UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Observations on chick embryo lens morphogenesis in vivo and in vitro McLean, Brian G. 1972

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1972_A6_7 M32_5.pdf [ 25.8MB ]
Metadata
JSON: 831-1.0101645.json
JSON-LD: 831-1.0101645-ld.json
RDF/XML (Pretty): 831-1.0101645-rdf.xml
RDF/JSON: 831-1.0101645-rdf.json
Turtle: 831-1.0101645-turtle.txt
N-Triples: 831-1.0101645-rdf-ntriples.txt
Original Record: 831-1.0101645-source.json
Full Text
831-1.0101645-fulltext.txt
Citation
831-1.0101645.ris

Full Text

c\ OBSERVATIONS ON CHICK EMBRYO LENS MORPHOGENESIS IN VIVO AND IN VITRO BRIAN G. McLEAN B.Sc., The U n i v e r s i t y o f B r i t i s h Columbia A THESIS SUBMITTED IN•PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f ZOOLOGY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1972 In p r e s e n t i n g t h i s t h e s i s In p a r t i a l f u l f i l m e n t o f the r equ i r ements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Co lumb ia , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r an t ed by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s unde r s tood t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l no t be a l l owed w i thou t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada \ i ABSTRACT U l t r a s t r u e t u r a l o b s e r v a t i o n s on t h e s i x - d a y c h i c k embryo l e n s r e v e a l t h a t f i b r e c e l l d i f f e r e n t i a t i o n , w h i c h i n v o l v e s e x t e n s i v e c e l l e l o n g a t i o n , i s c h a r a c t e r i z e d b y t h e p r e s e n c e o f numerous o r i e n t e d m i c r o t u b u l e s and by marked changes i n i n t e r c e l l u l a r r e l a t i o n s h i p s w h i c h a r e f e l t t o be i m p o r t a n t f o r c e l l e x t e n s i o n . I n c r e a s e d G o l g i a c t i v i t y i n c e l l s - i n i t i a t i n g e l o n g a t i o n appears t o be r e l a t e d t o t h e f o r m a t i o n o f i n t e r c e l l u l a r j u n c t i o n s and t h e e l a b o r a t i o n o f new s u r f a c e membrane. S t u d i e s c o n c e r n i n g t h e d i f f e r e n t i a t i o n i n v i t r o o f a n t e r i o r l e n s e p i t h e l i a d e m o n s t r a t e t h a t t h e i r c e l l s e l o n g a t e e i t h e r t o a g r e a t e x t e n t o r t o a l i m i t e d e x t e n t , depending on f a c t o r s d e r i v i n g f rom t h e e p i t h e l i a l c o n f o r m a t i o n . T h e conform-a t i o n o f an e x p l a n t e d a n t e r i o r l e n s e p i t h e l i u m can be manip-u l a t e d so t h a t i t s c e l l s e l o n g a t e c o n s i s t e n t l y t o e i t h e r a l i m i t e d o r a g r e a t e x t e n t . These c e l l s , w hether e l o n g a t e d t o a l i m i t e d o r t o a g r e a t e x t e n t , a r e u l t r a s t r u c t u r a l l y s i m i l a r w i t h r e s p e c t t o f e a t u r e s r e f l e c t i n g c y t o p l a s m i c d i f f e r e n t i a t i o n , i n c l u d i n g t h o s e i m p o r t a n t f o r morphogenesis. T h e i r f i n e s t r u c t u r e c l o s e l y r e s embles t h a t o f f i b r e c e l l s d i f f e r e n t i a t -i n g I n v i v o . The c e l l s o f a n t e r i o r l e n s e p i t h e l i a d i f f e r e n t -i a t i n g i_n v i t r o , w hether e l o n g a t i n g t o a l i m i t e d o r t o a g r e a t e x t e n t , a r e t h e same i n terms o f t h e n a t u r e and r e l a t i v e p r o p o r t i o n s o f t h e i r s o l u b l e p r o t e i n s as i n d i c a t e d by 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 . There i s a change i n t h e r e l -a t i v e p r o p o r t i o n s o f t h e s o l u b l e p r o t e i n s a c c u m u l a t e d by t h e a n t e r i o r l e n s e p i t h e l i a when they d i f f e r e n t i a t e i n v i t r o t h a t i s s i m i l a r t o t h a t w h i c h o c c u r s i n c e l l s u n d e r t a k i n g f i b r e c e l l d i f f e r e n t i a t i o n i n v i v o . I t i s c o n c l u d e d t h a t t h o s e f a c t o r s d e r i v i n g from t h e c o n f o r m a t i o n o f t h e a n t e r i o r l e n s e p i t h e l i u m t h a t a f f e c t t h e degree o f e l o n g a t i o n o f i t s c e l l s d i f f e r e n t i a t i n g in v i t r o a r e p h y s i c a l i n n a t u r e . S i n c e such p h y s i c a l f a c t o r s a r e i m p o r t a n t w i t h r e g a r d t o r e s t r a i n i n g o r e n c o u r a g i n g t h e e x p r e s s i o n o f morphogenetic p o t e n t i a l i n v i t r o , i t i s s u g g e s t e d t h a t s i m i l a r p h y s i c a l f o r c e s a r e i m p o r t a n t i n l e n s morphogenesis in v i v o v O b s e r v a t i o n s c o n c e r n i n g a n t e r i o r l e n s e p i t h e l i a e l o n g a t i n g t o a g r e a t e x t e n t i n v i t r o d e monstrate t h a t t h e i r c e l l s p r o g r e s s i v e l y e l o n g a t e f o r o n l y about t h r e e days. W e l l d i f f e r e n t i a t e d e x p l a n t s c u l t u r e d f o r l o n g e r p e r i o d s do n o t show g r e a t e r e l o n g a t i o n o r f u r t h e r c y t o p l a s m i c d i f f e r e n t i a t i o n . T h e i r : c e l l s , a t t h e u l t r a s t r u c t u r a l l e v e l , r e s e m b l e d i f f e r -e n t i a t i n g f i b r e c e l l s i n v i v o r a t h e r t h a n d i f f e r e n t i a t e d ones. I t i s c o n c l u d e d t h a t t h e a n t e r i o r l e n s e p i t h e l i u m has some c a p a c i t y f o r d i f f e r e n t i a t i o n i n d e p e n d e n t o f t h e o c u l a r e n v i r o n -ment, b u t t h a t t h e l a t t e r i s e s s e n t i a l f o r t h e e x p r e s s i o n o f i t s f u l l d e v e l o p m e n t a l p o t e n t i a l . TABLE OF CONTENTS INTRODUCTION MATERIALS & METHODS OBSERVATIONS I - O r g a n i z a t i o n o f t h e c h i c k embryo l e n s \ I I - F i n e s t r u c t u r e o f t h e c e n t r a l a n t e r i o r l e n s e p i t h e l i u m I I I - F i n e s t r u c t u r e o f t h e l a t e r a l l e n s e p i t h e l i u m IV- F i n e s t r u c t u r e o f t h e e q u a t o r i a l r e g i o n o f the l e n s V- F i n e s t r u c t u r e o f d i f f e r e n t i a t i n g l e n s f i b r e c e l l s V I - D i f f e r e n t i a t i o n o f a n t e r i o r l e n s e p i t h e l i a irt v i t r o V I I - F i n e s t r u c t u r e o f a n t e r i o r l e n s e p i t h e l i a d i f f e r e n t i a t i n g i n  v i t r o V I I I - 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 I - C h i c k embryo l e n s morphogenesis I I - C h i c k embryo l e n s morphogenesis i n v i t r o DISCUSSION SUMMARY BIBLIOGRAPHY LIST OF FIGURES Figure 1 Diagram of chick embryo lens 15. Figures 2-4 Photomicrograph of chick embryo lens defining regions for reference i n discussion of u l t r a s t r u c t u r e . 16. Figures 5-8 Ultrastructure of region I 17-19. Figures 9-11 Ultrastructure of region II 20-21. Figures 12-18 Ultrastructure of region III 22-25. Figures 19-26 Ultr a s t r u c t u r e of regions IV and V 25-29. Figure 27 Golgi of c e l l of region III to compare with figure 26 29. Figure 28-29 Ultrastructure of region V 30. Figures 30-39 Anterior lens e p i t h e l i a at d i f f e r e n t stages of _in v i t r o development * 33-34. Figures 40-42 Anterior lens e p i t h e l i a explanted by method 3 and cultured f o r 1% days 3 5. Figures 43-44 Anterior lens e p i t h e l i a explanted by method 2 and cultured f o r 3 days 35-36. Figures 45-47 Anterior lens e p i t h e l i a explanted by method 3 and cultured f o r 3 days 36. Figures 48-64 Ultrastructure of anterior lens e p i t h e l i a at d i f f e r e n t stages of i n v i t r o development 42-54. Figures 65-66 Ultrastructure of anterior lens epithelia,explanted by method 2, that elongated to a l i m i t e d extent in v i t r o . 55-56. Figures 67-69 Polyaery1amide gel electrophor-esis p r o f i l e s of proteins of anterior lens e p i t h e l i a _in vivo and i n v i t r o 59. V ACKNOWLEDGEMENTS I w o u l d l i k e t o e x p r e s s my s i n c e r e t h a n k s t o Dr, C.V. F i n n e g a n f o r h i s encouragement and s u p p o r t d u r i n g t h e c o u r s e o f t h i s work. A l s o , I am g r a t e f u l t o Denis L a r r i v e e f o r many i n v a l u a b l e d i s c u s s i o n s and t o Mr. L. Ve t o f o r c o n s i d e r a b l e good a d v i s e . I w o u l d l i k e t o thank Mrs. Maureen Douglas f o r h e r h e l p and c o o p e r a t i o n . I am i n d e b t e d t o Dr. A.B. A c t o n f o r h i s a d v i s e c o n c e r n i n g t h e p r e p a r a t i o n o f t h i s m a n u s c r i p t and t o Drs. H. K a s i n s k y and N. A u e r s p e r g f o r c r i t i c a l l y r e a d i n g i t . T h i s work was s u p p o r t e d i n p a r t by t h e N a t i o n a l R e s e a r c h C o u n c i l o f Canada. INTRODUCTION The V e r t e b r a t e l e n s has l o n g been s t u d i e d b y develop^-m e n t a l b i o l o g i s t s w i t h a v a r i e t y o f p o i n t s o f v i e w . Spemann's d e m o n s t r a t i o n , a t t h e t u r n o f t h e c e n t u r y , o f t h e r o l e o f t h e o p t i c v e s i c l e i n ' i n d u c i n g t h e l e n s i s a c l a s s i c example o f i n d u c t i o n (see Spemann, 1938). H i s work, as w e l l as more r e c e n t i n v e s t i g a t i o n s (Jacobson, 1966; McKeehan, 1954; Muthukkaruppan, 1965), have c o n t r i b u t e d much t o t h e c o n c e p t s o f i n d u c t i o n and d e t e r m i n a t i o n . The a d j u s t m e n t o f t h e growth o f t h e l e n s t o t h e r e s t o f t h e eye has r e c e i v e d some a t t e n t i o n ( T w i t t y , 1955; Coulombre and Coulombre, 1969).. B i o l o g i s t s i n t e r e s t e d i n t h e r e g u l a t i o n o f t h e c e l l c y c l e have a l s o d i r e c t e d t h e i r a t t e n t i o n t o t h e l e n s (see r e c e n t r e v i e w b y H a r d i n g _et al. , 1971). Workers c o n c e r n e d w i t h t h e problems o f c e l l d i f f e r e n t -i a t i o n have been a t t r a c t e d t o t h e l e n s f o r a number o f r e a s o n s . The o r g a n i s a t i o n o f t h i s s t r u c t u r e i s s uch t h a t one can s t u d y , i n a s i n g l e l e n s , c e l l s a t d i f f e r e n t s t a g e s o f d i f f e r e n t i a t i o n . And i t i s p o s s i b l e t o p h y s i c a l l y s e p a r a t e d i f f e r e n t r e g i o n s o f t h e l e n s t h a t c o r r e s p o n d t o d i f f e r e n t s t a g e s o f c e l l d i f f e r e n t -i a t i o n i n o r d e r t o do b i o c h e m i c a l s t u d i e s ( P a p a c o n s t a n t i n o u , 1967). An a d d i t i o n a l advantage i s t h a t t h e l e n s i s composed o f c e l l s o f a s i n g l e t y p e d e r i v e d from an i n i t i a l e c t o d e r m a l p o p u l a t i o n , t h e - l e n s p l a c o d e . F u r t h e r m o r e , l e n s c e l l d i f f e r e n t -i a t i o n i n v o l v e s marked m o r p h o l o g i c a l and b i o c h e m i c a l changes, t h e most s t r i k i n g o f w h i c h a r e e x t e n s i v e c e l l e l o n g a t i o n and t h e s y n t h e s i s o f l a r g e q u a n t i t i e s o f u n i q u e s t r u c t u r a l p r o t e i n s , t h e l e n s c r y s t a l l i n s . F o r a l l t h e s e r e a s o n s , c o n s i d e r a b l e e f f o r t has been d i r e c t e d t oward d e s c r i b i n g l e n s d i f f e r e n t i a t i o n , u l t r a s t r u c t u r a l l y and b i o c h e m i c a l l y , w i t h a v i e w t o l e a r n i n g something about t h e p r o c e s s e s o f c e l l d i f f e r e n t i a t i o n and t h e i r c o n t r o l . The l e n s a l s o r e p r e s e n t s a s t r u c t u r e i n t e r e s t i n g from t h e p o i n t o f v i e w o f morphogenesis inasmuch as i t s c e l l s a r e h i g h l y o r g a n i z e d and form a s y m m e t r i c a l s t r u c t u r e o f p a r t i c u l a r shape, w i t h a s p e c i f i c s p a t i a l r e l a t i o n s h i p t o o t h e r components o f t h e eye, t o w h i c h l e n s growth i s a p p r o p r i a t e l y r e g u l a t e d (Coulombre, 1969; Coulombre and Coulombre, 1969). The growth o f t h e l e n s i n v o l v e s t h e g e n e r a t i o n , t h r o u g h ..mitosis / o f new c e l l s w i t h i n t h e a n t e r i o r l e n s e p i t h e l i u m and t h e c o o r d i n a t e d d i f f e r e n t i a t i o n o f c e l l s a t t h e l e n s e q u a t o r . Here, d i f f e r e n t i a t i o n i n v o l v e s e x t e n s i v e c e l l e l o n g a t i o n so t h a t new f i b r e c e l l s a r e added t o t h e l e n s body u n i f o r m l y , g e n e r a t -i n g a f u n c t i o n a l l e n s o f a p p r o p r i a t e s i z e and shape. What i s o f i n t e r e s t i s t h e manner i n w h i c h morphogenesis i s a c h i e v e d and how i t i s c o n t r o l l e d . The t i s s u e s o f t h e eye i n t e r a c t w i t h each o t h e r i n some manner t h a t i s i m p o r t a n t f o r i t s development and i n t e g -r i t y (Coulombre, 1969; Coulombre and Coulombre, 1963; T a k e i c h i , 1970; McKeehan, 1951, .1954; Muthukkaruppan, 1965). I t w o u l d be o f i n t e r e s t t o be a b l e t o d i s t i n g u i s h t h e n a t u r e and r e l a t -i v e i m p o r t a n c e o f t h e r o l e s t h e o c u l a r e n v ironment and t h e p r o p e r t i e s o f t h e l e n s p l a y i n l e n s development. A r e p o r t r e l e v a n t i n t h i s c o n t e x t i s t h a t o f Coulombre and Coulombre (1971). These a u t h o r s d e m o n s t r a t e d t h a t i f t h e l e n s o f a c h i c k embryo were r e p l a c e d w i t h a n t e r i o r l e n s e p i t h e l i u m , t h e l a t t e r 3. w o u l d form a l e n s w i t h an e p i t h e l i u m , o f a p p r o p r i a t e s i z e and o r i e n t a t i o n , and o f a shape w h i c h , a l t h o u g h n o t p e r f e c t , more o r l e s s r e s e m b l e d t h a t o f a normal l e n s . On t h e o t h e r hand, when p l a c e d i n v i t r o , t h e c e l l s o f t h e a n t e r i o r l e n s e p i t h e l i u m were r e p r t e d ( P h i l p o t t and Coulombre, 1965, 1968) t o e l o n g a t e , i n c u l t u r e medium supplemented w i t h serum, t o o n l y a l i m i t e d degree. They d i d n o t e l o n g a t e a t a l l , however, i n unsupplemented medium. These f i n d i n g s r a i s e a number o f q u e s t i o n s , f i r s t w i t h r e s p e c t t o t h e r e a s o n s f o r l i m i t e d r a t h e r t h a n e x t e n s i v e c e l l e l o n g a t i o n , and second c o n c e r n i n g t h e r e l a t i o n s h i p between c e l l e l o n g a t i o n and c y t o p l a s m i c d i f f e r e n t i a t i o n . They a l s o i n d i c a t e d t h e p o s s i b i l i t y o f m a n i p u l a t i n g l e n s development i n v i t r o and t h e r e b y l e a r n i n g something about morphogenesis, c e l l d i f f e r e n t i a t i o n and t h e r e l a t i v e r o l e s o f t h e l e n s and t h e o c u l a r environment w i t h r e s p e c t t o t h e s e p r o c e s s e s . Because o f t h e s e c o n s i d e r a t i o n s i t was c o n s i d e r e d o f i n t e r e s t t o i n v e s t i g a t e t h e development o f t h e c h i c k embryo a n t e r i o r l e n s e p i t h e l i u m i n v i t r o . S i n c e t h e p u b l i s h e d 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 s ( P o r t e e t a l . . , 1968; B e y e r s and P o r t e r , 1964) appeared somewhat i n a d e q u a t e , my i n v e s t i g a t i o n was f i r s t c o n c e r n e d w i t h t h e f i n e s t r u c t u r e o f t h e c h i c k embryo l e n s i n o r d e r t o make o b s e r v a t i o n s r e l e v a n t t o m o r p h o g e n e t i c c o n s i d e r -a t i o n s , and n e c e s s a r y f o r u l t r a s t r u c t u r a l e v a l u a t i o n o f l e n s d i f f e r e n t i a t i o n i n - v i t r o . 