r n a t a n t was put i n the s t e r i l e l i d of the t i s s u e c u l t u r e d i s h , the t e s t s u b s t a n c e was added t o i t and the r e s u l t i n g s o l u t i o n was added back t o the s u p e r n a t a n t w i t h g e n t l e m i x i n g . S t a l k f o r m a t i o n was d e t e r m i n e d 48 hours a f t e r the i n i t i a l p l a t i n g . When s c o r i n g t h e s e e x p e r i m e n t s , the average number of c e l l s p e r microscope f i e l d was de t e r m i n e d and compared t o an u n p e r t u r b e d c o n t r o l t o ensure t h a t c e l l detachment had not o c c u r r e d d u r i n g the wash p r o c e d u r e . ( c ) E x p e r i m e n t s t o de t e r m i n e e x p r e s s i o n of d e v e l o p m e n t a l markers i n low d e n s i t y monolayers In e x p e r i m e n t s measuring the e x p r e s s i o n of d e v e l o p m e n t a l markers, 2.5x10^ washed c e l l s were p l a t e d i n 100 mm F a l c o n t i s s u e c u l t u r e d i s h e s c o n t a i n i n g 5 ml LDMS supplemented w i t h e i t h e r 1 mM c y c l i c AMP o r 450 u n i t s DIF as d e s c r i b e d i n the 41 text. At the indicated times, dishes were cooled to 4°C and 2 ml of supernatant was removed from each dish. C e l l s were detached in the remaining supernatant by gently scraping with a rubber policeman. The c e l l suspensions were pooled and centrifuged for 10 minutes at 5,000 rpm in an SW 41 rotor. 15 ul of either Imidazole buffer or SDS-sample buffer (see Media and Solutions) was added to the pell e t e d c e l l s . The c e l l s were resuspended and s o l u b i l i z e d by placing the tubes for 30 seconds in a bath sonicator f i l l e d with ice. Samples s o l u b i l i z e d in Imidazole buffer were immediately frozen at -20°C, whereas those in SDS-sample buffer were heat-denatured for 5 minutes at 95°C, then frozen at -20°C. These samples were subjected to electrophoresis within a few days for detection of antigens or enzyme a c t i v i t y . (d) High density monolayer experiments Washed c e l l s were plated at 10 cells/cm' in 65 mm Nunc tissue-culture dishes in 2 ml of HDMS (High density monolayer s a l t s , see Media and Solutions) containing 1 mM c y c l i c AMP. Under these conditions, >95% c e l l s formed stalk c e l l s within 48 hours. In experiments measuring the expression of acid phosphatase during d i f f e r e n t i a t i o n in monolayer, c e l l s from one or two dishes were harvested at the indicated times by resuspending into the supernatant with a rubber policeman. The c e l l suspension was centrifuged 42 for 5 minutes at 1,OOOg and the pelleted c e l l s were re-suspended in 100 pi ice-cold KK2 buffer and frozen at -20°C. After the protein concentration was determined for each of these samples, aliquots were s o l u b i l i z e d in 15% sucrose, 0.5% NP-40 to a f i n a l protein concentration of 4 pg/ul and samples were subjected to electrophoresis for detection of acid phosphatase a c t i v i t y . 5• Disaggregation experiments Ce l l s developing on Mi l l i p o r e f i l t e r s were harvested p e r i o d i c a l l y during development, washed once in Bonner's s a l t s , resuspended at 1-3x10^ cells/ml and gently t r i t u r a t e d for 5 minutes through a 23 G needle. The r e s u l t i n g single c e l l suspension was counted, d i l u t e d and plated at low c e l l densities in 65 mm Nunc tissue culture dishes containing 2 ml unsupplemented LDMS or LDMS containing 150 units DIF. In some experiments, disaggregated c e l l s were plated in LDMS containing the indicated concentration of c y c l i c AMP. After 2 hours incubation, monolayers were rinsed with Bonner's s a l t s and LDMS containing 150 DIF units was replenished. 43 6• Isolation of p r e - s t a l k and pre-spore c e l l s on Percoll  grad ients The i s o l a t i o n of pre-stalk and pre-spore c e l l s from disrupted slugs was performed using a modification of the protocol described by Ratner and Borth (1983). Slugs c o l l e c t e d a f t e r 16 to 18 hours d i f f e r e n t i a t i o n were disrupted by mixing vigorously in 20 to 30 ml buffer and centrifuged at 1,OOOg for 5 minutes. The pelleted slugs were mixed vigorously in 15 ml KK 2 buffer, f i l t e r e d through a fine nylon sieve (mesh size 20 um) to remove slime sheath and large c e l l clumps and centrifuged again. The c e l l s were, resuspended in 1.5 ml 5 mM MES, pH 6.2, 0.06% {l-mercaptoethanol and 1 mg/ml Sigma Type XIV protease (extracted from Proteus griseus) and incubated at room temperature for 10 minutes. Aliquots (0.3 ml) containing Q approximately 10 c e l l s were deposited onto ice-cold discontinuous Percoll gradients comprised of: 2.5 ml 48% Percoll in the bottom layer; 2.5 ml 33% Percoll in the middle layer and 2.5 ml 18% Percoll in the top layer, each dil u t e d in 20 mM MES, pH 7.0 and 20 mM EDTA, pH 7.0. Gradients were centrifuged at 4°C in an SW 41 rotor at 12,000 rpm for 5 minutes. Pre-stalk c e l l s were recovered at the interface between the top and middle layers of the gradient, whereas pre-spore c e l l s were recovered between the middle and bottom layers. Pre-spore c e l l s contained no appreciable pre-stalk s p e c i f i c acid phosphatase (Oohata, 44 1983) (see Figure 13 in Results section). The s p e c i f i c a c t i v i t y of the pre-spore s p e c i f i c enzyme UDP-galactosyl:mucopo1ysaccharide transferase (Newell e_t a l . , 1969a) for the pre-spore band was 4.4 nmol UDP-galactose incorporated/Hr/mg protein while that for the pre-stalk band was 0.026 nmol UDP-galactose incorporated/Hr/mg protein. C e l l s were c o l l e c t e d from the gradients with a Pasteur pipette, d i l u t e d with ice-cold KK2 buffer and centrifuged at 2,000g for 5 minutes. Cel l s were washed once in KK2 buffer and then resuspended in the buffer appropriate for determination of antigen or enzyme a c t i v i t y or monolayer d i f f e r e n t i a t i o n . 7. Protein determination Protein concentration was determined according to Bradford (1976), using the Coomassie B r i l l i a n t Blue reagent described in Media and Solutions. 100 pi aliquots from the test solution were mixed with 5 ml reagent and incubated for 5 to 45 minutes at room temperature. The absorbance at 595 nm against a reagent blank was measured and the protein concentration was determined r e l a t i v e to T - g l o b u l i n as standard. 45 8- Polyacrylaalde gel electrophoresis and el e c t r o b l o t t I n g onto nUroceUuiose. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s under d e n a t u r i n g c o n d i t i o n s was c a r r i e d out as d e s c r i b e d by Laemmli ( 1 9 7 0 ) . G e l s c o n s i s t i n g of a 3.0% a c r y l a m i d e s t a c k i n g g e l and 6.8% a c r y l a m i d e r u n n i n g g e l were p o l y m e r i z e d f o r 2 hours and c o o l e d t o 4°C b e f o r e use (see Media and S o l u t i o n s ) . P r o t e i n samples were e l e c t r o p h o r e s e d a t 5 m i l l i a m p s i n the s t a c k i n g g e l u n t i l the t r a c k i n g dye e n t e r e d the r u n n i n g g e l and th e n a t 15 m i l l i a m p s u n t i l the dye r e a c h e d the bottom of the g e l . A m o l e c u l a r weight s t a n d a r d mix was a l s o e l e c t r o p h o r e s e d and s i l v e r s t a i n e d u s i n g a c o m m e r c i a l l y a v a i l a b l e k i t ( B i o - R a d ) . T h i s s t a n d a r d mix c o m p r i s e d Ovalbumin ( 4 . 5 x l o S , Bovine serum a l b u m i n ( 6 . 6 x l o S , P h o s p h o r y l a s e b ( 9 . 7 x 1 0 ^ ) , fl-G a l a c t o s i d a s e (1.2x10^) and myosin ( 2 . 1 x 1 0 ^ ) . B e f o r e e l e c t r o b l o t t i n g , g e l s were e q u i l i b r a t e d i n e l e c t r o b l o t t i n g b u f f e r (see Media and S o l u t i o n s ) f o r 30 minutes and b l o t t e d i n the c o l d onto a d j a c e n t n i t r o c e l l u l o s e s h e e t s f o r 12 t o 16 hours a t 0.20 t o 0.22 amps, as d e s c r i b e d by Towbin e_t a l _ . (1979) 46 9. Enzyme a s s a y s (a) U D P - g a l a c t o s y l : m u c o p o l y s a c c h a r i d e t r a n s f e r a s e U D P - g a l a c t o s y l : m u c o p o l y s a c c h a r i d e t r a n s f e r a s e ( g a l . t r a n s f e r a s e ) was a s s a y e d by measuring the i n c o r p o r a t i o n of U D P - [ J H ] - g a l a c t o s e onto a m u c o p o l y s a c c h a r i d e a c c e p t o r as p r e v i o u s l y d e s c r i b e d by Kay ( 1 9 7 9 ) . 15 p i of t r a n s f e r a s e s t o c k s o l u t i o n (see Media and S o l u t i o n s ) was mixed w i t h 10 p i o f a s o l u t i o n c o n t a i n i n g m u c o p o l y s a c c h a r i d e a c c e p t o r a c t i v i t y p r e p a r e d as d e s c r i b e d by Sussman and Osborn ( 1 9 6 4 ) . D u p l i c a t e samples w i t h o u t i s o - a c c e p t o r were a l s o p r e p a r e d to det e r m i n e the enzyme a c t i v i t y a r i s i n g from the presence of endogenous a c c e p t o r , and t h i s a c t i v i t y was s u b s t r a c t e d from the above c o u n t s (Sussman and Osborn, 1964). C e l l e x t r a c t s c o n t a i n i n g 50 pg p r o t e i n s o l u b i l i z e d i n 50 p i 0.15% NP-40 and 20% g l y c e r o l were added t o the a s s a y mix and the m i x t u r e s were i n c u b a t e d on i c e . A f t e r 5 hours i n c u b a t i o n , 65 p i a l i q u o t s were s p o t t e d on Whatman 3MM f i l t e r d i s c s and washed i n i c e - c o l d 55% e t h a n o l 3 t i m e s f o r 10 minu t e s . F i l t e r s were washed once i n 100% e t h a n o l a t room temperature and once i n e t h e r . F i l t e r s were a i r d r i e d and JH i n c o r p o r a t i o n was d e t e r m i n e d i n 4 ml t o l u e n e s c i n t i l l a t i o n s o l u t i o n . 47 (b) A c i d Phosphatase A c i d phosphatase isozymes were s e p a r a t e d on n o n - d e n a t u r i n g g e l s as d e s c r i b e d by W e i j e r and D u r s t o n ( 1 9 8 5 ) . C e l l samples s o l u b i l i z e d i n I m i d a z o l e b u f f e r were l o a d e d onto 3-6% a c r y l a m i d e g r a d i e n t g e l s (see Media and S o l u t i o n s ) and r u n a t 100 V o l t s a t 4°C u n t i l the t r a c k i n g dye r e a c h e d the bottom. A c i d phosphatase a c t i v i t y was d e t e c t e d by i n c u b a t i n g g e l s a t room te m p e r a t u r e i n 100 mM c i t r a t e b u f f e r , pH 4.8 c o n t a i n i n g 0.5 mg Naphthol AS-MX phosphate and 1 mg/ml F a s t Garnet GBC s a l t (see Media and S o l u t i o n s ) . A c t i v i t y was u s u a l l y d e t e c t a b l e w i t h i n 5 m i n u t e s , a l t h o u g h up t o 60 minutes i n c u b a t i o n was n e c e s s a r y when a c t i v i t y was low. Serum p r e p a r a t i o n (a) P r e p a r a t i o n o f a r a b b i t immune serum a g a i n s t s t a l k c e l l e x t r a c t s F r u i t i n g b o d i e s from 6x10^ c e l l s were washed over t h r e e l a y e r s of c o a r s e n y l o n mesh w i t h s e v e r a l l i t e r s phosphate b u f f e r e d s a l i n e (PBS) u n t i l s p o r e s were u n d e t e c t a b l e i n the wash. The washed s t a l k s were resuspended i n 30 ml i c e - c o l d PBS c o n t a i n i n g 5 mM p-aminobenzamidine, 1 mM PMSF and 10 g a c i d washed g l a s s beads (0.45-0.50 mm), and were m e c h a n i c a l l y s h e a r e d by s t i r r i n g f o r 4 hours a t 4°C. Coarse 48 debris were removed by centrifuging the suspension for 5 minutes at l,OOOg and the supernatant was aliquoted and frozen at -20°C u n t i l needed. A stalk c e l l extract containing 2.5 mg protein was emulsified in 50% Freund's complete adjuvant <FCA) and injected subcutaneously into a New-Zealand white rabbit above each limb. Injections were repeated at 3 week intervals in 33% FCA. The antibody t i t e r against stalk c e l l proteins ceased to increase a f t e r the 6th inj e c t i o n , and 35 ml blood was obtained by cardiac puncture 10 days a f t e r a f i n a l 7th injection for preparation of an antibody solution. The blood sample was c l o t t e d for 1 hour at 37°C and for 24 hours at 4°C. The serum which had separated from the c l o t was c a r e f u l l y withdrawn and was stored at 4°C u n t i l needed for the preparation of the antibody solution described below. <b) Preparation of a rabbit immune serum against pre-stalk c e l l extracts c Approximately 5x10 pre-stalk c e l l s p u r i f i e d on a Percoll gradient were incubated in 0.25 ml PBS containing 0.5% SDS for 30 minutes at 4°C. An equal volume of rabbit antiserum raised against pre-spore and spore c e l l extracts was added to the s o l u b i l i z e d c e l l extracts and the mixture was 49 incubated for an additional 4 hours. The antigen-antibody complexes formed during t h i s incubation were removed using a commercial preparation of Staphy1ococcus aureus c e l l s (IgGsorb) and the remaining solution was recovered. This pre-stalk c e l l extract was used for a single i n j e c t i o n . A new pre-stalk antigen preparation was made shortly before each i n j e c t i o n . Due to the small amount of antigenic material obtained from each pre-stalk c e l l extract, 2.5 mg stalk c e l l extracts emulsified in 50% FCA were used to immunize a New-Zealand White rabbit for the f i r s t two injections at 3 week in t e r v a l . Thereafter, pre-stalk extracts in 33% FCA were injected at 3 week inter v a l s . Ten days afte r the 8th inje c t i o n , a 30 ml blood sample was co l l e c t e d and treated as described above to prepare an immune serum. (c) Preparation of a mouse immune serum against stalk c e l l e xtracts To immunize mice, stalk c e l l extracts were prepared using a protocol described by Wallace e_t al_. ( 1984). F r u i t i n g bodies q from 6x10 c e l l s were washed over three layers of coarse nylon mesh with several volumes of TBS (Tris buffered s a l t s , see Media and Solutions) u n t i l spores were undetectable in the wash. The washed stalk c e l l s were resuspended in 30 ml 50 ice-cold TBS containing 5 mM p-aminobenzamidine, 1 mM PMSF and 10 g acid washed glass beads (0.45-0.50 mm), and were mechanically sheared by s t i r r i n g at 4°C. After 4 hours, coarse debris were separated from the glass beads and col l e c t e d by centrifuging at 4°C at l,000g for 10 minutes. The pelleted coarse debris were washed three times in TBS and incubated over ice with TBS containing 0.5% NP-40. After 30 minutes, the s o l u b i l i z e d proteins were removed by centrifuging at lOOOg for 20 minutes and the p e l l e t was washed three times with TBS/NP-40. The washed p e l l e t was resuspended in 20 ml TBS containing 0.82% SDS and 10 mM d i t h i o t h r e i t o l (DTT) and placed in a b o i l i n g water bath for 10 minutes. The particulate matter was removed by centrifugation at lOOOg for 20 minutes and the s o l u b i l i z e d stalk proteins were precipitated with 4 volumes acetone cooled to -20°C. The prec i p i t a t e was centrifuged at 4°C at 5,000g for 10 minutes and the p e l l e t s were resuspended in 1-2 ml PBS and stored at -20 °C u n t i l required for i mmun i zat i on. A s o l u b i l i z e d stalk c e l l extract containing 1 mg protein was emulsified in 50% FCA and injected intra-peritonial1y and in the thighs of 6 week old Balb/c mice. Three further injections in 33% incomplete Freund's adjuvant ( I FA ) at 3 week intervals gave a serum with high t i t e r against stalk proteins. One week after the last injection, blood was obtained aft e r k i l l i n g the immunized mice by c e r v i c a l 51 d i s l o c a t i o n and mouse immune serum was p r e p a r e d as d e s c r i b e d above f o r r a b b i t serum. <d) A n t i b o d y p u r i f i c a t i o n P r o t e i n s were p r e c i p i t a t e d from r a b b i t o r mouse immune s e r a by a d d i n g an e q u a l volume of s a t u r a t e d i c e - c o l d ammonium s u l f a t e , pH 7.0 and i n c u b a t i n g f o r 4 t o 24 hours a t 4°C. P r e c i p i t a t e d p r o t e i n s were c e n t r i f u g e d a t 10,000g f o r 30 min u t e s , s o l u b i l i z e d i n a minimal volume of PBS c o n t a i n i n g 0.02% sodium a z i d e (PBS-NaN 3> and d i a l y z e d a t 4°C f o r 24 hours a g a i n s t 2 changes o f 2 l i t e r s PBS-NaN^. F o r the r a b b i t a n t i b o d y p r e p a r a t i o n s , the p r o t e i n s from the d i a l y s e d f r a c t i o n were a p p l i e d t o a DEAE-Sephacel column e q u i 1 i b r a t e d w i t h 10 mM T r i s - C l , pH 7.4. The column was washed w i t h s e v e r a l volumes of the same b u f f e r and p r o t e i n s were e l u t e d w i t h a 0 t o 500 mM NaCl g r a d i e n t i n the same b u f f e r . A n t i b o d i e s were d e t e c t e d i n the e l u t e d f r a c t i o n s u s i n g a l k a l i n e p h o s p h a t a s e - c o n j u g a t e d goat a n t i - r a b b i t a n t i b o d i e s i n an ELISA a s s a y . The peak a n t i b o d y f r a c t i o n s were p o o l e d and d i a l y z e d a t 4°C f o r 24 hours a g a i n s t 2 changes of 2 l i t e r s PBS. The d i a l y z e d a n t i b o d y s o l u t i o n was s t o r e d a t 4ttC a f t e r a d d i n g t h i m e r o s a l t o a f i n a l c o n c e n t r a t i o n of 0.0013% (w/v) . 52 (e) Antibody adsorption against cross-reactive antigens Non-specific antibodies were removed by adsorption of serum with c e l l extracts bound onto n i t r o c e l l u l o s e sheets as described by Dominov and Town (1986; Dominov, personal communication). The rabbit and mouse an t i - s t a l k antibody preparations were adsorbed against extracts of amoebae, spore and slug c e l l s , whereas the rabbit anti-pre-stalk antibody preparation was adsorbed against extracts of amoebae, pre-spore and spore c e l l s . Cell extracts were prepared by b o i l i n g c e l l s for 5 minutes or spores for 15 minutes in TBS containing 1% SDS and 0.1% /5-mercaptoethanol . The particulate matter was removed by centrifugation at 12,000g and the soluble f r a c t i o n , containing between 10 to 20 mg/ml protein, was dil u t e d with 9 volumes TBS. This solution was incubated for 2 hours at room temperature with n i t r o c e l l u l o s e sheets at about 100 ug protein/cm^. The sheets were washed with several changes PBS-NaN^ or TBS and were incubated 1-2 hours at room temperature with either 1% bovine T-globulin ( f a t t y acid free) /0.1% Tween 80 in TBS or 5% non-fat dried milk (Carnation) in PBS-NaN^ (PBS-NaN^/dry milk) to block the remaining reactive s i t e s . These sheets were washed again and incubated for 24 hours at 4°C with the appropriate antibody solution. The antibody solution remaining aft e r adsorption was tested on Western blots against appropriate c e l l 53 extracts and was re-adsorbed against freshly prepared c e l l extracts on n i t r o c e l l u l o s e sheets u n t i l a s a t i s f a c t o r y number of undesired antibodies were removed. 