DEVELOPMENT AL AND CELLULAR BIOLOGY OF COELENTERATES Proceedings of the 4th International Coelenterate Conference held in Interlaken, Switzerland, 4-8 September, 1979.

Editors P. Tardent and R. Tardent

1980

ELSEVIER/NORTH-HOLLAND BIOMEDICAL PRESS AMSTERDAM • NEW YORK • OXFORD

ix

CONTENTS P r e f a c e

Our l i n k w i t h the T r e m b l e y s - Abraham (1710-1784), M a u r i c e (1874-1942) and J e a n - G u s t a v e (1903-1977) H. L e n h o f f XVII

L I F E CYCLES AND REPRODUCTIVE BIOLOGY

L i f e c y c l e s o f t h e C n i d a r i a B. Werner 3

E a r l y l i f e h i s t o r y o f the s e a wasp, C h i r o n e x f l e c k e r i ( c l a s s Cubozoa) M- Yamaguchi, R. H a r t w i c k 11

R e p r o d u c t i o n and b r o o d i n g i n A c t i n i a M.A. C a r t e r , M.E. F u n n e i l 17

C o m p a r i s o n o f c i r c a s e m i l u n a r r h y t h m i c i t y i n e a s t and west c o a s t c n i d a r i a n s M.A. B r o c k 23

I n c r e a s e i n g o n o z o o i d f r e q u e n c y as an a d a p t i v e r e s p o n s e t o s t r e s s i n C a m p a n u l a r i a f l e x u o s a A.R.D. S t e b b i n g 27

R e p r o d u c t i o n s e a s o n s and d a y / n i g h t b a t h y m e t r i c d i s t r i b u t i o n o f t h r e e s p e c i e s o f D i p h y i n a e ( S i p h o n o p h o r a e ) , o f f C a l i f o r n i a and B a j a C a l i f o r n i a 7.. A l v a r i n o 33

GAMETOGENESIS AND SPAWNING

The a n t h o z o a n egg: T r o p h i c mechanisms and o o c y t e s u r f a c e s H. Schmidt, W.G. Schäfer 41

The a n t h o z o a n egg: D i f f e r e n t i a t i o n o f i n t e r n a l o o c y t e s t r u c t u r e W.G. Schäfer, H. Schmidt 47

P e c u l i a r i t i e s o f s p e r m a t o g e n e s i s and sperm i n A n t h o z o a II. S c hmidt, B. Höltken 53

U l t r a s t r u c t u r a l a s p e c t s o f gametogenesis i n A c t i n i a e q u i n a L. A. Larkman 61

Gametogenesis o f S e r t u l a r e l l a p o l y z o n i a s w i t h s p e c i a l r e f e r e n c e t o v i t e l l o g e n e s i s (Hydrozoa, A t h e c a t a ) T. Honegger 67

X

Darkness i n d u c e d m a t u r a t i o n and spawning i n S p i r o c o d o n s a l t a t r i x M. Y o s h i d a , N. H o n j i , S. Ikegami 75

L i g h t - c o n t r o l l e d spawning i n P h i a l i d i u m h e m i s p h a e r i c u m (Leptomedusae) T. Honegger, J . Achermann, R. S t i d w i l l , L. L i t t l e f i e l d , R. B a e n n i n g e r , P. T a r d e n t 83

S p e c i e s - s p e c i f i c i t y o f sperm Chemotaxis i n t h e Hydromedusae R.L. M i l l e r 89

EMBRYOGENESIS, LARVAL DEVELOPMENT AND METAMORPHOSIS

The r o l e o f c l e a v a g e i n t h e e s t a b l i s h m e n t o f t h e a n t e r i o r ­p o s t e r i o r a x i s o f t h e h y d r o z o a n embryo G. Freeman 97

A c o m p a r a t i v e s t u d y o f t h e embryonic development o f H y d r o z o a a t h e c a t a G. Van de V y v e r 109