4. I t was a p p a r e n t t h a t most u l t r a s t r u e t u r a l o b s e r -v a t i o n s r e p o r t e d were c o n c e r n e d w i t h c y t o d i ' f f e r e n t i a t i o n o f t h e l e n s . The l o s s o f c y t o p l a s m i c o r g a n e l l e ' s ^ t h a t accompan-i e s l e n s f i b r e c e l l d i f f e r e n t i a t i o n , and t h e ' f e a t u r e s a t t e n d -i n g p r o t e i n s y n t h e s i s (prominent n u c l e o l i , numerous polysomes, a c c u m u l a t i o n o f c y t o p l a s m i c m a t r i x ) a r e f a i r l y w e l l document-ed f o r a number o f V e r t e b r a t e l e n s e s (Wanko and G a y i n , 19 59; P o r t e jet _ a l , , 1968; Rabaey and Lagasse, 1965; E g u c h i , 1964; Cohen, 1965; see Smelser, 1965). Only two f i n e s t r u c t u r a l s t u d i e s c o n c e r n i n g t h e c h i c k embryo l e n s were p e r f o r m e d u s i n g g l u t a r a l d e h y d e f i x a t i o n (Beyers and P o r t e r , 1964; P o r t e e_t aT.,1968), and t h e former was c o n c e r n e d m a i n l y w i t h m i c r o -t u b u l e s i n t h e e a r l y l e n s r u d i m e n t . W i t h t h e e x c e p t i o n o f t h e p o s s i b l e r o l e o f m i c r o -t u b u l e s (Beyers and P o r t e r , 1964; Kuwabara, 1968) i n f i b r e c e l l e l o n g a t i o n , l i t t l e c o n s i d e r a t i o n has been g i v e n t o o b s e r v a t i o n s p e r t i n e n t t o l e n s morphogenesis. A l t h o u g h i n t e r -c e l l u l a r j u n c t i o n s ( t i g h t j u n c t i o n s and 1desmosomes 1) between f i b r e c e l l s have been r e p o r t e d i n a number o f V e r t e b r a t e l e n s -es, t h e l i t e r a t u r e does n o t p r o v i d e a c l e a r d e s c r i p t i o n o f t h e i r development. Nor does t h e l i t e r a t u r e p r o v i d e any o b s e r v a t i o n s c o n c e r n e d w i t h t h e manner i n w h i c h the new s u r f a c e membrane r e q u i r e d f o r c e l l e l o n g a t i o n i s e l a b o r a t e d / o r v e r y much i n f o r m a t i o n r e g a r d i n g t h e G o l g i a p p a r a t u s , a n o r g a n e l l e c o n s i d e r e d t o be c o n c e r n e d w i t h membrane s y n t h e s i s ( S j o s t r a n d , 1968; Whaley e t aT.,1971), i n c e l l s o f the l e n s . 5. W i t h r e s p e c t t o i n v e s t i g a t i n g _in v i t r o d i f f e r e n t -i a t i o n o f a n t e r i o r l e n s e p i t h e l i a , a p r e l i m i n a r y e x p e r i m e n t i n d i c a t e d t h a t c e l l s o f t h e e p i t h e l i a w o u l d undergo c o n s i d e r -a b l y g r e a t e r e l o n g a t i o n t h a n t h a t p r e v i o u s l y r e p o r t e d ( P h i l -p o t t and Coulombre, 1965, 1968). I ^ s u b s e q u e n t l y became appar-e n t t h a t t h e e p i t h e l i a l c o n f o r m a t i o n was i m p o r t a n t f o r t h i s e l o n g a t i o n , and t h a t by m a n i p u l a t i n g t h e e p i t h e l i a l conform-a t i o n i n c e r t a i n ways e o t h e r v e r y l i m i t e d o r v e r y g r e a t c e l l e l o n g a t i o n w o u l d r e s u l t . I t was th e n o f i n t e r e s t t o u n d e r t a k e u l t r a s t r u c t u r a l s t u d i e s w i t h r e s p e c t t o a n t e r i o r l e n s e p i t h e l -i a d i f f e r e n t i a t i n g i n v i t r o from t h e p o i n t s o f view o f morph-o g e n e s i s and c y t o d i f f e r e n t i a t i o n . MATERIALS & METHODS  G e n e r a l i n v i t r o p r o c e d u r e s . F e r t i l i z e d W h i te Leghorn eggs o f s i x days i n c u b a t i o n were o b t a i n e d from t h e Department o f P o u l t r y G e n e t i c s (U.B.C.). The embryos were removed and t r a n s f e r r e d t o a p e t r i d i s h c o n t -a i n i n g s t e r i l e Puck's S a l i n e - G f o r d i s s e c t i o n . The v i t r e o u s b o d i e s w i t h a d h e r i n g l e n s e s were p l u c k e d from t h e eye w i t h watchmaker's f o r c e p s and t h a a d h e r i n g i r i s and v i t r e o u s s e p a r -a t e d from t h e l e n s e s . The l e n s e p i t h e l i a were s e p a r a t e d from t h e l e n s body, and c a r e f u l l y t r a n s f e r r e d , by one o f t h r e e methods, t o e i t h e r a N u c l e p o r e ( W i n l e y - M o r r i s ) o r M i l l i p o r e ( t y p e TH) f i l t e r . T h e f i l t e r was s u p p o r t e d by e i t h e r a P l e x i g l a s s o r s t a i n -l e s s s t e e l mesh r a f t on t h e s u r f a c e o f a p p r o x i m a t e l y 1 m l . o f c u l t u r e medium (Ham's F-10, N o r t h A m erican B i o l o g i c a l Co.) supplemented w i t h 1 5 % f e t a l c a l f serum (Grand I s l a n d B i o l o g i c a l Co.) and a n t i b i o t i c s (lOOu/ml. p e n i c i l l i n , 0.25 meg./ml.fungi-zone, 100 meg./ml. s t r e p t o m y c i n , Grand I s l a n d B i o l o g i c a l Co.) i n an organ c u l t u r e d i s h ( F a l c o n P l a s t i c s ) . T r a n s f e r method 1. T h i s method o f t r a n s f e r r i n g e p i t h -e l i a was one t h a t i n v o l v e d no a t t e m p t t o o r i e n t t h e e x p l a n t , and r e s u l t e d i n t h e e p i t h e l i a h a v i n g a v a r i e t y o f c o n f o r m a t i o n s . T r a n s f e r method 2. T h i s method i n v o l v e d r e v e r s i n g t h e normal e p i t h e l i a l c u r v a t u r e so t h a t t h e e p i t h e l i a were f o l d e d w i t h t h e l e n s c a p s u l e i n s i d e . T r a n s f e r method 3. T h i s method o f e x p l a n t a t i o n was such as t o m a i n t a i n t h e normal c u r v a t u r e o f t h e e p i t h e l i a so t h a t t h e e p i t h e l i a were f o l d e d w i t h t h e l e n s c a p s u l e o u t s i d e . The c u l t u r e s were i n c u b a t e d a t 37°C. i n an atmosphere o f 9 6 % a i r and 4% CC^ ••, and a h u m i d i t y a p p r o a c h i n g 100%, f o r d i f f e r e n t p e r i o d s o f t i m e between 1-6 days, a f t e r w h i c h t h e y were p r o c e s s e d f o r m i c r o s c o p i c e x a m i n a t i o n o r f o r e l e c t r o p h o r -e s i s . M i c r o s c o p y . Lenses and e x p l a n t s were f i x e d i n 3% g l u t a r a l d e h y d e i n 0.1N sodium c a c o d y l a t e (pH 7.4) a t room t e m p e r a t u r e , washed i n b u f f e r ( p l u s 1 0 % s u c r o s e ) and p o s t - f i x e d i n c o l d OsO^ ( 1 % i n _ p h o s p h a t e b u f f e r , pH 7.2). S u b s e q u e n t l y t h e t i s s u e s were washed, d e h y d r a t e d and embedded i n Epon. S e c t i o n i n g was p e r f o r m e d w i t h a LKB u l t r a t o m e . T h i c k s e c t i o n s ( a p p r o x i m a t e l y lum) f o r l i g h t m i c r o s c o p e e x a m i n a t i o n were s t a i n e d w i t h 1% T o l u i d i n e B l u e ( F i s h e r S c i e n t i f i c ) i n 1% sodium b o r a t e . T h i n s e c t i o n s f o r e l e c t r o n m i c r o s c o p y Were s t a i n e d w i t h m e t h a n o l i c u r a n y l a c e t a t e and Reynold's l e a d c i t r a t e . T h i c k s e c t i o n s were examined and p h o t o g r a p h e d w i t h a Z e i s s photomicroscope.From t h e b l o c k s o f embedded t i s s u e examined i n t h i s manner, a p p r o p r i a t e ones were chosen f o r t h i n s e c t i o n i n g . E l e c t r o n m i c r o s c o p y was p e r f o r m e d w i t h t h e HU 7S, HU 11A,and AEI 801 e l e c t r o n m i c r o s c o p e s . 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 . E l e c t r o p h o r e s i s on 7% p o l y a c r y l a m i d e g e l s was p e r f o r m -ed as o u t l i n e d b y Truman (1968). Lenses o r e x p l a n t s were homogenized i n lOmM phosphate (pH 7.2), t h e homogenate c e n t r i f -uged (10,000 g. f o r 10 min.) and t h e s u p e r n a t a n t (mixed i n 4 0 % 8. s u c r o s e , 1:1), i n a p p r o p r i a t e q u a n t i t y , l a y e r e d on t h e . s t a c k -i n g g e l . E l e c t r o p h o r e s i s was c a r r i e d o u t a t 3 mA/tube f o r 2-4 h o u r s . The g e l s were s u b s e q u e n t l y f i x e d ( i n 1 2 % TCA), s t a i n e d ( i n a m i x t u r e c o n t a i n i n g 45ml. o f 0.2% Coomassie B r i l l i a n t B l u e (Mann Research L a b o r a t o r i e s ) , 45ml. a b s o l u t e e t h a n o l , 10 ml. g l a c i a l a c e t i c a c i d ) , d e s t a i n e d ( i n a m i x t u r e c o n t a i n i n g 65ml. w a t e r , 25 ml. a b s o l u t e e t h a n o l , 10 m l . g l a c i a l a c e t i c a c i d ) , s t o r e d ( i n 1 0 % a c e t i c a c i d ) , and scanned w i t h a G i l f o r d s p e c t r o p h o t o m e t e r a t 540 nm. OBSERVATIONS I - O r g a n i z a t i o n o f t h e 6-day c h i c k embryo l e n s . The 6-day c h i c k embryo l e n s i s i l l u s t r a t e d i n f i g -u r e s 1-4. The d e v e l o p i n g l e n s i s a p o p u l a t i o n o f e p i t h e l i a l c e l l s e n c l o s e d by a m o d i f i e d basement membrane, t h e l e n s cap-s u l e . The c e l l s o f t h e a n t e r i o r p o r t i o n ( a n t e r i o r l e n s e p i t h -e l i u m ) a r e u n d i f f e r e n t i a t e d a n d j n i t o . t i c . a l l y a c t i v e . Those o f t h e p o s t e r i o r p a r t ( f a c i n g t h e n e u r a l r e t i n a ) a r e h i g h l y d i f f -e r e n t i a t e d f i b r e c e l l s . I n t h e r e g i o n o f t h e e q u a t o r ( f i g u r e s 1, 4 ) , c e l l s o f t h e a n t e r i o r l e n s e p i t h e l i u m e l o n g a t e and c o n t r i b u t e t o t h e l e n s body. Here, one can o b s e r v e c e l l s a t d i f f e r e n t s t a g e s i n t h e i r r e m a r k a b l e t r a n s f o r m a t i o n i n t o l e n s f i b r e s . F i g u r e s 1-4 i l l u s t r a t e how v a r i o u s r e g i o n s o f t h e 6-day l e n s have been d e f i n e d , f o r purposes o f d i s c u s s i o n , as r e p r e s -e n t i n g d i f f e r e n t s t a g e s i n what, i s a c o n t i n u o u s p r o c e s s o f d i f f e r e n t i a t i o n . I I - Morphology o f the c e n t r a l a n t e r i o r e p i t h e l i u m ( r e g i o n I ) . The c e n t r a l a n t e r i o r e p i t h e l i u m ( r e g i o n I , f i g u r e 2) o f t h e s i x - d a y c h i c k embryo l e n s i s a s i m p l e c u b o i d a l l a y e r a d h e r e n t t o t h e l e n s c a p s u l e . The n a t u r e o f t h e a s s o c i a t i o n between c e l l s i s r e f l e c t e d by t h e numerous i n t e r c e l l u l a r spaces ( f i g u r e s 2, 7) and by t h e r e l a t i o n s h i p between a d j a c e n t memb-ranes w h i c h a r e n o t c l o s e l y apposed, b u t t h e c o n t o u r s o f w h i c h g e n e r a l l y conform ( f i g u r e s 5,7,8). T y p i c a l e p i t h e l i a l j u n c t i o n -a l complexes a r e e v i d e n t a t t h e a p i c a l and b a s a l b o u n d r i e s , each w i t h a s s o c i a t e d m i c r o f i l a m e n t s t r a v e r s i n g t h e c y t o p l a s m between j u n c t i o n s ( f i g u r e s 5,8). The a p i c a l j u n c t i o n a l comp-l e x e s a r e more p r o m i n e n t t h a n t h e b a s a l ones, and i n v o l v e 10. r e g i o n s o f t i g h t j u n c t i o n and z o n u l a e a d h a e r e n t e s , w h i l e t h e b a s a l ones i n v o l v e o n l y t h e l a t t e r . The m i t o c h o n d r i a , a l t h o u g h o f g e n e r a l d i s t r i b u t i o n , t e n d t o be c o n c e n t r a t e d b a s a l l y and a p i c a l l y ( f i g u r e s 5,7).They a r e o f t e n o b s e r v e d t o be a s s o c i a t e d w i t h l i p i d i n c l u s i o n s ( f i g -u r e 5) w h i c h a r e c o n s i s t e n t l y found, u s u a l l y i n t h e a p i c a l c y t o p l a s m . A m o d e r a t e l y d e v e l o p e d . p e r i n u c l e a r rough e n d o p l a s m i c r e t i c u l u m i s p r e s e n t ( f i g u r e s 5,6), and i t s elements always appear f i l l e d w i t h dense m a t e r i a l . Ribosomes, i n a d d i t i o n t o th o s e a s s o c i a t e d w i t h t h e en d o p l a s m i c r e t i c u l u m , appear i n t h e c y t o p l a s m , though polysomes a r e n o t prominent. A m o d e r a t e l y d e v e l o p e d G o l g i a p p a r a t u s i s c o n s i s t e n t l y found i n t h e a p i c a l c y t o p l a s m ( f i g u r e 6 ) . C e n t r i o l e s and c i l i a c an a l s o be o b s e r v e d i n t h i s r e g i o n . M i c r o t u b u l e s a r e p r e s e n t ( f i g u r e 8 ) , b u t r e l a t i v e l y s p a r s e ( e x c e p t i n m i t o t i c f i g u r e s ) , and a r e o f t e n randomly a r r a n g e d . The number o f m i c r o t u b u l e s and t h e i r degree o f o r i e n t -a t i o n i n c r e a s e t o w rd t h e l a t e r a l r e g i o n s o f t h e a n t e r i o r e p i t h -e l i u m . M i c r o f i b r e s ( f i g u r e 8 ) , a p p r o x i m a t e l y 100 i f i n d i a m e t e r , a r e c o n s i s t e n t l y found i n t h e c e n t r a l a n t e r i o r e p i t h e l i u m . They a r e l a r g e l y r e s t r i c t e d i n t h e i r d i s t r i b u t i o n t o t h e b a s a l c y t o p -l a s m and show no p a r t i c u l a r o r i e n t a t i o n . I l l - Morphology o f r e g i o n IT. The l a t e r a l a n t e r i o r e p i t h e l i u m i s m u l t i - l a y e r e d ( f i g -u r es 3-4). The i n t e r c e l l u l a r spaces f i r s t become r e s t r i c t e d t o th e b a s a l r e g i o n s and t h e n d i s a p p e a r . Toward t h e e q u a t o r , r e g i o n I I shows the i n i t i a l f e a t u r e s o f d i f f e r e n t i a t i o n t h a t w i l l be 11. p r o g r e s s i v e l y e l a b o r a t e d i n r e g i o n s I I I and IV, and t h e c e l l s o f r e g i o n I I ( f i g u r e s 9-11) i l l u s t r a t e a s t a g e o f d i f f e r e n t i a t i o n i n t e r m e d i a t e between t h a t o f r e g i o n s I and I I I . They c o n t a i n a more a c t i v e G o l g i a p p a r a t u s t h a n t h o s e o f t h e c e n t r a l e p i t h e l -ium. C i l i a a r e more f r e q u e n t l y o b s e r v e d i n t h i s r e g i o n , though l i p i d i n c l u s i o n s a r e fewer. Some degree o f c e l l e l o n g a t i o n , accompanied by a •red u c t i o n i n i n t e r c e l l u l a r s p a c e s , i-s a p p a r e n t . M i c r o t u b u l e s a r e more abundant and a r e o r i e n t e d w i t h t h e long, axes o f t h e c e l l s . S i n c e t h e s e s t r u c t u r e s appear t o occupy t h e p e r i p h e r a l a s p e c t s o f t h e c y t o p l a s m , t h e i r abundance can be a p p r e c i a t e d i n s e c t i o n s c u t t a n g e n t i a l l y t h r o u g h t h e l o n g axes o f a d j a c e n t c e l l s ( f i g u r e 1 0 ) . IV- Morphology o f r e g i o n I I I . C e l l s o f r e g i o n I I I ( f i g u r e 4 ) , i m m e d i a t e l y a d j a c e n t t o young f i b r e c e l l s , have s t a r t e d t o e l o n g a t e and c o n t a i n o r i e n t e d m i c r o t u b u l e s , p a r t i c u l a r l y i n t h e r e g i o n between t h e n u c l e u s and t h e a p i c a l s u r f a c e . C i l i a a r e f r e q u e n t l y o b s e r v e d a t t h e a p i c a l b o r d e r o f t h e s e c e l l s ( f i g u r e 16) and s a t e l l i t e s , a s s o c i a t e d w i t h b a s a l b o d i e s ( f i g u r e 17) appear t o be i n v o l v e d i n t h e e l a b o r a t i o n o f m i c r o t u b u l e s . I n t h e more b a s a l r e g i o n s o f t h e s e c e l l s , o r i e n t e d m i c r o f i b r e s a r e r e m a r k a b l y abundant ( f i g -u r e 1 2 ) . The i n t e r c e l l u l a r spaces o f r e g i o n I I I a r e few and b a s a l . A d j a c e n t c e l l membranes r u n more o r l e s s p a r a l l e l ( f i g -u r e s 12, 15. 