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UBC Theses and Dissertations

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

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c\  OBSERVATIONS ON CHICK EMBRYO LENS MORPHOGENESIS I N VIVO  AND  B R I A N G.  I N VITRO  McLEAN  B . S c . , The U n i v e r s i t y o f B r i t i s h  Columbia  A THESIS SUBMITTED I N • P A R T I A L FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department of 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 required  to th  standard  THE U N I V E R S I T Y OF B R I T I S H COLUMBIA S e p t e m b e r , 1972  In  presenting this  thesis  an a d v a n c e d d e g r e e a t the I  Library  further  for  agree  make i t  partial  fulfilment  of  University  of B r i t i s h  Columbia, I  freely  available  that permission for  the requirements  for reference  extensive  agree  this  representatives. thesis  for  It  financial  is  understood that  copying of t h i s  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  Columbia  \  that  thesis or  publication  g a i n s h a l l n o t be a l l o w e d w i t h o u t my  written permission.  Department  copying or  for  and s t u d y .  s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t  by h i s of  shall  the  In  i ABSTRACT Ultrastruetural observations embryo l e n s r e v e a l t h a t f i b r e c e l l involves extensive c e l l  on t h e s i x - d a y  differentiation,  which  elongation, i s characterized by the  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 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 important  f o rc e l l  -initiating  chick  extension.  Increased  t o be  Golgi activity  i n cells  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 formation  of i n t e r c e l l u l a r  j u n c t i o n s a n d t h e e l a b o r a t i o n o f new  surface  membrane. Studies  concerning  the differentiation i n vitro of  a n t e r i o r lens e p i t h e l i a demonstrate t h a t t h e i r c e l l s either to a great  extent o r to a l i m i t e d  extent,  elongate  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 conformation.The conforma 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 c a n be manip-  u l a t e d so t h a t i t s c e l l s limited or a great  elongate  extent.  consistently to either a  These c e l l s , whether e l o n g a t e d  a l i m i t e d o r t o a great extent,  are ultrastructurally  with respect t o features r e f l e c t i n g cytoplasmic i n c l u d i n g those important  ing  I n v i v o . The c e l l s  iating great  similar  differentiation,  f o r morphogenesis. T h e i r  s t r u c t u r e c l o s e l y resembles t h a t o f f i b r e c e l l s  to  fine  differentiat-  of a n t e r i o r lens e p i t h e l i a  different-  i_n v i t r o , w h e t h e r 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 extent,  proportions acrylamide  a r e t h e same i n t e r m s o f t h e n a t u r e  and r e l a t i v e  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 b y p o l y 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 -  ative proportions  o f the s o l u b l e p r o t e i n s accumulated by the  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 is  similar  cell  t o that which occurs  i n cells  undertaking  d i f f e r e n t i a t i o n i n vivo. I t i s concluded that  f a c t o r s d e r i v i n g from t h e conformation  that  fibre those  of the anterior lens  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 differentiating  in  v i t r o are p h y s i c a l i n nature.  