M e t a b o l i e p a t t e r n s and m o r p h o l o g i c a l p o l a r i z a t i o n i n the embryonic development and b u d d i n g o f some Hydrozoa T.V. Ostroumova, L.V. B e l o u s s o v 121

O c curence o f p o l y ( A ) RNA d u r i n g embryogenesis and meta-mo r p h o s i s o f H y d r a c t i n i a e c h i n a t a R. E i b e n 125

Embryonic development o f T u b u l a r i a c r o c e a A g a s s i z w i t h s p e c i a l r e f e r e n c e t o the f o r m a t i o n o f t h e i n t e r s t i t i a l c e l l s F . J . F e n n h o f f 127

An u l t r a s t r u c t u r a l O b s e r v a t i o n on t h e embryogenesis o f P e l m a t o h y d r a r o b u s t a , w i t h s p e c i a l r e f e r e n c e t o " g e r m i n a l dense b o d i e s " K. Nöda, C. K a n a i ^ 3 3

Metamorphosis and r e p r o d u c t i o n by t r a n s v e r s e f i s s i o n i n an E d w a r d s i i d anemone S. C r o w e l l , S. O ates 139

L o c a l i s a t i o n o f i n t r a c e l l u l a r c a l c i u m w i t h i n t h e e p i d e r m i s o f a c o o l temperate c o r a l D. K i n c h i n g t o n 143

The l a r v a l l i f e h i s t o r y o f C t e n o p h o r e s : A r e v i e w o f r e c e n t r e s e a r c h K. Stanlaw, M. Reeve, M.A. W a l t e r 149

GROWTH AND COLONIAL DIFFERENTIATION

The modular h a b i t - A r e c u r r i n g s t r a t e g y G. Chapman, A.R.D. S t e b b i n g 157

x i

Growth r a t e s o f A e q u o r e a medusae M . N . A r a i 163

Post-metamorphic l i f e h i s t o r y o f the s y m b i o t i c anemone C a l l i a c t i s p a r a s i t i c a (Couch) D.M. ROSS 171

Growth rhythms and s p e c i e s - s p e c i f i c shape i n T h e c a p h o r a h y d r o i d s L.V. B e l o u s s o v , L.A. Badenko, Ju.A. Labas 175

C u r r e n t i n d u c e d v a r i a t i o n s i n the growth and morphology o f h y d r o i d s R.G. Hughes 179

M e c h a n i c a l l y i n d u c e d s t o l o n b r a n c h i n g i n E i r e n e v i r i d u l a ( T h e c a t a , C a m p a n u l i n i d a e ) G. P l i c k e r t 185

C o l l a g e n d e p o s i t i o n i n b r a n c h t i p s o f t h e o c t o c o r a l L e p t o g o r g i a v i r g u l a t a G.J. L e v e r s e e 191

The f i n e s t r u c t u r e o f t h e " g o r g o n i n " s e c r e t i n g c e l l s o f t h e g o r g o n i a n c o r a l L e p t o g o r g i a v i r g u l a t a (Lam.) J.G. T i d b a l l 197

The e f f e c t o f seawater c h e m i s t r y on t h e growth o f some s c l e r a c t i n i a n c o r a l s P. Swart 203

ASEXUAL REPRODUCTION

E f f e c t s o f the t r o p h i c and p h y s i c a l e n v i r o n m e n t s on a s e x u a l r e p r o d u c t i o n and body s i z e i n the sea anemone M e t r i d i u m s e n i l e J.M. S h i c k , R.J. Hoffmann 211

Bud f o r m a t i o n and c o n t r o l o f p o l y p morphogenesis i n C a s s i o p e a a n d r o m e d a (Scyphozoa) R. Neumann, G. Schmahl, D.K. Hofmann 217

The r e g e n e r a t i o n o f p o l y p s and f r u s t u l e s o f V a l l e n t i n i a g a b r i e l l a e Mendes 1948 (Limnomedusae) B. Auberson, P. T a r d e n t 225