1 1 • B i o t i n y l a t i o n protocol for reporter antibodies and  enzymes The protocol used to b i o t i n y l a t e proteins was adapted from Bayer e_t al_. ( 1979). Alkaline phosphatase (Bovine mucosal, Sigma Type XX) and goat anti-rabbit T-globulin were bi o t i n y l a t e d following t h i s protocol. The protein to be bioti n y l a t e d was s o l u b i l i z e d in 1-2.5 ml PBS at a concentration of 1-3 mg/ml and reacted at room temperature in several tubes containing from 1:8 to 1:45 (mol:mol) biotinyl-€-aminocaproic acid N-hydroxysuccinimide (biotin-X-NHS). Biotin-X-NHS was prepared as a 7 mg/ml stock solution in dimethylformamide and kept in the dark at 4°C. After 4 hours incubation, glycine was added to a f i n a l concentration of 25 mM to bind unreacted biotin-X-NHS. After 30 minutes, the reaction mix was passed over a 5 ml Sephadex G-25 column and the protein f r a c t i o n was recovered in the void volume and dialyzed overnight at 4°C against 2 changes of 2 l i t e r s PBS-NaN^. The biotinyl a t e d protein preparation with the highest degree of modification which retained high a c t i v i t y was used for experimental work. 54 12. R e a c t i o n of n i t r o c e l l u l o s e b l o t s w i t h a n t i b o d i e s Proteins that had been electroblotted onto n i t r o c e l l u l o s e sheets were reacted with mouse antibodies using the following protocol. N i t r o c e l l u l o s e sheets with transferred proteins were fixed for 5 minutes in 10% acetic acid/25% iso-propanol, washed extensively with water and incubated for 2 hour at 37 °C in 1% bovine T-globulin in TBS containing 0.05% Tween 80 <TBS/1% BGG/Tween) to block remaining reactive s i t e s . The sheets were rinsed in TBS/0.1% BGG/Tween and incubated with a mouse a n t i - s t a l k antibody preparation diluted 1:100 in TBS/1% BGG/Tween for 2 hour at 37°C (Figure 21) , or incubated with a mouse a n t i - s t a l k antibody preparation d i l u t e d 1:10 for 20 hours at 4°C (Figure 22). The probed blot was washed at 37 QC four times for 15 minutes each with TBS/0.1% BGG/Tween (Figure 21), or for a tota l of 140 minutes with 3 changes of TBS/0.1% BGG/Tween (Figure 22) . An alkaline phosphatase-conjugated goat anti-mouse antibody solution diluted at 1:1000 in TBS/1% BGG/Tween was incubated for 2 hours at 37°C. The blot was washed again 5 times for 10 minutes each and developed at room temperature for alkaline phosphatase a c t i v i t y in 100 mM T r i s - C l , pH 9.4, 100 mM NaCl, 50 mM MgCl 2, 0.16 mg/ml 5'bromo-4'chlore-s' indoyl phosphate (BCIP) and 0.33 mg/ml Nitro Blue 55 Tetrazolium for 30 minutes (Figure 21) or 2 hours (Figure 22) . A d i f f e r e n t protocol was used for n i t r o c e l l u l o s e blots reacted with rabbit antibodies. N i t r o c e l l u l o s e sheets with transferred proteins were fixed for 5 minutes in 10% acetic acid/25% iso-propanol, washed extensively with water and incubated for 1 hour at room temperature in PBS-NaN^/dry milk (Johnson et. al.-> 1984) to block remaining reactive s i t e s . The sheets were rinsed in PBS-NaN-j and incubated with the desired antibody solution either undiluted (Figure 19) or di l u t e d 1:10 (Figures 18 and 20) in PBS-NaN3/dry milk containing 0.1% Triton X-100 (PBS-NaN3/dry milk/TX-100), for either 1 hour at 37°C or for 24 hours at 4°C. The probed blot was washed 5 times for 5 minutes each with PBS-NaN3 at room temperature and once for 30 minutes at 37°C in PBS-NaN3/dry milk/TX-100. A b i o t i n conjugated goat a n t i -rabbit d i l u t e d at 1:1000 in PBS-NaN3/dry milk/TX-100 was incubated for 1 hour at 37°C. The blot was washed as above and incubated at 37°C for 1 hour with alkaline phosphatase-conjugated strepavidin d i l u t e d 1:3000 in PBS-NaN3/dry milk/TX-100. In 2 experiments (Figures 18 and 19), bio t i n y l a t e d alkaline phosphatase was added to this solution a f t e r 30 minutes incubation to y i e l d a 3:1 r a t i o (mol:mol) with strepavidin. The blot was f i n a l l y washed 6 times for 10 56 minutes in PBS-NaN 3/dry milk/TX-100 and developed f o r a l k a l i n e phosphatase a c t i v i t y as d e s c r i b e d above. 5 7 RESULTS PART I CHARACTERIZATION OF STALK CELL FORMATION IN  LOW DENSITY MONOLAYERS Brookman e_t a l _ . showed p r e v i o u s l y t h a t the p r o d u c t i o n of DIF was d e v e l o p m e n t a l l y r e g u l a t e d , but they a l s o r e p o r t e d t h a t t h e i r DIF e x t r a c t s c o n t a i n e d i n h i b i t o r y a c t i v i t y (Brookman e_t al . . , 1982). In o r d e r t o e l i m i n a t e t h i s i n h i b i t o r y a c t i v i t y , DIF was e x t r a c t e d w i t h heptane, a more s e l e c t i v e s o l v e n t ( S o b o l e w s k i e_t a l . . , 1983), and i t s p r o d u c t i o n d u r i n g development was r e a s s e s s e d . I t was found t h a t DIF l e v e l s remained low thr o u g h o u t the a g g r e g a t i o n phase (the f i r s t 8 hours of de v e l o p m e n t ) , and i t began to accumulate r a p i d l y s h o r t l y t h e r e a f t e r ( F i g u r e 2 ) . The marked i n c r e a s e i n DIF l e v e l s preceded the e s t a b l i s h m e n t of the w e l l d e f i n e d p r e - s t a l k / p r e - s p o r e p a t t e r n a t the f i n g e r stage and was c o n s i s t e n t w i t h the view t h a t DIF might f u n c t i o n as the a c t i v a t o r of p r e - s t a l k c e l l s . However, the time of DIF a c t i o n i s more r e l e v a n t than the time of i t s p r o d u c t i o n and a s e r i e s of monolayer e x p e r i m e n t s were und e r t a k e n to t r y to determine t h i s . 58 TIME (Hours) F i g . 2. DIF a c t i v i t y presen t in c e l l masses and lower pad s o l u t i o n d u r i n g d i f f e r e n t i a t i o n on M i l l i p o r e f i l t e r s . C e l l s were p l a t e d on a 4 cm M i l l i p o r e f i l t e r supported above and main ta ined in c o n t a c t wi th 6.7 ml lower pad s o l u t i o n by a 3.7 cm p l e x i g l a s s r i n g . At the i n d i c a t e d t i m e s , the amount of DIF presen t in the c e l l s masses (O.) and the lower pad s o l u t i o n (•) was de termined as d e s c r i b e d under Methods. Data are averages from three s eparate e x p e r i m e n t s . 59 A. Factor removal experiments. A l l these experiments were carried out using the in  v i tro monolayer d i f f e r e n t i a t i o n conditions elaborated by Gross and co-workers (Town et al.. , 1976; Gross et al_. , 1981; Kay, 1982). In th i s system, amoebae of s t r a i n V12 M2 co l l e c t e d from a p a r t i a l l y cleared growth plate are washed free of bacteria and plated at 10J cells/cnr onto the surface of tissue culture dishes, submerged under a buffered s a l t solution containing 1 mM c y c l i c AMP. At this c e l l density, amoebae are separated from each other by about 10 c e l l diameters and remain undifferentiated during prolonged incubation. If a heptane extract of e x t r a c e l l u l a r material containing the d i f f e r e n t i a t i o n - i n d u c i n g factor (DIF) is provided, the c e l l s d i f f e r e n t i a t e within 48 hours into highly vacuolated c e l l s (Town and Stanford, 1979) very sim i l a r in appearance to the stalk c e l l s of mature f r u i t i n g bodies (Bonner, 1970). In this system, stalk c e l l formation is proportional to the amount of DIF added (Gross e_t a l . , 1981). In monolayers at low c e l l d e n s ities, stalk c e l l formation is t o t a l l y dependent on c y c l i c AMP and DIF. The time at which they act was determined through factor removal experiments. I n i t i a l l y , amoebae were plated in the presence of both factors. 60 At regular intervals the supernatant bathing the c e l l s was removed, the c e l l monolayers were rinsed and LDMS (Low-Density Monolayer Salts) lacking either c y c l i c AMP or DIF was replenished. The proportion of stalk c e l l s formed was assessed 48 hours after the i n i t i a t i o n of the experiment and was compared with that obtained in an unperturbed dish. The results from a t y p i c a l experiment are shown on figure 3. The removal of c y c l i c AMP or DIF during the f i r s t 8 hours of incubation t o t a l l y prevented stalk c e l l formation. This indicates that the factors had been removed p r i o r to the time when they acted on c e l l s . Removal of either factor after 24 hours did not a f f e c t stalk c e l l formation, indicating that these factors were no longer required by that time. During the period between 10 and 24 hours, there was an increasing proportion of c e l l s committed to stalk c e l l formation in the absence of c y c l i c AMP or DIF, defining this as the period for th e i r requirement. The appearance of stalk c e l l s in an unperturbed plate began at 18 hours (Figure 3), close to the time when they normally begin to appear i n vivo. Stalk c e l l formation in monolayers continued u n t i l 32 hours, indicating that c e l l d i f f e r e n t i a t i o n in. v i tro took somewhat longer than the 24 hours normally required by V12 M2 during f r u i t i n g body formation in vivo. 61 £0 Time (h) F i g . 3. The e f f e c t of DIF removal and c y c l i c AMP removal on s t a l k c e l l f ormation d u r i n g d i f f e r e n t i a t i o n i n low d e n s i t y monolayers. C e l l s were p l a t e d a t 10 c e l l s / c m i n 2 ml LDMS c o n t a i n i n g 1 mM c y c l i c AMP and 36 u n i t s DIF. Supernatants were removed at the i n d i c a t e d times, c e l l monolayers were r i n s e d three times with Bonner's s a l t s and medium without DIF ( O ) or without c y c l i c AMP (•> was r e p l e n i s h e d . The percentage of s t a l k c e l l s in the p o p u l a t i o n was determined a f t e r 48 hours. In a d d i t i o n , the p r o p o r t i o n of s t a l k c e l l s formed in an unperturbed i n c u b a t i o n ( A ) was determined at the i n d i c a t e d times. The data shown in the above f i g u r e and those which f o l l o w ( u n l e s s otherwise s t a t e d ) are from a s i n g l e experiment showing a t y p i c a l r e s u l t . The experiments shown in t h i s t h e s i s were g e n e r a l l y repeated three times with s i m i l a r r e s u l t s . 62 T h e s e d a t a s u g g e s t e d t h a t t h e p e r i o d o f c y c l i c AMP r e q u i r e m e n t was s l i g h t l y i n a d v a n c e o f t h e p e r i o d o f D I F r e q u i r e m e n t ( F i g u r e 3 ) , a r e s u l t t h a t was h i g h l y r e p r o d u c i b l e . To c o n f i r m t h a t c y c l i c AMP a c t e d b e f o r e D I F , c e l l s were i n c u b a t e d w i t h c y c l i c AMP a l o n e f o r 24 h o u r s f o l l o w e d by D I F f o r 24 h o u r s , o r w i t h D I F f o r 24 h o u r s f o l l o w e d by c y c l i c AMP. The r e s u l t s i n T a b l e I show t h a t s t a l k c e l l f o r m a t i o n o c c u r r e d when c e l l s were i n c u b a t e d w i t h c y c l i c AMP b e f o r e D I F , b u t n o t when c e l l s were f i r s t i n c u b a t e d w i t h D I F f o l l o w e d by c y c l i c AMP. T h e s e d a t a n o t o n l y c o n f i r m e d t h a t t h e c y c l i c AMP r e q u i r e m e n t p r e c e d e d t h e r e q u i r e m e n t f o r D I F , t h e y a l s o d e m o n s t r a t e d t h a t t h e c o n t i n u e d p r e s e n c e o f c y c l i c AMP was n o t e s s e n t i a l f o r i n d u c t i o n o f s t a l k c e l l s by D I F , s u g g e s t i n g t h a t t h e two i n d u c t i v e e v e n t s c a n t a k e p l a c e s e p a r a t e l y u n d e r t h e s e c o n d i t i o n s . H o w e v e r , T a b l e I a l s o shows t h a t i n c u b a t i o n o f c e l l s w i t h b o t h f a c t o r s t o g e t h e r r e s u l t e d i n more s t a l k c e l l s b e i n g f o r m e d t h a n when t h e f a c t o r s were a d d e d s e q u e n t i a l l y , s u g g e s t i n g a t l e a s t a c e r t a i n amount o f i n t e r a c t i o n b e t w e e n c y c l i c AMP a n d D I F . Table I The r e l a t i o n s h i p between the dependence of c y c l i c AMP and DIF for stalk formation in low density monolayers. Experimental treatment 0 Stalk c e l l formation* 3 C y c l i c AMP + DIF 67 Cyc l i c AMP < 1 DIF < 1 followed by DIF for 24 h c 37 Cyc l i c AMP for 24 h, for DIF for 24 h, followed by c y c l i c AMP for 24 h c < 1 a C e l l s were plated in LDMS containing 1 mM c y c l i c AMP or 67 units DIF, as indicated. ''The percentage of stalk c e l l s formed following each treatment was determined aft e r 48 hours. c A t 24 hours the supematants were removed, c e l l monolayers were rinsed three times with Bonner's s a l t s and 2 ml LDMS containing either c y c l i c AMP or DIF, as indicated, was replenished for 24 hours. To determine the amount of time r e q u i r e d f o r i n d u c t i o n of s t a l k c e l l s by D I F , c e l l s p r e - i n c u b a t e d w i t h c y c l i c AMP for 20 hours were incubated wi th DIF for s p e c i f i c p e r i o d s of time and the degree of s t a l k c e l l f o r m a t i o n r e s u l t i n g from t h i s treatment was a s s e s s e d . Table II shows tha t a 2 hour i n c u b a t i o n wi th DIF was s u f f i c i e n t to g ive complete i n d u c t i o n under these c o n d i t i o n s . As noted p r e v i o u s l y , s e q u e n t i a l i n c u b a t i o n wi th c y c l i c AMP and DIF r e s u l t e d in c o n s i d e r a b l y l e s s s t a l k c e l l f o r m a t i o n than tha t a c h i e v e d wi th c y c l i c AMP and DIF t o g e t h e r . 65 Table II Determination of the minimum time required for the induction of stalk c e l l formation by DIF in low density monolayers. Experimental treatment 0 Stalk c e l l formation^ Cyclic.AMP + DIF for 48 h 62 Cyc l i c AMP + DIF for 20 h 41 Cy c l i c AMP for 20 h < 1 Cyc l i c AMP for 20 h, followed by DIF for 1 h c 7 followed by DIF for 2 h c 15 followed by DIF for 4 h c 15 followed by DIF for 28 h c 15 °Cells were plated in LDMS containing 1 mM c y c l i c AMP or 60 units DIF, as indicated. ^The percentage of stalk c e l l s formed following each treatment was determined a f t e r 48 hours. c A f t e r 20 hours, supematants were removed, c e l l monolayers were rinsed three times with Bonner's s a l t s , and 2 ml LDMS containing 60 units DIF was added for the indicated times. Supematants were then removed, monolayers were rinsed as above and LDMS was added for the remaining incubation period. 66 B. F a c t o r a d d i t i o n experiments. The data shown in f i g u r e 3 d e f i n e d the p e r i o d when c e l l s became independent of e i t h e r c y c l i c AMP or DIF d u r i n g the d i f f e r e n t i a t i o n p r o c e s s , but they d i d not r e v e a l when these f a c t o r s were f i r s t r e q u i r e d . To e s t a b l i s h the time when c y c l i c AMP was f i r s t r e q u i r e d f o r s t a l k c e l l formation, i t was added to separate s e t s of d i s h e s at p r o g r e s s i v e l y l a t e r times a f t e r c e l l s were p l a t e d and the e f f e c t of delayed a d d i t i o n was determined by a s s e s s i n g the appearance of c y c l i c AMP independent c e l l s , as d e s c r i b e d p r e v i o u s l y . Figure 4 shows that very s i m i l a r k i n e t i c s of s t a l k c e l l i n d u c t i o n were obtained i f c y c l i c AMP was added at 0, 6, or 8 hours, but there was a marked d e l a y when c y c l i c AMP was added at 10 hours. I t i s t h e r e f o r e apparent that s t a l k c e l l formation does not r e q u i r e the presence of c y c l i c AMP e a r l i e r than 8 hours in. v i t r o . A f t e r the a d d i t i o n of c y c l i c AMP at 8 hours, c y c l i c AMP independent c e l l s were f i r s t d e t e c t a b l e 2 hours l a t e r . A s i m i l a r experiment was conducted with DIF, and the r e s u l t s are shown in f i g u r e 5. DIF c o u l d be added at any time up to 10 hours of i n c u b a t i o n without a s i g n i f i c a n t change in the k i n e t i c s of s t a l k c e l l formation, whereas a d d i t i o n at 12 hours r e s u l t e d in a delayed response. 75 Time (h) F i g . 4. The e f f e c t of varying the time of c y c l i c AMP a d d i t i o n and i t s subsequent removal on s t a l k c e l l formation in low d e n s i t y monolayers. C e l l s were p l a t e d in 2 ml LDMS c o n t a i n i n g 60 u n i t s DIF. 1 mM c y c l i c AMP was added at 0 hours (•), 6 hours (•), 8 hours (O) or 10 hours (A). At the i n d i c a t e d times, supernatants were removed, the monolayers r i n s e d three times with Bonner's s a l t s and LDMS c o n t a i n i n g 60 u n i t s DIF was added. The percentage of s t a l k c e l l s in the population was determined 48 hours a f t e r the beginning of the experiment. 50 Time (hJ F i g . 5. The e f f e c t of varying the time of DIF a d d i t i o n and i t s subsequent removal on s t a l k c e l l formation in low den s i t y monolayers. C e l l s were p l a t e d in 2 ml LDMS c o n t a i n i n g 1 mM c y c l i c AMP. 45 u n i t s DIF were added to supematants at 0 hours <•), 10 hours <A> or 12 hours (O) . At the i n d i c a t e d times, the supematants were removed, monolayers were r i n s e d three times with Bonner's s a l t s and LDMS c o n t a i n i n g 1 mM c y c l i c AMP was r e p l e n i s h e d . The percentage of s t a l k c e l l s was determined 48 hours a f t e r the beginning of the experiment. 69 These re s u l t s indicated that c y c l i c AMP induction of stalk c e l l formation started between 8 and 10 hours in low density monolayers, followed by DIF induction between 10 and 12 hours and were consistent with re s u l t s of the factor removal experiments which showed that DIF acted immediately after c y c l i c AMP (Figure 3). C. Experiments with c y c l i c AMP analogues. The observation that c e l l s did not respond to c y c l i c AMP u n t i l a f t e r an 8 hour incubation in monolayers suggested the p o s s i b i l i t y that a binding protein activated by exogenously supplied c y c l i c AMP might appear at that time. It has also been suggested that the high c y c l i c AMP concentration used in monolayers is required so that i t can permeate c e l l s and act upon an internal binding protein (Sampson e_t al.. , 1978), but there is no strong evidence supporting t h i s idea. Three developmentally regulated c y c l i c AMP binding proteins have been described that might possibly mediate the response to c y c l i c AMP: the membrane-bound c y c l i c AMP-specific phosphodiesterase, the c y c l i c AMP c e l l surface receptor and the i n t r a c e l l u l a r c y c l i c AMP-dependent prote in k inase. 70 Analogues of c y c l i c AMP wi th d i f f e r e n t a f f i n i t i e s f or these p r o t e i n s (Tab le I I I , taken from Van H a a s t e r t and K i e n , 1983; Schaap and Van D r i e l , 1985) were used to c h a r a c t e r i z e the b i n d i n g p r o t e i n i n v o l v e d in s t a l k c e l l i n d u c t i o n . F i g u r e 6 shows that c y c l i c AMP was e f f e c t i v e at micromolar c o n c e n t r a t i o n s , a l e v e l c l e a r l y f a r more p h y s i o l o g i c a l (Merkle e_t a l . . , 1984) than the 1 mM c o n c e n t r a t i o n used in the i n i t i a l s t u d i e s . 