18) and engage i n j u n c t i o n s o n l y toward t h e i r a p i c a l and b a s a l s u r f a c e s . The G o l g i a p p a r a t u s appears t o be v e r y a c t i v e ( f i g -u r e s 15, 27) and extends t h r o u g h o u t t h e a p i c a l c y t o p l a s m , 12. o c c a s i o n a l l y from two o r t h r e e c e n t r e s . V e s i c l e s , a p p a r e n t l y o f G o l g i o r i g i n , a r e p a r t i c u l a r l y e v i d e n t i n t h e a p i c a l c y t o p l a s m o f c e l l s i n t h i s r e g i o n ( f i g u r e s 14-16). These v e s i c l e s appear empty and a r e f r e q u e n t l y a s s o c i a t e d w i t h t h e plasma membrane, o c c a s i o n a l l y a p p e a r i n g t o f u s e w i t h i t ( f i g u r e 1 4 ) . Elements o f th e rough endoplasmic r e t i c u l u m , a l t h o u g h t e n d i n g t o be concen-t r a t e d j u s t above and j u s t below t h e n u c l e u s , a r e o f t e n d i s t r i -u t e d a l o n g t h e l e n g t h o f t h e c e l l . The q u a n t i t y o f rough endo-p l a s m i c r e t i c u l u m ( f i g u r e s 13, 15) does n o t appear t o be g r e a t e r t h a n i n c e l l s o f r e g i o n I . However, numerous f r e e polysomes pervade t h e c y t o p l a s m ( f i g u r e s 15, 1 8 ) . The n u c l e i o f t h i s / r e g i o n o f t h e d e v e l o p i n g l e n s have an e l l i p t i c a l shape, p r o m i n e n t n u c l e o l i , and many n u c l e a r p o r e s , a l l f e a t u r e s w h i c h become more f u l l y e l a b o r a t e d i n young f i b r e c e l l s ( f i g u r e s 22, 2 3 ) . V- Morphology o f r e g i o n s IV & V. The c e l l s o f r e g i o n IV a r e s p e c t a c u l a r l y e l o n g a t e d young f i b r e c e l l s ( f i g u r e 20) i n w h i c h m i c r o t u b u l e s a r e remark-a b l y p l e n t i f u l ( f i g u r e 1 9 ) . However, i n more mature f i b r e c e l l s , w h i c h p c c u p y a more c e n t r a l p o s i t i o n i n t h e l e n s body, m i c r o -t u b u l e s a r e much l e s s abundant ( f i g u r e s 21,25,26,28,29). M i c r o -f i b r e s , so abundant i n r e g i o n I I I , a r e much l e s s so i n d i f f e r -e n t i a t i n g f i b r e c e l l s o f r e g i o n IV, and i n r e g i o n V o n l y s c a t t e r e d m i c r o f i b r e s a r e o b s e r v e d ( f i g u r e 2 5 ) . W h i l e t h e a d j a c e n t plasma membranes o f c e l l s i n r e g i o n I I I a r e not i n v o l v e d i n j u n c t i o n s , e x c e p t f o r a p i c a l and b a s a l complexes, t h e r e l a t i o n s h i p o f a d j a c e n t c e l l membranes o f 13. Young f i b r e c e l l s i s d i f f e r e n t i n two ways. One m o d i f i c a t i o n i s t h e a s s o c i a t i o n o f p l a q u e s o f dense m a t e r i a l w i t h t h e mem-br a n e s o f a d j a c e n t c e l l s a t v a r i o u s p o i n t s a l o n g t h e i r l e n g t h s ( f i g u r e 20)..The i n t e r c e l l u l a r space o f t h e s e j u n c t i o n s c o n t a i n s amorphous m a t e r i a l . S i m i l a r j u n c t i o n s i n t h e c h i c k embryo nervous system have been termed 'adhesion p l a q u e s ' CPannese, *1"9'68). "These j u n c t i o n s appear t o b e m a i n t a i n e d as t h e c e l l s d i f f e r e n t i a t e , s i n c e t h e y a r e e v i d e n t i n r e g i o n V ( f i g u r e s 21,28,29). P o r t e e t _ a l . (1968) o b s e r v e d t h e s e j u n c t -i o n s between f i b r e c e l l s o f t h e c h i c k embryo l e n s and r e f e r -r e d t o them as desmosomes. However, i n t h e i n t e r c e l l u l a r s spaces o f t h e s e j u n c t i o n s amorphous m a t e r i a l r a t h e r t h a n t h e c h a r a c t e r i s t i c s t r u c t u r e o f desmosomes i s e v i d e n t . Coated v e s i c l e s a r e o f t e n o b s e r v e d i n a s s o c i a t i o n w i t h and f u s e d w i t h c e l l membranes a t v a r i o u s p o i n t s ( f i g u r e 2 4 ) } s u g g e s t i n g t h a t t h e s e j u n c t i o n s may be d e r i v e d from t h e G o l g i . A n o t h e r s t r i k -i n g m o d i f i c a t i o n o f young f i b r e c e l l membranes i s t h e d e v e l -opment o f t i g h t j u n c t i o n s . W i t h d i f f e r e n t i a t i o n , t h e e l a b o r -a t i o n o f t i g h t j u n c t i o n s appears more and more e x t e n s i v e , as i l l u s t r a t e d by c e l l membranes i n r e g i o n V, where t i g h t j u n c t -i o n s a r e q u i t e e x t e n s i v e , b u t n o t c o n t i n u o u s ( f i g u r e s 21,28,29). W h i l e t h e G o l g i a p p a r a t u s i s w e l l d e v e l o p e d i n c e l l s i n i t i a t i n g e l o n g a t i o n , and many G o l g i d e r i v e d v e s i c l e s a r e o b s e r v e d i n t h e a p i c a l c y t o p l a s m o f t h e s e c e l l s ( f i g u r e 2 0 ) , G o l g i a c t i v i t y appears t o d e c l i n e w i t h f u r t h e r d i f f e r e n t i a t i o n . I n f i b r e c e l l s o f r e g i o n V, a v e r y r e d u c e d G o l g i body i s t y p -i c a l l y f ound i n t h e a p i c a l c y t o p l a s m ( f i g u r e 2 6 ) . The rough 14. e n d o p l a s m i c r e t i c u l u m a l s o becomes re d u c e d , t h e v e s i c l e s ap-p e a r i n g t o fragment, so t h a t i n r e g i o n V o n l y s m a l l , s c a t t e r e d elements a r e o b s e r v e d . M i t o c h o n d r i a a l s o become r e d u c e d i n number. D e g e n e r a t i n g m i t o c h o n d r i a can be o b s e r v e d ( f i g u r e s 28, .29). Some m i t o c h o n d r i a undergo e x t e n s i v e e l o n g a t i o n ( f i g u r e 7 2 9 ) , y e t a l s o show s i g n s o f d e g e n e r a t i o n . Polysomes, w h i c h a r e v e r y abundant i n young f i b r e c e l l s , become p r o g r e s s i v e l y d i s -p e r s e d a m i d s t an a c c u m u l a t i n g m a t r i x w i t h d i f f e r e n t i a t i o n ( f i g -u r e s 21,26,28,29) and d e c l i n e i n number. C i l i a and b a s a l bod-i e s a r e o f t e n seen i n t h e a p i c a l c y t o p l a s m o f c e l l s i n r e g i o n s IV and V. I n summary, i t may be c o n s i d e r e d t h a t t h e u l t r a s t r u c -t u r a l d i f f e r e n c e s between t h e c e l l s o f r e g i o n s I , I I , and I I I r e p r e s e n t t h e changes t h a t o c c u r d u r i n g t h e i n i t i a l s t a g e s of. l e n s c e l l d i f f e r e n t i a t i o n . Between r e g i o n s I and I I I t h e c e l l s become m o d e r a t e l y e l o n g a t e d , and i n t e r c e l l u l a r spaces a r e e l i m -i n a t e d . O r i e n t e d m i c r o t u b u l e s i n c r e a s e i n number and abundant m i c r o f i b r e s come t o occupy t h e b a s a l c y t o p l a s m . G o l g i a c t i v i t y i n c r e a s e s . N u c l e i become e l l i p t i c a l i n shape, d e v e l o p p r o m i n e n t n u c l e o l i , and many n u c l e a r p o r e s . Polysomes i n c r e a s e i n number. Marked e l o n g a t i o n o f c e l l s i n r e g i o n IV i s c h a r a c t e r i z e d by t h e p r e s e n c e o f numerous o r i e n t e d m i c r o t u b u l e s and t h e development o f i n t e r c e l l u l a r j u n c t i o n s , f i r s t a d h e s i o n p l a q u e s and t h e n t i g h t j u n c t i o n s . N u c l e o l i a r e p r o m i n e n t and n u c l e a r p o r e s num-er o u s . Polysomes pervade t h e c y t o p l a s m . M i c r o f i b r e s appear t o be g r e a t l y r e d u c e d i n number. C e l l s i n r e g i o n V i l l u s t r a t e t h a t w i t h f i b r e c e l l d i f f e r e n t i a t i o n t h e r e i s a p r o g r e s s i v e l o s s o f o r g a n e l l e s and t h e d e p o s i t i o n o f c y t o p l a s m i c m a t r i x . 15. F i g u r e .1.- Diagram o f t h e 6-day c h i c k embryo l e n s . D i f f e r e n t r e g i o n s o f t h e l e n s a r e i l l u s t r a t e d i n f i g u r e s 2-4. ANTER IOR ANT. LENS EP I THEL IUM POSTER IOR 16. F i g u r e s 2-4. - S i x day c h i c k embryo ilens i n mid s e c t i o n showing d i f f e r e n t r e g i o n s . These a r e d e f i n e d (I-V) f o r r e f e r e n c e t o u l t r a s t r u c t u r a l i l l u s t r a t i o n s , x 300 F i g u r e 2.- A n t e r i o r p o r t i o n o f t h e l e n s showing t h e c e n t r a l a n t e r i o r l e n s e p i t h e l i u m ( r e g i o n I ) . Note t h e i n t e r c e l l u l a r s p a c e s . F i g u r e 3a.- L a t e r a l r e g i o n o f t h e a n t e r i o r e p i t j e l i u m . Note t h a t i t becomes m u l t i - l a y e r e d . F i g u r e 3b.- That a r e a o f t h e l a t e r a l e p i t h e l i u m d e f i n e d as r e g i o n I I . Note t h a t t h e i n t e r c e l l u l a r spaces become r e s t r i c t e d t o t h e b a s a l a r e a and t h e n d i s a p p e a r . F i g u r e 4.- The e q u a t o r i a l r e g i o n o f t h e l e n s . Note c e l l s a t d i f f e r e n t s t a g e s o f e l o n g a t i o n . Regions I I I , IV and V a r e d e f i n e d . N u c l e o l i i n r e g i o n IV a r e v e r y p r o m i n e n t . 16a .17. F i g u r e 5.- The a p i c a l p o r t i o n o f r e g i o n I . L i p i d i n c l u s i o n s a r e e v i d e n t w i t h a s s o c i a t e d m i t o c h o n d r i a , J u n c t i o n a s s o c i a t -ed m i c r o f i l a m e n t s can be seen a d j a c e n t t o t h e plasmalemma. Note t h a t t h e a d j a c e n t c e l l membranes a r e p a r a l l e l w i t h no i n t e r c e l l u l a r j u n c t i o n s e x c e p t a t t h e a p i c a l b o r d e r , x 25,000. F i g u r e 6 . - S i m i l a r t o f i g u r e 5. Note t h e rough e n d o p l a s m i c r e t i c u l u m f i l l e d w i t h dense m a t e r i a l and t h e elements o f t h e g o l g i a p p a r a t u s . The l o w e r r i g h t hand c o r n e r i s t h e a p i c a l p o r t i o n o f an apposed f i b r e c e l l . x 3 6 , 0 0 0 17a .18. F i g u r e 7.- Low m a g n i f i c a t i o n o f r e g i o n I. Note t h e i n t e r -c e l l u l a r spaces and t h e d i s t r i b u t i o n o f m i t o c h o n d r i a and rough endoplasmic r e t i c u l u m . The l e n s c a p s u l e c a n be seen on t h e l e f t and apposed f i b r e c e l l s on t h e r i g h t , x 10,000. 18a 19. F i g u r e 8.- B a s a l a s p e c t o f a c e l l i n r e g i o n I.Note m i c r o f i b r e s t h r o u g h o u t c y t o p l a s m . CAP, l e n s c a p s u l e , x 44,000. 19a 20. F i g u r e 9&10.- A p i c a l r e g i o n s o f c e l l s i n r e g i o n I I . Note o r i e n t e d m i c r o t u b u l e s and t h e c o n t r a s t between t h e c y t o p l a s m o f t h e e p i t h e l i a l c e l l s and t h e a d j a c e n t a p i c a l p o r t i o n s o f apposed f i b r e c e l l s . The a p i c a l c e l l membranes o f f i b r e c e l l s and c e l l s o f t h e a n t e r i o r e p i t h e l i u m can be seen t o engage i n j u n c t i o n s a t c e r t a i n p o i n t s . Arrows i n t h e l o w e r r i g h t hand c o r n e r o f f i g u r e 10 p o i n t t o c i l i a . G o l g i a c t i v i t y appears " g r e a t e r t h a n i n c e l l s o f ' r e g i o n I . x 14,500. 20a 21.. F i g u r e 11.- S i m i l a r t o f i g u r e s 9&10. Note t h e g o l g i and a s s o c i a t e d v e s i c l e s . - x 32,000 21a 22. F i g u r e 1 2 . - B a s a l a s p e c t o f c e l l o f r e g i o n I I I , i l l u s t r a t i n g numerous o r i e n t e d m i c r o f i b r e s and m i c r o t u b u l e s , x 48,000. F i g u r e 1 3 . - C e l l o f r e g i o n I I I showing rough e n d o p l a s m i c r e t i c u l u m . A l s o n o t e m i c r o t u b u l e s a d j a c e n t t o c e l l membranes x 48,000. 2 2a. 23 . F i g u r e 1 4 . - A p i c a l b o r d e r o f c e l l o f r e g i o n I I I . Note a p p a r e n t f u s i o n o f c y t o p l a s m i c v e s i c l e w i t h plasma membrane, x 70,000. F i g u r e 15.-Mid a p i c a l p o r t i o n o f a c e l l s i m i l a r t o t h a t i n f i g u r e 14 showing g o l g i and a s s o c i a t e d v e s i c l e s . Note a g g r e g a t i o n o f v e s i c l e s n e a r l a t e r a l c e l l b o r d e r . x 3 7,000 23 a 24. F i g u r e 1 6 . - A p i c a l p o r t i o n o f c e l l o f r e g i o n I I I i l l u s t r a t i n g c i l i u m . Note a l s o numerous c y t o p l a s m i c v e s i c l e s , x 64,000 F i g u r e 17.- A p i c a l p o r t i o n o f c e l l o f r e g i o n I I I showing p a r t o f a c i l i u m , b a s a l body and a s s o c i a t e d s a t e l l i t e and . m i c r o t u b u l e s , x 64,000. 24a 25. F i g u r e 18.-Mid b a s a l p o r t i o n o f c e l l o f r e g i o n I I I . Note o r i e n t e d m i c r o t u b u l e s and m i c r o f i b r e s . x 45,000. F i g u r e 19.-Mid a p i c a l a s p e c t o f young f i b r e c e l l o f r e g i o n IV.Note m i c r o t u b u l e s , x 64,000. 25a F i g u r e .20.-. Zone o f t r a n s i t i o n between r e g i o n s I I I and IV showing a p i c a l p o r t i o n s o f c e l l s . N o t e t h e numerous m i c r o t -u b u l e s i n t h e young f i b r e c e l l s as w e l l as t h e polysomes and t h e dense m a t e r i a l a s s o c i a t e d w i t h a d j a c e n t c e l l membranes a t v a r i o u s p o i n t s ( a d h e s i o n p l a q u e s ) . Note a l s o t h e a p i c a l j u n c t i o n a l complexes and. t h e numerous c y t o p l a s m i c v e s i c l e s i n t h e a p i c a l a s p e c t s o f t h e c e l l s o f b o t h r e g i o n s , x 10,000. F i g u r e 21.- Young f i b r e c e l l s between r e g i o n s IV and V d e m o n s t r a t i n g numerous polysomes and e x t e n s i v e t i g h t j u n c t -i o n s . x 17,000. 26a 27. F i g u r e 22 & 23.- Young f i b r e c e l l s o f r e g i o n IV showing prominent n u c l e o l u s and n u c l e a r p o r e s , x 38,000. 27a 28. F i g u r e 24.-Young f i b r e c e l l d e m o n s t r a t i n g a p p a r e n t f u s i o n w i t h c e l l membrane o f c o a t e d v e s i c l e , x 64,000. F i g u r e 2 5.- A more mature f i b r e c e l l . Note r e l a t i v e s p a r s i t y o f m i c r o t u b u l e s and m i c r o f i b r e s and t h e d i s p e r s i o n o f polysomes a m i d s t t h e c y t o p l a s m i c m a t r i x , x 64,000 28a »5 1 29. F i g u r e s 26 & 27.- F i g u r e 26 i s t h e a p i c a l p o r t i o n o f a f i b r e c e l l i n r e g i o n V showing r e d u c e d g o l g i a p p a r a t u s compared t o t h a t , i n f i g u r e 27 w h i c h shows o n l y a p o r t i o n t h e g o l g i i n a c e l l o f r e g i o n I I I . x 57,000. 29a F i g u r e s 28 & 29.- C e l l s o f r e g i o n V showing e l o n g a t i o n o f n u c l e u s , e x t e n s i v e r e g i o n s o f t i g h t j u n c t i o n and g r e a t l y e l o n g a t e d m i t o c h o n d r i a w i t h a p p a r e n t a r e a s o f d e g e n e r a t i o n . Note t h a t some a d h e s i o n p l a q u e s a r e e v i d e n t , x 12,000. 30a 29 3 1 . V I - D i f f e r e n t i a t i o n o f a n t e r i o r l e n s e p i t h e l i a i n v i t r o . E x a m i n a t i o n o f t h e 6-day a n t e r i o r l e n s e p i t h e l i a e x p l a n t e d by p r o c e d u r e 1 and c u l t u r e d f o r 3,4,5, o r 6 days demonstrated t h a t i n 87% (105/121) o f t h e ca s e s a s i g n i f i c a n t p o r t i o n o f t h e e x p l a n t was d i f f e r e n t i a t e d . T w e n t y - s i x e x p l a n t s were h i g h l y o r g a n i z e d ( f i g u r e s 34-39). S i x t e e n were d i f f e r e n t -i a t e d t o a l i m i t e d degree, t h e . c e l l s h a v i n g e l o n g a t e d t o t h e e x t e n t i l l u s t r a t e d i n f i g u r e s 43-44. A n o t h e r 16 were p o o r l y d i f f e r e n t i a t e d . I n 11 o f t h e r e m a i n i n g 79 c a s e s , a p o r t i o n o f t h e e x p l a n t was e v i d e n t i n w h i c h t h e c e l l s had e l o n g a t e d o n l y t o a l i m i t e d degree. I n a l l , 26 e x p l a n t s were h i g h l y o r g a n i z e d , 47 ( i n c l u d i n g t h e 16 w i t h poor d i f f e r e n t i a t i o n ) p o o r l y o r g a n -i z e d , and t h e r e m a i n i n g 48 o r g a n i z e d t o an i n t e r m e d i a t e degree. Those c u l t u r e s w i t h a h i g h degree o f o r g a n i z a t i o n and h i s t o g e n e s i s had a common f e a t u r e : t h e e p i t h e l i u m was so o r i e n t -ed t h a t i t s c u r v a t u r e tended t o e n c l o s e t h e c e l l s w i t h i n t h e c a p s u l e . S i m i l a r l y , i n t h o s e e x p l a n t s w i t h l e s s o r g a n i z a t i o n , t h e r e g i o n s o f maximum d i f f e r e n t i a t i o n t e n d e d t o be i n areas more o r l e s s e n c l o s e d by t h e l e n s c a p s u l e (see f i g u r e s 33, 3 7 ) . On t h e o t h e r hand, i n t h o s e e x p l a n t s o r r e g i o n s o f e x p l a n t s w i t h l i m i t e d d i f f e r e n t i a t i o n , t h e arrangement was one o f l i t t l e e p i t h e l i a l f o l d i n g , o r w i t h i t so f o l d e d t h a t t h e e p i t h e l i u m e n c l o s e d t h e c a p s u l e , as seen i n f i g u r e s 43 and 44. The c e l l s o f the e p i t h e l i a e x p l a n t e d by p r o c e d u r e 2, w i t h t h e e p i t h e l i u m e v e r t e d and t h e c a p s u l e f o l d e d on i t s e l f , c o n s i s t e n t l y (15 ca s e s ) d i f f e r e n t i a t e d t o a l i m i t e d e x t e n t .32. ( f i g u r e s 43-44) d u r i n g t h r e e days i n c u l t u r e . However, when t h e e p i t h e l i a were e x p l a n t e d b y p r o c -edure 3, w i t h t h e normal c u r v a t u r e m a i n t a i n e d , t h e i r c e l l s c o n s i s t e n t l y e l o n g a t e d t o a g r e a t e x t e n t ( 8 . c a s e s ) d u r i n g t h r e e days i n c u l t u r e ( f i g u r e s 45-47). Even a f t e r 1-1% days i n c u l t -u r e (7 c a s e s ) a h i g h degree o f c e l l e l o n g a t i o n was a p p a r e n t i n e p i t h e l i a e x p l a n t e d i n t h i s manner ( f i g u r e s 4 0 - 4 2). E x a m i n a t i o n o f c u l t u r e d e p i t h e l i a f i x e d a t d i f f e r e n t i n t e r v a l s (1-6 days) i n d i c a t e d t h a t t h e i r c e l l s e l o n g a t e p r o g -r e s s i v e l y f o r a p e r i o d o f about t h r e e days. The e x p l a n t s c u l t -u r e d f o r l o n g e r p e r i o d s (see f i g u r e s 38-39) d i d n o t show g r e a t e r c e l l e l o n g a t i o n . E l o n g a t i o n t e n d e d t o be i n a d i r e c t i o n perpend-i c u l a r t o t h e r a f t , as though r e f l e c t i n g t h e p o l a r i t y imposed by t h e medium-air i n t e r f a c e . I n summary, 6-day c h i c k embryo l e n s a n t e r i o r e p i t h e l i a w i l l undergo l e n s h i s t o g e n e s i s i n v i t r o . The e x t e n s i v e c e l l e l o n g a t i o n i n v o l v e d i n t h i s p r o c e s s appears t o be encouraged by an e p i t h e l i a l c u r v a t u r e whereby t h e c e l l s f o r m t h e l e s s e r c u r v a t u r e and t h e l e n s c a p s u l e t h e g r e a t e r c u r v a t u r e . Lack o f e p i t h e l i a l c u r v a t u r e , o r c u r v a t u r e o p p o s i t e t o t h e above seemi t o d i s c o u r a g e c e l l e l o n g a t i o n . F i g u r e 30 & 31.