Can Kydra count? S. Shostak 231

R e g u l a t i o n o f bud i n d u c t i o n and s i t e o f t e n t a c l e s p r o u t i n g i n a nonbudding s t r a i n o f H y d r a v i r i d i s P. Novak, H.M. L e n h o f f 237

MEDUSA DEVELOPMENT

Morphogenetic p a t t e r n s o f schyphozoan s t r o b i l a t i o n K.-I. Kato, T. Tomioka, K. Sakagami 245

x i i

Morphogenesis i n i s o l a t e d a p i c a l and b a s a l p a r t s o f s t r o b i l a t i n g s c y p h i s t o m a e i n A u r e l i a a u r i t a and C h r y s a o r a sp. (Scyphozoa) G. Schmahl

Organ d i f f e r e n t i a t i o n i n A u r e l i a a u r i t a ( E f f e c t o f t h e a n a e s t h e t i c MS 222 on d i f f e r e n t i a t i o n o f t h e s c y p h i s t o m a t o s t r o b i l a i n A u r e l i a a u r i t a ) Y. Kakinuma, Y. S u g i u r a

S t r o b i l a t i o n a b e r r a t i o n s i n A u r e l i a i n d u c e d by p e t r o l e u m -r e l a t e d a n i l i n e and p h e n o l D.B. Spangenberg, K. I v e s , M. P a t t e n

REGENERATION

The f a t e and r e g e n e r a t i o n c a p a c i t y o f i s o l a t e d e c t o - and endoderm i n p o l y p s o f P o d o c o r y n e c a r n e a M. S a r s (Hydrozoa, A t h e c a t a ) J . Achermann

The i n f l u e n c e o f p r e d a t i o n upon T u b u l a r i a e r o c e a J.W. Cooper

The e f f e c t o f a c t i n o m y c i n D on RNA b i o s y n t h e s i s and morphogenesis i n Hydra J.R. Voland,G.E. L e s h - L a u r i e

DIFFERENTIATION AND TRANSDIFFERENTIATION

An SEM p o r t r a i t o f Hydra L.G. Epp

C o n t r o l o f stem c e l l p r o l i f e r a t i o n i n H y d r a a t t e n u a t a C.N. D a v i d

C o n t r o l o f i n t e r s t i t i a l c e l l d i v i s i o n and d i f f e r e n t i a t i o n i n H . a t t e n u a t a H.R. Bode

An u l t r a s t r u c t u r a l and k i n e m a t o g r a p h i c s t u d y o f t h e nematocyst morphogenesis i n s e l e c t e d c o e l e n t e r a t e s p e c i e s T. H o l s t e i n

Nematocyte d i f f e r e n t i a t i o n from i n t e r s t i t i a l c e l l s newly-i n t r o d u c e d i n t o i n t e r s t i t i a l c e l l - d e f i c i e n t Hydra T. F u j i s a w a , T. Sugiyama

The f i n e s t r u c t u r e o f d e v e l o p i n g and mature c n i d o b l a s t s o f E i r e n e v i r i d u l a T. Germer, M. Hündgen

About t h e f u n c t i o n o f s t e n o t h e l e s i n H y d r a a t t e n u a t a P a l l . P. T a r d e n t , T. Honegger, R. B a e n n i n g e r

The e l e m e n t a l c o m p o s i t i o n o f n e m a t o c y sts as d e t e r m i n e d by X - r a y m i c r o a n a l y s i s R.N. M a r i s c a l 337

D i f f e r e n t i a t i o n o f o c e l l i i n ephyrae o f A u r e l i a a u r i t a M. Y o s h i d a , Y. Y o s h i n o 343

S t r u c t u r a l Organization, ontogeny and r e g e n e r a t i o n o f t h e eye o f C l a d o n e m a r a d i a t u m (Anthomedusae) C. Weber 347

Manubrium r e g e n e r a t i o n " i n v i t r o " V. Schmid, R. B a e n n i n g e r 353