2 ' - d e o x y - c y c l i c AMP was n e a r l y as e f f e c t i v e in i n d u c i n g s t a l k c e l l f o r m a t i o n as c y c l i c AMP, whereas 8 - B r o m o - c y c l i c AMP was ten t imes l e s s e f f e c t i v e and c y c l i c IMP was a very poor i n d u c e r . T h i s o r d e r of p o t e n c i e s on ly c o r r e l a t e s w i th tha t f o r the c y c l i c AMP c e l l s u r f a c e r e c e p t o r s u g g e s t i n g that t h i s p r o t e i n most l i k e l y mediates the i n d u c t i o n of s t a l k c e l l f o r m a t i o n by exogenous c y c l i c AMP in low d e n s i t y monolayers . Other a u t h o r s a l s o found tha t these c y c l i c AMP analogues showed the same r e l a t i v e p o t e n c i e s in the i n d u c t i o n of p r e - s t a l k and p r e - s p o r e s p e c i f i c components, i m p l y i n g tha t the same c e l l s u r f a c e r e c e p t o r might a l s o mediate the f o r m a t i o n of p r e - s t a l k and p r e - s p o r e c e l l s (Schaap and Van D r i e l , 1985; Oyama and Blumberg , 1986; Gomer et a l . , 1986a). 71 Table III The r e l a t i v e a f f i n i t y of three c y c l i c AMP-binding proteins for c y c l i c AMP and c y c l i c AMP analogues. Cell surface 0 I n t r a c e l l u l a r chemotactic c y c l i c AMP-receptor dependent protein kinase Plasma membrane phosphod iesterase c y c l i c AMP 8-bromo-c y c l i c AMP 2'-deoxy-c y c l i c AMP 1 0.005 0. 14 1 2.5 0.0004 1 0. 15 0.22 c y c l i c IMP 0.004 0.025 0.08 "Values were taken from Schaap and Van Driel (1985) and are expressed as a f f i n i t y r e l a t i v e to c y c l i c AMP. 72 CONCENTRATION (>iM) Fig. 6. Comparison of the effect of various c y c l i c AMP analogues on stalk formation in low density monolayers. Cell s were plated in monolayers in 2 ml LDMS containing 150 units DIF. After 8 hours incubation, c y c l i c AMP (O), 2'-deoxy-cyclic AMP (•> , 8-bromo-cyclic AMP (•) or c y c l i c IMP <•) were added at the indicated concentrations. Stalk formation was assessed aft e r 48 hours. Values are averaged from two determinations. 73 D. Experiments with potential antagonists of c y c l i c AMP and DIF. < i) . Ammon ia. Ammonia has been shown to enhance spore formation and to i n h i b i t stalk c e l l formation in a number of in. v i tro systems (Wilcox and Sussman, 1978; Sternfeld and David, 1979; Gross et al.. , 1981). It is known to be secreted at high levels during development (Gregg e_t al.. , 1954) and Gross and co-workers have suggested that i t might play a role in pattern regulation as a DIF antagonist. Ammmonium chloride was inhibitory at pH 7.5, but not at pH 6.2 suggesting that free ammonia was the active species (Gross et al_. , 1981 ) . When tested in monolayers, ammonia i n h i b i t i o n was found to be independent of DIF concentration (Table IV). Stalk c e l l formation was inhibited to approximately 60% by 1 mM NH^Cl, 84% by 3 mM NH^Cl and 95% by 10 mM NH^Cl at a l l three levels of DIF examined. T r i s - C l , pH 7.5 was not i t s e l f i nhibitory compared with MES, pH 6.2. Table IV Effect of varying NH^Cl and DIF concentrations on stalk formation in low density monolayers. DIF a MES, pH 6.2 T r i s - C l , pH 7.5 Added NH^Cl <mM) 0 1 3 10 1 Pi 14 14 7 2 1 2 P 1 28 30 18 5 1 3 pi 42 43 26 8 3 a C e l l s were plated in LDMS (buffered with 5 mM MES, pH 6.2 or 5 mM T r i s - C l , pH 7.5) containing I mM c y c l i c AMP and DIF and NH4CI as indicated. Stalk c e l l formation was assessed afte r 48 hours. 75 Removal experiments were ca r r i e d out at pH 7.5 to determine the time when ammonia inhibited stalk c e l l formation in low density monolayers. Under these conditions, stalk c e l l formation was found to be slower than at pH 6.2, and the period of DIF requirement followed the period of c y c l i c AMP requirement by 3 to 6 hours (Figure 7). To determine the time when ammonia acted in monolayers, 2 mM or 8 mM ammonium chloride was added at regular intervals during development (Figure 8). Maximum i n h i b i t i o n by NH^Cl occurred during the f i r s t 6 hours in monolayers and gradually lessened as c e l l s met the i r requirement for c y c l i c AMP. Although ammonia was s t i l l i n hibitory when added as late as 18 hours, the i n h i b i t i o n c l e a r l y preceded the period of DIF requirement by several hours, again suggesting that i t did not act by antagonizing DIF. Other experiments showed that ammonia i n h i b i t i o n could not be reversed by addition of up to 10 mM c y c l i c AMP (data not shown) suggesting that ammonia did not i n h i b i t stalk c e l l formation by a d i r e c t interaction with the c e l l surface c y c l i c AMP receptor. 76 2 2 « 12 24 36 48 TIME (H.) F i g . 7. The e f f e c t of c y c l i c AMP or DIF removal on s t a l k c e l l f o r m a t i o n d u r i n g d i f f e r e n t i a t i o n i n low d e n s i t y monolayers a t pH 7.5. C e l l s were p l a t e d i n 2 ml LDMS, b u f f e r e d w i t h T r i s - C l , pH 7.5 c o n t a i n i n g 1 mM c y c l i c AMP and 18 to 55 u n i t s DIF. S u p e m a t a n t s were removed a t the i n d i c a t e d t i m e s , monolayers were r i n s e d 3 t i m e s w i t h Bonner's s a l t s and LDMS w i t h o u t e i t h e r c y c l i c AMP <0> o r DIF <•) was r e p l e n i s h e d . S t a l k f o r m a t i o n was a s s e s s e d a f t e r 48 h o u r s . I n a d d i t i o n , the p r o p o r t i o n of s t a l k c e l l s i n an u n p e r t u r b e d i n c u b a t i o n was d e t e r m i n e d a t the i n d i c a t e d t i m e s (A,). The r e s u l t s are e x p r e s s e d as the p e r c e n t a g e of s t a l k c e l l s r e l a t i v e t o the maximum number i n d u c e d , and they r e p r e s e n t the average f o r 3 s e p a r a t e e x p e r i m e n t s . The maximum l e v e l of s t a l k c e l l f o r m a t i o n a t t a i n e d f o r each e x p e r i m e n t was 18%, 44% and 55%. <0 8 1 6 2 4 3 6 T I M E (H.) F i g . 8. The period of s e n s i t i v i t y to NH^Cl i n h i b i t i o n during d i f f e r e n t i a t i o n in low density monolayers. Cell s were plated in 2 ml LDMS with T r i s - C l , pH 7.5 containing 1 mM c y c l i c AMP and 35 units DIF. At the indicated times, NH^Cl was added to produce a f i n a l concentration of 2 mM (A) or 8 mM <•). In another set of plates, supernatants were removed at the indicated times, monolayers were rinsed 3 times with Bonner's s a l t s and LDMS at pH 7.5 containing 35 units DIF was replenished (O) . Results are expressed r e l a t i v e to the maximum level of stalk c e l l formation attained, 35%. For a l l samples, stalk c e l l formation was determined a f t e r 48 hours of incubation. 78 ( i i ) Caffeine. Caffeine was shown to s p e c i f i c a l l y i n h i b i t the well characterized c y c l i c AMP a c t i v a t i o n of adenylate cyclase in aggregation competent c e l l s (Brenner and Thorns, 1984). The adaptation of c e l l s to stimulation by c y c l i c AMP was not affected by th i s drug (Theibert and Devreotes, 1983). Although the calcium ionophore A23187 was shown to be toxic to c e l l s , i t was also found to i n h i b i t the c y c l i c AMP ac t i v a t i o n of adenylate cyclase, suggesting that the i n h i b i t i o n by caffeine might r e s u l t from an ef f e c t on c y t o s o l i c levels of calcium (Brenner and Thorns, 1984). Figure 9 shows that caffeine markedly inhibited stalk c e l l formation in monolayers. However, varying the external calcium concentration over a 100 fo l d range had very l i t t l e e f f e c t on stalk c e l l formation or on the inhibitory e f f e c t of caffeine (Figure 10), suggesting that external calcium was not a l i m i t i n g factor for stalk c e l l formation. 79 S T A L K I N D U C T I O N (%) 100 0 1 2 C O N C E N T R A T I O N ( m M ) F i g . 9. The e f f e c t o f d e n s i t y m o n o l a y e r s . c a f f e i n e on s t a l k f o r m a t i o n i n low C e l l s were p l a t e d i n LDMS c o n t a i n i n g 1 mM c y c l i c AMP, 150 u n i t s D I F and c a f f e i n e a t t h e i n d i c a t e d c o n c e n t r a t i o n s . S t a l k c e l l f o r m a t i o n was a s s e s s e d a f t e r 48 h o u r s . V a l u e s a r e a v e r a g e d f r o m t h r e e e x p e r i m e n t s a nd a r e shown w i t h s t a n d a r d d e v i a t i o n s . 80 STALK INDUCTION (%) 100 80-1 60 4 0 20- -1 I I i 1 1 0.1 0-5 2.0 5.0 100 C O N C E N T R A T I O N (mM) Fig. 10. The influence of calcium levels on caffeine i n h i b i t i o n of stalk formation in low density monolayers. Cell s were plated in 2 ml medium containing: 10 mM NaCl, 10 mM KCI, 5 mM MES, pH 6.2, 100 ug/ml streptomycin sulfate, 1 mM c y c l i c AMP and 150 units DIF. Calcium was added at the indicated concentrations for incubations containing no caffeine (I I) or 5 mM caffeine <^). Stalk formation was assessed aft e r 48 hours. Values are averaged from three determinations and are shown with standard deviations. 81 In contrast, figure 11 shows that the c y c l i c AMP analogue 8-Bromo-cyclic AMP p a r t i a l l y reversed the in h i b i t i o n of stalk c e l l formation by caffeine. Though exogenous c y c l i c AMP was present at saturating concentrations in a l l the caffeine experiments (Figures 9, 10 and 11), i n h i b i t i o n of stalk c e l l formation was only relieved when 8-Bromo-cyclic AMP was present (Figure 11), despite the lower a f f i n i t y of th i s c y c l i c AMP analogue for the c e l l surface c y c l i c AMP receptor (Table I I I ) . Since this analogue has been reported to cross the plasma membrane of mammalian c e l l s (Free ejt. al.., 1971; Rubin e_t al.., 1971) the reversal of caffeine i n h i b i t i o n is very l i k e l y due to the uptake of 8-Bromo-cyclic AMP, circumventing the block in c y c l i c AMP synthesis. Consistent with this view is the fact that 8-Bromo-cyclic AMP alone induced stalk c e l l formation at micromolar concentrations (Figure 6) whereas the reversal of caffeine i n h i b i t i o n required millimolar concentrations (Figure 11). Caffeine inhibited the induction of stalk c e l l s by both c y c l i c AMP and DIF in experiments where both factors were added independently (Figure 12), suggesting that the act i v a t i o n of adenylate cyclase was possibly involved in both inductive events. STALK INDUCTION (%) 80 60 40 20 0 - 4 0.0 0.2 0.4 0.6 0.8 1.0 8 _ B r - c A M P CONCENTRAT ION (mM) Fig. 11. The e f f e c t of 8-Bromo-cyclic AMP on the i n h i b i t i o n of stalk c e l l formation by caffeine in low density mono 1ayers. Cells were plated in LDMS containing 1 mM c y c l i c AMP, 150 units DIF, 5 mM caffeine and the indicated lev/els of 8-bromo-cyclic AMP. Stalk formation was determined a f t e r 48 hours. The amount of stalk c e l l formation in a control incubation without added caffeine was 85% and is denoted by the dotted l i n e . Values are averaged from three determinations and are shown with standard deviations. 83 STALK INDUCTION (%) 8 10 CONCENTRAT ION (mM) Fig. 12. The e f f e c t of caffeine on stalk c e l l induction by c y c l i c AMP and by DIF in low density monolayers. Cell s were plated for 24 hours in LDMS containing 1 mM c y c l i c AMP and the indicated level of caffeine (A), rinsed three time with Bonner's s a l t s and replenished with LDMS containing 150 units DIF; or plated for 24 hours in LDMS containing 1 mM c y c l i c AMP, followed by LDMS containing 150 units DIF and the indicated level of caffeine (O) . Stalk c e l l formation was determined 48 hours a f t e r the beginning of the experiment. Values are averaged from two experiments and are shown with standard deviations. SUMMARY OF PART I In the f i r s t part of this thesis, the res u l t s from factor addition and removal experiments suggested that, st a r t i n g between 8 and 10 hours in low density monolayers, c y c l i c AMP and DIF acted sequentially to induce stalk c e l l formation. The k i n e t i c s of induction indicated that they acted within two hours of each other. C y c l i c AMP was found to be active at micromolar concentrations, and experiments with c y c l i c AMP analogues and with caffeine suggested that i t s binding to the c e l l surface c y c l i c AMP surface receptor might cause an increase in the c e l l u l a r concentration of c y c l i c AMP. Although DIF could induce stalk c e l l s independently from c y c l i c AMP, a number of experiments indicated a possible interaction between these two factors. The timing of the inductive events in low density monolayers suggests that the formation of pre-stalk c e l l s might be the i r s i t e of action. However, these experiments did not ruled out the p o s s i b i l i t y that these two components might instead be involved in the conversion of pre-stalk to stalk c e l l s . The experiments described in the Part II were performed to try to resolve this question. 85 PART II THE REQUIREMENTS OF CYCLIC AMP AND DIF FOR  EXPRESSION OF PRE-STALK AND STALK CELL MARKERS In order to e s t a b l i s h the degree of c o r r e l a t i o n between development in v i t r o and development in vivo and to try to determine i f c y c l i c AMP and DIF were required e i t h e r for pre-stalk formation or the conversion of pre-stalk to stalk c e l l s , components unique to pre-stalk and stalk c e l l s were needed as markers for c e l l d i f f e r e n t i a t i o n . This presented considerable d i f f i c u l t y because r e l a t i v e l y few pre-stalk and stalk c e l l s s p e c i f i c components have been reported that can be detected with s u f f i c i e n t s e n s i t i v i t y in low density monolayers. E f f o r t s to adapt and u t i l i z e previously i d e n t i f i e d c e l l components and to e s t a b l i s h new ones as d i f f e r e n t i a t i o n markers are described below. A. Acid Phosphatase Acid phosphatase II (AP II) is the only enzyme that has thus far been shown to be unique to pre-stalk and stalk c e l l s (the other enzymes detected only in pre-stalk c e l l s are a fl-galactosidase isozyme, Ooohata and Takeuchi, 1977, and a fl-mannosidase isozyme, Devine and Loomis, 1985). This isozyme can be separated by non-denaturing polyacrylamide gel electrophoresis from acid phosphatase I (AP I ) , an 86 enzyme present in a l l ce l l - t y p e s (Oohata, 1983). AP II was reported to be f i r s t detectable at the finger stage during development in s t r a i n V12 M2, 2 hours aft e r a pre-spore c e l l marker UDP-galactosyl:mucopolysaccharide transferase was f i r s t detected (Oohata, 1983). It has been postulated that AP II arises by post-translational modification of AP I because the two isozymes share antigenic determinants and because mutants with altered AP I a c t i v i t y show identical changes in AP II a c t i v i t y (Loomis and Kuspa, 1984; Tasaka et al . , 1986). Weijer and Durston ( 1985) and more recently Oohata (1986) have i d e n t i f i e d another isozyme with a mobility intermediate between AP I and AP II that has been designated AP IV (Weijer and Durston, 1985; Oohata, 1986). AP IV is also developmentally regulated, but i t is present in both pre-stalk and pre-spore c e l l s (Weijer and Durston, 1985). Figure 13 shows that pre-stalk c e l l extracts contain the low mobility acid phosphatase a c t i v i t y (AP II) whereas this a c t i v i t y is absent in pre-spore c e l l extracts, as reported e a r l i e r by Oohata (1983). Although not resolved as a d i s t i n c t band, some enzyme a c t i v i t y with a mobility intermediate between the pre-stalk and the vegetative isozymes was also detectable in both c e l l - t y p e s . 87 8 j j g 9/jg TOP BOT 16/jg rsjjg TOP BOT O AP 2 < AP4 <3 AP 1 F i g . 13. A c i d phosphatase a c t i v i t y of s l u g c e l l s s e p a r a t e d by d e n s i t y c e n t r i f u g a t i o n on P e r c o l l g r a d i e n t s . C e l l s from e n z y m a t i c a l 1 y d i s r u p t e d s l u g s were s e p a r a t e d on P e r c o l l g r a d i e n t s as d e s c r i b e d under Methods and s o l u b i l i z e d i n 0.5% NP-40. The i n d i c a t e d amounts of p r o t e i n e x t r a c t of l i g h t d e n s i t y c e l l s (TOP) or heavy d e n s i t y c e l l s (BOT) were e l e c t r o p h o r e s e d and s t a i n e d f o r a c i d phosphatase as d e s c r i b e d under Methods. The t h r e e isozymes of a c i d phosphatase are i n d i c a t e d (AP 1, 2, 4 ) . 88 The mobility of this enzyme a c t i v i t y corresponds to the AP IV a c t i v i t y reported by Weijer and Durston (1985) and by Oohata (1986) and i t w i l l t e n t a t i v e l y be referred to as AP IV in the following sections. The expression of acid phosphatase a c t i v i t y during development on M i l l i p o r e f i l t e r s was investigated. Figure 14 shows that AP II was f i r s t detected at 12 hours (the finger stage), confirming previously published r e s u l t s (Oohata, 1983). Enzyme a c t i v i t y of intermediate mobility, which probably corresponds to AP IV, was f i r s t detected in tipped aggregates at 9 hours of development, prior to the establishment of the discrete pre-stalk and pre-spore pattern. Despite e a r l i e r indications that developmentally regulated acid phosphatase a c t i v i t i e s were not expressed in  v i tro (Dominov and Town, 1986; Kay, personal communication), acid phosphatase isozymes were detectable in c e l l s d i f f e r e n t i a t i n g in monolayers. Figure 15 shows that developmentally regulated enzyme a c t i v i t y was f i r s t detected between 6 to 8 hours in high density monolayers as a f a i n t l y staining region with lower mobility than AP I. Acid phosphatase a c t i v i t y of gradually decreasing mobility accumulated u n t i l 18 hours development. 89 0 3 6 9 12 15 18 21 24 v i l l i O AP 2 <3 AP4 <3 AP 1 F i g . 14. Acid phosphatase a c t i v i t y in c e l l s developing on Mi l l i p o r e f i l t e r s . C ells plated on M i l l i p o r e f i l t e r s were sampled during development at the times (Hours) indicated above. Cel l s were washed off f i l t e r s , c o l l e c t e d by centrifugation and s o l u b i l i z e d in 0.5% NP-40. Ce l l extracts containing 13 ug protein were electrophoresed and stained for acid phosphatase a c t i v i t y as described under Methods. The three isozymes of acid phosphatase are indicated (AP 1, 2, 4). 0 2 4 6 8 10 12 14 16 18 20 22 24 ••••• MlMM F i g . 15. A c i d p h o s p h a t a s e a c t i v i t y i n e x t r a c t s o f c e l l s d e v e l o p i n g i n m o n o l a y e r s a t h i g h c e l l d e n s i t y . C e l l s p l a t e d i n m o n o l a y e r s a t 1 0 d c e l l s / c m were h a r v e s t e d the i n d i c a t e d t i m e s and s o l u b i l i z e d i n 0.5% NP-40. C e l l e x t r a c t s c o n t a i n i n g 8 ug p r o t e i n were e l e c t r o p h o r e s e d and s t a i n e d f o r a c i d p h o s p h a t a s e a c t i v i t y as d e s c r i b e d under Me t h o d s . 