- A n t e r i o r l e n s e p i t h e l i a a f t e r 1 day jLn v i t r o . E x p l a n t e d by method 1. B a r ( v e r t i c a l ) r e p r e s e n t s 50 m i c r a . F i g u r e s 32 & 3 3 . - A n t e r i o r l e n s e p i t h e l i a a f t e r 2 days i n v i t r o . E x p l a n t e d b y method 1, B a r r e p r e s e n t s 100 m i c r a . F i g u r e s 34 & 3 5 . - A n t e r i o r l e n s e p i t h e l i a c u l t u r e d f o r 3 days. ..Explanted by .method 1. Bar r e p r e s e n t s .10.0 micra,. 33a 34-F i g u r e s 3 6 & 37.- F i g u r e 36 r e p r e s e n t s a l e n s a n t e r i o r e p i t h e l i u m e x p l a n t e d by method 1 and c u l t u r e d f o r 3% days. F i g u r e 37 i s s i m i l a r , b e i n g c u l t u r e d ' f o r t h r e e days. B a r r e p r e s e n t s 100 m i c r a . F i g u r e 3 8 . - A n t e r i o r l e n s e p i t h e l i u m c u l t u r e d f o r 5 days. B a r r e p r e s e n t s 100 m i c r a . F i g u r e 39.- Same as f i g u r e 38, b u t c u l t u r e d f o r 6 days. 34a F i g u r e 4 0 . - A n t e r i o r l e n s e p i t h e l i u m e x p l a n t e d b y method 3 and c u l t u r e d f o r 1^ days. B ar r e p r e s e n t s 50 m i c r a . F i g u r e 41.- Enlargement o f f i g u r e 40. B a r r e p r e s e n t s 50 m i c r a . F i g u r e 42.- S i m i l a r t o f i g u r e 41. B a r r e p r e s e n t s 50 m i c r a . F i g u r e 43.- A n t e r i o r l e n s e p i t h e l i u m e x p l a n t e d by method 2 and c u l t u r e d f o r t h r e e days. B a r r e p r e s e n t s 50 m i c r a . 3 5a 36. F i g u r e 44.- A n t e r i o r l e n s e p i t h e l i u m c u l t u r e d f o r 3 days. E x p l a n t e d by method 2. Bar r e p r e s e n t s 50 m i c r a . F i g u r e s 45-47.- A n t e r i o r l e n s e p i t h e l i a e x p l a n t e d by method 3 and c u l t u r e d f o r t h r e e days. B a r r e p r e s e n t s 100 m i c r a . 3 6a 37. V I I - F i n e s t r u c t u r e o f a n t e r i o r l e n s e p i t h e l i a d i f f e r e n t i a t i n g ' i n v i t r o . Those e p i t h e l i a c u l t u r e d f o r p e r i o d s o f 1-6 days t h a t appeared t o be w e l l o r g a n i z e d and d i f f e r e n t i a t e d were examined w i t h t h e e l e c t r o n m i c r o s c o p e i n o r d e r t o e v a l u a t e t h e i r f i n e s t r u c t u r e i n terms o f t h a t o f c e l l s i n d i f f e r e n t r e g i o n s o f t h e normal embryonic l e n s . U l t r a s t r u c t u r a l e x a m i n a t i o n o f t h e s e e x p l a n t s i l l u s t r a t e d a number o f f e a t u r e s c e n t r a l t o t h e i r d i f f e r e n t i a t i o n w h i c h , i n g e n e r a l , c o r r e s p o n d t o t h o s e o b s e r v e d i n normal l e n s f i b r e c e l l d i f f e r e n t i a t i o n . W i t h d i f f e r e n t i a t i o n , n u c l e i come t o assume an e l l i p -t i c a l shape, w i t h t h e i r l o n g axes a l i g n e d w i t h t h o s e o f t h e c e l l s . As t h e c e l l s e l o n g a t e , t h e i r n u c l e i become more e l o n g a t e d and narrow, r e s e m b l i n g c l o s e l y t h o s e o f d i f f e r e n t i a t i n g f i b r e c e l l s , w i t h p r o m i n e n t n u c l e o l i and numerous n u c l e a r p o r e s ( f i g - ^ u r e s 48,60). Polysomes, d i s p e r s e d t h r o u g h o u t t h e c y t o p l a s m , a r e most p r o m i n e n t i n c e l l s c u l t u r e d f o r two days ( f i g u r e s 50, 58), a p p e a r i n g t o s u b s e q u e n t l y become somewhat l e s s numerous. W i t h d i f f e r e n t i a t i o n , t h e c e l l s accumulate a f i n e c y t o p l a s m i c " m a t r i x t h a t appears s i m i l a r t o t h a t o f l e n s f i b r e c e l l s ( f i g -u r e 6 3 ) . C e l l e l o n g a t i o n i s accompanied by t h e p r e s e n c e o f numerous o r i e n t e d m i c r o t u b u l e s ( f i g u r e s 57-58) w h i c h t e n d t o occupy t h e . p e r i p h e r a l c y t o p l a s m . They a r e t y p i c a l l y more abund-a n t a p i c a l l y t h a n b a s a l l y , p a r t i c u l a r l y d u r i n g t h e i n i t i a l s t a g e s o f e l o n g a t i o n . As i n t h e l e n s , t h e y appear t o be e l a b -o r a t e d by s a t e l l i t e s a s s o c i a t e d w i t h b a s a l b o d i e s ( f i g u r e 53). 38. M i c r o t u b u l e s g e n e r a l l y p e r s i s t i n w e l l d i f f e r e n t i a t e d e x p l a n t s , even a f t e r s i x days i n v i t r o , n o t d e c l i n i n g i n number t o t h e e x t e n t t h e y do i n l e n s f i b r e c e l l s o f r e g i o n V in v i v o . O r i e n t e d 100 A m i c r o f i b r e s a r e e v i d e n t i n a l l t h e c u l t u r e d e p i t h e l i a examined, b u t d i f f e r i n number and d i s t r i b -u t i o n a t d i f f e r e n t s t a g e s o f xn v i t r o development. The appear-ance o f c y t o p l a s m i c m i c r o f i b r e s a f t e r 1 day i n c u l t u r e i s i l l u s t r a t e d i n f i g u r e s 48-49. They a r e much more numerous i n c e l l s o f e x p l a n t s d i f f e r e n t i a t i n g d u r i n g t h e i r second and t h i r d day i n c u l t u r e ( f i g u r e s 50, 59). The r e l a t i v e l y h i g h c o n c e n t -r a t i o n o f m i c r o f i b r e s shown i n t h e s e f i g u r e s i s t y p i c a l o n l y o f t h e c y t o p l a s m b a s a l t o t h e n u c l e u s . E x a m i n a t i o n o f w e l l d i f f e r -e n t i a t e d expJ.ants c u l t u r e d f o r t h r e e o r more days i n d i c a t e d m i c r o f i b r e s t o d e c l i n e i n number d u r i n g t h i s p e r i o d , a l t h o u g h n o t t o t h e same degree as i n f i b r e c e l l s i n v i v o , and assume a more g e n e r a l d i s t r i b u t i o n . W h i l e i n most s e c t i o n s t h e l e n g t h o f m i c r o f i b r e s i s i n d e t e r m i n a t e , i n some i t i s e v i d e n t t h e y may be q u i t e l o n g ( f i g u r e 6 4 ) . A r e l a t i v e i n c r e a s e i n G o l g i a c t i v i t y accompanies t h e i n i t i a l s t a g e s o f c e l l e l o n g a t i o n ( f i g u r e 55). As i n t h e l e n s , t h e G o l g i g e n e r a t e s c o a t e d v e s i c l e s , some o f w h i c h appear t o be i n v o l v e d i n t h e e s t a b l i s h m e n t o f a d h e s i o n p l a q u e s ( f i g u r e s 49,50,52,55),and o t h e r v e s i c l e s w h i c h a r e o f t e n a s s o c i a t e d w i t h t h e c e l l membrane ( f i g u r e s 52,54). The G o l g i appears most dev-e l o p e d i n c e l l s u n d e r g o i n g e l o n g a t i o n d u r i n g t h e second day i n v i t r o ( f i g u r e 55) and appears t o s u b s e q u e n t l y d e c l i n e i n a c t i v -i t y ( f i g u r e 6 2 ) . M i t o c h o n d r i a a r e , i n t h e i n i t i a l s t a g e s o f c e l l e l o n g a t i o n , o f at .• . g e n e r a l : d i s t r i b u t i o n . D u r i n g f u r t h e r e l o n g a t i o n , t h e m i t o c h o n d r i a appear t o become somewhat r e d u c e d i n number, and a c o n s i d e r a b l e p o r t i o n o f t h e m i t o c h o n d r i a l p o p u l a t i o n o c c u p i e s t h e b a s a l c y t o p l a s m ( f i g u r e 59) i m m e d i a t e l y a d j a c e n t t o t h e l e n s c a p s u l e . W i t h i n i t i a l c e l l e l o n g a t i o n , t h e m i t o c h o n d r i a t e n d t o show moderate e l o n g a t i o n . W i t h f u r t h e r e l o n g a t i o n o f t h e c e l l s , some m i t o c h o n d r i a e l o n g a t e t o a g r e a t e x t e n t , as i n f i b r e c e l l e l o n g a t i o n in v i v o . M i t o c h o n d r i a l d e g e n e r a t i o n , s i m i l a r t o t h a t d e s c r i b e d - i n t h e l e n s , i s a l s o e v i d e n t . However, a p p a r e n t l y h e a l t h y m i t o c h o n d r i a seem t o be r a t h e r more numerous i n d i f f e r e n t i a t i n g f i b r e c e l l s i n v i t r o as compared t o t h o s e in v i v o . A moderate rough endoplasmic r e t i c u l u m i s p r e s e n t a t t h e i n i t i a l s t a g e s o f i n v i t r o development. W h i l e elements o f t h i s o r g a n e l l e t e n d i n i t i a l l y t o be c o n c e n t r a t e d i n t h e p e r i -n u c l e a r r e g i o n , t h e y become d i s p e r s e d t h r o u g h o u t t h e c y t o p l a s m , o f t e n a l i g n e d w i t h t h e l o n g a x i s o f t h e c e l l ( f i g u r e s 50,59,60). As t h e c e l l s d i f f e r e n t i a t e , many elements o f t h e e n d o p l a s m i c r e t i c u l u m appear t o be q u i t e s m a l l , as i f r e p r e s e n t i n g f r a g -m e n t a t i o n p r o d u c t s o f l a r g e r ones ( f i g u r e 58). A l t h o u g h t h e rough endoplasmic r e t i c u l u m becomes r e d u c e d i n e x t e n t w i t h c e l l d i f f e r e n t i a t i o n , , some elements g e n e r a l l y p e r s i s t , so t h a t t h e o r g a n e l l e does n o t d i s a p p e a r as i t does i n t h e l e n s . W i t h i n v i t r o d i f f e r e n t i a t i o n , i n t e r c e l l u l a r r e l a t i o n s h i p s a r e m o d i f i e d . D u r i n g t h e f i r s t day i n c u l t u r e , t h e i n t e r c e l l u l a r spaces o f t h e a n t e r i o r l e n s e p i t h e l i u m a r e l a r g e l y e l i m i n a t e d , 40. ( f i g u r e s 40-42). As t h e c e l l s d i f f e r e n t i a t e , a d h e s i o n p l a q u e s a r e formed between a d j a c e n t c e l l s ( f i g u r e s 48-50), p o s s i b l y by means o f c o a t e d v e s i c l e s w h i c h a r e o f t e n o b s e r v e d i n a s s -o c i a t i o n w i t h c e l l membranes ( f i g u r e s 49, 50, 52, 5 5 ) . A l s o , r e g i o n s o f t i g h t j u n c t i o n a r e e l a b o r a t e d ( f i g u r e s ^ 5 9 - 6 0 ) . However, t h e y do n o t become as e x t e n s i v e as t h o s e between / . f i b r e c e l l s in. v i v o . B a s a l j u n c t i o n a l complexes a r e m a i n t -a i n e d and a p i c a l j u n c t i o n a l complexes a r e p r o m i n e n t , w i t h bands o f m i c r o f i l a m e n t s c o u r s i n g between a d j a c e n t j u n c t i o n s ( f i g u r e s 51-52). I n summary, a n t e r i o r l e n s e p i t h e l i a d i f f e r e n t i a t -i n g jLn v i t r o undergo m o r p h o l o g i c a l changes s i m i l a r t o t h o s e . e v i d e n t i n the l e n s . C e l l e l o n g a t i o n i s accompanied b y t h e l o s s o f i n t e r c e l l u l a r s p a c e s , i n c r e a s e d G o l g i a c t i v i t y , and t h e f o r m a t i o n o f i n t e r c e l l u l a r j u n c t i o n s . T i g h t j u n c t i o n s do n o t become as e x t e n s i v e as t h e y do i n the f i b r e c e l l s o f t h e l e n s . M i c r o t u b u l e s , a p p a r e n t l y e l a b o r a t e d from s a t e l l i t e s i n t h e a p i c a l c y t o p l a s m , a r e numerous. N u c l e i e l o n g a t e and c o n t a i n numerous pores and p r o m i n e n t n u c l e o l i . Polysomes become abundant. Numerous o r i e n t e d m i c r o f i b r e s appear i n t h e b a s a l c y t o p l a s m . W i t h d i f f e r e n t i a t i o n , t h e l o s s o f c y t o -p l a s m i c o r g a n e l l e s i s n o t n e a r l y as marked as i t i s i n t h e l e n s . Those e p i t h e l i a e x p l a n t e d by method 2, w h i c h e l o n g a t e d o n l y t o a l i m i t e d e x t e n t d u r i n g t h r e e days _in v i t r o ( f i g u r e s 43-44) were examined f i r s t i n o r d e r t o d e t e r m i n e i f t h e r e were any u l t r a s t r u e t u r a l f e a t u r e s t h a t m i g h t a c c o u n t 41. f o r t h e i r n o t e l o n g a t i n g f u r t h e r , and second t o compare t h e i r c y t o p l a s m i c d i f f e r e n t i a t i o n w i t h t h a t o f e x p l a n t s t h a t e l o n g a t e d t o a g r e a t e x t e n t j l n v i t r o . U l t r a s t r u c t u r a l e x a m i n a t i o n o f t h e s e e x p l a n t s , however, d e m o n s t r a t e d them t o be i n no o t h e r way r e m a r k a b l y d i f f e r e n t . from t h o s e t h a t had—-,. e l o n g a t e d t o a g r e a t e r degree (see f i g u r e s 65-66). The c e l l s . . / c o n t a i n e d numerous m i c r o t u b u l e s , m i c r o f i b r e s and polysomes. They had t y p i c a l j u n c t i o n a l complexes, "with a s s o c i a t e d p r o m i n e n t m i c r o f i l a m e n t s , and were t y p i c a l l y c i l i a t e d . I n t e r -c e l l u l a r r e l a t i o n s h i p s , i n v o l v i n g a d h e s i o n p l a q u e s and t i g h t j u n c t i o n s were v e r y much the same as t h o s e i n e p i t h e l i a i n w h i c h t h e c e l l s had e l o n g a t e d t o a much g r e a t e r e x t e n t . 4 2 . F i g u r e 48.- A n t e r i o r l e n s e p i t h e l i u m a f t e r 1 day j i n v i t r o . Note m i c r o t u b u l e s and m i c r o f i b r e s . Arrows p o i n t t o n u c l e a r p o r e s o f a t a n g e n t i a l l y s e c t i o n e d n u c l e a r membrane, x 20,000. NOTE: F i g u r e s 48-64 a r e m i c r o g r a p h s o f e p i t h e l i a c u l t u r e d f o r v a r i o u s p e r i o d s a f t e r h a v i n g been e x p l a n t e d b y method l.T h e m i c r o g r a p h s a r e o f e x p l a n t s chosen f o r t h e i r h i g h degree o f o r g a n i s a t i o n and d i f f e r e n t i a t i o n and i n c l u d e t h o s e i l l u s t r a t e d i n f i g u r e s 30-39. 42a 4 3 . F i g u r e 49.- S i m i l a r t o f i g u r e 48 showing mid b a s a l a s p e c t o f c e l l . Note m i c r o t u b u l e s and m i c r o f i l a m e n t s and dense m a t e r i a l a s s o c i a t e d w i t h a d j a c e n t c e l l membranes (arrows) termed a d h e s i o n p l a q u e s , x 20,000. 44. F i g u r e 50.- C e l l s o f a n t e r i o r l e n s e p i t h e l i u m a f t e r 2 days i n v i t r o . Note numerous polysomes, and numerous m i c r o f i b r e s . x 20,000. 44a 45. F i g u r e 51.- A p i c a l r e g i o n o f c e l l s o f a n t e r i o r l e n s e p i t h e l i u m a f t e r 1 day i n v i t r o d e m o n s t r a t i n g p r o m i n e n t m i c r o f i l a m e n t s e x t e n d i n g between j u n c t i o n a l complexes, x 9,000 F i g u r e 52.-Enlargement o f f i g u r e 51.Note t h e c o a t e d v e s i c l e s i n what appears t o be v a r i o u s s t a g e s 'of f u s i o n w i t h t h e plasma membranes, x 40,000 45a 46. F i g u r e 53.- I l l u s t r a t i o n d e m o n s t r a t i n g c i l i u m and s a t e l l i t e w i t h a s s o c i a t e d m i c r o t u b u l e s a t a p i c a l b o r d e r o f c e l l a f t e r 2 days _in v i t r o , x 40,000. F i g u r e 54.- A p i c a l r e g i o n o f c e l l a f t e r 1 day i n v i t r o . Note arrangement o f m i c r o t u b u l e s and m i c r o f i b r e s . The-arrows p o i n t t o some o f t h e numerous v e s i c l e s w h i c h occupy t h e c y t o p l a s m toward t h e l a t e r a l c e l l b o r d e r , x 20,000. 46a 47j. F i g u r e 55.- I l l u s t r a t i o n o f p a r t o f g o l g i a p p a r a t u s i n e l o n g a t i n g c e l l a f t e r 2 days i n v i t r o . N o t e numerous m i c r o t u b u l e s . x 34,000 48. F i g u r e 56.