PATTERN FORMATION AND MORPHOGENS

Hydra as a model f o r p h y s i c a l c o n c e p t s o f b i o l o g i c a l p a t t e r n f o r m a t i o n A. G i e r e r 363

M o r p h o g e n e t i c models f o r h y d r a n t h development T.C. L a c a l l i 373

A n a l y s i s o f m o r p h o g e n e t i c p r o c e s s e s i n Hydra by means o f an endogeneous i n h i b i t o r S. B e r k i n g 377

P o l a r i t y r e v e r s a l i n H y d r a c t i n i a by head a c t i v a t o r s and n e u r o t r a n s m i t t e r s , and c o m p a r a t i v e s t u d i e s i n Hydra W.A. Müller, H.M. Meier-Menge 383

M o r p h o g e n e t i c s u b s t a n c e s i n n e r v e - f r e e Hydra H.C. S c h a l l e r , T. Schmidt, C.J.P. G r i m m e l i k h u i j z e n T. Rau, H. Bode 389

M o r p h o g e n e t i c s u b s t a n c e s i n Hydra T. Schmidt, C.J.P. G r i m m e l i k h u i j z e n , H.C. S c h a l l e r 395

P u r i f i c a t i o n and a n a l y s i s o f Hydra morphogen G.E. L e s h - L a u r i e , V.R. F l e c h t n e r , R.L. Hood 401

CELLULAR DYNAMICS AND INTERACTIONS

Growth r a t e and c e l l c y c l e o f Hydra J . Takano, T. F u j i s a w a , T. Sugiyama 409

The d i s t r i b u t i o n o f p r o l i f e r a t i n g c e l l s i n an anthozoan p o l y p , H a l i p l a n e l l a l u c i a e ( A c t i n i a r i a : A c o n t i a r i a ) , as i n d i c a t e d by ^ T - t h y m i d i n e i n c o r p o r a t i o n L .L. M i n a s i a n , J r . 415

R o l e o f muscle p r o c e s s e s i n Hydra morphogenesis R.D. Campbell 421

xiv

A mutant Hydra s t r a i n (SF-1) c o n t a i n i n g t e m p e r a t u r e - s e n s i t i v e i n t e r s t i t i a l c e l l s B. A. Marcum, T. F u j i s a w a , T. Sugiyama 429

E p i d e r m a l c e l l movement d u r i n g p o d o c y s t f o r m a t i o n i n C h r y s a o r a q u i n q u e c i r r h a J . E . Magnusen 4 35

C e l l i n t e r a c t i o n and form i n r e g e n e r a t i n g and b u d d i n g Hydra F. K l i m e k , L. G r a f , G. Hansmann, A. G i e r e r 441

F r e e z e f r a c t u r e s t u d i e s on c e l l j u n c t i o n f o r m a t i o n i n r e g e n e r a t i n g Hydra R.L. Wood 447

The gap j u n c t i o n s i n Hydra do not p e r m i t e l e c t r i c a l and c h e m i c a l t r a n s m i s s i o n C. J.P. G r i m m e l i k h u i j z e n , J . L e p a u l t , A.W. McDowall, S.W. de L a a t , L.G.J. T e r t o o l e n , C . J . Weyer 453

P h y s i o l o g i c a l and u l t r a s t r u c t u r a l b a s e s f o r a s t u d y o f the o n t o g e n e s i s o f I n t e g r a t i o n i n P o d o e o r y n e o a r n e a c u l t u r e s M. Pavans de C e c c a t t y 459

S u r v i v a l , b u d d i n g and o s m o t r o p h i c n u t r i t i o n i n H y d r a v i r i d i s M. Rahat, V. R e i c h 465

A v o i d a n c e o f h o s t - d i g e s t i o n by s y m b i o t i c a l g a e T.L. O ' B r i e n 471

C o m p a t i b i l i t i e s and i n c o m p a t i b i l i t i e s i n P o d o e o r y n e c a r n e a (Anthomedusae)