91 Although the developmentally regulated a c t i v i t y did not resolve into d i s t i n c t bands, i t s mobility corresponded to that of a mixture of AP II and AP IV. The overall pattern was similar to that observed i_n vivo, except that the formation of AP II appears to be somewhat delayed. Figure 16 shows that in low density monolayers the pre-stalk AP II was only expressed in c e l l s incubated in the presence of both c y c l i c AMP and DIF (compare lanes 1 and 5), suggesting that i t s expression in monolayers required both factors. C e l l s incubated with c y c l i c AMP alone expressed AP IV a c t i v i t y (lane 4) but only AP I was detectable in c e l l s incubated in unsupplemented LDMS (lane 3). These data indicate that that the expression of AP IV, which f i r s t appeared in. vivo in tipped aggregates (see figure 14), was dependent on c y c l i c AMP and the expression of the pre-stalk s p e c i f i c AP 11 was DIF-dependent. The time of appearance of the acid phosphatase isozymes during c e l l d i f f e r e n t i a t i o n in low density monolayers was examined (Figure 17). AP IV was f i r s t f a i n t l y detectable in 14 hour extracts of c e l l s in the presence of either c y c l i c AMP alone (lane 7) or c y c l i c AMP and DIF (lane 8). The pre-stalk s p e c i f i c AP II was also f i r s t detectable at 14 hours (lane 8) but only in c e l l s incubated with both c y c l i c AMP and DIF, and i t became increasingly d i s t i n c t at later times. 92 F i g . 16. DlF-dependence of the e x p r e s s i o n of the p r e - s t a l k s p e c i f i c a c i d phosphatase a c t i v i t y i n low d e n s i t y monolayers . C e l l s were p l a t e d in 5 ml unsupplemented LDMS ( lane 3 ) , or LDMS c o n t a i n i n g 1 mM c y c l i c AMP ( lane 4 ) , or 1 mM c y c l i c AMP and 450 u n i t s DIF ( lane 5 ) . A f t e r 2 4 . h o u r s , they were c o l l e c t e d and s o l u b i l i z e d in 0.5% NP-40. S o l u b i l i z e d e x t r a c t s from 5x10^ c e l l s were e l e c t r o p h o r e s e d a t the same time as 6 pg e x t r a c t s of p r e - s t a l k p r o t e i n ( lane 1) and p r e -spore p r o t e i n ( lane 2) and s t a i n e d f o r a c i d phosphatase a c t i v i t y as d e s c r i b e d under Methods. The three isozymes of a c i d phosphatase are i n d i c a t e d (AP 1, 2, 4 ) . 93 1 2 3 4 5 6 7 8 9 10 11 12 F i g . 17. Acid phosphatase 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 in low density monolayers. Cells were plated in 5 ml LDMS containing 1 mM c y c l i c AMP (lanes 1, 3, 5, 7, 9, 11) or 1 mM c y c l i c AMP and 450 units DIF (lanes 2, 4, 6, 8, 10, 12). They were harvested aft e r 10 hours (lanes 1,2), 12 h (lanes 3,4), 14 h (lanes 5,6), 16 h (lanes 7,8), 20 h (lanes 9,10) and 24 h (lane 11,12) and so l u b i l i z e d in 0.5% NP-40. S o l u b i l i z e d extracts equivalent to 2.5x10^ c e l l s were electrophoresed and stained for acid phosphatase as described under Methods. The three isozymes of acid phosphatase are indicated (AP 1, 2, 4). These r e s u l t s show that the c y c l i c AMP-dependent AP IV and the DIF-dependent AP II appeared at the same time during d i f f e r e n t i a t i o n in low density monolayers. This correlates well with the factor removal experiment indicating that c y c l i c AMP independent c e l l s appear very shortly before DIF independent c e l l s (Figure 3). However, t h e i r appearance in low density monolayers is somewhat delayed r e l a t i v e to the time when they appear in. vivo (Figure 15). B. Rabbit a n t i - s t a l k and anti-pre-stalk immune sera A rabbit immunized by i n j e c t i n g crude stalk c e l l extracts produced antibodies that recognized s t a l k - s p e c i f i c antigens. The immune serum also detected a large number of vegetative and spore antigens, and extracts of amoebae, slugs and spores were used to adsorb non-specific antibodies. An immunoblot of separated stalk and spore c e l l extracts probed with the adsorbed serum revealed two stalk c e l l s p e c i f i c components, one at 310,000 Mr (ST 310) migrating as a diffuse band and a 41,000 Mr component (ST 41), as well as a number of spore antigens (Figure 18A). ST 41 has an apparent molecular weight close to that of a stalk s p e c i f i c antigen (ST 40) described by Dominov and Town (1986), suggesting that these two antigens may in fact be the same. 95 To determine i f thi s was the case, stalk c e l l extracts on a separate immunoblot were stained with an adsorbed preparation of Dominov and Town's B 11 serum (kindly donated by Dr Town) and compared with the blot probed with the adsorbed rabbit a n t i - s t a l k serum. Figures 18A (Rabbit serum) and 18B (B 11 serum) show that the stalk antigen migrating at 41,000 Mr stained i d e n t i c a l l y with both immune sera, suggesting that they might be detecting the same stalk c e l l component. Only ST 41 was reproducibly detected in low density monolayers, and i t f i r s t appeared at 16 hours when c e l l s were incubated with both c y c l i c AMP and DIF (lane 5, Figure 19). The expression of t h i s antigen increased at l a t e r times, but i t was never found in extracts of c e l l s incubated with c y c l i c AMP alone indicating that i t s expression was DIF-dependent. Dominov and Town (1986) detected c e l l s s taining for ST 40 as early as 12 hours in high density monolayers. However, they could not determine whether i t s expression was dependent on DIF because stalk c e l l formation was not dependent on exogenously supplied DIF under t h e i r conditions. ST 41 was not detectable u n t i l late f r u i t i n g body formation during in. v i vo development (lane 12, Figure 19), whereas Dominov and Town reported that ST 40 was expressed during early f r u i t i n g body formation. This r e s u l t may r e f l e c t greater s e n s i t i v i t y of the B 11 serum, or that the two immune sera in fact detect d i f f e r e n t antigens. F i g . 18. S p e c i f i c i t y of the a n t i g e n s d e t e c t e d by the adsorbed r a b b i t a n t i - s t a l k a n t i b o d y p r e p a r a t i o n . A. E x t r a c t s of s t a l k c e l l s ( l a n e 1) and spore c e l l s ( l a n e 2) c o n t a i n i n g 40 ug p r o t e i n were s o l u b i l i z e d i n SDS by h e a t i n g a t 95°C. They were e l e c t r o p h o r e s e d on an SDS p o l y a c r y l a m i d e g e l as d e s c r i b e d under Methods and the p r o t e i n bands were e l e c t r o b l o t t e d onto a sheet of n i t r o c e l l u l o s e . The b l o t was f i r s t i n c u b a t e d w i t h a r a b b i t a n t i - s t a l k a n t i b o d y p r e p a r a t i o n t h a t had been adsorbed a g a i n s t v e g e t a t i v e , s l u g and spore c e l l e x t r a c t s , and the bound r a b b i t a n t i b o d i e s were d e t e c t e d u s i n g a b i o t i n y l a t e d goat a n t i - r a b b i t serum, a l k a l i n e p h o s p h a t a s e - c o n j u g a t e d s t r e p a v i d i n and b i o t i n y l a t e d a l k a l i n e phosphatase a s d e s c r i b e d under Methods. B. An i d e n t i c a l immunoblot i s shown, except t h a t i t was i n i t i a l l y i n c u b a t e d w i t h the B 11 s t a l k a n t i s e r u m adsorbed as the above serum. See t e x t f o r f u r t h e r d e t a i l . 97 1 2 3 4 5 6 7 8 9 1011 12 ST41 F i g . 19. Detection of a stalk antigen by an adsorbed rabbit an t i - s t a l k antibody preparation during c e l l d i f f e r e n t i a t i o n in vivo and in low density monolayers. Cells were plated on M i l l i p o r e f i l t e r s (lanes 3, 6, 9, 12) or as monolayers in 5 ml LDMS containing 1 mM c y c l i c AMP (lanes 1, 4, 7, 10) or 1 mM c y c l i c AMP and 450 units DIF (lanes 2, 5, 8, 11). They were sampled at 12 h (lanes 1-3), 16 h (lanes 4-6), 20 h (lanes 7-9) and 24 h (lanes 10-12) and s o l u b i l i z e d in SDS by heating at 95°C. S o l u b i l i z e d extracts containing 24 pg protein for c e l l s developing in vivo or from 10° c e l l s from monolayers were electrophoresed on a SDS polyacrylamide g e l . Antigens were detected as described in F i g . 18A. 98 A rabbit anti-pre-stalk immune serum was raised using pre-stalk c e l l extracts that had been adsorbed with an immune serum raised against pre-spore and spore c e l l s . Unique pre-stalk components could be detected by this serum af t e r i t had been adsorbed extensively against extracts of vegetative, pre-spore and spore c e l l s , and two such components were detectable in low density monolayers (Figure 20). Expression of a pre-stalk s p e c i f i c 110,000 Mr (PST 110) antigen in low density monolayers required the presence of c y c l i c AMP and DIF (lane 3, Figure 20). A 166,000 Mr pre-stalk s p e c i f i c antigen (PST 166) was detectable in extracts of c e l l s incubated with c y c l i c AMP alone (lane 4) but i t s level was considerably enhanced by the addition of DIF (lane 3). This r e s u l t showed that both c y c l i c AMP and DIF were essential for the f u l l expression of two pre-stalk components, PST 110 and PST 166, in low density monolayers and is consistent with the hypothesis that DIF is necessary for the expression of pre-stalk c e l l s p e c i f i c components. This experiment was performed using a small batch of serum which had been extensively adsorbed. Unfortunately, the remaining serum became contaminated before i t could be adsorbed s u f f i c i e n t l y to extend this finding, and the expression of PST 110 and PST 166 during d i f f e r e n t i a t i o n in vivo and in v i t r o could not be established. 99 1 2 3 4 ^ B M W • F i g . 20. P r e - s t a l k a n t i g e n s d e t e c t e d by an adsorbed r a b b i t a n t i - p r e - s t a l k a n t i b o d y p r e p a r a t i o n in c e l l s d e v e l o p i n g in low d e n s i t y monolayers . S o l u b i l i z e d e x t r a c t s (24 ug p r o t e i n ) of p r e - s t a l k ( lane 2) _ or p r e - s p o r e c e l l s ( lane 1) , or e x t r a c t s e q u i v a l e n t to 5x10 c e l l s d i f f e r e n t i a t i n g in monolayers f o r 24 hours in LDMS c o n t a i n i n g I mM c y c l i c AMP ( lane 4) or 1 mM c y c l i c AMP and 450 u n i t s DIF ( lane 3) were e l e c t r o p h o r e s e d on a SDS p o l y a c r y l a m i d e g e l . The p r o t e i n bands were e l e c t r o b l o t t e d o n a sheet of n i t r o c e l l u l o s e . The b l o t was f i r s t incubated wi th a r a b b i t a n t i - p r e - s t a l k a n t i b o d y p r e p a r a t i o n adsorbed a g a i n s t v e g e t a t i v e , p r e - s p o r e and spore c e l l e x t r a c t s , and the bound r a b b i t a n t i b o d i e s were d e t e c t e d u s i n g a b i o t i n y l a t e d goat a n t i - r a b b i t serum and a l k a l i n e p h o s p h a t a s e - c o n j u g a t e d s t r e p a v i d i n as d e s c r i b e d under Methods. 100 C. Mouse a n t i - s t a l k immune serum An additional polyclonal serum was prepared by immunizing mice with stalk c e l l extracts. This serum also required extensive adsorption with extracts of vegetative, slug and spore c e l l s before i t could detect developmentally regulated antigens in low density monolayers. The adsorbed serum detected a high molecular weight antigen (approximately 260,000 Mr) which appeared at 18 hours at the culmination stage during development Ui vivo (Figure 21). It appeared at 16 hours during d i f f e r e n t i a t i o n in low density monolayers (Figure 22) and i t s expression was t o t a l l y dependent on the presence of DIF (compare lanes 6 and 8 with lanes 5 and 7). Furthermore, i t was detectable within 2 hours of adding DIF to c e l l s incubated for 20 hours with c y c l i c AMP (lane 9), c l e a r l y indicating that i t s expression was DlF-dependent. This serum also detected a lower molecular weight component which f i r s t appeared at 20 hours in v i tro, and was dependent upon the presence of c y c l i c AMP and DIF. However, this component was undetectable in extracts of c e l l s developing in vivo. 101 2 4 6 8 10 12 14 16 18 2022 24 " M W ST260 F i g . 21. D e t e c t i o n of d e v e l o p m e n t a l l y r e g u l a t e d a n t i g e n s by an adsorbed mouse a n t i - s t a l k a n t i b o d y p r e p a r a t i o n . C e l l s d e v e l o p i n g on M i l l i p o r e f i l t e r s were sampled a t the i n d i c a t e d times and s o l u b i l i z e d i n SDS by h e a t i n g a t 95°C. E x t r a c t s c o n t a i n i n g 32 ug p r o t e i n were e 1 e c t r o p h o r e s e d on an SDS-gel and the p r o t e i n bands were e l e c t r o b l o t t e d on a sheet of n i t r o c e l l u l o s e . The b l o t was f i r s t i n c u b a t e d w i t h a mouse a n t i - s t a l k a n t i b o d y p r e p a r a t i o n adsorbed a g a i n s t e x t r a c t s of v e g e t a t i v e , s l u g and spore c e l l s , and the bound a n t i b o d i e s were d e t e c t e d w i t h an a l k a l i n e p h o s p h a t a s e - c o n j u g a t e d goat anti-mouse serum as d e s c r i b e d under Methods. 102 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 ' MW F i g . 22. Detection of developmentally regulated antigens by an adsorbed mouse a n t i - s t a l k antibody preparation during c e l l d i f f e r e n t i a t i o n in low density monolayers. Cell s were plated in 5 ml LDMS containing 1 mM c y c l i c AMP (lanes 1, 3, 5, 7) or 1 mM c y c l i c AMP and 450 units DIF (lanes 2, 4, 6, 8). They were sampled at 0 hours (lane 1), 8 hours (lanes 2,3), 12 h (lanes 4,5), 16 h (lanes 6,7) and 20 h (lanes 8,12). In addition, some c e l l s were incubated in LDMS containing 1 mM c y c l i c AMP for 20 hours and were sampled afte r incubation with DIF for 2 hours (lane 9), 4 hours (lane 10), or 6 hours (lane 11). C e l l s were s o l u b i l i z e d in SDS by heating at 95°C, and s o l u b i l i z e d extracts from 10^ monolayer c e l l s were electrophoresed on a SDS polyacrylamide g e l . Antigens were detected as described in F i g . 21. 103 SUMMARY OF PART II Several experiments were ca r r i e d out to examine the requirements of c y c l i c AMP and DIF for expression of pre-stalk and stalk c e l l markers in low density monolayers and they are summarized in figure 23. Two major conclusions can be drawn from these studies. Two pre-stalk c e l l s p e c i f i c components (AP II and PST 110) were t o t a l l y dependent on the presence of DIF for t h e i r expression in low density monolayers, providing support for the hypothesis that DIF is an inducer of pre-stalk c e l l formation. DIF was also essential for the expression of stalk c e l l antigens in low density monolayers, and one of these components was detectable 2 hours a f t e r the addition of DIF in monolayers. A comparison of the time of appearance of the various components (Figure 23) reveals that d i f f e r e n t i a t i o n is considerably compressed in monolayers compared to in. v i vo development. The pre-stalk s p e c i f i c AP II and stalk s p e c i f i c ST 41 and ST 260 are expressed within 2 hours of each other in v i.trp whereas t h e i r appearance in v i vo is separated by 6 to 10 hours. Thus the appearance of stalk c e l l antigens during d i f f e r e n t i a t i o n in monolayers seems to be accelerated compared with the time of t h e i r appearance in. vivo, while 104 the appearance of AP II in, v i t r o is more c l o s e l y correlated to the time of i t s appearance in. vivo. 105 A P E AP LT IN. VIVO C IN. VITRO IN VIVO 1M.VITRQ ST 41 ST 260 IN VITRO I N V I V O 0 IN VITRO IN VIVO 4 8 12 16 20 24 T I M E ( H o u r s ) F i g . 23. E x p r e s s i o n of p r e - s t a l k and s t a l k c e l l components d u r i n g development .in v i v o and i n low d e n s i t y monolayers. The times when the i n d i c a t e d p r e - s t a l k and s t a l k c e l l components were d e t e c t e d i n c e l l s d e v e l o p i n g on M i l l i p o r e f i l t e r s or i n low d e n s i t y monolayers are r e p r e s e n t e d by the above bar graph. A s t i p p l e d bar i n d i c a t e s t i m e s when components were f a i n t l y d e t e c t e d and a p l a i n bar i n d i c a t e s times when components were e a s i l y d e t e c t e d . 106 PART III EXPERIMENTS WITH DISRUPTED DEVELOPING CELLS A. Requirements of i n v i vo d e v e l o p i n g c e l l s f or s t a l k c e l l f o r m a t i o n in monolayers . Exper iments were c a r r i e d out to e s t a b l i s h the t imes when c e l l s d e v e l o p i n g in . v i vo become independent of c y c l i c AMP and DIF f o r s t a l k c e l l f o r m a t i o n i n v i t r o . At r e g u l a r i n t e r v a l s d u r i n g development on M i l l i p o r e f i l t e r s , c e l l s were h a r v e s t e d , r e - s u s p e n d e d in 3 ml B o n n e r ' s s a l t s and d i s a g g r e g a t e d by g e n t l e t r i t u r a t i o n . The d i s a g g r e g a t e d c e l l s were d i l u t e d and p l a t e d in LDMS in the presence or absence of D I F , in o r d e r to determine whether they r e q u i r e d DIF and c y c l i c AMP f o r i n v i t r o d i f f e r e n t i a t i o n . C e l l s h a v i n g met t h e i r requ irement f o r c y c l i c AMP d u r i n g i n v i vo d i f f e r e n t i a t i o n s h o u l d be ab le to form s t a l k c e l l s in low d e n s i t y monolayers in the presence of DIF a l o n e , whereas those h a v i n g met t h e i r requ irement for both c y c l i c AMP and DIF shou ld be ab le to form s t a l k c e l l s in t h e i r absence . F i g u r e 24A shows tha t c y c l i c AMP independent c e l l s f i r s t appeared at 12 h o u r s , a time o n l y s l i g h t l y d e l a y e d compared to that i n d i c a t e d by the f a c t o r removal exper iments in monolayers ( F i g u r e 3 ) . 107 a 8 16 24 TIME (h.) F i g . 24. The c y c l i c AMP and DIF requirements for stalk c e l l formation in low density monolayers for c e l l s developing in  vivo. A. C e l l s developing on M i l l i p o r e f i l t e r s were harvested in Bonner's s a l t s at the indicated times, disaggregated by gentle t r i t u r a t i o n as described in Methods and plated in: LDMS containing 150 units DIF (O), LDMS alone (•) or LDMS containing 1 mM c y c l i c AMP for 2 hours, followed by LDMS containing 150 units DIF for the remainder of the incubation (•). Stalk c e l l formation was assessed 48 hours a f t e r the beginning of the experiment. Values are averaged from three experiments and are shown with standard deviations. Stalk c e l l formation in unsupplemented LDMS was < 1% for a l l samples, and for c e l l s plated in LDMS containing 1 mM c y c l i c AMP and 150 units DIF for 48 hours was 90.7 ± 1.9%. B. C e l l s were sampled during development as above and plated in LDMS containing 150 units DIF (O), or containing 1 mM c y c l i c AMP (•) or 25 pM c y c l i c AMP (•) for 2 hours followed by LDMS containing 150 units DIF for the remainder of the incubation. Stalk c e l l formation was assessed 48 hours a f t e r the beginning of the experiment. The data are averaged from two determinations. 