p h y s i c a l f a c t o r s are important encouraging the expression it  with regard  Since  to restraining or  o f morphogenetic p o t e n t i a l i n v i t r o ,  i s suggested that s i m i l a r p h y s i c a l forces are  i n l e n s m o r p h o g e n e s i s in Observations elongating  to a great  concerning extent  explants  a n t e r i o r lens  epithelia  i n v i t r o demonstrate that f o r o n l y about three  cultured f o r longer  elongation or further cytoplasmic  Their: c e l l s ,  at the ultrastructural  level,  their  days.  periods  show g r e a t e r  entiating fibre cells  important  vivov  c e l l s p r o g r e s s i v e l y elongate differentiated  such  Well  do n o t  differentiation.  resemble  differ-  i n v i v o rather than 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 h a s some c a p a c i t y f o rd i f f e r e n t i a t i o n independent o f the o c u l a r ment, b u t t h a t t h e l a t t e r its  full  environ-  i s e s s e n t i a l f o rthe expression  developmental p o t e n t i a l .  of  TABLE OF CONTENTS INTRODUCTION MATERIALS & METHODS OBSERVATIONS I- Organization lens  of thechick  embryo \  II-  Fine structure o f thec e n t r a l anterior lens epithelium  III-  Fine structure o f the l a t e r a l lens epithelium  IV-  Fine structure o f the equatorial region o f the lens  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 lens f i b r e c e l l s VI- 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 lens e p i t h e l i a irt v i t r o VII-  VIII-  Fine structure o f a n t e r i o r lens epithelia differentiating i n vitro Polyacrylamide  g e l electrophoresis  DISCUSSION I- Chick IISUMMARY BIBLIOGRAPHY  embryo l e n s  morphogenesis  C h i c k embryo l e n s in vitro  morphogenesis  LIST OF FIGURES Figure 1  Diagram o f c h i c k embryo l e n s  15.  F i g u r e s 2-4  Photomicrograph o f c h i c k embryo l e n s d e f i n i n g regions f o r reference i n discussion of u l t r a s t r u c t u r e .  16.  F i g u r e s 5-8  Ultrastructure of region I  17-19.  F i g u r e s 9-11  Ultrastructure of region II  20-21.  F i g u r e s 12-18  Ultrastructure of region I I I  22-25.  F i g u r e s 19-26  U l t r a s t r u c t u r e of regions  25-29.  F i g u r e 27  Golgi of c e l l of region I I I to  IV and V  compare w i t h f i g u r e 26  29.  F i g u r e 28-29  Ultrastructure of region V  30.  F i g u r e s 30-39  A n t e r i o r lens e p i t h e l i a at d i f f e r e n t stages o f _in v i t r o development * A n t e r i o r l e n s e p i t h e l i a explanted by method 3 and c u l t u r e d f o r 1% days  33-34.  F i g u r e s 40-42  F i g u r e s 43-44 A n t e r i o r l e n s e p i t h e l i a explanted method 2 and c u l t u r e d f o r 3 days F i g u r e s 45-47 F i g u r e s 48-64  F i g u r e s 65-66  F i g u r e s 67-69  A n t e r i o r l e n s e p i t h e l i a explanted method 3 and c u l t u r e d f o r 3 days  3 5.  by 35-36. by 36.  U l t r a s t r u c t u r e of a n t e r i o r lens e p i t h e l i a a t d i f f e r e n t stages o f i n v i t r o development  42-54.  U l t r a s t r u c t u r e o f a n t e r i o r lens e p i t h e l i a , e x p l a n t e d by method 2, t h a t elongated t o a l i m i t e d e x t e n t in v i t r o .  55-56.  Polyaery1amide g e l e l e c t r o p h o r esis p r o f i l e s of proteins of a n t e r i o r l e n s e p i t h e l i a _in v i v o and i n v i t r o  59.  V  ACKNOWLEDGEMENTS I would l i k e  t o express  my s i n c e r e t h a n k s t o D r ,  C.V. F i n n e g a n f o r h i s e n c o u r a g e m e n t a n d s u p p o r t course for  of this  work. A l s o ,  I am g r a t e f u l  during the  t o Denis L a r r i v e e  many i n v a l u a b l e d i s c u s s i o n s a n d t o Mr. L. V e 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 would l i k e  t o t h a n k 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  A.B.  Acton  thepreparation of  this  m a n u s c r i p t and t o Drs.  f o r h i s advise concerning  H. K a s i n s k y  t o Dr.  a n d N. A u e r s p e r g f o r  c r i t i c a l l y reading i t . T h i s w o r k was s u p p o r t e d R e s e a r c h C o u n c i l o f Canada.  