M. B u e h r e r , P. T a r d e n t 4 77

A d d r e s s l i s t o f p a r t i e i p a n t s 481

A u t h o r i n d e x 487

S u b j e c t i n d e x 489

© 1 9 8 0 ElsevierINor th-Holland Biomedical Press Developmental and Cellular Biology of Coelenterates

P. Tardent and R . Tardent, editors 301

CONTROL OF ST EM CELL PROLIFERATION IN HYDRA ATTENUATA

CHARLES N. DAVID, Dept. of Molecular Biology, Albert Einstein College of Medicine, Bronx , N.Y. 10461 (U.S.A.) INTRODUCTION

I n t e r s t i t i a l stem c e l l s i n Hydra constitute a rapidly p r o l i f e r a t i n g Pop­ulation of c e l l s which continuously gives r i s e to differentiated nerves and nematocytes} Under conditions of assexual growth 60% of stem c e l l daughters divide to yi e l d more stem c e l l s (seif-renew), 30% d i f f e r e n t i a t e nematocytes

2 and 107» dif f e r e n t i a t e nerves per stem c e l l generation. The i n t e r s t i t i a l c e l l 3 2 System makes up 10% of t o t a l Hydra c e l l s ; about 47« are stem c e l l s .

Analysis of the stem c e l l System has been f a c i l i t a t e d by the maceration 4

technique for dissociating tissue quantitatively into Single c e l l s or small Clusters in the case of i n t e r s t i t i a l c e l l s and nematoblasts held together by

5 3 cytoplasmic bridges. By combining appropriate pulse and continuous H-thymidine labeling techniques with tissue maceration i t has been possible to determine the c e l l cycle and d i f f e r e n t i a t i o n kinetics of stem c e l l s , d i f f e r e n -

3 6 t i a t i n g nematoblasts and d i f f e r e n t i a t i n g nerves.' The results are shown schematically i n Fig. 1. Stem c e l l s p r o l i f e r a t e with a 24 hr c e l l cycle. Stem c e l l s committed to nematocyte pathway divide several times (18 hr c e l l cycle) to yield nests of 4,8 or 16 nematoblasts; each c e l l i n a nest differentiates

Fig. 1. Schematic representation of stem c e l l p r o l i f e r a t i o n and nerve and nematocyte d i f f e r e n ­t i a t i o n in Hydra.

DIFFERENTIATION >

302

NM HYDRA

REAGGREGATE 105 NM CELLS

AGGREGATE REGENERATION

LIVE HYDRA •100 LIVE CELLS CLONE GROWTH W

¥ Fig. 2. Procedure for cloning stem c e l l s i n (•) i n NM aggregates.

a nematocyte capsule i n 2-3 days. Stem c e l l s committed to nerve pathway divide once and both daughters c e l l s d i f f e r e n t i a t e as nerves in about 6 hours.

CULTURE OF STEM CELLS IN FEEDER LAYERS To investigate factors affecting stem c e l l p r o l i f e r a t i o n and d i f f e r e n t i a t i o n

we have developed a technique for culturing stem c e l l s i n feeder layers of nitrogen mustard (NM) inactivated Hydra tissue. NM treatment eliminates

g i n t e r s t i t i a l c e l l s from Hydra tissue and thus provides an empty host i n which growth and d i f f e r e n t i a t i o n of added i n t e r s t i t i a l c e l l s can be followed.

9 I n t e r s t i t i a l c e l l s are added to feeder layers using the aggregation technique shown i n Fig. 2. Normal Hydra and NM treated Hydra are dissociated to Single c e l l suspensions i n c e l l culture medium. Aliquots containing 10^ NM c e l l s and a small number of normal c e l l s are mixed together and centrifuged. The c e l l p e l l e t s are then incubated during which time they regenerate normal Hydra structures. Stem c e l l s added to the NM aggregates proliferate and d i f f e r e n t i a t e normally. Individual stem c e l l s form clones which grow to contain several hundred c e l l s after 1-2 weeks. NM aggregates are e a s i l y manipulated and pro-vide a v e r s a t i l e technique for culturing and analyzing stem c e l l s and stem c e l l d i f f e r e n t i a t i o n . Several examples are given below. MULTIPOTENCY OF STEM CELLS