1 0 8 The number of stalk c e l l s produced in this assay increased with time as ui vivo development progressed, suggesting a ce r t a i n amount of heterogeneity for the c y c l i c AMP requirement within the d i f f e r e n t i a t i n g c e l l population. There was l i t t l e stalk c e l l formation in the absence of DIF, even when culminating c e l l s were disrupted (Figure 24A) and i t is l i k e l y that the stalk c e l l s scored late in development arose from c e l l s that were p a r t i a l l y vacuolated at the time of disaggregation. This result was quite unexpected in view of the finding that c e l l s d i f f e r e n t i a t i n g in monolayers became independent of DIF only a short time afte r they become independent of c y c l i c AMP (Figure 3). Considering the data indicating that induction of stalk c e l l formation by c y c l i c AMP was mediated by the c e l l surface c y c l i c AMP receptor (Figure 6), a component that appears early in development (Devreotes, 1982), i t was pertinent to ask why c e l l s do not become independent of c y c l i c AMP u n t i l 12 hours of development. To determine whether c e l l s developing in, vivo would respond to e x t r a c e l l u l a r c y c l i c AMP j_n v i tro e a r l i e r than 12 hours of development, disaggregated c e l l s were plated in monolayers in the presence of 1 mM c y c l i c AMP for 2 hours and then with DIF for the remainder of the incubation period. 109 Figure 24A shows that c e l l s disaggregated as early as 6 hours of development could be induced to form stalk c e l l s a f t e r a 2 hour incubation with c y c l i c AMP. Since aggregation was f u l l y in progress at 6 hours of development, the c y c l i c AMP surface receptor must have been present at that time, further supporting a role for this receptor in stalk c e l l formation. The dose dependence for this response was examined. Figure 24B shows that while 1 mM c y c l i c AMP could induce the formation of stalk c e l l s in monolayers afte r c e l l s had developed for 6 hours in. vivo, 25 uM c y c l i c AMP did not induce c e l l s u n t i l 8 hours of development in. vivo. The number of c e l l s which could be induced by 25 pM c y c l i c AMP rose gradually during development in. v i vo whereas the maximum number of c e l l s inducible by 1 mM c y c l i c AMP was attained from the onset at 6 hours. B. Studies of isolated pre-stalk and pre-spore c e l l s . The r e s u l t s from the disaggregation experiments suggested the p o s s i b i l i t y that DIF may be necessary for the pre-stalk to stalk c e l l conversion since disaggregated slugs, which contain pre-stalk c e l l s , were found to require DIF for stalk c e l l formation in monolayers. This was further investigated by determining the c y c l i c AMP and DIF requirements of isolated pre-stalk and pre-spore c e l l s for stalk c e l l formation in low density monolayers. 110 Table V shows that pre-stalk c e l l s only required DIF to form stalk c e l l s in monolayers, although the presence of c y c l i c AMP markedly enhanced the overall level of stalk c e l l formation. On the other hand, pre-spore c e l l s had an absolute requirement for c y c l i c AMP and DIF for stalk c e l l formation. Pre-stalk and pre-spore c e l l s also d i f f e r in the i r response to increasing amounts of DIF (Figure 25): pre-stalk c e l l s consistently formed 30% more stalk c e l l s than pre-spore c e l l s in response to the same doses of DIF, up to saturating l e v e l s . Table V Requirements of pre-stalk and pre-spore c e l l s for stalk formation in low density monolayers. Ce11-type Treatment q % Stalk c e l l formation Pre-stalks c y c l i c AMP + DIF 90.0 DIF 19.0 Pre-spores c y c l i c AMP + DIF 81.0 DIF 1.0 0Enzymatical1y disrupted slug c e l l s were separated into pre stalk and pre-spore fractions on Percoll density gradients. Ce l l s were washed and plated in LDMS containing 1 mM c y c l i c AMP and 150 units DIF as indicated. ^Stalk c e l l formation was assessed a f t e r 48 hours. STALK INDUCTION (%) 80 60 40 20 DIF (Units) F i g . 25. S t a l k c e l l f o r m a t i o n by i s o l a t e d p r e - s t a l k and p r e -spore c e l l s i n response t o i n c r e a s i n g amounts of DIF. E n z y m a t i c a l 1 y d i s r u p t e d s l u g c e l l s were s e p a r a t e d i n t o p r e -s t a l k and p r e - s p o r e c e l l s on P e r c o l l d e n s i t y g r a d i e n t s . The p r e - s t a l k c e l l s «0) and p r e - s p o r e c e l l s (A) were washed and p l a t e d i n LDMS c o n t a i n i n g 1 mM c y c l i c AMP and the i n d i c a t e d amounts of DIF. S t a l k c e l l f o r m a t i o n was a s s e s s e d a f t e r 48 h o u r s . 113 The requirement of pre-spore c e l l s for both c y c l i c AMP and DIF may indicate a c y c l i c AMP-dependent conversion of pre-spore to pre-stalk c e l l s p r i o r to stalk formation. To determine i f the conversion of pre-stalk c e l l s to stalk c e l l s occurs more rapidly than the conversion of pre-spore c e l l s to stalk c e l l s , the DIF requirement for isolated pre-stalk and pre-spore c e l l s was determined by DIF removal experiments. Figure 26 shows that the conversion of some pre-stalk c e l l s to stalk c e l l s required only one hour of incubation with DIF, whereas stalk c e l l formation from pre-spore c e l l s required a minimum incubation of 4 hour with both c y c l i c AMP and DIF. This 4 hour delay might be indicative of a conversion of pre-spore c e l l s to pre-stalk c e l l s , and is consistent with the 2-4 hour delay reported for the loss of a pre-spore antigen during the incubation of isolated pre-spore c e l l s in low density monolayers in the presence of DIF (Kay and Jermyn, 1983). The e f f e c t of caffeine on stalk c e l l formation from isolated pre-stalk and pre-spore c e l l s was also investigated. Figure 27 shows that stalk c e l l formation from both pre-stalk and pre-spore c e l l s was inhibited by caffeine. However, the pre-spore c e l l to stalk conversion was s i g n i f i c a n t l y more sensitive to caffeine 'inhibition, perhaps r e f l e c t i n g the fact that there was an absolute requirement for both c y c l i c AMP and DIF. 1 14 STALK INDUCTION (°/o) 0 1 2 3 4 B. 50 30 10 « 5 0 1 2 3 4 48 T IME (HOURS) F i g . and 26. K i n e t i c s of s t a l k i n d u c t i o n by DIF f o r p r e - s t a l k p r e - s p o r e c e l l s i n low d e n s i t y m o n olayers. E n z y m a t i c a l 1 y d i s r u p t e d s l u g c e l l s were s e p a r a t e d on P e r c o l l d e n s i t y g r a d i e n t s i n t o p r e - s t a l k and p r e - s p o r e c e l l s . The p r e - s t a l k «3> and p r e - s p o r e (A) c e l l s were washed and p l a t e d i n LDMS c o n t a i n i n g A) 1 mM c y c l i c AMP and 150 u n i t s DIF, or B) 150 u n i t s DIF. At the i n d i c a t e d t i m e s , s u p e r n a t a n t s were removed, monolayers were r i n s e d 3 t i m e s w i t h Bonner's s a l t s and LDMS was added. S t a l k c e l l f o r m a t i o n was a s s e s s e d 48 hours a f t e r the b e g i n n i n g of the e x p e r i m e n t . V a l u e s are aver a g e d from t h r e e e x p e r i m e n t s and are shown w i t h s t a n d a r d de v i a t i ons. STALK INDUCTION (%) 100 F i g . 27. Caffeine i n h i b i t i o n of stalk c e l l formation from isolated pre-stalk and pre-spore c e l l s in low density monolayers. Enzymatical1y disrupted slug c e l l s were separated on Percoll density gradients into pre-stalk and pre-spore c e l l s . The pre-stalk (O) and pre-spore (A) c e l l s were washed and plated in LDMS containing 1 mM c y c l i c AMP, 150 units DIF and the indicated concentrations of c a f f e i n e . Stalk c e l l formation was assessed a f t e r 48 hours. Values are averaged from t r i p l i c a t e s and are shown with standard deviations. SUMMARY OF PART III Cells that were independent for the c y c l i c AMP requirement for stalk c e l l formation in monolayers did not appear u n t i l about 12 hours of in. vivo development (the finger stage), although they acquired the potential to be induced by c y c l i c AMP e a r l i e r during aggregation (Summarized in Figure 28). Only few c e l l s which acquired independence from DIF could be detected late during culmination, when stalk c e l l formation occurs in. vivo, suggesting that either DIF independence is a late developmental event a f f e c t i n g a small number of c e l l s in the total population or that c e l l s lose the DIF-activated state upon disaggregation of developing c e l l masses. Both pre-stalk and pre-spore c e l l s p u r i f i e d from disrupted migrating slugs formed stalk c e l l s in low density monolayers, but they d i f f e r e d in a number of ways. Pre-stalk c e l l s required only DIF, though c y c l i c AMP increased the number of stalk c e l l s formed, whereas the pre-spore c e l l s required both c y c l i c AMP and DIF. Stalk c e l l formation from both c e l l - t y p e s was inhibited by c a f f e i n e , suggesting again that elevated i n t r a c e l l u l a r c y c l i c AMP levels might be necessary for the induction of stalk c e l l formation of c y c l i c AMP independent c e l l s by DIF. 1 17 CYCLIC AMP RESPONSE 1 1 mM I I 25/JM CYCLIC AMP INDEPENDENCE I I I AP H APPEARANCE I I DIF INDEPENDENCE I I STALK CELL APPEARANCE I I 0 4 8 12 16 20 24 T I M E ( H o u r s ) F i g . 28. The r e q u i r e m e n t s f o r c y c l i c AMP and DIF i n c e l l s d e v e l o p i n g j j i v i v o : Summary o f d a t a . The t i m e s when c e l l s became r e s p o n s i v e t o c y c l i c AMP and when c y c l i c AMP i n d e p e n d e n t a nd DIF i n d e p e n d e n t c e l l s were d e t e c t e d d u r i n g d e v e l o p m e n t a r e r e p r e s e n t e d i n t h e above b a r g r a p h . The t i m e s when c e l l s e x p r e s s i n g t h e p r e - s t a l k s p e c i f i c AP I I and when s t a l k c e l l s a p p e a r e d d u r i n g d e v e l o p m e n t a r e a l s o i n d i c a t e d . DISCUSSION Two d i f f e r e n t approaches were used in t h i s thesis to e s t a b l i s h whether c y c l i c AMP and DIF were required for the formation of pre-stalk c e l l s or for the pre-stalk to stalk c e l l conversion. One approach was to determine the time at which c e l l s became independent of c y c l i c AMP and DIF for stalk c e l l formation during d i f f e r e n t i a t i o n in monolayers and during development i n vivo and to correlate these periods with the times of cytod i f f erent iat ion i.n v i vo . The second approach was to examine the c y c l i c AMP and DIF requirements for the expression of pre-stalk and stalk c e l l s p e c i f i c components. These experiments w i l l be discussed below, f i r s t in r e l a t i o n to other work on the role of c y c l i c AMP and DIF in stalk c e l l formation and subsequently in r e l a t i o n to various models that have been proposed tc explain the process of c e l l d i f f e r e n t i a t i o n and the establishment of the pre-stalk/pre-spore pattern in Dictyostelium disco ideum. 119 The i n i t i a l f a c t o r r e m o v a l e x p e r i m e n t i n low d e n s i t y m o n o l a y e r s showed t h a t t h e r e q u i r e m e n t s f o r c y c l i c AMP and DIF f o r s t a l k c e l l f o r m a t i o n o c c u r r e d a t s i m i l a r t i m e s ( F i g u r e 3 ) . The c y c l i c AMP i n d u c t i o n p e r i o d b e g a n b e t w e e n 8 and 10 h o u r s o f d e v e l o p m e n t i n m o n o l a y e r s a nd p r o c e e d e d u n t i l 21 t o 24 h o u r s ( F i g u r e s 3 and 4 ) , i n d i c a t i n g some h e t e r o g e n e i t y i n t h e p o p u l a t i o n f o r t h e c y c l i c AMP r e q u i r e m e n t . D I F was i n i t i a l l y r e q u i r e d i n m o n o l a y e r s b e t w e e n 10 and 12 h o u r s a n d t h i s r e q u i r e m e n t p r o c e e d e d u n t i l 23 t o 26 h o u r s ( F i g u r e s 3 a n d 5 ) . These r e s u l t s s u g g e s t e d t h a t c y c l i c AMP i n d e p e n d e n t c e l l s a p p e a r e d b e f o r e DIF i n d e p e n d e n t c e l l s a n d t h i s was c o n f i r m e d by t h e f i n d i n g s t h a t c e l l s i n c u b a t e d s e q u e n t i a l l y w i t h c y c l i c AMP and DIF f o r m e d s t a l k c e l l s w h e r e a s c e l l s f i r s t i n c u b a t e d w i t h D I F f o l l o w e d by c y c l i c AMP d i d n o t ( T a b l e I ) . A two h o u r t r e a t m e n t w i t h D I F gave t h e maximum l e v e l o f s t a l k c e l l f o r m a t i o n i n c e l l s p r e - i n c u b a t e d w i t h c y c l i c AMP f o r 20 h o u r s ( T a b l e I I ) , s u g g e s t i n g t h a t i n d u c t i o n o f s t a l k c e l l s by D I F o c c u r r e d w i t h i n t h i s r e l a t i v e l y s h o r t t i m e p e r i o d . V i s i b l e s t a l k c e l l s b e g a n t o a p p e a r 8 h o u r s a f t e r c e l l s h a d s t a r t e d t o become i n d e p e n d e n t o f D I F ( F i g u r e 3 ) . A l t h o u g h s t a l k c e l l f o r m a t i o n i n l o w d e n s i t y m o n o l a y e r s a nd d u r i n g d e v e l o p m e n t in. v i v o b egan a t a p p r o x i m a t e l y t h e same t i m e , t h i s p r o c e s s t o o k c o n s i d e r a b l y l o n g e r i r i v i t r o t h a n i n . v i v o b e c a u s e o f t h e h e t e r o g e n e i t y w i t h i n t h e d e v e l o p i n g m o n o l a y e r p o p u l a t i o n . 120 Since the pre-stalk/pre-spore pattern becomes established during the t r a n s i t i o n between the tipped aggregates and the finger stage in s t r a i n NC 4 (Hayashi and Takeuchi, 1976), which occurs from 9 to 12 hours during in. v ivo development in s t r a i n V12 M2, these monolayer experiments suggest that c y c l i c AMP and DIF are required for the induction of pre-stalk c e l l s rather than the pre-stalk to stalk c e l l conversion. The r e s u l t s from experiments which determined the factor requirements of c e l l s developing i n vivo for stalk c e l l formation in monolayers contrasted markedly with those from the above experiments. While c y c l i c AMP independent c e l l s began to appear during development i n vivo close to the time when they f i r s t appear during monolayer d i f f e r e n t i a t i o n , DIF independent c e l l s did not appear u n t i l very late during the culmination stage (Figure 24A). This r e s u l t suggested that DIF was required for the conversion of pre-stalk c e l l s to stalk c e l l s late in development, and this conclusion was substantiated- by the finding that pre-stalk c e l l s isolated on Percoll gradients required DIF for stalk c e l l formation in monolayers (Table V). In view of the discrepancy between the appearance of DIF independent c e l l s during monolayer d i f f e r e n t i a t i o n and i n v i vo development, i t was necessary to examine the expression of individual pre-stalk and stalk c e l l s p e c i f i c components in monolayers. 121 The expression of AP IV, an enzyme present in both pre-stalk and pre-spore c e l l s (Figure 13), required only c y c l i c AMP in low density monolayers (Figures 16 and 17). This enzyme a c t i v i t y was f i r s t detected at the tipped aggregate stage (9 hours) during development in vivo and i t s appearance preceded that of the pre-stalk s p e c i f i c AP II by 3 hours (Figure 14). These results indicated that c y c l i c AMP alone did not induce pre-stalk c e l l formation in low density monolayers. AP IV a c t i v i t y was f i r s t detectable aft e r 14 hours of development in low density monolayers, somewhat lat e r than the time when c y c l i c AMP independent c e l l s begin to appear in monolayers and _iri vivo (Figures 4 and 24). This s l i g h t discrepancy is not e n t i r e l y unexpected, given that the c e l l d i f f e r e n t i a t i o n assay is probably more sensitive than the enzyme assay and that there is c e l l heterogeneity for the c y c l i c AMP requirement (Figures 3 and 4). It is quite possible that AP IV was i n i t i a l l y expressed at e a r l i e r times but was not detectable. In many respects, the expression of AP IV resembles that of glycogen phosphorylase, a postaggregative enzyme present in both pre-stalk and pre-spore c e l l s (Rutherford and Harris, 1976; Tsang and Bradbury, 1981; Weijer e_t a l . , 1984) which f i r s t appears during the tipped aggregate stage (Jones and Wright, 1970; F i r t e l and Bonner, 1972) and for which the expression in v i t r o is dependent on c y c l i c AMP (Town and Gross, 1978; Sobolewski e_t al.. , 1983; Schaap and Van D r i e l , 1985). 122 The expression of the pre-stalk s p e c i f i c AP II (Figures 16 and 17) and two pre-stalk s p e c i f i c antigens (Figure 20) in low density monolayers required the presence of DIF, suggesting that DIF acts as a pre-stalk inducer. This r e s u l t is consistent with the observation that DIF is required for the expression of AP II in high density monolayers of HM 44, a mutant impaired in the production of DIF (Kopachik et 'al.., 1983). In addition, Williams ejt al_. ( 1987) recently reported that a pre-stalk s p e c i f i c mRNA is expressed within 15 minutes of adding DIF to high density monolayers of HM 44. Despite the p o s s i b i l i t y that a r t i f a c t s might arise from using a poorly characterized mutant s t r a i n or high density monolayer conditions, these observations and the above data strongly support the view that DIF acts as an inducer of pre-stalk c e l l s . AP II was f i r s t detected together with AP IV a f t e r 14 hours of development in low density monolayers, a r e s u l t that agrees with the close times of appearance of c y c l i c AMP and DIF independent c e l l s in low density monolayers (Figure 3). However, AP II was f i r s t detected at 12 hours of development i n v ivo, 3 hours a f t e r AP IV f i r s t appeared (Figure 14). This difference may r e f l e c t the fact that DIF does not accumulate to high levels within the developing c e l l masses u n t i l a f t e r the tipped aggregate stage (Figure 2), whereas in monolayers i t is present at saturating concentrations from the beginning of the incubation. Since 123 AP II appears at the finger stage, whereas overt pre-spore c e l l d i f f e r e n t i a t i o n occurs e a r l i e r at the tipped aggregate stage (See Introduction), these r e s u l t s and the data of Williams e_t al_. ( 1987) suggest that pre-stalk c e l l formation takes place a f t e r the formation of pre-spore c e l l s . The appearance of several stalk c e l l s p e c i f i c antigens was also induced by DIF in low density monolayers (Figures 19, 22). This re s u l t agrees with the report of DIF-dependent induction of stalk c e l l s p e c i f i c proteins in high density monolayers of HM 44 (Kopachik ejb al_. , 1985). The appearance of the stalk c e l l antigens in low density monolayers was always advanced, sometimes quite s u b s t a n t i a l l y , compared with t h e i r appearance in. v i vo (Figure 23). This finding, taken together with the fact that AP II was s l i g h t l y delayed i.J0L v i tro compared with in. vivo, suggests that stalk c e l l formation in monolayers might occur immediately following the formation of pre-stalk c e l l s (Figure 23). This idea was strengthened by the finding that a 2 hour incubation in the presence of DIF was s u f f i c i e n t to induce the expression of a stalk c e l l antigen in c e l l s pre-incubated with c y c l i c AMP for 20 hours (Figure 22), indicating that both pre-stalk and stalk c e l l formation are i n i t i a t e d in monolayers within the short time required for induction by DIF (Table I I ) . 