- S e c t i o n t h r o u g h e l o n g a t i n g c e l l s a f t e r 1% days i n v i t r o showing h e x a g o n a l p a c k i n g , x 10,000. F i g u r e 57.- s i m i l a r t o f i g u r e 56, b u t a more t a n g e n t i a l ^ s e c t i o n showing -numerous - m i c r o t u b u l e s o c c u p y i n g t h e l a t e r a l c y t o p l a s m , x 40,000 49. F i g u r e 58.- A p i c a l p o r t i o n s o f c e l l s a f t e r 2 days i n c u l t u r e d e m o n s t r a t i n g m i c r o t u b u l e s and polysomes, x 20,000 49a 50. F i g u r e 5 9 . - B a s a l a s p e c t o f c e l l s a f t e r 3 days i n c u l t u r e . Note t h e numerous m i c r o f i b r e s i n t h e c y t o p l a s m , t h e elements o f e n d o p l a s m i c r e t i c u l u m and g o l g i , and t h e d i s t r i b u t i o n o f m i t o c h o n d r i a . The l e n s c a p s u l e i s i n t h e l o w e r r i g h t hand c o r n e r , x 20,000 50a 5 1 . F i g u r e 60.- C e l l s a f t e r t h r e e days i n v i t r o . Note r e g i o n o f t i g h t j u n c t i o n , x 20,000 51a ''SB. F i g u r e 61..- C e l l s a f t e r 4 days i n c u l t u r e . Note t h e numerous m i c r o t u b u l e s and m i t o c h o n d r i a . x 26,000. 52a 53,. Figure 62,- Apical region of c e l l a f t e r 3% days i n culture demonstrating the reduced extent of the g o l g i . x 30,000. Figure 63.- Cel l s a f t e r 5 days i n v i t r o . Note the cytoplasmic matrix, the microtubules, and the lack of t i g h t junctions, x 35,000. 54. F i g u r e 64.- C e l l o f an e x p l a n t c u l t u r e d f o r 6 days. Note t h e m i c r o t u b u l e s and m i c r o f i b r e s . x 54,000 54a if 55. F i g u r e 65.- Region o f an e x p l a n t . s h o w i n g l i m i t e d d i f f e r e n t -i a t i o n a f t e r t h r e e days i n v i t r o a f t e r b e i n g e x p l a n t e d b y method 2. Note polysomes and i n t e r c e l l u l a r j u n c t i o n s , x '20/000. 55a 56. F i g u r e 66.- S i m i l a r t o f i g u r e 65. Note t h e numerous polysomes and m i c r o t u b u l e s . x 40,000. 56a •57. V I I I - 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 . E l e c t r o p h o r e s i s o f t h e s o l u b l e p r o t e i n s o f c u l t u r e d and u n c u l t u r e d a n t e r i o r l e n s e p i t h e l i a was p e r f o r m e d t o d e t -ermine i f c e l l e l o n g a t i o n i n v i t r o i s accompanied by a change i n t h e p r o p o r t i o n o f s o l u b l e p r o t e i n s and t o compare t h e s o l -u b l e p r o t e i n s o f c e l l s e l o n g a t e d t o a g r e a t e x t e n t i n v i t r o ' W - i t h t h o s e -of c e l l s - . e l o n g a t e d , - o n l y t o a l i m i t e d degree. As p o i n t e d o u t i n t h e i n t r o d u c t i o n , one i n t e r e s t i n g f e a t u r e o f l e n s c e l l d i f f e r e n t i a t i o n i s t h e s y n t h e s i s o f l a r g e q u a n t i t i e s o f what a r e c o n s i d e r e d u n i q u e s t r u c t u r a l p r o t e i n s , t h e l e n s 1 c r y s t a l l i n s ' . The d i f f e r e n t l e n s c r y s t a l -l i n s have t r a d i t i o n a l l y been d e f i n e d b y e l e c t r o p h o r e t i c mob-i l i t y . Most l e n s p r o t e i n i s l e n s c r y s t a l l i n p r o t e i n . And i n t h e 6-day c h i c k embryo l e n s t h e o n l y major c r y s t a l l i n i s t h e F i r s t I m p o r t a n t S o l u b l e C r y s t a l l i n (FISC) ( C l a y t o n , 1970; Y o s h i d a and Katoh, 1971a,b). The e s s e n t i a l d i f f e r e n c e between t h e c e l l s o f t h e a n t e r i o r e p i t h e l i u m and t h e f i b r e c e l l s , a t t h i s s t a g e , w i t h r e s p e c t t o t h e i r s o l u b l e p r o t e i n s i s t h a t t h e f i b r e c e l l s s y n t h e s i z e a l m o s t e x c l u s i v e l y FISC, w h i l e t h e c e l l s o f t h e a n t e r i o r e p i t h e l i u m s y n t h e s i z e a d d i t i o n a l p r o t -e i n s . ( C l a y t o n , 1970; Y o s h i d a and Katoh, 1 9 7 1 a , b ; P i a t i g o r s k y , 1972). Thus, t h e p r o p o r t i o n o f FISC r e l a t i v e t o a l l t h e p r o t -e i n s i s v e r y h i g h i n f i b r e c e l l s and r e l a t i v e l y l o w e r i n c e l l s o f t h e a n t e r i o r e p i t h e l i u m . E l e c t r o p h o r e s i s o f t h e s o l u b l e p r o t e i n s o f e p i t h -e l i a e x p l a n t e d by methods 2 and 3 and c u l t u r e d f o r t h r e e days d e m o n s t r a t e d t h e s e two groups t o be t h e same by t h i s c r i t e r -58. .ion ( f i g u r e s 68,69), s i n c e t h e y showed i d e n t i c a l p r o f i l e s . The d i f f e r e n c e between c u l t u r e d ( f i g u r e s 68,69) and un-c u l t u r e d ( f i g u r e 67) e p i t h e l i a w i t h r e s p e c t t o t h e i r s o l -u b l e p r o t e i n s i s t h a t t h e major peak (which w o u l d be e x p e c t -ed t o i n c l u d e FISC) o f t h e l a t t e r group c o m p r i s e s a c o n s i d e r a b l y s m a l l e r p r o p o r t i o n o f t h e t o t a l p r o t e i n on t h e - g e l .in-comparison t o t h a t ..of t h e f o r m e r .group. That t h e c u l t u r e d e p i t h e l i a have a h i g h e r p r o p o r t i o n o f t h e i r s o l -u b l e p r o t e i n s i n t h e m ajor peak r e l a t i v e t o u n c u l t u r e d e p i t h e l i a s u g g e s t s them t o be d e m o n s t r a t i n g a c h a r a c t e r i s t i c o f f i b r e c e l l d i f f e r e n t i a t i o n . .59. F i g u r e 67.- Scan o f p o l y a c r y l a m i d e g e l on w h i c h t h e s o l u b l e p r o t e i n s o f 6-day a n t e r i o r l e n s e p i t h e l i a have been e l e c t r o p h o r e s e d . Note t h e p r o p o r t i o n t h e major peak c o m p r i s e s o f t h e t o t a l g e l ; p r o t e i n . O r i g i n a t t h e cathode. F i g u r e s 68 & 69.- A s i m i l a r s c a n comparing t h e s o l u b l e p r o t e i n s o f a n t e r i o r l e n s e p i t h e l i a c u l t u r e d f o r 3 days a f t e r h a v i n g been e x p l a n t e d by method 2 ( f i g u r e 68) and by method 3 ( f i g u r e 6 9 ) . E q u i v a l e n t amounts o f sample, i n terms o f numbers o f e x p l a n t s , were a p p l i e d t o t h e g e l . Note t h a t t h e p r o p o r t i o n t h e major peak c o m p r i s e s o f t h e t o t a l p r o t e i n on t h e g e l i s c o n s i d e r a b l y g r e a t e r t h a n t h a t i n f i g u r e 67. 59a I 67 o 60. DISCUSSION I - C h i c k embryo l e n s morphogenesis. A. Plasma membrane. Lens f i b r e d i f f e r e n t i a t i o n i n v o l v e s e x t e n s i v e c e l l e l o n g a t i o n . T h i s change i n shape r e q u i r e s t h e a d d i t i o n o f new s u r f a c e membrane and t h e q u e s t i o n a r i s e s ^ a s t o how t h i s r e q u i r e m e n t i s accommodated. U l t r a s t r u c t u r a i ' o b s e r v a t -i o n s s u g g e s t t h e G o l g i a p p a r a t u s t o be i n v o l v e d i n the s y n t h -e s i s o f new c e l l membrane. The G o l g i i n c r e a s e s i n a c t i v i t y i n t h o s e c e l l s i n i t i a t i n g e l o n g a t i o n , and numerous c y t o p l a s m -i c v e s i c l e s , p r o b a b l y o f G o l g i o r i g i n , a r e e v i d e n t i n t h e s e c e l l s . These a r e o f t e n o b s e r v e d i n a s s o c i a t i o n w i t h c e l l membranes w i t h w h i c h t h e y sometimes appear t o f u s e . C y t o p l a s m i c v e s i c l e s have been d e m o n s t r a t e d t o c o n t r i b u t e new c e l l membrane d u r i n g m i t o s i s i n b o t h p l a n t (Whaley e t aJL. , 1966) and a n i m a l (Buck and T i s d a l e , 1962; Hay and Low, 1972) c e l l s . W h i l e Buck and T i s d a l e (1962) s u g g e s t t h a t such v e s i c l e s a r e d e r i v e d from smooth e n d o p l a s m i c r e t i c -ulum, t h e i r d e r i v a t i o n from t h e G o l g i i n o t h e r systems i s c l e a r l y e s t a b l i s h e d (Whaley e t a_l. , 1971), and t h e r e a r e a few r e p o r t s i n the l i t e r a t u r e w h i c h d i r e c t l y r e l a t e G o l g i a c t i v i t y t o the a d d i t i o n o f new plasma membrane. H i c k s (1966) r e p o r t e d t h a t i n t h e r a t t r a n s i t i o n a l e p i t h e l i u m , membrane s p e c i a l i z a t i o n s ( r e g i o n s o f a s y m m e t r i c a l t h i c k e n i n g ) were o b s e r v e d i n t h e G o l g i as w e l l as a t t h e c e l l s u r f a c e , and t h a t such membrane s p e c i a l i z a t i o n s " a p p e a r e d t o be t r a n s p o r t e d t h e r e from t h e G o l g i a p p a r a t u s i n the form o f f u s i f o r m v e s i c l e s . F a l k (1969) a s s i g n e d a s i m i l a r 61. ' o r i g i n and f a t e t o f u s i f o r m v e s i c l e s i n c e r t a i n A l g a e . Bonne-v i l l e and W e i n s t o c k (1970). d i s c u s s e d what t h e y termed ' s u r f a c e f o r m i n g v e s i c l e s ' i n t h e c e l l s o f t h e s m a l l i n t e s t i n e o f metamorphosing Xenopus. These v e s i c l e s a r e a p p a r e n t l y i n v o l v e d i n t h e e x p a n s i o n o f t h e a p i c a l s u r f a c e and t h e e l o n g a t i o n o f m i c r o v i l l i . Whaley erf a l . (1971) o b s e r v e d t h e G o l g i t o be v e r y a c t i v e d u r i n g p o l l e n t u b e e x t e n s i o n . A r n o l d (1967) r e p o r t e d t h a t s q u i d i r i d o p h o r e d i f f e r e n t i a t i o n i n v o l v e s t h e e l a b o r a t -i o n o f c o n s i d e r a b l e new c e l l membrane w h i c h i s c o n t r i b u t e d by G o l g i d e r i v e d v e s i c l e s . These appear empty, and a r e r a t h e r s i m i l a r t o t h o s e o b s e r v e d i n t h e p r e s e n t i n v e s t i g a t i o n . Thus, t h e a v a i l a b l e e v i d e n c e has l e d S j o s t r a n d =' (1968) and more r e c e n t l y Whaley et a l . (1971) t o v i e w t h e G o l g i a p p a r a t u s as a p o s s i b l e c e n t r e o f membrane d i f f e r e n t -i a t i o n . Indeed, Whaley et a l . (1971) s u g g e s t , . . . i t seems l i k e l y t h a t much plasma membrane i s , i n one form o r a n o t h e r , t r a n s f e r r e d from t h e G o l g i a p p a r a t u s , (p. 28) Wirth r e g a r d t o l e n s development, t h e o b s e r v a t i o n s d e s c r i b e d i n s e c t i o n s IV and V s u g g e s t t h a t t h e G o l g i a c t i v i t y o b s e r v e d i n c e l l s i n i t i a t i n g f i b r e d i f f e r e n t i a t i n g i s c o n c e r n -ed w i t h t h e s y n t h e s i s o f new membrane, and t h a t c y t o p l a s m i c v e s i c l e s o b s e r v e d i n t h e s e c e l l s c o n t r i b u t e t o t h e plasma-lemma, p o s s i b l y t h r o u g h f u s i o n w i t h i t . That t h e G o l g i i s m o d e r a t e l y a c t i v e i n t h e c e l l s o f t h e a n t e r i o r l e n s e p i t h e l -ium, and t h a t v e s i c l e s s i m i l a r t o t h e above a r e seen i n t h i s r e g i o n , a r e o b s e r v a t i o n s a l s o c o n s i s t e n t w i t h t h e s t a t e d view, s i n c e t h e s e c e l l s a r e m i t o t i c a l l y a c t i v e and must, t h e r e f o r e , 62. e l a b o r a t e new membrane. B- C e l l j u n c t i o n s . Other v e s i c l e s g e n e r a t e d b y t h e G o l g i a r e c o a t e d v e s i c l e s . These have been s t u d i e d i n a number o f t i s s u e s w h e r e i n t h e y a r e c o n s i d e r e d t o be i n v o l v e d i n s e c r e t i o n and uptake (see d i s c u s s i o n by S h e f f i e l d , 1970). Coated v e s i c l e s a r e o f i n t e r e s t r e g a r d i n g l e n s morphogenesis s i n c e ray 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 s s u g g e s t them t o be i n v o l v e d i n t h e f o r m a t -i o n o f t h e 'adhesion p l a q u e s ' ( a c c o r d i n g t o t h e t e r m i n o l o g y o f Pannese, 1968; a l s o termed 'maculae a d h a e r e n t i a d i m i n u t a e ' by Hay, 1968; S h e f f i e l d , 1970; and S h e f f i e l d and Fischman, 1970) e s t a b l i s h e d between d i f f e r e n t i a t i n g f i b r e c e l l s . S h e f f -i e l d (1970) h y p o t h e s i z e d , on t h e b a s i s o f s t u d i e s w i t h embryonic n e u r a l r e t i n a , t h a t c o a t e d v e s i c l e s form such j u n c t -i o n s b y f u s i n g w i t h t h e plasma membrane. My o b s e r v a t i o n s on t f i b r e c e l l d i f f e r e n t i a t i o n ' s u p p o r t s u c h an h y p o t h e s i s . J u n c t i o n s o f t h i s t y p e have been o b s e r v e d i n a number o f t i s s u e s , b o t h embryonic ( S h e f f i e l d , 1970; S h e f f i e l d and Fischman, 1970; Pannese, 1968; Hay, 1968; Aoki,1967) and a d u l t ( R a v i o l a and R a v i o l a , 1967; P a l a y , 1967). They appear t o r e p r e s e n t p o i n t s o f s t r o n g a d h e s i o n between c e l l s (Pannese, 1968), and t h e i r f u n c t i o n i s c o n s i d e r e d ( S h e f f i e l d and Fischman, 1970; A o k i , 1967) t o be one o f s t a b i l i z i n g i n t e r -c e l l u l a r r e l a t i o n s h i p s d u r i n g morphogenesis. I t i s o f i n t e r e s t t o n o t e t h a t t h e f o r m a t i o n o f a d h e s i o n p l a q u e s p r e c e d e s t h e development o f s y n a p t i c c o n n e c t i o n s i n t h e embryonic r e t i n a S h e f f i e l d and Fischman 1970) s i n c e , i n t h e l e n s , t h e f o r m a t i o n 63. o f a d h e s i o n p l a q u e s precedes t h e development o f t i g h t j u n c t -i o n s . P o s s i b l y a d h e s i o n p l a q u e s i n t h e l e n s may s e r v e t o s t a b i l i z e t h e r e l a t i o n s h i p between d i f f e r e n t i a t i n g f i b r e c e l l s i n a manner t h a t f a c i l i t a t e s t h e subsequent development o f e x t e n s i v e r e g i o n s o f t i g h t j u n c t i o n . The r o l e t h e s e j u n c t i o n s m i g h t p l a y i n l e n s c e l l e l o n g a t i o n w i l l be d i s c u s s e d i n a l a t e r s e c t i o n . A t t h i s p o i n t , however, i t may be p o i n t e d o u t t h a t t i g h t j u n c t i o n s may be i m p o r t a n t i n a n o t h e r way i n l e n s development. L o w e n s t e i n ' s ( 1968) work has demonstrated t h a t t i g h t j u n c t i o n s a r e perm-e a b l e t o s m a l l e r m o l e c u l e s so t h a t t o some e x t e n t t h e i n t r a -c e l l u l a r environment o f a f i b r e c e l l i s s h a r e d by i t s n e i g h -b o u r s . L o w e n s t e i n has p o i n t e d o u t t h a t s uch j u n c t i o n s may be one b a s i s f o r t h e i n t e r c e l l u l a r communication f u n d a m e n t a l t o d e v e l o p m e n t a l p r o c e s s e s , and i t i s p o s s i b l e t h a t t h i s k i n d o f communication f a c i l i t a t e s t h e degree o f u n i f o r m i t y o f d i f f e r -e n t i a t i o n so v e r y c h a r a c t e r i s t i c o f t h e l e n s . C. M i c r o t u b u l e s . T h i s c o n s i d e r a t i o n o f l e n s morphogenesis must i n c l u d e a r e f e r e n c e t o m i c r o t u b u l e s s i n c e t h e p r e s e n c e o f abundant, o r i e n t e d m i c r o t u b u l e s i n t h e c e l l s o f t h e c h i c k embryo l e n s a t t h e s t a g e a t w h i c h t h e y undergo g r e a t e l o n g a t i o n i m p l i c a t e s them i n t h i s p r o c e s s . M i c r o t u b u l e s a r e u b i q u i t o u s o r g a n e l l e s and t h e i r appearance and d i s t r i b u t i o n c o r r e s p o n d w i t h changes i n c e l l shape i n a number o f t i s s u e s ( T i l n e y and Gibbons, 1969; Gibbons _et _ a l . , 1969; T i l n e y , 1971) i n c l u d i n g t h e l e n s (Kuwabara, 1968; P o r t e e t a l . , 1968; B eyers and P o r t e r , 1964). Y e t t h e 64. c i r c u m s t a n c e s and manner i n w h i c h m i c r o t u b u l e s a r e o r g a n i z e d i s n o t e n t i r e l y c l e a r , though t h e r e i s c o n s i d e r a b l e e v i d e n c e t h a t t h e y a r e composed o f s u b u n i t s t h a t can be r e v e r s i b l y a s s o c i a t e d as p o s t u l a t e d by Inoue and S a t o (1967) (see T i l n e y , 1971). The m i c r o t u b u l e s o b s e r v e d by me i n p r e s u m p t i v e and d i f f e r e n t i a t i n g f i b r e c e l l s appear t o be e l a b o r a t e d b y s a t e l -l i t e s a s s o c