The a b i l i t y to clone stem c e l l s i n NM aggregates has allowed a rigorous test of the d i f f e r e n t i a t i o n potential of individual stem c e l l s . When clones derived from Single stem c e l l s were examined, a l l were found to contain both dif f e r e n t i a t e d nerves and differentiated nematocytesj No clones were found which contained only nerves or nematocytes. Thus, the stem c e l l population is homogeneous and multipotent with regard to nerve and nematocyte d i f f e r e n t i a t i o n .

303

Fig. 3. Distribution of stem c e l l s and ls+2s i n Hydra. Stem c e l l s were determined as clone-forming units (CFU) in NM aggregates. About 137, of stem c e l l s y i e l d clones under these conditions. ls+2s are a morphological class of large i n t e r s t i t i a l c e l l s occurring as Single c e l l s or in pairs. ls+2s consist of stem c e l l s and early committed precursors to nerve and nematocyte d i f f e r e n t i a t i o n (Fig. 1). The concentration of stem c e l l s and ls+2s is expressed per e p i t h e l i a l c e l l (EP1).

DISTRIBUTION OF STEM CELLS IN HYDRA Using the NM culture System we have found that stem c e l l s are uniformly

distributed along the body column in the gastric region and Upper peduncle (Fig. 3)}^ The concentration of stem c e l l s , expressed as clone-forming units (CFU) per e p i t h e l i a l c e l l , i s about 0.02. In the hypostome and basal disk, however, the concentration of stem c e l l s is 20-fold lower consituting only about 0.001 CFU/epithelial c e l l .

Stem c e l l s and early committed nerve and nematocyte precursors constitute a morphologically d i s t i n c t class of i n t e r s t i t i a l c e l l s which occur as Single c e l l s and in pairs (see Fig. 1). We refer to this class as ls+2s. The d i s t r i -bution of ls+2s i n Hydra i s similar but not i d e n t i c a l to that of stem c e l l s . In p a r t i c u l a r , the r a t i o of stem cells/ls+2s drops in the hypostome and basal disk compared to the gastric region (Fig. 3) indicating an increase i n these regions i n early committed c e l l s . Changes in the proportions of stem c e l l s and early committed c e l l s result from changes in the proportion of stem c e l l s which self-renew versus d i f f e r e n t i a t e . From the observed decrease in the CFU/ ls+2s r a t i o (Fig. 3) i t i s possible to estimate that the frac t i o n of stem c e l l s undergoing self-renewal i n the hypostome and basal disk has decreased to <107o

compared to 607, i n the gastric region (see below).

304

2000

1000

UJ 500

o üJ

% 200

«3 wo 50

20

10

, A O X

Y

I 2 3 4 5 6 7 DAYS

10000

5000

2000

1000

400 K

200

100

/ 6

2 3 4 5 6 7 DAYS

1.0-

0.9 -

0.8

0.7

0.6

0.5-

100 500 1000 ls-2s/AGGREGATE

5000

Fig. 4. Growth of i n t e r s t i t i a l c e l l s i n NM aggregates. NM aggregates were seeded with 30 or 400 CFU. After 4-7 days incu-bation, aggregates were macerated and scored for ls+2s.

Fig. 5. Dependence of self-renewal pro-b a b i l i t y (P ) on i n t e r s t i t i a l c e l l density

P g was calculated from the growth rate of i n t e r s t i t i a l c e l l pop­in NM aggregates. the growth rate o i ulations as shown i n Fig .