124 The above evidence provides support for the hypothesis, not yet considered in this thesis, that DIF induces both pre-stalk c e l l formation and the conversion of pre-stalk to stalk c e l l s . In low density monolayers, these two processes seem to occur close together in time and only require a short incubation in the presence of DIF, whereas during development in. vivo these two processes are separated in time. If the above view is correct, the finding that DIF-independent c e l l s do not appear u n t i l late in culmination during _in. vivo d i f f e r e n t i a t i o n (Figure 24) indicates that the conversion of pre-stalk to stalk c e l l s is restrained u n t i l culmination by some as yet unknown inhibitory mechanism. Given that DIF is produced continuously during late development (Figure 2, Brookman et al_. , 1982, 1987), i t is apparent that some mechanism must be acting to prevent the DIF-mediated conversion of pre-stalk c e l l s to stalk c e l l s during slug migration. The observation that developing c e l l masses at the finger stage can either culminate immediately or enter prolonged periods of slug migration (Newell e_t al_., 1969b) suggests that the pre-stalk to stalk c e l l conversion may be r e s t r i c t e d by the elements c o n t r o l l i n g the choice of morphogenetic pathway. 125 It is proposed that this i n h i b i t i o n might be due to a factor l o c a l i z e d in t i p s and present at high concentrations during slug migration. This is consistent with the observation that stalk c e l l formation does not occur u n t i l a f t e r pre-stalk c e l l s have migrated into the stalk tube away from the t i p of culminating f r u i t i n g bodies (Raper and Fennell, 1952). Under the standard monolayer conditions, the pre-stalk to stalk c e l l conversion appears to be unrestrained (Figure 26), presumably because the s p e c i f i c c e l l - c e l l interactions in the t i p region are absent or because the i n h i b i t o r is not l o c a l i z e d and never builds up to i n h i b i t o r y l e v e l s . This hypothesis is appealing because i t implies the existence of a signal l i n k i n g c y t o d i f f e r e n t i a t i o n with morphogenesis during development. Ammonia might be the hypothetical i n h i b i t o r , since i t has been shown to control the t r a n s i t i o n from the migrating slug stage to culmination (Schindler and Sussman, 1977) and since i t is the only substance present in c e l l extracts that can i n h i b i t stalk c e l l formation in monolayers (Table IV and Figure 8; Gross et a l . , 1981, 1983). The period of ammonia i n h i b i t i o n in low density monolayers for the most part preceded the period during which c y c l i c AMP independent c e l l s appeared (Figure 8), suggesting that ammonia probably antagonizes the requirement for c y c l i c AMP rather than DIF. 126 However, Gross and co-workers have given evidence suggesting that ammonia antagonizes the action of DIF (Gross et a l • , 1981 , 1983) and the p o s s i b i l i t y that ammonia might inh i b i t stalk c e l l formation at l a t e r times in monolayers cannot be excluded. It has also been shown that 3 to 15 mM ammonia prevents the accumulation and release of c y c l i c AMP in aggregation competent c e l l s (Schindler and Sussman, 1979; Williams ejt. al_. , 1984), suggesting a possible mechanism for the ammonia i n h i b i t i o n of stalk c e l l formation in monolayers. A requirement for increased i n t r a c e l l u l a r levels of c y c l i c AMP for stalk c e l l formation is also implied by the caffeine i n h i b i t i o n data, as w i l l be discussed l a t e r . DIF has been measured in the pre-spore region of slugs at twice the levels present in the pre-stalk region (Brookman et. al.. , 1987). Some evidence indicates that the majority of the DIF is associated with the slime sheath (Neave e_t al_. , 1985) and i t might therefore be present in a morphogenetical1y inactive form. Nevertheless, since DIF mediated the conversion of pre-spore to stalk c e l l s in high density monolayers (Kay and Jermyn, 1983) and in low density monolayers (Figures 25 and 26), i t s p r e f e r e n t i a l l o c a l i z a t i o n with pre-spore c e l l s suggests that this process is a l s o constrained in slugs. Clearly, some factor must be present in migrating s l u g s t o r e s t r a i n the DIF-mediated conversion of pre-spore to stalk c e l l s . 127 The choice of AP II as a pre-stalk s p e c i f i c marker might appear somewhat a r b i t r a r y since other components present only in pre-stalk c e l l s during slug migration are expressed e a r l i e r during development (See Introduction). However, these c e l l components, the CI antigen for example (Tasaka et al_., 1983; Noce and Takeuchi, 1985a), are expressed before any overt c e l l commitment to the stalk or spore pathway of d i f f e r e n t i a t i o n can be detected, and their subsequent disappearance in pre-spore c e l l s probably indicates that they are s e l e c t i v e l y degraded in these c e l l s . Thus, they very l i k e l y cannot be used to detect the appearance of pre-stalk c e l l s nor could they distinguish the presence of undifferentiated amoebae in monolayers. It seems improbable that pre-stalk c e l l induction occurs during aggregation since DIF levels do not r i s e appreciably u n t i l a f t e r aggregation (Figure 2, Brookman et al_. , 1982) and since production of the CI antigen cannot be induced by d i e t h y 1 s t i l b e s t r o l , a ster o i d compound which mimics the action of DIF in low density monolayers (Gross e_t al_., 1983; Noce and Takeuchi, 1985b). The fact that pre-stalk c e l l s r e t a i n many ch a r a c t e r i s t i c s of aggregating c e l l s suggests that there is a continuity between these two c e l l types, whereas pre-spore c e l l s are formed as a c l e a r l y d i s t i n c t c e l l type during the tipped aggregate stage. Many of the mRNAs that have been 128 designated as pre-stalk s p e c i f i c may therefore be expressed non-specifical1y in most or a l l aggregating c e l l s . In contrast, DIF-dependent c e l l components (Kopachik e_t a l . , 1985; Williams et al.. , 1987; this thesis) appear only aft e r the formation of tipped aggregates and during culmination, suggesting that pre-stalk c e l l formation occurs a f t e r the formation of pre-spore c e l l s . The idea that the elements c o n t r o l l i n g the t r a n s i t i o n from slug migration to culmination also prevent the pre-stalk to stalk c e l l conversion suggest that pre-stalk c e l l s may a c t u a l l y be better viewed as postaggregative c e l l s arrested during stalk c e l l formation. In the absence of a migratory stage, pre-stalk c e l l formation would not occur as a d i s t i n c t process but would proceed uninterrupted to the formation of stalk c e l l s . A s i m i l a r concept, derived from comparative studies of development in d i f f e r e n t c e l l u l a r slime mold species, was recently put forward by Schaap (1986). The DIF-activated genes in pre-stalk c e l l s may thus represent a subset of the t o t a l genes of the stalk pathway, and the remaining DIF-activated genes which are expressed in stalk c e l l s may be repressed u n t i l culmination. The interplay between DIF and the presumptive i n h i b i t o r would determine which of the DIF-inducible genes are expressed during slug migration on the one hand and during culmination on the other. Evidence suggesting that this concept is 129 plausible has recently been presented (Jermyn et a l . , 1987* Development in. press). These authors have examined the expression of eight mRNAs found in pre-stalk and stalk c e l l s , and they have shown that the mRNAs which are inducible by DIF in. v i tro are f i r s t expressed either in pre-stalk or stalk c e l l s during development i n vivo, whereas the mRNAs present in pre-stalk c e l l s which are not DIF-dependent i n v i t r o always appear p r i o r to the formation of tipped aggregates. Invariably, the DIF-dependent mRNAs remained expressed in stalk c e l l s whereas the DIF-independent mRNAs did not. There is a p o s s i b i l i t y that the experiments with disaggregated developing c e l l s and with pre-stalk c e l l s are misleading because these c e l l s lose t h e i r DIF induced state during t h e i r i s o l a t i o n . However, i f such d e d i f f e r e n t i a t i o n occurs i t is far from complete, since there were d i s t i n c t differences between pre-stalk and pre-spore c e l l s for stalk c e l l formation in monolayers. Pre-stalk c e l l s formed more stalk c e l l s with a given dose of DIF than did pre-spore c e l l s (Figure 25) and they required a short incubation with DIF to form stalk c e l l s whereas pre-spore c e l l s required a longer incubation in the presence of both c y c l i c AMP and DIF (Figure 26). Moreover, the mere removal of DIF did not result in the loss of DIF independence, since pre-stalk c e l l s treated b r i e f l y with DIF were able to form stalk c e l l s in monolayers (Figure 26). It is therefore unlikely that 130 c e l l s d e differentiated in the interval between the i r sampling and t h e i r deposition in low density monolayers. In addition to i t s role in the aggregation process, c y c l i c AMP is required for the expression of cell-type s p e c i f i c and non-specific postaggregative genes (See Introduction). C y c l i c AMP is required before DIF for stalk c e l l formation in low density monolayers (Figure 3, Table I ) , suggesting that i t renders c e l l s responsive to DIF. Several experiments were performed in an attempt to elucidate i t s mode of action in monolayers. The experiment with c y c l i c AMP analogues (Figure 6) suggested that stalk c e l l induction by c y c l i c AMP in low density monolayers is mediated by the c e l l surface c y c l i c AMP receptor, and that c y c l i c AMP is active in micromolar concentrations. Other workers have recently reported s i m i l a r analogue experiments which suggested that t h i s receptor mediates the induction of a pre-spore c e l l antigen (Schaap and Van D r i e l , 1985), postaggregative mRNA (Haribabu and Dottin, 1986) and pre-stalk or pre-spore s p e c i f i c mRNAs (Oyama and Blumberg, 1986; Gomer et. a l . , 1986a). In these experiments, c y c l i c AMP gave f u l l induction at 10 uM or less (Figure 6; Oyama and Blumberg, 1986; Gomer et al_. , 1986a). 131 Ce l l s independent of c y c l i c AMP for stalk c e l l formation in monolayers began to appear between 8 and 10 hours during d i f f e r e n t i a t i o n in low density monolayers (Figure 4), presumably r e f l e c t i n g the a c t i v a t i o n of the c e l l surface c y c l i c AMP receptor. The i n i t i a l appearance of this receptor during development in. vivo was determined by incubating disaggregated c e l l s in LDMS containing c y c l i c AMP for 2 hours followed by DIF. No induction was observed when c e l l s were disaggregated before 6 hours, but c e l l s induced by 1 mM c y c l i c AMP appeared from that time onwards whereas c e l l s induced by 25 pM c y c l i c AMP began to appear at 8 hours (Figure 24B). The number of c e l l s induced by 25 uM c y c l i c AMP gradually increased u n t i l about 12 hours of development, whereas induction with the higher dose was maximal from the onset. Clearly, developing c e l l s are p o t e n t i a l l y inducible by c y c l i c AMP as early as 6 hours, but c e l l s that had become c y c l i c AMP independent were not detected u n t i l l a t e r in development (Figures 24A and B). This suggests that the ambient c y c l i c AMP concentrations in aggregation streams are too low for stalk c e l l induction and that they do not reach s u f f i c i e n t levels u n t i l tipped aggregates are formed. Ambient c y c l i c AMP concentrations during aggregation range from 10~8 to lO -^ M in aggregation streams (Tomchik and Devreotes, 1981) and then r i s e s u b s t a n t i a l l y at the mound and tipped mound stages (Merkle et al_. , 1984). Furthermore, 132 changes in c e l l physiology have been detected that accompany the increase in c y c l i c A M P l e v e l : chemotactic c e l l s respond to nanomolar concentrations of c y c l i c AMP during the aggregation stage (Reviewed by Devreotes, 1982) and to micromolar concentrations in slugs (Matsukuma and Durston, 1979). Several postaggregative c e l l u l a r functions can be induced by micromolar concentrations of c y c l i c AMP (Town and Gross, 1978; Brookman et al.. , 1982; Sobolewski et. al.. , 1983; Mehdy e_t al_., 1983; Chisholm et al.., 1984; Schaap and Van D r i e l , 1985; Oyama and Blumberg, 1986; Haribabu et a l . , 1986; Gomer et a l . , 1986a; Kumagai and Okamoto, 1986), suggesting that the t r a n s i t i o n from aggregative to postaggregative gene expression is triggered by the b u i l d up of c y c l i c AMP concentrations within the developing c e l l mass and the a c t i v a t i o n of the c e l l surface c y c l i c AMP receptor. The induction of aggregation stage gene expression requires pulses of nanomolar concentrations of c y c l i c AMP, and is prevented by continuous exposure to c y c l i c AMP (Yeh et al.., 1978), ostensibly because of an adaptation response exhibited by the c e l l surface c y c l i c AMP receptor (Devreotes, 1982; Devreotes and Sherring, 1985). In contrast, c y c l i c AMP can be given either as pulses or as a single dose to induce postaggregative enzymes (Figure 6, Schaap and Van D r i e l , 1985; Haribabu and Dottin, 1986; Oyama and Blumberg, 1986; Gomer e_t al.. , 1986a), suggesting that the c e l l surface c y c l i c AMP receptor of postaggregative 133 c e l l s has d i f f e r e n t properties from the receptor of aggregation stage c e l l s . The postaggregative form of the c e l l surface c y c l i c AMP receptor is poorly characterized, but i t is reported to have altered binding properties (Coukell, 1981; Schaap and Spek, 1984). Devreotes and Sherring (1985) have shown that the c e l l surface c y c l i c AMP receptor is present in aggregation stage c e l l s in two interconvertible forms. The R form is the excitable form of the receptor and is present in c e l l s exposed to nanomolar concentrations of c y c l i c AMP, whereas the D form is the adapted form of the receptor and is associated with exposure to micromolar concentrations. These data suggest that the c e l l surface c y c l i c AMP receptor is predominantly in the R form during aggregation and in the D form aft e r aggregation, implying that postaggregative functions are induced by c y c l i c AMP via the D form. A possible explanation for the difference in the induction of disaggregated developing c e l l s by the 2 doses of c y c l i c AMP (Figure 24B) is that 1 mM c y c l i c AMP induces the D form in monolayers but 25 uM does not, suggesting that the appearance of c e l l s inducible by 25 uM c y c l i c AMP r e f l e c t s the appearance of the D form during development. 134 The c e l l u l a r response to the a c t i v a t i o n of the c e l l s u r f a c e c y c l i c AMP r e c e p t o r by exogenous c y c l i c AMP was i n v e s t i g a t e d u s i n g the drug c a f f e i n e . In a g g r e g a t i o n stage c e l l s , c y c l i c AMP e l i c i t s a t r a n s i e n t a c t i v a t i o n o f a d e n y l a t e c y c l a s e and the s y n t h e s i s and r e l e a s e of c y c l i c AMP as p a r t of the s i g n a l r e l a y response (Reviewed by D e v r e o t e s , 1982). C a f f e i n e i n h i b i t s t h i s response i n a g g r e g a t i o n competent c e l l s ( B r e n n e r and Thorns, 1984), and i t a l s o i n h i b i t e d s t a l k c e l l f o r m a t i o n from v e g e t a t i v e ( F i g u r e 9) and s l u g stage c e l l s i n low d e n s i t y monolayers ( F i g u r e 2 7 ) . T h i s r e s u l t s u g g e s t s t h a t the a c t i v a t i o n of a d e n y l a t e c y c l a s e or an i n c r e a s e i n i n t e r n a l c y c l i c AMP may be i n v o l v e d i n s t a l k c e l l d i f f e r e n t i a t i o n . 1 mM c a f f e i n e i n h i b i t e d the p r e - s p o r e t o s t a l k c e l l c o n v e r s i o n to the same e x t e n t as 8 mM c a f f e i n e f o r s t a l k c e l l f o r m a t i o n from p r e -s t a l k c e l l s ( F i g u r e 2 7 ) , perhaps a r e f l e c t i o n of the f a c t t h a t the former r e q u i r e d b o t h c y c l i c AMP and DIF f o r s t a l k c e l l f o r m a t i o n i n monolayers whereas the l a t t e r o n l y r e q u i r e d DIF ( T a b l e V ) . Spore c e l l f o r m a t i o n was found to be more s e n s i t i v e to c a f f e i n e than s t a l k c e l l f o r m a t i o n i n sporogenous mutants d i f f e r e n t i a t i n g i n monolayers ( R i l e y and B a r c l a y , 1986), but the i n d u c t i o n of p r e - s p o r e c e l l s i n w i l d - t y p e c e l l s was not s i g n i f i c a n t l y a f f e c t e d by 3 mM c a f f e i n e i n shaken s u s p e n s i o n (Schaap e t al _ . , 1986). The l a t t e r r e s u l t s u g g e s t s t h a t the spore pathway o f d i f f e r e n t i a t i o n might d i f f e r from the s t a l k pathway w i t h r e s p e c t to c a f f e i n e i n h i b i t i o n . The f i n d i n g t h a t 1.5 mM 135 caffeine reduced the c y c l i c AMP-dependent induction of the pre-stalk s p e c i f i c cathepsin by 50% is also consistent with the above data, although the lack of i n h i b i t i o n by 2.5 mM cannot be explained (Gomer e_t al.. , 1986a), and these authors argue that i t is having no e f f e c t on pre-stalk gene expression. The permeable c y c l i c AMP analogue 8-Bromo-cyclic AMP p a r t i a l l y reversed the i n h i b i t i o n of stalk c e l l formation by caffeine (Figure 11). Since millimolar concentrations of 8-Bromo-cyclic AMP were required to reverse caffeine i n h i b i t i o n whereas micromolar concentrations were s u f f i c i e n t to induce stalk c e l l formation in the absence of caffeine (Figure 6), and since c y c l i c AMP was present at saturating concentrations in this experiment, i t is very l i k e l y that the caffeine i n h i b i t i o n was reversed by uptake of this analogue into c e l l s . Uptake of this analogue probably mimics an increase in c y c l i c AMP levels inside c e l l s , suggesting that caffeine might i n h i b i t stalk c e l l formation by preventing the i n t r a c e l l u l a r accumulation of c y c l i c AMP. Since caffeine inhibited both the c y c l i c AMP requirement and the DIF requirement for stalk c e l l formation in low density monolayers (Figure 11), i t is probable that the elevation of internal c y c l i c AMP levels in c e l l s is necessary for both requ i rements. 