i n part by the National  INTRODUCTION The  Vertebrate  l e n s has  long been s t u d i e d by  mental b i o l o g i s t s w i t h a v a r i e t y of points of view. demonstration, at the  t u r n of the century,  Spemann's  of the r o l e of  o p t i c v e s i c l e i n ' i n d u c i n g the lens i s a c l a s s i c induction  develop^-  example  the  of  ( s e e Spemann, 1 9 3 8 ) . H i s w o r k , as w e l l as more  investigations  ( J a c o b s o n , 1966;  M c K e e h a n , 1954;  recent  Muthukkaruppan,  1965), h a v e 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 determination. r e s t of the  The  adjustment of the growth of the  eye h a s  C o u l o m b r e and  lens to  r e c e i v e d some a t t e n t i o n ( T w i t t y ,  C o u l o m b r e , 1969).. B i o l o g i s t s i n t e r e s t e d i n  of the 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  to the lens  (see r e c e n t  review  by  attention  different-  i a t i o n h a v e b e e n 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  in And  a single lens, cells  s t r u c t u r e i s s u c h t h a t one at d i f f e r e n t stages  i t i s p o s s i b l e to p h y s i c a l l y separate  of  1 9 6 7 ) . An cells  t o do b i o c h e m i c a l  studies  can  study,  differentiation.  of c e l l  of  different-  (Papaconstantinou,  a d d i t i o n a l a d v a n t a g e i s t h a t t h e l e n s i s composed  of a s i n g l e type derived  population,  the-lens  placode.  f r o m an  the most s t r i k i n g o f w h i c h are  initial  and  extensive  biochemical cell  For  differentchanges,  elongation  of l a r g e q u a n t i t i e s of unique s t r u c t u r a l  the lens c r y s t a l l i n s .  of  ectodermal  Furthermore, lens c e l l  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  synthesis  reasons.  d i f f e r e n t regions  the lens t h a t correspond t o d i f f e r e n t stages i a t i o n i n order  the  H a r d i n g _et al. , 1 9 7 1 ) .  Workers concerned w i t h the problems of c e l l  organisation of t h i s  the  1955;  regulation  The  and  and  the  proteins,  a l l these reasons, considerable  effort  has been d i r e c t e d toward d e s c r i b i n g l e n s  differentiation,  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 processes o f c e l l  d i f f e r e n t i a t i o n and  their control. The l e n s a l s o r e p r e s e n t s  a structure interesting  from t h e p o i n t o f view o f morphogenesis are h i g h l y organized  inasmuch as i t s c e l l s  and f o r m a s y m m e t r i c a l  p a r t i c u l a r shape, w i t h  a specific  structure of  s p a t i a l r e l a t i o n s h i p t o other  components o f t h e eye, t o w h i c h l e n s g r o w t h i s a p p r o p r i a t e l y regulated  (Coulombre, 1969; Coulombre  and Coulombre,  The g r o w t h 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 ,  1969).  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 a n d t h e coordinated  differentiation of cells  d i f f e r e n t i a t i o n involves extensive new f i b r e c e l l s ing  a t t h e l e n s equator. Here,  cell  elongation  so t h a t  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 -  a f u n c t i o n a l lens of appropriate  s i z e a n d s h a p e . 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 m o r p h o g e n e s i s  i s achieved  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 e y e i n t e r a c t w i t h some m a n n e r t h a t i s i m p o r t a n t rity  each o t h e r i n  f o r i t s development  (Coulombre, 1969; Coulombre  and Coulombre,  1970; McKeehan, 1 9 5 1 , .1954; M u t h u k k a r u p p a n ,  and i n t e g -  1963; T a k e i c h i ,  1965). I t would  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 ive  and r e l a t -  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 i r o n m e n t and t h e  p r o p e r t i e s o f t h e lens p l a y i n l e n s development. A relevant i n t h i s context (1971).  