i a t e d w i t h b a s a l b o d i e s . S a t e l l i t e s a r e s m a l l dense s t r u c t u r e s c o n s i d e r e d t o s e r v e as s i t e s f o r i n i t i a t i n g p o l y -m e r i z a t i o n o f m i c r o t u b u l a r s u b u n i t s ( T i l n e y , 1971). That m i c r o t u b u l e s a r e e l a b o r a t e d from a p o i n t i n t h e a p i c a l c y t o -p l a s m may e x p l a i n why m i c r o t u b u l e s a r e more abundant a p i c a l l y t h a n b a s a l l y d u r i n g i n i t i a l s t a g e s o f c e l l e l o n g a t i o n , and may h e l p t o a c c o u n t f o r t h e i r p a r t i c u l a r d i s t r i b u t i o n and o r i e n t a t i o n i n 'lens morphogenesis. D. C e l l e l o n g a t i o n . W h i l e m i c r o t u b u l e s a r e i m p l i c a t e d i n c e l l e l o n g a t i o n , t h a t i n t e r c e l l u l a r j u n c t i o n s a r e formed by c e l l s u n d e r g o i n g e x t e n s i o n s u g g e s t s t h e s e a r e a l s o i m p o r t a n t i n t h i s p r o c e s s . There i s good e v i d e n c e t h a t ,. i n c e r t a i n systems a t l e a s t , i n t e r c e l l u l a r r e l a t i o n s h i p s a s w e l l as m i c r o t u b u l e s a r e i m p o r t -ant i n morphogenesis. Beyers and P o r t e r (1964) r e p o r t e d o r i e n t -ed m i c r o t u b u l e s i n t h e c e l l s o f t h e c h i c k embryo l e n s p l a c o d e , and s u g g e s t e d them t o be r e s p o n s i b l e f o r t h e p a l i s a d i n g o f t h i s p r e s u m p t i v e s t r u c t u r e . P e a r ce and Zwaan (1970) r e p o r t e d t h a t t h e a p p l i c a t i o n o f c o l c e m i d t o t h e c h i c k embryo l e n s 65. p l a c o d e , w h i l e r e d u c i n g s i g n i f i c a n t l y t h e number o f m i c r o t u b -u l e s o b s e r v e d i n t h e c e l l s , d i d n o t r e s u l t i n t h e l o s s o f t h e i r columnar form. These a u t h o r s s u g g e s t e d m i c r o t u b u l e s t o be i n v o l v e d i n t h e i n i t i a t i o n , b u t n o t t h e maintenance o f c e l l e l o n g a t i o n . Somewhat s i m i l a r c o n c l u s i o n s were r e a c h e d b y T i l n e y and h i s a s s o c i a t e s (see T i l n e y , 1971) on t h e b a s i s o f a number o f e l e g a n t e x p e r i m e n t s i n v o l v i n g t h e e f f e c t s o f c a l c i u m - f r e e sea w a t e r , D 2 O , h y d r o s t a t i c p r e s s u r e and c o l c h i c i n e on sea u r c h i n morphogenesis. The p r i m a r y mesenchyme c e l l s o f t h e sea u r c h i n embryo undergo, a t t h e t i m e o f g a s t r u l a t i o n , a number o f changes i n c e l l shape t h a t c a n be c o r r e l a t e d w i t h t h e d i s t -r i b u t i o n o f m i c r o t u b u l e s . A l l t h e above t r e a t m e n t s p r e v e n t e d t h e morphogenesis o f t h e s e c e l l s , t h e £>y s t a b i l i z i n g m i c r o -t u b u l e s i n t h e i r i n i t i a l d i s t r i b u t i o n and t h e o t h e r s by d i s -a s s e m b l i n g them. W h i l e t h e c o l c h i c i n e c a u sed o n l y p a r t i a l r e d u c t i o n o f e c t o d e r m a l c e l l e l o n g a t i o n , t r e a t m e n t w i t h c a l c i u m -f r e e sea w a t e r , w h i c h n o t o n l y d i s a s s e m b l e s m i c r o t u b u l e s b u t i n t e r f e r e s w i t h i n t e r c e l l u l a r j u n c t i o n s and t h e i n t e g r i t y o f th e basement membrane and h y a l i n e l a y e r , r e s u l t e d i n t h e i r s p h e r u l a t i o n . The a p p l i c a t i o n o f h y d r o s t a t i c p r e s s u r e , w h i c h e f f e c t s t h e d i s a s s e m b l y o f m i c r o t u b u l e s and i n t e r f e r e s some-what w i t h i n t e r c e l l u l a r j u n c t i o n s and basement membrane i n t -e g r i t y , r e s u l t e d i n p a r t i a l s p h e r u l a t i o n o f t h e c e l l s . T i l n e y has c o n c l u d e d t h a t w h i l e m i c r o t u b u l e s a r e r e q u i r e d t o produce changes i n c e l l shape, a d d i t i o n a l f a c t o r s ( i n t e r c e l l u l a r j u n c t -i o n s and attachments t o e x t r a c e l l u l a r m a t e r i a l ) a r e i m p o r t a n t i n i t s maintenance. 66. W h i l e m i c r o t u b u l e s appear t o be i n v o l v e d i n t h e p r o c e s s o f c e l l e l o n g a t i o n i n t h e l e n s , t h e y do n o t f u l l y a c c o u n t f o r i t . Nor do m i c r o t u b u l e s p r o v i d e any e x p l a n a t i o n f o r t h e maintenance o f c e l l e l o n g a t i o n , p a r t i c u l a r l y s i n c e t h e number o f m i c r o t u b u l e s i s g r e a t l y r e d u c e d i n d i f f e r e n t i a t e d f i b r e c e l l s . I t i s s u g g e s t e d h e r e t h a t i n t e r c e l l u l a r j u n c t i o n s p l a y a r o l e b o t h i n c e l l e l o n g a t i o n and i t s maintenance, and f u r t h e r t h a t i t i s i m p o r t a n t t o r e c o g n i z e t h e e n e r g e t i c s t a b -i l i t y o f t h e l e n s body i n t h e c o n t e x t o f t h e s e p r o c e s s e s . E x a m i n a t i o n o f the' l e n s s u g g e s t s t h a t t h e l e n s body i n f l u e n c e s t h e degree o f e l o n g a t i o n o f d i f f e r e n t i a t i n g f i b r e c e l l s . The g r e a t e x t e n t o f t i g h t j u n c t i o n s between f i b r e c e l l s r e i n f o r c e s t h e view t h a t t h e l e n s body i s composed o f c e l l s t h a t a r e t i g h t l y a d h e r e n t t o one a n o t h e r . The shape o f t h e l e n s body i s m a i n t a i n e d i n t h e absence o f t h e l e n s c a p s u l e , w h i c h s u g g e s t s i t t o have a s t a b l e c o n f o r m a t i o n t h a t t e n d s , as t h e l e n s d e v e l o p s , toward t h e s p h e r i c a l . W i t h c o n t i n u e d growth o f t h e l e n s , new f i b r e c e l l s wrap around t h e l e n s body. They p r o c e e d t o e l o n g a t e somewhat more t h a n t h o s e c e l l s l a i d down p r e v i o u s l y s i n c e t h e y have a l a r g e r c i r c u m f e r e n c e t o a t t a c h t o and o v e r w h i c h e x t e n d i n o r d e r t o m a i n t a i n t h e e n e r -g e t i c a l l y s t a b l e c o n f o r m a t i o n o f t h e l e n s body as t h e y become an i n t e g r a l p a r t o f t h e s t r u c t u r e . I t i s s u g g e s t e d , t h e n , t h a t t h e l e n s body p l a y s a r o l e i n d e t e r m i n i n g t o what degree new f i b r e c e l l s e l o n g a t e , i . e . t h a t p h y s i c a l f a c t o r s a r e imp-o r t a n t i n l e n s morphogenesis. 67. W h i l e m i c r o t u b u l e s p r o b a b l y make a major c o n t r i b -u t i o n t o l e n s c e l l e x t e n s i o n , i n t e r c e l l u l a r j u n c t i o n s a r e p r o b a b l y i m p o r t a n t t o t h i s p r o c e s s as w e l l . W o l p e r t (1967) has p o i n t e d out on s i m p l e t h e o r e t i c a l grounds t h e i n f l u e n c e c e l l a d h e s i o n s m i g h t have i n t h e development o f c e l l shape. I f one c o n s i d e r s a s h e e t o f c e l l s w i t h b a s a l and l a t e r a l a t -tachments, t h e . r e i n f o r c e m e n t o f t h e l a t t e r and t h e r e d u c t i o n o f t h e f o r m e r w i l l f a v o u r t h e e l o n g a t i o n o f t h e c e l l s . T h i s i s p r e c i s e l y what happens i n t h e l e n s , f o r as t h e young f i b r e c e l l s d i f f e r e n t i a t e , t h e y become s t r o n g l y a d h e r e n t a t t h e i r l a t e r a l a s p e c t s and l o s e t h e i r a t t a c h m e n t t o t h e l e n s cap-^ s u l e b a s a l l y . That c e l l s i n i t i a t i n g e l o n g a t i o n become l a t e r -a l l y a d h e r e n t might w e l l be i m p o r t a n t i n terms o f t h e shape t h e y u n d e r t a k e . These l a t e r a l a d h e s i o n s may a l s o "serve t o s t a b i l i z e c e l l e l o n g a t i o n as t h e l a t t e r p r o c e e d s . Thus b o t h m i c r o t u b u l e s and i n t e r c e l l u l a r j u n c t i o n s may be c o n s i d e r e d i m p o r t a n t f o r f i b r e c e l l e l o n g a t i o n . A f t e r c e l l e l o n g a t i o n has been a c h i e v e d , i n t e r c e l l u l a r j u n c t i o n s c o n t r i b u t e t o t h e maintenance o f c e l l shape i n t h e sense t h a t t h e y m a i n t a i n each c e l l as an i n t e g r a l p a r t o f t h e l e n s body. The e s s e n t i a l s u g g e s t i o n h e r e i s t h a t t h e i n t e r c e l -l u l a r j u n c t i o n s formed d u r i n g l e n s c e l l e l o n g a t i o n a r e n o t m e r e l y a r e f l e c t i o n o f t h e t r a n s f o r m a t i o n o f u n d i f f e r e n t i a t -ed c e l l s i n t o d i f f e r e n t i a t e d ones. R a t h e r , t h e i n t e r c e l l u l a r j u n c t i o n s formed c o n t r i b u t e t o t h e p r o c e s s o f c e l l e x t e n s i o n . 68. I n summary, my 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 s on t h e embryonic l e n s s u g g e s t t h e f o l l o w i n g view o f f i b r e c e l l e l o n -g a t i o n i n l e n s morphogenesis.New plasmalemma, d e r i v e d from G o l g i a c t i v i t y , i s c o n t r i b u t e d t o t h e c e l l s u r f a c e . M i c r o -t u b u l e s , e l a b o r a t e d from s a t e l l i t e s i n t h e a p i c a l .-cytoplasm, c o n t r i b u t e t o c e l l e l o n g a t i o n . T h i s e l o n g a t i o n i s ^ a s s i s t e d -through t h e f o r m a t i o n o f s u r f a c e a d h e s i o n s l a t e r a l l y w i t h a d h e s i o n p l a q u e s and t h e n t i g h t j u n c t i o n s , w h i l e b a s a l a t t a c -hments t o : t h e l e n s c a p s u l e a r e l o s t . These d e v e l o p i n g l e n s c e l l s e x t e n d a l o n g o t h e r p r e v i o u s l y d i f f e r e n t i a t e d c e l l s t o an e x t e n t d e t e r m i n e d by t h e degree t o w h i c h t h e l a t t e r have e l o n g a t e d . T h i s extended s t a t e i s i n t u r n i n f l u e n c e d b y t h e s t a b i l i t y o f t h e o v e r a l l c o n f o r m a t i o n o f t h e l e n s body. E. C i l i a . T hat c i l i a have been o b s e r v e d i n t h i s i n v e s t i g a t i o n i n c e l l s o f a l l r e g i o n s o f t h e l e n s may be o f i n t e r e s t w i t h r e s p e c t t o t h e c e l l c y c l e . Fonte ejt aJL. (1971) have r e p o r t e d t h a t w h i l e a l l c e l l s a r e m i t o t i c a l l y a c t i v e i n t h e c h i c k emb-r y o l i m b bud ( s t . 1 9 ) , a h i g h p r o p o r t i o n a r e c i l i a t e d , and t h e y c o n c l u d e t h a t t h e c e l l s i n t h e G^, S, G 2 phases o f t h e c e l l c y c l e may have c i l i a . S i m i l a r l y , i n t h e l e n s , c i l i a t e d , c e l l s have been o b s e r v e d i n t h e p r e s e n t i n v e s t i g a t i o n i n r e g -i o n s i n w h i c h a l l t h e c e l l s a r e r e p o r t e d (Persons and Modak, 1970) t o be m i t o t i c a l l y a c t i v e . The p r e s e n c e o f a c i l i u m may r e f l e c t a l e n s c e l l i n something o t h e r t h a n t h e M phase o f t h e c e l l c y c l e so t h a t i n t h e c e l l s o f r e g i o n s I I - V c i l i a 69. w o u l d be e x p e c t e d i n t h o s e c e l l s i n v o l v e d i n e l o n g a t i o n , s i n c e t h e c e l l s i n m i t o s i s round up n e a r t h e a p i c a l b o r d e r . The f a c t t h a t c i l i a appeared t o be more f r e q u e n t l y o b s e r v e d i n c e l l s o f r e g i o n I I as compared t o r e g i o n I may be t h e r e s u l t o f h a v i n g examined t h e a p i c a l b o r d e r s o f c e l l s o f t h e m u l t i - l a y e r e d l a t e r a l e p i t h e l i u m , a method t h a t w o u l d s e l e c t f o r t h o s e c e l l s n o t i n m i t o s i s . The f r e q u e n t o b s e r v a t i o n o f c i l i a i n c e l l s o f r e g i o n s I I I - I V , c e l l s w h i c h have e n t e r e d t h e t e r m i n a l c e l l c y c l e (Modak and Purdue, 1970), s u g g e s t s t h a t as t h e c e l l s c omplete t h e i r f i n a l m i t o s i s t h e y become c i l i a t e d and p r o c e e d t o d i f f e r e n t i a t e . T h e r e f o r e , t h a t some c e l l s o f t h e a n t e r i o r l e n s e p i t h e l i u m a r e c i l i a t e d i s c o n s i s t e n t w i t h t h e view t h a t t h e p r e s e n c e o f c i l i a r e f l e c t s t h e s t a t u s o f t h e c e l l c y c l e (Fonte et a l . , 1971) r a t h e r t h a n m i t o t i c a c t i v i t y (Rash e t a l . , 1969). When columnar c e l l s o f t h e l e n s p l a c o d e e n t e r t h e m i t o t i c phase o f t h e c e l l c y c l e , t h e y round up n e a r t h e a p i c a l s u r f a c e , undergo d i v i s i o n , and s u b s e q u e n t l y r e - e l o n g a t e (Zwann, 1969). The appearance and d i s t r i b u t i o n o f c y t o p l a s m i c m i c r o -t u b u l e s c o r r e s p o n d t o t h i s change i n shape. Thus t h e c e l l c y c l e imposes a c o n s t r a i n t on t h e o r g a n i z a t i o n o f c y t o p l a s m i c m i c r o t u b u l e s , and t h e r e f o r e on morphogenesis. Many c e l l s o f t h e l a t e r a l a n t e r i o r l e n s e p i t h e l i u m ( r e g i o n I I ) have a t e n d -ency t o be somewhat e l o n g a t e d , w i t h an a p p r o p r i a t e d i s t r i b u t -i o n o f m i c r o t u b u l e s , b u t s i n c e t h e s e c e l l s undergo d i v i s i o n , 7Q. t h e y undergo r e c u r r e n t changes i n c e l l shape. I t w o u l d be o f i n t e r e s t t o know i f the tendency o f t h e c e l l s o f r e g i o n I I t o be somewhat e l o n g a t e d i s a r e f l e c t i o n o f a g r e a t e r d u r a t -i o n o f t h e growth phase:of t h e i r c e l l c y c l e s as compared t o t h a t o f t h e c e l l s o f r e g i o n I . C e l l s o f r e g i o n I I I c o r r e s p o n d t o t h o s e demonstrated (Persons and Modak, 1970) t o be m i t o t -i c a l l y i n a c t i v e . Thus i t seems f a i r l y c l e a r t h a t l e n s c e l l s show t h e i r tendency t o e l o n g a t e o n l y when i n an a p p r o p r i a t e phase o f t h e c e l l c y c l e . However, whether o r n o t c y t o p l a s m i c m i c r o t u b u l e s a r e formed i n a s u f f i c i e n t q u a n t i t y and o r g a n i z e d i n a manner as i n t h e e x t e n s i v e l y e l o n g a t i n g c e l l s o f r e g i o n I I I and IV may depend on o t h e r f a c t o r s i n a d d i t i o n t o t h e c e l l c y c l e . Such a d d i t i o n a l f a c t o r s would seem t o d e r i v e from t h e p o s i t i o n o f t h e c e l l s r e l a t i v e t o t h e o c u l a r environment,, p a r t i c u l a r l y t h e n e u r a l r e t i n a (Coulombre, 1969). Y e t , c o n s i d -e r i n g t h a t one e s s e n t i a l theme o f l e n s development i s t h e r e g u l a t i o n o f l e n s growth by means o f r e g u l a t i n g t h e c e l l c y c l e s o f t h e c e l l s o f t h e a n t e r i o r l e n s e p i t h e l i u m , i t i s v e r y t e m p t i n g t o s p e c u l a t e t h a t the i n f l u e n c e m e d i a t e d by t h e n e u r a l r e t i n a a f f e c t s t h e c e l l c y c l e o f t h e c e l l s o f r e g i o n I I I such t h a t the l a t t e r a r e ' l o c k e d ' i n t h e growth phase o f t h e c e l l c y c l e and t h e r e b y p e r m i t t e d t o d i f f e r e n t i a t e . That i s , r a t h e r t h a n a t t r i b u t i n g t h e i n a b i l i t y o f t h e c e l l s o f r e g i o n I I I t o d i v i d e t o t h e i r commitment t o d i f f e r e n t i a t i o n , i t may be more p r o f i t a b l e t o view t h e i r commitment t o d i f f e r e n t i a t i o n as a r e s u l t , a t l e a s t i n p a r t , o f c e l l c y c l e r e g u l a t i o n . 