136 Both the i n t r a c e l l u l a r pools of c y c l i c AMP and the basa l a c t i v i t y of adenylate c y c l a s e r i s e d u r i n g development and these increases have been r e p e a t e d l y i m p l i c a t e d in the r e g u l a t i o n of gene e x p r e s s i o n ( K l e i n , 1976; Roos ejt a l . , 1977; Sampson e_t al.., 1978; Yeh ejt al.. , 1978; Pahl i c and Ruth e r f o r d , 1979; Coukell and Chan, 1980). However, a causal r e l a t i o n has not yet been e s t a b l i s h e d , and the above r e s u l t s are the f i r s t to s u b s t a n t i a t e the hypothesis that increased adenylate c y c l a s e a c t i v i t y and c y c l i c AMP l e v e l s might lead to c e l l d i f f e r e n t i a t i o n . Oyama and Blumberg (1986) r e c e n t l y p r o v i d e d evidence that the c y c l i c AMP i n d u c t i o n of some p r e - s t a l k and pre-spore mRNAs i n shaken suspension can occur without the a c t i v a t i o n of adenylate c y c l a s e . I t i s p o s s i b l e , t h e r e f o r e , that c a f f e i n e might reduce i n t r a c e l l u l a r c y c l i c AMP l e v e l s in p o s t a g g r e g a t i v e and monolayer d i f f e r e n t i a t i n g c e l l s without a f f e c t i n g adenylate c y c l a s e a c t i v i t y . It has been proposed that exogenous c y c l i c AMP s t a b i l i z e s the e x p r e s s i o n of post a g g r e g a t i v e mRNAs (Landfear and L o d i s h , 1980; Chung e_t al.. , 1981; M a n g i a r o t t i e_t a l . , 1985). In Fr-17, u n l i k e f o r wild-type c e l l s , the mRNA f o r UDP-glucose pyrophosphorylase d i d not become l a b i l e upon d i s r u p t i o n o f l a t e d e veloping c e l l masses in the absence o f c y c l i c AMP (Newell et al.. , 1971; Brackenbury, 1975, Figure 3). T h i s r a p i d l y d e veloping mutant c a r r i e s a s i n g l e mutation 137 (Kessin, 1977) which causes a premature a c t i v a t i o n of adenylate cyclase and elevation of i n t r a c e l l u l a r c y c l i c AMP levels during development (Coukell and Chan, 1980) and this a l t e r a t i o n might have resulted in the s t a b i l i z a t i o n of the mRNA in the disrupted c e l l masses. This suggests that the c y c l i c AMP a c t i v a t i o n of the postaggregative c e l l surface c y c l i c AMP receptor leads to elevated i n t r a c e l l u l a r c y c l i c AMP levels which s t a b i l i z e postaggregative mRNAs. Ce l l s pre-incubated with c y c l i c AMP required a 2 hour incubation with DIF to be maximally induced, but induction of stalk c e l l s under these conditions consistently reached a f r a c t i o n of the level attained when c e l l s were incubated with c y c l i c AMP and DIF together (Table I and I I ) . Though induction by c y c l i c AMP and by DIF have been shown to be d i s t i n c t events, these findings suggest that c y c l i c AMP and DIF can s t i l l interact during stalk c e l l d i f f e r e n t i a t i o n . The preceding discussion suggests that exogenous c y c l i c AMP might do this by s t a b i l i z i n g postaggregative function during DIF induction. Thus in pre-stalk c e l l s or vegetative c e l l s pre-incubated with c y c l i c AMP, internal levels of c y c l i c AMP would decrease below a c r i t i c a l level in the majority of c e l l s before induction by DIF was complete. Ce l l s incubated with c y c l i c AMP and DIF together would form more stalk c e l l s because c y c l i c AMP would maintain elevated internal c y c l i c AMP concentrations while DIF induces stalk c e l l formation. 138 Stalk c e l l formation from pre-stalk c e l l s required only DIF, but i t was also considerably enhanced by c y c l i c AMP (Table V). The above argument may also explain this enhancement by c y c l i c AMP. However, the c y c l i c AMP enhancement of stalk c e l l formation in this case might also r e f l e c t the presence of pre-stalk c e l l sub-populations in the l i g h t density f r a c t i o n of Percoll gradients. One such sub-population might be the 'an t e r i o r - l i k e c e l l s ' o r i g i n a l l y characterized by Sternfeld and David (1981, 1982) as neutral red staining c e l l s located in the posterior region of slugs. In many respects, these c e l l s appear to be pre-stalk c e l l s since they express several proteins t y p i c a l of pre-stalk c e l l s , including AP II (Devine and Loomis, 1985). Nonetheless, minor differences between a n t e r i o r — l i k e and pre-stalk c e l l s have also been noted (Devine and Loomis, 1985). They appear to be located among pre-spore c e l l s because they are prevented from sorting to the anterior region of slugs by a d i f f u s i b l e i n h i b i t o r which is secreted by c e l l s from the anterior of slugs (Sternfeld and David, 1981). Their requirements for stalk c e l l formation in monolayers has not been s p e c i f i c a l l y established. Another d i s t i n c t pre-stalk c e l l sub-population are t i p c e l l s . These c e l l s are morphologically d i s t i n c t from non-tip pre-stalk c e l l s (Kopachik, 1982b) and they can be d i f f e r e n t i a t e d from these and a n t e r i o r - l i k e c e l l s by their a b i l i t y to be stained with an anti-cathepsin antiserum 139 (Gomer e_t al_., 1986b). Since tips emit c y c l i c AMP pulses in slugs (Rubin and Robertson, 1975), i t seems plausible that ti p c e l l s might be independent of c y c l i c AMP for stalk c e l l formation. Indeed, t i p c e l l s have been shown to form stalk c e l l s in the absence of c y c l i c AMP in high density monolayers (Town and Stanford, 1977) and they may represent a c y c l i c AMP independent pre-stalk sub-population. Cells independent of c y c l i c AMP for stalk c e l l formation in low density monolayers were f i r s t detected at 12 hours of development, well aft e r the mound and tipped aggregate stages when c y c l i c AMP-dependent postaggregative gene expression is i n i t i a t e d (Figure 24). Their number rose gradually u n t i l culmination despite the fact that c e l l masses developing on M i l l i p o r e f i l t e r s exhibited a high degree of morphological synchrony. It might seem reasonable to assume a priori that this heterogeneity for the c y c l i c AMP requirement is analogous to that for c e l l s developing in low density monolayers (Figure 3). However, th i s seems unlikely because c y c l i c AMP is present at saturating concentrations from the onset of development in low density monolayers whereas c y c l i c AMP levels r i s e throughout development (Merkle e_t al.. , 1984). C y c l i c AMP concentrations are highest in the anterior portion of slugs (Pan e_t a 1 ., 1974; Brenner, 1977) and i t is possible that c y c l i c AMP levels r i s e progressively throughout the slug thereby inducing the pre-stalk c e l l population gradually. 140 Indirectly, this heterogeneity for c y c l i c AMP independence provides further support for the view that d i s t i n c t pre-stalk sub-populations exist within developing c e l l masses. Factors other than DIF , c y c l i c AMP and ammonia have been shown to a f f e c t c e l l d i f f e r e n t i a t i o n in. v i tro. Several reports have shown that soluble factors present in conditioned media can induce spore d i f f e r e n t i a t i o n i n v i tro (Wilcox and Sussman, 1978; Sternfeld and David, 1979; Ishida, 1980; Kay, 1982; Weeks, 1984; Mehdy and F i r t e l , 1985; Wilkinson ejb aj_. , 1985; Kumagai and Okamoto, 1986). One such factor is required in addition to c y c l i c AMP for the induction of pre-spore c e l l formation in low density monolayers (Kay, 1982; Weeks, 1984; Mehdy and F i r t e l , 1985; Kumagai and Okamoto, 1986). This factor has recently been characterized as a developmentally regulated, heat stable, low molecular weight metabolite designated PIF (Kumagai and Okamoto, 1986). PIF accumulates rapidly in developing c e l l masses at the mound stage, and i t s synthesis i n v i tro, like DIF, is c y c l i c AMP-dependent. These c h a r a c t e r i s t i c s are consistent with those expected for an inducer of pre-spore c e l l formation. A factor c a l l e d SPIF has been implicated in the conversion of pre-spore to spore c e l l s (Wilkinson e_t aj.., 1985). This factor is also present in conditioned media, but it s a c t i v i t y d i f f e r s from that of PIF in that i t induces the 1 4 1 expression of terminal stage spore proteins only in the presence of c e l l - c e l l contacts, whereas PIF a c t i v i t y is demonstrated in low density monolayers (Kumagai and Okamoto, 1986). Thus, i t appears that these two factors act at di f f e r e n t times during spore c e l l d i f f e r e n t i a t i o n . Other factors can modulate c e l l d i f f e r e n t i a t i o n without exhibiting any inductive a c t i v i t y in. v i t r o . Ammonia affects terminal d i f f e r e n t i a t i o n hi v i t r o by enhancing spore c e l l formation though i t is not capable of inducing spore c e l l s alone (Wilcox and Sussman, 1978; Sternfeld and David, 1979; Gross e_t al_. , 1983; Weeks, 1984). Ammonia i n h i b i t i o n of stalk c e l l formation appears to be associated with the requirement for c y c l i c AMP rather than the DIF requirement (Figure 8) and i t also i n h i b i t s the c y c l i c AMP-dependent synthesis of DIF in high density monolayers (Neave e_t a 1 ., 1983). Adenosine has an eff e c t opposite to that of ammonia, preventing the formation of pre-spore c e l l s in clumps of pre-stalk c e l l s shaken in suspension while c y c l i c AMP favours pre-spore c e l l formation at the expense of pre-stalk c e l l s (Weijer and Durston, 1985). The ef f e c t of adenosine on pre-spore c e l l d i f f e r e n t i a t i o n may result from an i n h i b i t i o n of c y c l i c AMP binding (Newell, 1982; Van Lookeren Campagne e t a l . , 1986). Furthermore, treatment of slugs with adenosine deaminase, which reduces ambient concentrations of 142 a d e n o s i n e , d i m i n i s h e d the p r o p o r t i o n of p r e - s t a l k c e l l s (Schaap and Wang, 1986). These exper iments suggest t h a t the l e v e l s of adenos ine and c y c l i c AMP w i t h i n c e l l masses might r e g u l a t e the p r o p o r t i o n s of p r e - s t a l k and p r e - s p o r e c e l l s . It is noteworthy that o n l y those c e l l u l a r s l ime mold s p e c i e s which use c y c l i c AMP f o r t h e i r a c r a s i n e x h i b i t a d i s t i n c t p r e - s t a l k / p r e - s p o r e p a t t e r n (Bonner , 1982). However, s i n c e p r e - s t a l k and p r e - s p o r e c e l l s d i f f e r in t h e i r l e v e l s of c y c l i c AMP-dependent p h o s p h o d i e s t e r a s e a c t i v i t y , in t h e i r a b i l i t y to chemotax to c y c l i c AMP and in t h e i r a b i l i t y to produce c y c l i c AMP (Tsang and B r a d b u r y , 1981; K o p a c h i k , 1982b; Ratner and B o r t h , 1983; Schaap and Spek, 1984; Mee ejt al_. , 1986), the above r e s u l t s must be i n t e r p r e t e d c a u t i o u s l y because of the p o s s i b i l i t y of s e v e r a l u n d e t e c t a b l e feedback systems o p e r a t i n g in the above systems. A consequence of these d i f f e r e n t p r o p e r t i e s of p r e -s t a l k and p r e - s p o r e is tha t the a n t e r i o r and p o s t e r i o r r e g i o n s of s l u g s may r e p r e s e n t c o n s i d e r a b l y d i f f e r e n t m i c r o e n v i r o n m e n t s , which might r e - e n f o r c e the p r e - s t a l k or p r e - s p o r e s t a t e s . Such i n t e r a c t i o n s might be c e n t r a l a spec t s of the mechanism f o r p a t t e r n r e g u l a t i o n but they were not c o n s i d e r e d in the above e x p e r i m e n t s . F o r i n s t a n c e , DIF and PIF s y n t h e s i s or the a b i l i t y of c e l l s to respond to these i n d u c i n g f a c t o r s might be a f f e c t e d by l e v e l s of c y c l i c AMP, 5'AMP or a d e n o s i n e . 143 Various attempts to detect some ef f e c t of adenosine in low density monolayers were unsuccessful (Kwong and Weeks, personal communication; Kay, personal communication). Possibly, adenosine exerts i t s effects iri vivo by modulating c e l l - c e l l interactions that do not occur in low density monolayers. Thus, while factors such as adenosine may play a s i g n i f i c a n t role in the regulation of the pre-sta1k/pre-spore pattern in m u l t i c e l l u l a r aggregates, there is no evidence from in. v i tro systems that they can induce c e l l d i f f e r e n t i a t i o n by themselves. In the following analysis of the various models that have been proposed for the d i f f e r e n t i a t i o n of D. d i sco i deum, a d i s t i n c t i o n has been made between primary inducers essential for in, v i tro d i f f e r e n t i a t i o n , such as DIF, c y c l i c AMP, PIF and SPIF, and molecules such as ammonia and adenosine that modulate the ef f e c t s of the primary inducers. Such a d i s t i n c t i o n may prove to be i n v a l i d eventually, but i t allows for a simpler analys i s . Many models have been developed to explain the formation and the regulation of the pre-stalk/pre-spore pattern. Several are purely mathematical, and a number of these have put forward useful working hypotheses about the properties expected of morphogens (eg. L a c a l l i and Harrison, 1978; Meinhardt, 1983; Grinfel d and Segel, 1986). However, they have been formulated for the most part without consideration of the known properties of factors such as 144 DIF, ammonia or c y c l i c AMP. Only those models that include the e f f e c t s of such molecules (Gross e_t al_. , 1981; Sussman, 1982; Loomis, 1985; MacWilliams et a l . , 1986; Schaap, 1986; Williams ejt al.. , 1986) w i l l be discussed. Sussman and Schindler (1978) and more recently Sussman (1982) posit that the v a r i a b i 1 i t y of slug migration, from it s absence to i t s prolonged duration, necessitates the involvement of internal signals which coordinate c y t o d i f f e r e n t i a t i o n and morphogenesis. Morphogenesis is postulated to be organized primarily by the chemotactic movement of c e l l s towards c y c l i c AMP s i g n a l l i n g centers, whose location (and, presumably, level of a c t i v i t y ) is determined by ambient ammonia concentrations. This is supported by evidence that slug c e l l s are chemotactic (Durston and Vork, 1979) and that exogenous c y c l i c AMP can disrupt slug migration and formation of f r u i t i n g bodies (Nestle and Sussman, 1972). The t r a n s i t i o n between developmental stages would be controlled by the balance of c y c l i c AMP and ammonia concentrations, for which there is some evidence (Schindler and Sussman, 1977). It has been argued that the predominant determinants of ammonia levels in c e l l masses are environmental factors, the metabolic a c t i v i t y of developing c e l l s and the shape of the c e l l mass. 145 Although t h i s model provides an i n t u i t i v e l y plausible description of the morphogenetic sequence of development, the known inductive properties of DIF and PIF are not considered. Thus, ammonia and c y c l i c AMP alone are said to - control the c e l l u l a r levels of c y c l i c AMP which in turn determines whether a c e l l d i f f e r e n t i a t e s along the spore or stalk pathway. Ammonia has been shown to a f f e c t the choice of d i f f e r e n t i a t i o n pathways (Gross et al.., 1981 , 1983) and to prevent the internal accumulation of c y c l i c AMP in aggregation competent c e l l s (Schindler and Sussman, 1979). The r e s u l t s from the caffeine experiments in th i s thesis also lend support for thi s idea, but more studies are needed to determine how internal c y c l i c AMP concentrations might be involved in the control of c e l l d i f f e r e n t i a t i o n . Other factors such as DIF are said to be accessory and only a l t e r the a c t i v i t i e s of c y c l i c AMP and ammonia (Sussman, 1982). It is presumed that the pre-stalk/pre-spore pattern is regulated by a combination of d i f f e r e n t i a l chemotaxis and l o c a l i z e d d i s t r i b u t i o n of c y c l i c AMP and ammonia. However, the only evidence presented in support of t h i s claim is the observation that mutants unusually sensitive to ammonia have abnormally high proportions of pre-spore c e l l s in slugs (Sussman, 1982). Unfortunately, the current form of th i s model f a l l s short of providing a comprehensive explanation for d i f f e r e n t i a t i o n .in. vivo and i t does not account for a l l the factors that are known to affe c t c e l l d i f f e r e n t i a t i o n .in. v i t r o . 146 Loomis (1975) o r i g i n a l l y developed the concept of a step-wise a c t i v a t i o n of genes in a dependent sequence which accompanies the t r a n s i t i o n through each of the morphogenetic stages of development. The idea that the entry of c e l l s into each new developmental stage is accompanied by a new round of gene expression is r e f l e c t e d in the observation that proteins are predominantly synthesized at s p e c i f i c periods rather than continually during development (Morrissey ejt al . , 1984; C a r d e l l i e_t al.. , 1985). In a more recent elaboration of his model, Loomis (1985) proposed that the signals responsible for the t r a n s i t i o n between the major developmental stages were starvation, the i n i t i a l secretion of c y c l i c AMP at the onset of aggregation, i t s build up within nascent m u l t i c e l l u l a r aggregates and a drop in ammonia levels during culmination. The induction of c e r t a i n aggregation stage components and the i n h i b i t i o n of a starvation-induced protein by nanomolar pulses of c y c l i c AMP is the most convincing evidence presented in support of the idea that c y c l i c AMP pulsing e f f e c t s the t r a n s i t i o n between the interphase and aggregation stages of development (Yeh e_t a l . , 1978). The high levels of c y c l i c AMP required for the induction of pre-stalk and pre-spore c e l l components (Mehdy et al . , 1983; Chisholm ejt al.. , 1984; Mehdy and F i r t e l , 1985) lead Loomis to suggest that induction of these components In v i tro is caused by uptake of c y c l i c AMP and that multice 11ularity must therefore trigger a r i s e in internal 147 c y c l i c AMP. Although recent findings indicate that lower levels of exogenous c y c l i c AMP w i l l induce postaggregative c e l l components by act i v a t i n g the c e l l surface c y c l i c AMP receptor (Figure 6, Schaap and Van D r i e l , 1985; Haribabu and Dottin, 1986; Oyama and Blumberg, 1986; Gomer e_t a 1 . , 1986a), the res u l t s presented in this thesis suggest that a c t i v a t i o n of thi s receptor by exogenous c y c l i c AMP res u l t s in increased internal c y c l i c AMP l e v e l s . Loomis proposed that high concentrations of c y c l i c AMP are s u f f i c i e n t for spore c e l l formation since c e l l s submerged in monolayers at 3 ? 