CONTROL OF STEM CELL POPULATION GROWTH The growth of stem c e l l populations depends on both the c e l l generation

time and the fraction of daughter c e l l s remaining stem c e l l s (the self-renewal fr a c t i o n , Pg)» Several independent experiments have now made i t clear that

11 12 13 regulation of stem c e l l growth i n Hydra occurs by changing P g. ' ' In a l l these experiments the length of the stem c e l l generation was not observed to change s i g n i f i c a n t l y . These experiments also suggested that the parameter Controlling P g was the density of stem c e l l s i n tissue. We have now confirmed this d i r e c t l y using the NM culture System. Fig. 4 shows that the growth rate of stem c e l l populations (scored as ls+2s) i n NM aggregates depends on the

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13 number of stem c e l l s seeded. When 30 stem c e l l s are seeded per aggregate, the growth rate i s 4-fold faster than when 400 stem c e l l s are seeded per aggregate. The value of P g can be d i r e c t l y calculated from the doubling time. Fig. 5 shows the dependence of P on stem c e l l density in NM aggregates. As stem c e l l

S 13 density increases i n feeder layers, P g decreases from 0.7 to 0.5.

MODEL FOR CONTROL OF STEM CELL PROLIFERATION A model for the control of stem c e l l p r o l i f e r a t i o n must explain the growth

as well as the d i s t r i b u t i o n of stem c e l l s i n Hydra. The observed dependence of P g on stem c e l l density i n tissue (Fig. 5) indicates that P g is regulated by negative feedback from neighboring stem c e l l s . Because stem c e l l s are spread out i n tissue at some distance from each other, the feedback Signal appears to be mediated by a d i f f u s i b l e factor secreted by stem c e l l s and to

12 which stem c e l l s are also sensitive. Low stem c e l l concentration leads to low factor concentration and high P g; high stem c e l l concentration leads to high factor concentration and low Pg. Such a model w i l l regulate the stem c e l l concentration to a s p e c i f i c level since low concentrations w i l l raise P and

s increase the population growth rate while high concentrations w i l l lower P g

and decrease the population growth rate. Thus, the model explains the observed 3 14

homeostasis of stem c e l l population density in Hydra.' The negative feedback model predicts that stem c e l l s should f i l l a l l

available ectodermal space uniformly. Any areas of low stem c e l l density w i l l have l o c a l l y higher P g and tend to f i l l up. This prediction agrees well with the observed uniform stem c e l l concentration throughout the gastric region. It does not, however, explain the depletion of stem c e l l s i n hypostome and basal disk (Fig. 3). Thus, i n hypostome and basal disk other factors in addition to stem c e l l density must affect P .

s 2 15 16 Nerve d i f f e r e n t i a t i o n i s localized i n the hypostome and basal disk. ' ' These same regions are depleted in stem c e l l s and enriched in early committed c e l l s . The simplest Interpretation of these observations i s , therefore, that l o c a l l y enhanced nerve d i f f e r e n t i a t i o n e f f e c t i v e l y removes stem c e l l s from the self-renewal pathway. If this Interpretation is correct, i t i s the f i r s t direct evidence that self-renewal and d i f f e r e n t i a t i o n compete for the same target stem c e l l population.

In summary, the results indicate that stem c e l l growth i n the gastric region i s regulated by negative feedback from neighboring stem c e l l s . Expansion of the epithelium due to p r o l i f e r a t i o n of e p i t h e l i a l c e l l s spreads stem c e l l s apart, thereby lowering stem c e l l density and increasing P . Growth of the stem c e l l population then f i l l s i n the gaps to maintain a uniform density of

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stem c e l l s . Stem c e l l s carried into hypostome and basal disk by e p i t h e l i a l tissue movements"^ are forced to d i f f e r e n t i a t e nerves by morphogenetic Signals lo c a l i z e d in these regions} Extensive nerve d i f f e r e n t i a t i o n essentially eliminates the stem c e l l population from the epithelium as i t moves into the hypostome and basal disk, thereby creating the empty zones observed there.

ACKNOWLEDGEMENTS This research was supported by grants from the National Institute of Health

(GM11301), National Science Foundation (77-25426) and a Career Development Award (FRA-132) from the American Cancer Society.

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