71. F. M i c r o f i b r e s . I t i s n o t c l e a r whether t h e 100 A m i c r o f i b r e s a r e i n v o l v e d i n morphogenesis o r c y t o d i f f e r e n t i a t i o n . S h e i r appear-ance i n l a r g e numbers i n an o r i e n t a t i o n c o i n c i d e n t w i t h t h a t o f c e l l e l o n g a t i o n d u r i n g t h e i n i t i a t i o n o f c e l l e l o n g a t i o n c o u l d be i n t e r p r e t e d t o s i g n i f y m o rphogenetic i m p o r t , o r t h e . o r i e n t a t i o n c o u l d b e - m e r e l y a r e f l e c t i o n o f c e l l p o l a r i t y , t h e abundance o f m i c r o f i b r e s r e f l e c t i n g c y t o p l a s m i c d i f f e r e n t i a t -i o n . P o r t e e t _ a l . (1968), who o b s e r v e d t h e s e f i b r e s , s p e c u l a t e d t h a t t h e y might r e p r e s e n t a s p e c i f i c l e n s p r o t e i n , and t h a t t h e i r subsequent d i s a p p e a r a n c e m i g h t be due t o t r a n s f o r m a t i o n i n t o c e l l m a t r i x , i . e . l e n s 1 c r y s t a l l i n * p r o t e i n . On t h e o t h e r hand, t h e 100 A m i c r o f i b r e s a r e o f t e n found t o g e t h e r w i t h m i c r o t u b u l e s (which a l s o s u b s e q u e n t l y d i s a p p e a r ) , and t h e r e i s some c i r c u m s t a n t i a l e v i d e n c e ( W i s n i e w s k i and T e r r y , 1968$ Wis-n i e w s k i jet a l . , 1968) i n d i c a t i n g t h a t m i c r o t u b u l e s may r e v e r s -i b l y c o n v e r t t o 100 A m i c r o f i b r e s when t r e a t e d w i t h m i t o t i c i n h i b i t o r s , as though d e m o n s t r a t i n g a common b a s i s t o t h e s e u n i t s . A t t h e moment, t h e q u e s t i o n o f whether t h e s e m i c r o f i b r e s r e p r e s e n t t h e 100 A c l a s s o f f i b r e found i n many c e l l s , o r whether t h e y a r e i n some way unique t o l e n s c e l l s , i s n o t r e s o l v e d . The problem and t h e s e s t r u c t u r e s w a r r a n t f u r t h e r s t u d y . 72. I I - C h i c k embryo l e n s morphogenesis i n v i t r o . The _in v i t r o d i f f e r e n t i a t i o n o f s i x - d a y a n t e r i o r l e n s e p i t h e l i a i n d i c a t e s t h a t t h e i r c e l l s c a n undergo g r e a t e r e l o n g -a t i o n t h a n t h e ' l i m i t e d e l o n g a t i o n ' p r e v i o u s l y r e p o r t e d ( P h i l -p o t t and Coulombre, 1965; 1968). T h i s i s p r o b a b l y due t o my d i f f e r e n t method o f e x p l a n t a t i o n , f o r t h e c i t e d a u t h o r s c u l t -u r e d t h e e p i t h e l i a f l a t t e n e d o u t on t h e b o t t o m o f a P e t r i d i s h . I t w o u l d seem from t h e p r e s e n t e x a m i n a t i o n t h a t t h e e p i t h e l i u m has a c a p a c i t y t o d i f f e r e n t i a t e i n supplemented medium, b u t t h a t t h e degree o f e l o n g a t i o n depends on c o n d i t i o n s r e l a t i n g t o t h e e p i t h e l i a l c o n f o r m a t i o n , a view s u p p o r t e d by t h e o b s e r v a t i o n t h a t t h e e p i t h e l i u m shows e i t h e r l i m i t e d o r g r e a t e r c e l l e l o n g a t i o n a c c o r d i n g t o whether i t i s e x p l a n t e d b y t h e t h i r d o r second method. T h i s view i s a l s o c o n s i s t e n t w i t h t h e o b s e r v a t i o n t h a t t h o s e e p i t h e l i a e x p l a n t e d by t h e f i r s t method show b o t h l i m i t e d and g r e a t e r c e l l e l o n g a t i o n , s i n c e t h e s e end up i n v a r i o u s c o n f o r m a t i o n s . A l t h o u g h t h e l e n s e p i t h e l i u m seems t o be c a p a b l e o f g r e a t e r d i f f e r e n t i a t i o n i n v i t r o t h a n r e p o r t e d by P h i l p o t t and Coulombre (1965; 1968), my r e s u l t s a r e c o n s i s t e n t w i t h t h e i r o b s e r v a t i o n t h a t e l o n g a t i o n t a k e s p l a c e f o r o n l y about t h r e e days. Of f u r t h e r i n t e r e s t i s t h e i r o b s e r v a t i o n t h a t e p i t h e l i a c u l t u r e d i n unsupplemented medium p r o g r e s s i v e l y l o s t o v e r a p e r i o d o f t h r e e days t h e a b i l i t y t o d i f f e r e n t i a t e when s u b s e q u e n t l y p l a c e d i n supplemented medium. A p p a r e n t l y t h e i s o l a t e d e p i t h e l i u m l o s t a f a c t o r n e c e s s a r y f o r d i f f e r e n t i a t -i o n , b u t t h e l o s s c o u l d be compensated f o r , i n t h e i r work, i f 73. t h e e p i t h e l i u m were s u b s e q u e n t l y p l a c e d i n t h e eye o f a l e n t -e c t o m i z e d h o s t , o r i n a s s o c i a t i o n w i t h embryonic mesoderm i n c u l t u r e . 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 s on t h e c e l l s o f a n t -e r i o r l e n s e p i t h e l i a t h a t d i f f e r e n t i a t e d t o a h i g h degree i n  v i t r o d emonstrate them t o have f e a t u r e s e s s e n t i a l l y s i m i l a r t o t h o s e o f l e n s f i b r e c e l l s d i f f e r e n t i a t i n g _in v i v o . C e l l \ e l o n g a t i o n i s accompanied by the p r e s e n c e o f numerous o r i e n t - \ ed m i c r o t u b u l e s ; a m o d i f i c a t i o n o f i n t e r c e l l u l a r r e l a t i o n s h i p s i n v o l v i n g a d h e s i o n p l a q u e s , t i g h t j u n c t i o n s and a p i c a l j u n c t -i o n a l complexes w i t h a s s o c i a t e d p r o m i n e n t bands o f m i c r o f i l -aments; the appearance o f o r i e n t e d m i c r o f i b r e s i n t h e more b a s a l c y t o p l a s m ; a r e l a t i v e i n c r e a s e and subsequent d e c l i n e i n G o l g i a c t i v i t y ; t h e e l a b o r a t i o n o f numerous f r e e polysomes and a c y t o p l a s m i c m a t r i x s i m i l a r i n appearance t o t h a t o f l e n s f i b r e c e l l s . Thus, o b s e r v a t i o n s a t t h e l e v e l s o f b o t h the l i g h t and e l e c t r o n m i c r o s c o p e s i n d i c a t e t h a t t h e c e l l s o f the a n t e r i o r l e n s e p i t h e l i u m undergo l e n s f i b r e 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 . The d i f f e r e n c e s a t t h e u l t r a s t r u c t u r a l l e v e l between f i b r e c e l l d i f f e r e n t i a t i o n i_n v i t r o and _in v i v o , i n v o l v i n g l e s s e r degrees o f t i g h t j u n c t i o n f o r m a t i o n and o f l o s s o f c y t o p l a s m i c o r g a n e l l e s i n t h e former, do n o t d e t r a c t from t h i s c o n c l u s i o n . R a t h e r , t h e y may be i n t e r p r e t e d t o s i g n i f y t h a t the c e l l s d i f f e r e n t i a t i n g i n v i t r o come t o r e s -emble d i f f e r e n t i a t i n g f i b r e c e l l s more t h a n d i f f e r e n t i a t e d ones. T h i s i n t e r p r e t a t i o n i s c o n s i s t e n t w i t h t h e r e p o r t ( P i a t i g o r s k y e t a l . , 1972) t h a t l e n s e p i t h -74. e l i a d i f f e r e n t i a t i n g jLn v i t r o show a p a t t e r n w i t h r e s p e c t t o p o l y p e p t i d e s y n t h e s i s i n t e r m e d i a t e between t h a t o f a n t e r i o r l e n s e p i t h e l i a and f i b r e c e l l s o f s i x - d a y embryonic l e n s e s . The c e l l s o f e x p l a n t s e l o n g a t e i n c u l t u r e f o r o n l y about t h r e e days. These c e l l s , a t t h e u l t r a s t r u e t u r a l l e v e l , do n o t show f u r t h e r c y t o p l a s m i c m a t u r a t i o n a f t e r t h i s t i m e i n terms o f d e v e l o p i n g more e x t e n s i v e t i g h t j u n c t i o n s and a l m o s t c o m p l e t e l y l o s i n g c y t o p l a s m i c o r g a n e l l e s , as f i b r e c e l l s do i n t h e l e n s . T h i s s u g g e s t s a common b a s i s t o t h e s e o b s e r v a t i o n s . P o s s i b l y , t h e l o s s o f t h e h y p o t h e t i c a l f a c t o r o f P h i l p o t t and Coulombre (1965; 1968) p r e v i o u s l y r e f e r r e d t o d u r i n g t h r e e days jLn v i t r o a f f e c t s n o t o n l y t h e a b i l i t y o f t h e c e l l s t o f u r t h e r e l o n g a t e , b u t a l s o t h e i r a b i l i t y t o undergo f u r t h e r c y t o p l a s m i c d i f f e r e n t i a t i o n t h a t w o u l d r e s u l t i n t h e c h a r a c t e r -i s t i c morphology o f t h e mature l e n s f i b r e c e l l . I t i s s u g g e s t e d , t h e n , t h a t w h a t e v e r i s l o s t d u r i n g a p e r i o d o f t i m e i n c u l t u r e a c t u a l l y c o n c e r n s p r o c e s s e s f u n damental t o c e l l d i f f e r e n t i a t i o n r a t h e r t h a n o n l y t h o s e i n v o l v e d i n c e l l e l o n g a t i o n . C o n s i d e r i n g t h i s , t h e r e a s o n t h e a n t e r i o r l e n s e p i -t h e l i u m d i f f e r e n t i a t e s i n v i t r o i s n o t because l e n s d e v e l o p -ment i s indepe n d e n t o f t h e o c u l a r e n v i ronment's i n f l u e n c e , o r because t h e i n f l u e n c e s o f t h e o c u l a r environment a r e somehow d u p l i c a t e d i n v i t r o , b u t because t h e l e n s e p i t h e l i u m has a c q u i r e d , t h r o u g h i t s d e v e l o p m e n t a l h i s t o r y , some measure o f c a p a c i t y t o d i f f e r e n t i a t e i n d e p e n d e n t l y o f t h e s e i n f l u e n c e s ( c f . McKeehan, 1954). A c e r t a i n p e r i o d o f i s o l a t i o n , however, demonstrates a c e r t a i n measure o f dependency. This i s further i l l u s t r a t e d by the observation (Coulombre and Coulombre, 1971) that when the anterior lens epithelium i s placed i n the ocular environment, i t does not immediately undertake to d i f f e r e n t i a t e , a s i t does i n v i t r o . Rather, i t becomes m i t o t i c a l l y active and forms a.vesicle that develops into a lens, with f i b r e c e l l s p o s t e r i o r l y and -a germinative .epithelium .anteriorly. -Thus the ocular .envir-onment i s es s e n t i a l f o r i t s influence on the lens with resp-ect to mitotic a c t i v i t y , which permits growth, and to the encouragement of continued d i f f e r e n t i a t i o n (cf. McKeehan, 1951, 1954; Muthukkaruppan, 1965). The u l t r a s t r u c t u r a l observations regarding those explants i n which the c e l l s had elongated only to a l i m i t e d degree were made to determine i f there were any morphological features that might account for t h e i r not Elongating further. However, i n terms of the u l t r a s t r u c t u r e of these moderately elongating c e l l s , there proved to be nothing remarkably d i f f -erent from that of those elongating to a great extent. Neither was there any difference apparent between these groups with respect to the polyacrylamide g e l l electrophoresis p r o f i l e s of t h e i r soluble proteins. These observations are consistent with the view that an explanation of l i m i t e d c e l l elongation i s probably related to physical factors r e s u l t i n g from the conformation of the epithelium. It i s necessary, then, to consider how to in t e r p r e t the r e l a t i o n s h i p between an epith-elium's conformation and the degree to which i t s c e l l s extend. 76. I n o r d e r t o e l o n g a t e , c e l l s g e n e r a l l y must have a s u b s t r a t e t o adhere t o and o v e r w h i c h t o e x t e n d t h e m s e l v e s . F o r l e n s c e l l s , t h e s u b s t r a t e i s o t h e r c e l l s , and i t has been p o i n t e d o u t p r e v i o u s l y i n t h i s d i s c u s s i o n t h a t i n t h e l e n s new f i b r e c e l l s e x t e n d o v e r t h o s e p r e v i o u s l y l a i d down, wrap-p i n g around t h e l e n s body so t h a t t h e newer f i b r e c e l l s dep-•osi-ted -during -growth u l t i m a t e l y become l o n g e r t h a n t h e o l d e r ones, h a v i n g t o e x t e n d o v e r a g r e a t e r c i r c u m f e r e n c e . S i n c e d u r i n g growth new f i b r e c e l l s a r e c o n t i n u o u s l y added, t h e r e e x i s t s , a t a g i v e n t i m e a t t h e p e r i p h e r y o f t h e l e n s body, a number o f a s s o c i a t e d d i f f e r e n t i a t i n g f i b r e c e l l s w i t h a p r o g -r e s s i o n o f l e n g t h s . Each c e l l , t h e n , i s somewhat s h o r t e r t h a n t h e one p r e c e d i n g i t and m a r g i n a l l y l o n g e r t h a n t h e one f o l l o w -i n g . T h i s s u g g e s t s t h a t e l o n g a t i o n t a k e s p l a c e i n an i n c r e m e n t -a l f a s h i o n , so t h a t as t h e ' s u b s t r a t e ' e x t e n d s , t h e n e x t younger c e l l c an a l s o e x t e n d , and so on. One can c o n c e i v e t h a t c e l l e l o n g a t i o n i n v i t r o may w e l l depend on t h e e s t a b l i s h m e n t o f a s u b s t r a t e o f one o r a few c e l l s w h i c h a t a g i v e n time a r e m a r g i n a l l y more e l o n g a t e d t h a n t h e i r n e i g h b o u r s , so t h a t t h e c e l l s can e l o n g a t e a l o n g each o t h e r , y e t more o r l e s s t o g e t h e r . I n such a v i e w , d i f f e r -e n t i a t i n g c e l l s c o u l d a l t e r n a t e between s e r v i n g as s u b s t r a t a and e l o n g a t i n g i n a s s o c i a t i o n w i t h n e i g h b o u r i n g c e l l s so t h a t a l l t h e c e l l s c o u l d e l o n g a t e more o r l e s s t o g e t h e r . A l s o , a group o f c e l l s p r o c e e d i n g i n t h i s way c o u l d s e r v e c o n t i n u o u s l y as s u b s t r a t a f o r c e l l s a t t h e i r p e r i p h e r y , g i v i n g t h e appearance o f c e n t r e s o f r e l a t i v e l y g r e a t e r e l o n g a t i o n s u r r o u n d e d by 77. a r e a s o f r e l a t i v e l y l e s s e l o n g a t i o n . . I t i s p o s s i b l e t h a t my t h i r d method o f e x p l a n t i n g e p i t h e l i a r e s u l t e d i n a h i g h degree o f c e l l e l o n g a t i o n because the e p i t h e l i a l c o n f o r m a t i o n was s uch t h a t t h e c e l l s c o n v e r g e d and s u b s t r a t a were g e n e r a t e d . On t h e o t h e r hand, e x p l a n t i n g e p i t h e l i a b y t h e second method r e s u l t e d i n o n l y l i m i t e d c e l l e l o n g a t i o n , and may have done so because w i t h a f l a t e p i t h -e l i u m c e l l s a r e n o t c o n v e r g e n t , and when t h e e p i t h e l i u m i s c u r v e d , w i t h t h e c a p s u l e f o r m i n g t h e l e s s e r c u r v a t u r e , t h e c e l l s w o u l d t e n d t o d i v e r g e . I n o t h e r words, a c e r t a i n amount o f l a t e r a l t e n s i o n on t h e s h e e t o f c e l l s w o u l d work a g a i n s t c e l l e l o n g a t i o n , w h i l e a r e d u c t i o n i n l a t e r a l t e n s i o n would encourage c e l l e l o n g a t i o n . T h i s i n v e s t i g a t i o n i n d i c a t e s t h a t e p i t h e l i a l con-f o r m a t i o n i s i m p o r t a n t f o r f i b r e c e l l morphogenesis and s u g g e s t s t h a t t h e t e ndency o f t h e l e n s p r i m o r d i u m t o form a v e s i c l e d u r i n g normal development i s i m p o r t a n t , n o t o n l y f o r t h e c o n s t r u c t i o n o f a g e r m i n a t i v e a n t e r i o r e p i t h e l i u m , b u t a l s o f o r t h e morphogenesis i f f i b r e c e l l s i n t h e p o s t e r i o r p a r t . 78. SUMMARY 1. A s t u d y w i t h the l i g h t and e l e c t r o n m i c r o s c o p e s o f t h e i n v i t r o d i f f e r e n t i a t i o n o f 6-day c h i c k embryo l e n s a n t e r -i o r e p i t h e l i a was u n d e r t a k e n t o e v a l u a t e l e n s c e l l d i f f e r -e n t i a t i o n and t o a s c e r t a i n what f a c t o r s m i g h t be i n v o l v e d i n c e l l e l o n g a t i o n . ' ' '\ 2... I t was found n e c e s s a r y t o examine the u l t r a s t r u c t u r e o f t h e normal ( i n v i v o ) 6-day c h i c k embryo l e n s t o p r o p e r l y e v a l u a t e _in v i t r o l e n s c e l l d i f f e r e n t i a t i o n . T h i s s t u d y r e v e a l e d t h a t i n i t i a l s t a g e s o f l e n s c e l l d i f f e r e n t i a t i o n i n v i v o a r e accompanied by i n c r e a s e d G o l g i a c t i v i t y . I t i s s u g g e s t e d t h a t t h i s a c t i v i t y i s r e l a t e d t o t h e s y n t h e s i s o f new s u r f a c e membrane r e q u i r e d f o r c e l l e l o n g a t i o n . E x t e n s i v e c e l l e l o n g a t i o n i n the l e n s I s accompanied by t h e p r e s e n c e o f numerous o r i e n t e d m i c r o t u b u l e s and b y t h e development o f j u n c t i o n s ( a d h e s i o n p l a q u e s and t i g h t j u n c t i o n s ) between c e l l s a l o n g t h e i r l e n g t h s . I t i s s u g g e s t e d t h a t m i c r o t u b u l e s and i n t e r c e l l u l a r j u n c t i o n s may be i m p o r t a n t f o r c e l l e l o n g -a t i o n . 