4x10 cells/cm* - form spore coat proteins in the presence, but not in the absence, of c y c l i c AMP. This re s u l t contrasts with the findings that a conditioned media factor (PIF) is required for pre-spore c e l l formation in monolayers (Kay, 1982; Weeks, 1984; Mehdy and F i r t e l , 1985; Kumagai and Okamoto, 1986). It is possible that the c e l l density used by Loomis was s u f f i c i e n t l y high to allow for c e l l a c t i v a t i o n by an endogenously secreted factor. In his proposed model, Loomis does not mention DIF, PIF, or other factors which d i r e c t l y a f f e c t c e l l d i f f e r e n t i a t i o n . 148 The models put forward i n i t i a l l y by Gross and co-workers (Gross e_t al_. , 1981) and more recently elaborated by Williams and co-workers (Williams e_t a_l_. , 1986) are derived from th e i r extended studies of soluble factors which control c e l l d i f f e r e n t i a t i o n . C y c l i c AMP, DIF, CMF (for conditioned media factor) and SPIF are the only factors required for c e l l d i f f e r e n t i a t i o n in t h e i r model: c e l l - c e l l contacts are e x p l i c i t l y excluded as inducers of c e l l d i f f e r e n t i a t i o n . Considerable evidence is presented indicating that c y c l i c AMP s i g n a l l i n g e f f e c t s the t r a n s i t i o n between the pre-aggregation and aggregation stage. S p e c i f i c a l l y , a causal rela t i o n s h i p is postulated between the repression by c y c l i c AMP s i g n a l l i n g of two starvation induced pre-aggregation proteins, Discoidin I and the c y c l i c AMP phosphodiesterase i n h i b i t o r , and the induction of three aggregation stage proteins, contact s i t e s A, c y c l i c AMP phosphodiesterase and cysteine proteinase I, (Williams e_t al.. , 1986). It is proposed that c y c l i c AMP s i g n a l l i n g increases i n t r a c e l l u l a r c y c l i c AMP levels and this s t a b i l i z e s developmentally regulated mRNAs, for which there is supportive evidence (Landfear and Lodish, 1980; Chung et a l . , 1981; Barklis and Lodish, 1983). It is postulated that DIF induces c e l l s within tipped aggregates to form pre-stalk c e l l s , whereas either ammonia or SPIF induces pre-spore c e l l d i f f e r e n t i a t i o n . This induction is postulated to result from a reduction or an elevation, respectively, of i n t r a c e l l u l a r 149 pH, as indicated by the inductive properties of the proton pump i n h i b i t o r d i e t h y l s t i l b e s t r o l and the ef f e c t s of weak acids and weak bases on spore and stalk c e l l formation in monolayers of sporogenous mutants (Gross et al.., 1983). However, attempts to demonstrate such pH changes within c e l l s during c e l l d i f f e r e n t i a t i o n have thus far been negative (Jentoft e_t al.., 1985; Kay et al_. , 1986), indicating that the e f f e c t of weak acids and weak bases does not involve internal pH or that the postulated pH change may be r e s t r i c t e d to a compartment not discernible by current techniques. No attempt was made to reconcile the disparate observations that high concentrations of ammonia stimulate pre-spore and spore formation (Gross et al_., 1983) and that reduced ammonia concentrations signal the t r a n s i t i o n between slug migration and culmination (Schindler and Sussman, 1977). Otherwise, the available evidence provides good support for the main features of t h e i r model. While the above model was mainly derived from studies on the active induction of c e l l d i f f e r e n t i a t i o n in. v i tro, the models developed by MacWilliams (1984, 1985, 1986) stress predominantly mechanisms involving inhibitory feedback loops. He postulates that the level of secreted inhibitory factors r i s e s as the number of d i f f e r e n t i a t e d c e l l s increases u n t i l concentrations are attained which i n h i b i t further c e l l d i f f e r e n t i a t i o n . C y c l i c AMP was proposed as the pre-stalk i n h i b i t o r in an e a r l i e r model 150 (MacWilliams, 1984; MacWilliams and David, 1985) and l a t e r as the i n h i b i t o r of the conversion of a n t e r i o r - l i k e to pre-stalk c e l l s (MacWilliams et. al.., 1986). DIF is proposed as the i n h i b i t o r of pre-spore c e l l induction (MacWilliams and David, 1985) or the i n h i b i t o r of the a n t e r i o r - l i k e to pre-spore c e l l conversion (MacWilliams e_t al.., 1986). It was o r i g i n a l l y postulated that d i f f e r e n t i a l adhesion plays a central role in pattern formation (MacWilliams, 1984), but this idea was reconsidered and subsequently omitted from l a t e r models (MacWilliams and David, 1985; MacWilliams et a l . , 1986). The experiments indicating that c y c l i c AMP s t a b i l i z e s pre-spore c e l l s and favours the conversion of pre-stalk to pre-spore c e l l s , and that adenosine has the opposite e f f e c t (Weijer and Durston, 1985; Schaap and Wang, 1986) suggest that the pre-stalk/pre-spore pattern may be regulated through feed-back loops involved these factors. However, the proposition that c y c l i c AMP acts as a feed-back i n h i b i t o r for pre-stalk c e l l d i f f e r e n t i a t i o n cannot yet be distinguished from the p o s s i b i l i t y that the r a t i o of c y c l i c AMP to 5'AMP or adenosine in a microenvironment plays t h i s r o l e . The emphasis on the possible negative feed-back aspects of DIF action do not account for the findings that DIF induces pre-stalk c e l l s (Williams et a l . , 1987; th i s thesis) and mediates the interconversion of pre-spore to stalk c e l l s (Kay and Jermyn, 1983; this t h e s i s ) . 151 Furthermore, there is no mention in these models of pre-spore inducing factors. These models therefore f a i l to account for the known properties of positive effectors of c e l l d i f f e r e n t i a t i o n . The model recently proposed by Schaap (1986) in many respects represents a synthesis of the two preceding models and of ideas obtained from comparative studies among c e l l u l a r slime mold species. The a c t i v a t i o n of developmentally regulated gene products by starvation, c y c l i c AMP s i g n a l l i n g and r i s i n g c y c l i c AMP levels within nascent c e l l aggregates proposed in other models is re i t e r a t e d . The observation that pre-spore to stalk r e d i f f e r e n t i a t i o n takes place near the t i p in D. minutum and in other species (Schaap ejb al.., 1985) is taken as evidence that in D. d i sco ideum pre-spore and pre-stalk c e l l s are maintained into an anterior-posterior pattern by a pos i t i o n -dependent mechanism, whereas the regulation of pre-spore to a n t e r i o r - l i k e interconversion occurs independently of t h e i r p o s i t i o n . In thi s model, Schaap refers to pre-stalk and an t e r i o r - l i k e c e l l s as non pre-spore c e l l s which express postaggregative functions rather than as a d i s t i n c t c e l l -type opposite to pre-spore c e l l s . 152 It is proposed that differences between c e l l s within the population of aggregating c e l l s predispose them to form either pre-stalk or pre-spore c e l l s during the establishment of the pre-stalk/pre-spore pattern. These differences are said to r e f l e c t some i n t r i n s i c properties of c e l l s at the onset of starvation which somehow dictate c e l l fate. There is evidence that vegetative c e l l s can p r e f e r e n t i a l l y form pre-stalk or pre-spore c e l l s depending on th e i r phase of the c e l l - c y c l e at starvation (Weijer e_t al.. , 1984). S i m i l a r l y , axenic c e l l s grown on d i f f e r e n t metabolizable sugars w i l l p r e f e r e n t i a l l y form pre-stalk or pre-spore (Noce and Takeuchi, 1985a). However, these correlations do not represent a test for this hypothesis, and the way by which such i n t r i n s i c properties of vegetative c e l l s can lead to a propensity to form one or the other cell-type is at present unknown. It is postulated that a pre-pattern becomes established by these c e l l s within nascent c e l l mounds by d i f f e r e n t i a l adhesion and c e l l sorting. Thereafter, r i s i n g c y c l i c AMP levels within the c e l l mass are said to induce c e l l s to d i f f e r e n t i a t e into pre-stalk or pre-spore c e l l s according to thei r o r i g i n a l propensity. Although there is supportive evidence for morphological differences between aggregating c e l l s (Schaap, 1983), there is also some contradictory evidence indicating that c e l l s are di s t r i b u t e d at random within mounds immediatly p r i o r to the i n i t i a l formation of a 153 pre-stalk/pre-spore pattern at the tipped aggregate stage (Tasaka and Takeuchi, 1981). No factors such as DIF or PIF are implicated in the formation of the pre-stalk/pre-spore pattern, and there is no mention of the o r i g i n of anterior-li k e c e l l s in t h i s scheme. Once the pre-stalk/pre-spore pattern is established, several mechanisms are proposed to explain the regulation of pre-stalk, anterior-1ike and pre-spore c e l l s . The maintenance of a low c y c l i c AMP to adenosine r a t i o in the anterior of slugs is said to prevent pre-spore s p e c i f i c d i f f e r e n t i a t i o n . The demonstration that adenosine promotes the formation of pre-stalk c e l l s from clumps of isolated pre-spore c e l l s (Weijer and Durston, 1985) and that i t prevents the induction of pre-spore c e l l components in. v i tro (Van Lookeren Campagne ejt al.., 1986) are consistent with this hypothesis, but d i r e c t measurements of c y c l i c AMP and adenosine levels within slugs are s t i l l needed to substantiate t h i s hypothesis. Rising c y c l i c AMP levels in c e l l aggregates are said to induce the secretion of DIF which is proposed to mediate the conversion of pre-spore to a n t e r i o r - l i k e c e l l s in the posterior region of slugs. Though not stated e x p l i c i t l y in this model, DIF is also believed to mediate the pre-stalk to stalk c e l l conversion during culmination. However, DIF is not implicated in the formation of pre-stalk c e l l s , contrary 154 to the findings in this thesis and those of Williams and co-workers (1987). Ammonia is postulated as an antagonist of DIF action, both by opposing the pre-spore to a n t e r i o r - l i k e conversion and by prevented the pre-stalk to stalk' c e l l conversion. Evidence for the l a t t e r i n h i b i t i o n is provided by unpublished r e s u l t s , and Schaap suggests that this conversion would be rel i e v e d during development _in_ v i vo following a drop in ammonia levels in t i p s at the culmination stage. Many ideas presented by Schaap are consistent with published data, and her model is one of the most up-to-date in the l i t e r a t u r e . While some of the re s u l t s presented in this thesis are in f u l l agreement with i t , others do not f i t well with this model. The discussion which w i l l follow describes a d i f f e r e n t model based on the data from this thesis, and thi s model is contrasted with Schaap's model in Figure 29. Amoebae ^ A g g r e g a t i n g c A M P C e l l s ( n M ) c A MP (JJM) P o s t -^agg rega t i on C e l l s Pre - spo re C e l l s N H 3 © 155 .Stalk ^ P r e - s t a l k _ Cel Is D I F ' C e l l s Adenosine S P I F ? _> Spore Ce l l s T I M E ( H o u r s ) 0 12 24 B. NH 3? P o s t - © Amoebae > Aggregat ing ^agg regat ion >Pre - s ta lk * >Stalk c A M P (nM) C e l l s c A M P c e l l s (/J M) P IF c A M P DIF C e l l s D i p Cel l s c A M P Pre-spore Ce l l s A M P SPI F ? Spore C e l l s F i g . 29. C e l l d i f f e r e n t i a t i o n in Dictyoste1ium d i s c o ideum; a comparison between Schaap's model (1986) and a model d e r i v e d from t h i s t h e s i s . The two models above d e p i c t the p r i n c i p a l steps in c e l l d i f f e r e n t i a t i o n and the f a c t o r s which are b e l i e v e d to c o n t r o l them d u r i n g the development of D i c t y o s t e l i u m  d i sco ideum. A time s c a l e r e p r e s e n t i n g hours of development is drawn (not to s c a l e ) to i n d i c a t e when these processes take p l a c e . A. Schaap's model proposed in 1986, d e s c r i b e d e a r l i e r . B. A model d e r i v e d from the r e s u l t s of t h i s t h e s i s and from p u b l i s h e d data, which i s d e s c r i b e d below. 156 It is generally agreed that starvation i n i t i a t e s the developmental program in a l l c e l l s and that spontaneous pulsing of c y c l i c AMP at nanamolar concentrations activates the c e l l surface c y c l i c AMP receptor and induces the genes necessary for aggregation (Devreotes, 1982; Williams e_t al . , 1986). At the mound stage, r i s i n g c y c l i c AMP concentrations within the c e l l aggregate, perhaps concurrently with a change in the p e r i o d i c i t y of c y c l i c AMP pulses (Gross et a l . , 1977) would activate postaggregative genes and s p e c i f i c a l l y inactivate aggregation stage s p e c i f i c functions. The data from the disaggregated developing c e l l s exposed to 1 mM and 25 pM c y c l i c AMP in monolayers for 2 hours (Figure 24B) suggest that these functions may be induced through modified c e l l surface c y c l i c AMP receptors. The synthesis of cell-type non-specific enzymes such as glycogen phosphorylase or AP IV is probably i n i t i a t e d at that time (Figure 14) as well as the synthesis and secretion of DIF and PIF (Figure 2, Brookman ejb a l . , 1982; Kumagai and Okamoto, 1986). However," since PIF levels r i s e rapidly at the mound stage whereas DIF does not begin to accumulate s i g n i f i c a n t l y u n t i l the tipped aggregate stage, i t is also possible that DIF does not begin to be a c t i v e l y synthesized u n t i l a f t e r the induction of pre-spore c e l l s . 157 The factors d i c t a t i n g which c e l l becomes induced by PIF in c e l l masses remain to be determined, but they might include some i n t r i n s i c c e l l properties such as the position in the c e l l cycle at the onset of starvation (Weijer e_t al . , 1984). It is also possible that e x t r a c e l l u l a r c y c l i c AMP and adenosine levels a f f e c t t h i s induction and these factors might be involved in the establishment of the pre-stalk/pre-spore pattern as proposed by Schaap (1986). Pre-spore c e l l induction apparently involves the ac t i v a t i o n of spore pathway genes and the repression of aggregation stage genes such as the pre-stalk s p e c i f i c a c t i n (Tsang e_t al.. , 1982) or the CI antigen (Tasaka et al_. , 1983). The available evidence suggests that pre-spore c e l l s are less chemotactic to c y c l i c AMP than pre-stalk c e l l s (Kopachik, 1982b; Ratner and Borth, 1983; Mee e_t al_. , 1986), suggesting that most of the postaggregative c e l l s which do not form pre-spore c e l l s would sort chemotactical1y and form a t i p . However, some c e l l s remain dispersed among pre-spore c e l l s as a n t e r i o r - l i k e c e l l s (Tasaka and Takeuchi, 1981; Sternfeld and David, 1981; MacWilliams, 1984), apparently being prevented from sorting towards the t i p by a factor secreted from the t i p region i t s e l f (Sternfeld and David, 1981, 1982). 158 C e l l u l a r c y c l i c AMP l e v e l s may p l a y an important r o l e in d e t e r m i n i n g the pathway of c e l l d i f f e r e n t i a t i o n , an idea championed by Sussman (1982) and suppor ted by the f i n d i n g that 8-Bromo-c.ycl i c AMP r e v e r s e d c a f f e i n e i n h i b i t i o n ( F i g u r e 11). Both p r e - s t a l k c e l l f o r m a t i o n and the p r e - s t a l k to s t a l k c e l l c o n v e r s i o n were found to be i n h i b i t e d by c a f f e i n e ( F i g u r e s 9, 12 and 27) , whereas p r e - s p o r e c e l l f o r m a t i o n is i n s e n s i t i v e to c a f f e i n e i n h i b i t i o n (Schaap e_t al_. , 1986), s u g g e s t i n g that p r e - s p o r e and p r e - s t a l k c e l l s might d i f f e r in t h e i r i n t e r n a l l e v e l s of c y c l i c AMP. T h i s d i f f e r e n c e might a l s o e x p l a i n why ammonia i n h i b i t s s t a l k c e i l f o r m a t i o n and enhances spore c e l l f o r m a t i o n , s i n c e ammonia has been shown to prevent the a c c u m u l a t i o n of i n t r a c e l l u l a r c y c l i c AMP ( S c h i n d l e r and Sussman, 1979). I t i s proposed that DIF a c t i v a t e s the p o s t a g g r e g a t i v e , n o n - p r e - s p o r e c e l l s to the s t a l k pathway d u r i n g the t r a n s i t i o n between the t i p p e d aggregate and f i r s t f i n g e r s t a g e s . T h i s i s suppor ted by r e s u l t s i n d i c a t i n g tha t the DIF-dependent enzyme AP II is f i r s t d e t e c t e d at that time ( F i g u r e s 15 and 16; Oohata , 1983) and by the f a c t tha t t h i s enzyme i s p r e s e n t in both a n t e r i o r - l i k e and p r e - s t a l k c e l l s (Devine and Loomis , 1985). S ince c e l l s do not b e g i n to become c y c l i c AMP independent u n t i l 12 hours of development in v i vo ( F i g u r e 24A), and c y c l i c AMP and DIF ac t w i t h i n a s h o r t time of each o ther in monolayers ( F i g u r e 3 ) , c y c l i c AMP may a l s o be i n v o l v e d in t h i s i n d u c t i o n . A s i g n i f i c a n t 159 contribution from this thesis is the evidence suggesting that the pre-stalk to stalk conversion is mediated by DIF and that this process is restrained by a signal which might control the t r a n s i t i o n from slug migration to culmination. This signal might be a reduction of ammonia levels within c e l l masses, or possibly a c e l l u l a r function affected by the presence of ammonia. Ostensibly, the gradual increase in the number of c y c l i c AMP independent c e l l s observed during development in. v i vo (Figure 24A) might r e f l e c t a progressive r e l i e f from th i s i n h i b i t i o n , u n t i l culmination eventually becomes triggerred. The pre-spore to spore conversion is the process for which there is the least information .available. It may be mediated by c e l l - c e l l contacts or by a positive e f f e c t o r acting during culmination, for which SPIF is the most l i k e l y candidate (Wilkinson et a i . , 1985). The conversion of pre-spore to stalk c e l l s in monolayers has been shown to occur in the presence of DIF (Table V and Figure 26; Kay and Jermyn, 1983)) and since DIF is found p r e f e r e n t i a l l y in the pre-spore region of slugs (Brookman et a l . , 1987), there must also be elements present in this region which r e s t r i c t this process. Conceivably, ammonia might also r e s t r a i n the l a t t e r conversion during slug migration. 160 Whether the whole population secretes factors which r e s t r a i n terminal d i f f e r e n t i a t i o n or whether speci a l i z e d regions such as the t i p c e l l s produce these presumptive inhibi t o r s is a matter of conjecture. 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