3. In v i t r o s t u d i e s d emonstrated t h a t c e l l s o f t h e a n t e r i o r l e n s e p i t h e l i u m undergo e i t h e r e x t e n s i v e o r l i m i t e d e l o n g -a t i o n , depending on f a c t o r s d e r i v i n g from t h e e p i t h e l i a l c o n f o r m a t i o n . Experiments were performed t h a t i n v o l v e d exp-l a n t i n g e p i t h e l i a w i t h t h e i r normal c u r v a t u r e e i t h e r r e t a i n -ed o r r e v e r s e d . The former p r o c e d u r e c o n s i s t e n t l y r e s u l t e d i n e x t e n s i v e c e l l e l o n g a t i o n w h i l e t h e l a t t e r always r e s u l t -79. ed i n l i m i t e d c e l l e l o n g a t i o n , i n d i c a t i n g t h e s e conform-a t i o n s encouraged o r d i s c o u r a g e d c e l l e l o n g a t i o n . Exam-i n a t i o n o f e x p l a n t s i n w h i c h th e c e l l s had e l o n g a t e d e x t e n s i v e l y d u r i n g 1-6 days i n v i t r o i n d i c a t e d t h a t c e l l e l o n g a t i o n p r o g r e s s e s f o r o n l y about t h r e e days. 4. F i n e s t r u c t u r a l s t u d i e s on l e n s e p i t h e l i a t h a t e l o n g a t e d e x t e n s i v e l y jLn v i t r o d e m o n s t r a t e d t h a t t h e i r c e l l s were, , i n g e n e r a l , u l t r a s t r u e t u r a l l y s i m i l a r t o l e n s f i b r e c e l l s \ d i f f e r e n t i a t i n g i n v i v o . However, t h e y d i d n o t come t o resemble, even a f t e r s i x days _in v i t r o , f u l l y d i f f e r e n t i a t -ed f i b r e c e l l s i n terms o f t h e e x t e n t o f t i g h t j u n c t i o n f o r m a t i o n and t h e l o s s o f c y t o p l a s m i c o r g a n e l l e s . Indeed, t h e y d i d n o t appear t o show f u r t h e r c y t o p l a s m i c d i f f e r e n t -i a t i o n , i n t h e s e terms, a f t e r t h r e e days i n v i t r o . 5. U l t r a s t r u c t u r a l s t u d i e s r e g a r d i n g c e l l s o f t h e a n t e r i o r l e n s e p i t h e l i u m t h a t e l o n g a t e d o n l y t o a l i m i t e d degree i n v i t r o d e monstrated them t o have f e a t u r e s s i m i l a r t o t h o s e t h a t e l o n g a t e d e x t e n s i v e l y _in v i t r o . 6. A n t e r i o r l e n s e p i t h e l i a e x p l a n t e d w i t h r e v e r s e d o r normal c u r v a t u r e were the same i n terms o f t h e i r s o l u b l e p r o t e i n s as i n d i c a t e d by 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 . 7. I t was c o n c l u d e d t h a t c o n t i n u e d l e n s development dep-ends on as y e t u n d i s c l o s e d i n f l u e n c e s o f t h e o c u l a r e n v i r -onment, b u t t h a t t h e degree o f l e n s c e l l e l o n g a t i o n appears t o be dependent upon p h y s i c a l f a c t o r s i n h e r e n t i n t h e e p i t h e l i a l c o n f o r m a t i o n and enhanced by m i c r o t u b u l e s and 80. i n t e r c e l l u l a r j u n c t i o n s . I t i s s u g g e s t e d t h a t the normal c u r v a t u r e o f the _in v i v o l e n s i s i m p o r t a n t i n t h i s r e g a r d . 81. BIBLIOGRAPHY A o k i , A. ( 1 9 6 7 ) . Temporary c e l l j u n c t i o n s i n t h e d e v e l o p i n g r e n a l g l o m e r u l u s . D e v elopmental B i o l . , 1 _ 5 : 1 5 6 - 6 4 . A r n o l d , J . ( 1 9 6 7 ) . O r g a n e l l o g e n e s i s o f th e Cephalopod I r i d -ophore: cytomembranes i n development. J . U l t r a -s t r u c t u r a l Res., 2 j O : 4 1 0 - 2 1 . B e y e r s , B. and K. P o r t e r . ( 1 9 6 4 ) ; O r i e n t e d m i c r o t u b u l e s i n e l o n g a t i n g c e l l s o f th e d e v e l o p i n g l e n s r u d i m e n t a f t e r i n d u c t i o n . Proc. N a t i o n a l Acad. S c i e n c e (U.S.:-) , 52: 1 0 9 1 - 9 9 . B o n n e v i l l e , M. and M. Wein s t o c k . ( 1 9 7 0 ) . B r u s h b o r d e r d e v e l -opment i n the i n t e s t i n a l a b s o r p t i v e c e l l s o f Xenopus d u r i n g metamorphosis. J . C e l l B i o l . , 4 4 : 1 5 1 - 7 1 . Buck, R. and J . T i s d a l e . ( 1 9 6 2 ) . An e l e c t r o n m i c r o s c o p e s t u d y o f t h e development o f the c l e a v a g e f u r r o w i n Mammalian c e l l s . J . C e l l B i o l . , 1 3 _ : 1 1 7 - 2 5 . C l a y t o n , R. ( 1 9 7 0 ) . Problems o f d i f f e r e n t i a t i o n i n t h e V e r t -e b r a t e l e n s . C u r r e n t T o p i c s i n D e v elopmental B i o l o g y , _5_ : 1 1 5 ^ - 7 2 . Cohen, A. ( 1 9 6 5 ) . The e l e c t r o n m i c r o s c o p y o f the normal human l e n s . I n v e s t . Ophthalmol., 4: 4 3 3 - 4 6 . Coulombre, A . J . ( 1 9 6 9 ) . R e g u l a t i o n o f o c u l a r morphogenesis. I n v e s t . Ophthalmol., 8: 2 5 - 3 1 . Coulombre, J. L . and A . J . Coulombre. ( 1 9 6 3 ) . Lens d e v e l o p -ment: f i b r e e l o n g a t i o n and l e n s o r i e n t a t i o n . S c i e n c e , 1 4 2 : 1 4 8 9 - 9 0 . Coulombre, Coulombre, E g u c h i , G. J.L . and A . J . Coulombre. ( 1 9 6 9 ) . Lens development IV. S i z e , shape and O r i e n t a t i o n . I n v e s t . Ophthalmol., 8: 2 5 1 - 5 7 . J.L. and A . J . Coulombre. ( 1 9 7 1 ) . Lens development V. H i s t o l o g i c a l a n a l y s i s o f the mechanism o f l e n s r e c o n s t i t u t i o n from i m p l a n t s o f l e n s e p i t h -e l i a . J . E x p e r i m e n t a l Zoology, 1 7 6 : 1 5 - 2 4 . ( 1 9 6 4 ) . E l e c t r o n m i c r p s c o p e s t u d i e s on l e n s r e g e n e r a t i o n I I . F o r m a t i o n and g r o w t h o f the l e n s v e s i c l e and d i f f e r e n t i a t i o n o f l e n s f i b r e s . Embryologia,'. 8 _ : 2 4 7 - 8 7 . 82. F a l k , H. (1969). F u s i f o r m v e s i c l e s i n p l a n t c e l l s . J . C e l l B i o l . , 43:167-74. Fo n t e , V., R. S e a r l s and S. H i l f e r . (1971). The r e l a t i o n -s h i p o f c i l i a w i t h 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 . J . C e l l B i o l . , 49:226-29. Gibbons, J.R., L..G* T i l n e y and K. P o r t e r . (1969). M i c r o -t u b u l e s i n t h e f o r m a t i o n and development o f t h e p r i m a r y mesenchyme i n A r b a c i a p u n c t u l a t a I . The d i s t r i b u t i o n o f m i c r o t u b u l e s . J . C e l l B i o l . , 41:201-26. H a r d i n g , C.V., J.R. Reddan, N.J. Unakar and M. B a g c h i . (1971), The c o n t r o l o f c e l l d i v i s i o n i n t h e o c u l a r l e n s . I n t e r n a t i o n a l Rev. C y t o l o g y , 31:-215-300. Hay, E. (1968). O r g a n i z a t i o n and f i n e s t r u c t u r e o f e p i t h e l -ium and mesenchyme i n t h e d e v e l o p i n g c h i c k embryo, pp. 31-55 i n " E p i t h e l i a l - M e s e n c h y m a l I n t e r a c t i o n s " , R. F l e i s c h m a j e r and R. B i l l i n g h a m , eds. W i l l i a m s and W i l k i n s Co., B a l t i m o r e . Hay, D. and F. Low. (1972). The f i n e s t r u c t u r e o f p r o g r e s s -i v e s t a g e s o f m y o c a r d i a l m i t o s i s i n c h i c k embryos. Amer. J . Anat., 134^175-202. H i c k s , R. (1966). The f u n c t i o n o f t h e G o l g i complex i n t r a n s i t i o n a l e p i t h e l i u m . S y n t h e s i s o f t h e t h i c k c e l l membrane. J . C e l l B i o l . , 30:623-43. Inoue, S. and H. Sa t o . (1967). C e l l m o t i l i t y b y l a b i l e a s s o c -i a t i o n o f m o l e c u l e s : t h e n a t u r e o f m i t o t i c s p i n d l e f i b r e s and t h e i r r o l e i n chromosome movement. J . Gen. P h y s i o l . , 50 (6) .-259-84. Jacobson, A.G. (1966). I n d u c t i v e p r o c e s s e s i n embryonic development. S c i e n c e , 152:25-34. Kuwabara, T. (1968). M i c r o t u b u l e s i n t h e l e n s . A.M.A. A r c h . Ophthalmol., 79:189-95. L o w e n s t e i n , W.R. (1968). Communication t h r o u g h c e l l j u n c t i o n s . I m p l i c a t i o n s i n growth, c o n t r o l , and d i f f e r e n t i a t -i o n . D evelopmental B i o l . , s u p p l . 2:151-83. McKeehan, M.S. (1951). C y t o l o g i c a l a s p e c t s o f embryonic l e n s i n d u c t i o n i n t h e c h i c k . J.. E x p e r i m e n t a l Z o o l o g y , 117:31-64. 83. McKeehan, M.S. (1954). A q u a l i t a t i v e s t u d y o f s e l f d i f f e r -e n t i a t i o n o f t r a n s p l a n t e d l e n s p r i m o r d i a i n t h e c h i c k . J . E x p e r i m e n t a l Zoology, 126: 157-75. Modak, S.P. and S.W. Perdue. (1970). T e r m i n a l l e n s c e l l d i f f e r e n t i a t i o n I . H i s t o l o g i c a l and m i c r o -s p e c t r o p h o t o m e t r i c a n a l y s i s o f n u c l e a r degen-e r a t i o n . E x p e r i m e n t a l C e l l Res., _59: 43-56. Muthukkaruppan, V. (1965). I n d u c t i v e t i s s u e i n t e r a c t i o n i n the development o f t h e mouse l e n s _in v i t r o . J . E x p e r i m e n t a l Zoology, 1 5 9 2 6 9 - 8 8 . P a l a y , S.L. (1967). P r i n c i p l e s o f c e l l u l a r o r g a n i z a t i o n , i n t h e nervous system. I n " The N e u r o s c i e n c e s , a Study Program ", G.C. Quarton, T. Melnechuk and F.O. S c h m i t t , eds., R o c k e f e l l e r U n i v e r s i t y P r e s s , pp.24-34. Pannese, E. (1968). Temporary j u n c t i o n s between n e u r o b l a s t s i n t h e d e v e l o p i n g s p i n a l g a n g l i a o f t h e d o m e s t i c f o w l . J . U l t r a s t r u c t u r a l Res., _21_: 233-50. P a p a c o n s t a n t i n o u , J . (1967). M o l e c u l a r a s p e c t s o f l e n s c e l l d i f f e r e n t i a t i o n . S c i e n c e , 156: 338-45. Pearce, T. and J . Zwaan. (1970). A l i g h t and e l e c t r o n m i c r o -scope s t u d y o f c e l l b e h a v i o u r and m i c r o t u b u l e s i n t he embryonic c h i c k e n l e n s u s i n g c o l c e m i d . J . Embryology and E x p e r i m e n t a l Morph. , 23_: 491-507. P e r s o n s , B. and S. Modak. (1970). The p a t t e r n o f DNA s y n t h -e s i s i n the l e n s e p i t h e l i u m and the a n n u l a r pad d u r i n g development and growth o f t h e c h i c k l e n s . E x p e r i m e n t a l Eye Res., 9_: 144-51. P h i l p o t t , G.W. (1970). Growth and c y t o d i f f e r e n t i a t i o n o f embryonic c h i c k l e n s e p i t h e l i a l c e l l s in v i t r o . E x p e r i m e n t a l C e l l Res., _59: 57-68. P h i l p o t t , G.W. and A . J . Coulombre. (1965). The d i f f e r e n t -i a t i o n o f embryonic c h i c k l e n s e p i t h e l i a l c e l l s i n v i t r o and in v i v o . E x p e r i m e n t a l C e l l Res., 38: 635-44. P h i l p o t t , G.W. and A . J . Coulombre. (1968). C y t o d i f f e r e n t i a t -i o n o f p r e - c u l t u r e d embryonic c h i c k e n l e n s e p i t h e l i a l c e l l s ^ n v i t r o and _in v i v o . Exper-i m e n t a l C e l l Res.,- 52; 140-46. 84. P i a t i g o r s k y , J.,H. Webster, and S, C r a i g . (1972). P r o t e i n s y n t h e s i s and u l t r a s t r u c t u r e d u r i n g t h e f o r m a t i o n o f embryonic c h i c k l e n s f i b r e s i r i v i v o and i n  v i t r o . D evelopmental B i o l . , 27_: 176-89. P o r t e , A., M.E. S t o e c k e l e t A. B r i n i . (1968). F o r m a t i o n de 1'ebauche o c u l a i r e e t d i f f e r e n c i a t i o n du c r i s t a l l i n chez 1 1embryon de p o u l e t . A r c h i v e s d 1 O p h t h a l m o l o g i c , 28: 681-706. Rabaey, M. and A. Lagasse. (1965). E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s on t h e d e v e l o p i n g l e n s o f t h e Golden Hamster.. T h e i r r e l a t i o n t o p r o t e i n s y n t h e s i s . I n "Eye S t r u c t u r e " , J.W. Rohen ,ed. S c h a t t a u e r -V e r l a g , S t u t t g t . pp. 371-82. Rash, J . , J . Shay, and J . B i e s e l e . (1969). C i l i a i n c a r d i a c d i f f e r e n t i a t i o n . J . U l t r a s t r u c t u r a l Res., 29: 470-84. R a v i o l a , G., and E. R a v i o l a , . (1967). L i g h t and e l e c t r o n m i c r o s c o p e o b s e r v a t i o n s on t h e i n n e r p l e x i f o r m l a y e r o f t h e r a b b i t r e t i n a . Amer. J , Anat., 120: 402-26. S h e f f i e l d , J.B. (1970)„ S t u d i e s on a g g r e g a t i o n o f embryonic c e l l s : i n i t i a l c e l l a d h e s i o n s and t h e f o r m a t i o n o f i n t e r c e l l u l a r j u n c t i o n s . J . Morph., 132: 245-63. S h e f f i e l d , J.B. and D.A. Fischman. (1970). I n t e r c e l l u l a r j u n c t i o n s i n t h e d e v e l o p i n g n e u r a l r e t i n a o f the c h i c k embryo. Z. Z e l l f o r s c h . , 104: 405-18. S j o s t r a n d , F.S.. (1968) . U l t r a s t r u c t u r e and f u n c t i o n o f c e l l -u l a r membranes. In "The Membranes", A . J . D a l t o n and F. Haguenau, eds. Academic P r e s s , N.Y. pp. 151-210. Smelser, G.K. (1965). Embryology and morphology o f t h e l e n s . I n v e s t . Ophthalmol., 4: 398-410. Spemann, H. (1938). "Embryonic Development and I n d u c t i o n " . Y a l e U n i v e r s i t y P r e s s , New Haven. T a k e i c h i , M. (1970). Growth o f t h e c h i c k e n embryonic l e n s t r a n s p l a n t e d o n t o th e c h o r i o - a l l a n t o i c membrane. Development, Growth and D i f f e r e n t i a t i o n , JL2: 21-30. T i l n e y , L.G. (1971). O r i g i n and c o n t i n u i t y o f m i c r o t u b u l e s . I n " O r i g i n and C o n t i n u i t y o f C e l l O r g a n e l l e s " , J . R e i n e r t and H. U r s prung, eds, S p r i n g e r - V e r l a g , N.Y. pp. 222-56. 85; T i l n e y , L.G. and J.R. G i b b o n s . (1969). M i c r o t u b u l e s i n t h e f o r m a t i o n and development o f t h e p r i m a r y mesench-yme i n A r b a c i a p u n c t u l a t a I I . An e x p e r i m e n t a l a n a l y s i s o f t h e i r r o l e i n t h e development and I maintenance o f c e l l shape. J . C e l l B i o l . , 4 1: 227-50. Truman, D.E.S. (1968). G e l f i l t r a t i o n o f c h i c k l e n s p r o t e i n . E x p e r i m e n t a l Eye Res., _7: 3 58-68. Truman, D.E.S., A.G. Brown, and J.C. Campbell (1972). The r e l a t i o n s h i p between t h e ontogeny o f a n t i g e n s and o f t h e p o l y p e p t i d e c h a i n s o f t h e c r y s t a l l i n s d u r i n g c h i c k l e n s development. E x p e r i m e n t a l Eye Res,, 13: 58-69. T w i t t y , V.C. (1955). The Eye. I n " A n a l y s i s o f Development", B. W i l l i e r , P. W e i s s , and V. Hamburger, eds, W.B. Saunders Co., P h i l a d e l p h i a , pp. 402-14. Wanko, T. and M. G a v i n . (1959). An e l e c t r o n m i c r o s c o p e s t u d y o f l e n s f i b r e s . J . C e l l B i o l . , 6: 97-102. Whaley, W.G., M. Dauwalder and J.E. K e p h a r t . (1966). The G o l g i a p p a r a t u s and an e a r l y s t a g e i n c e l l p l a t e f o r m a t i o n . J . U l t r a s t r u c t u r a l Res., 1_5: 159-70. Whaley, W.G., M. Dauwalder and J.E. K e p h a r t . (1971). Assembly, c o n t i n u i t y and exchange i n c e r t a i n c y t o p l a s m i c membrane- systems. I n " O r i g i n and C o n t i n u i t y o f C e l l O r g a n e l l e s " , J . R e i n e r t and H. U r s p r u n g , eds. S p r i n g e r - V e r l a g , , N.Y. pp. 1-38. W i s n i e w s k i , H. and R. T e r r y . (1968). E x p e r i m e n t a l c o l c h i c i n e e n c e p h a l o p a t h y . Lab. I n v e s t . , 17: 577-87. W i s n i e w s k i , H., M.L. S h e l a n s k i and R.D. T e r r y . (1968). E f f e c t s o f m i t o t i c s p i n d l e i n h i b i t o r s on n e u r o t u b u l e s and n e u r o f i l a m e n t s i n a n t e r i o r h o r n c e l l s . J . C e l l B i o l . , 3_8: 224-30. W o l p e r t , L. (1967). C e l l movement and c e l l c o n t a c t i n s e a u r c h i n morphogenesis. Endeavour, XXVI: 85-90. Y o s h i d a , K. and A. Katoh. (1971a). C r y s t a l l i n s y n t h e s i s b y c h i c k l e n s II.Changes i n s y n t h e t i c a c t i v i t y o f e p i t h e l i a l and f i b r e c e l l s d u r i n g embryonic development. E x p e r i m e n t a l Eye Res., 11:184-94. 66 . Y o s h i d a , K. and A. Katoh. (1971b). C r y s t a l l i n s y n t h e s i s b y c h i c k l e n s I . A n a l y s i s o f r a d i o a c t i v e c r y s t a l l i n s by c e l l u l o s e a c e t a t e membrane e l e c t r o p h o r e s i s . E x p e r i m e n t a l Eye Res,, L I : 122-31. Zwaan, J . (1969). I n t e r k i n e t i c n u c l e a r m i g r a t i o n d u r i n g t h e e a r l y s t a g e s o f l e n s f o r m a t i o n i n t h e c h i c k embryo. J . Embryology and E x p e r i m e n t a l Morphology, 21-71-83. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0101645/manifest

Comment

Related Items