i s t h a t o f Coulombre  and  report Coulombre  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 w e r e r e p l a c e d w i t h  anterior lens epithelium,  the latter  3. would form a lens w i t h orientation,  and  an  epithelium,  of appropriate  o f a shape w h i c h , a l t h o u g h  o r l e s s r e s e m b l e d t h a t o f a n o r m a l l e n s . On when p l a c e d were r e p r t e d in  in vitro,  the c e l l s  ( P h i l p o t t and  of the  c u l t u r e medium s u p p l e m e n t e d w i t h  d e g r e e . They d i d not  elongate  and  n o t p e r f e c t , more the other  anterior lens  C o u l o m b r e , 1965,  size  1968)  hand, epithelium  to  serum, t o o n l y a  elongate, limited  a t a l l , however, i n unsupplemented  medium. T h e s e 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 , with respect  to the reasons f o r l i m i t e d r a t h e r than  cell  elongation,  cell  elongation  and and  second concerning cytoplasmic  the  v i t r o and  thereby  extensive  r e l a t i o n s h i p between  differentiation.  i n d i c a t e d the p o s s i b i l i t y of manipulating  first  They a l s o  l e n s development i n  l e a r n i n g something about morphogenesis,  d i f f e r e n t i a t i o n and  the r e l a t i v e r o l e s of the  o c u l a r environment w i t h respect  to these  Because of these c o n s i d e r a t i o n s  l e n s and  the  processes. i t was  considered  of i n t e r e s t t o i n v e s t i g a t e the development of the c h i c k anterior lens epithelium i n v i t r o . s t r u c t u r a l observations 1964)  the  and  ultra-  B e y e r s and  i n v e s t i g a t i o n was  r e l e v a n t to morphogenetic  Porter, first  in-vitro.  in  consider-  necessary f o r u l t r a s t r u c t u r a l evaluation of  differentiation  embryo  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  t o make o b s e r v a t i o n s  ations,  the published  ( P o r t e e t a l . . , 1968;  a p p e a r e d somewhat i n a d e q u a t e , my  concerned w i t h order  Since  cell  lens  4. I t was a p p a r e n t t h a t m o s t u l t r a s t r u e t u r a l  obser-  v a t i o n s r e p o r t e d were concerned 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 ies  lens f i b r e c e l l  ing  protein synthesis  accumulation  organelle's^ t h a t  differentiation, (prominent  ed f o r a number o f V e r t e b r a t e  and t h e ' f e a t u r e s  nucleoli,  of cytoplasmic matrix)  accompan-  numerous  polysomes,  a r e f a i r l y w e l l document-  lenses  (Wanko a n d G a y i n ,  P o r t e j e t _ a l , , 1968; R a b a e y a n d L a g a s s e , 1 9 6 5 ; E g u c h i , Cohen, 1 9 6 5 ; s e e S m e l s e r , 1 9 6 5 ) . O n l y two f i n e studies concerning glutaraldehyde  19 59; 1964;  structural  t h e c h i c k embryo l e n s w e r e p e r f o r m e d  fixation  (Beyers  a T . , 1 9 6 8 ) , a n d t h e f o r m e r was tubules  attend-  i n the e a r l y lens  using  a n d P o r t e r , 1 9 6 4 ; P o r t e e_t  concerned mainly w i t h  micro-  rudiment.  With the exception of the p o s s i b l e r o l e of microtubules cell  (Beyers  and P o r t e r , 1964; K u w a b a r a , 1968) i n f i b r e  elongation, l i t t l e  observations cellular  c o n s i d e r a t i o n has been g i v e n t o  p e r t i n e n t t o lens morphogenesis. Although  junctions  inter-  ( t i g h t j u n c t i o n s and desmosomes ) b e t w e e n 1  1  f i b r e c e l l s h a v e b e e n r e p o r t e d i n a number o f V e r t e b r a t e es,  lens-  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 observations  c o n c e r n e d w i t h t h e manner  s u r f a c e membrane r e q u i r e d f o r c e l l  any  i n w h i c h t h e new  elongation i s elaborated /  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 organelle considered  t o b e 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  of the lens.  5. With respect iation of anterior indicated  to investigating  lens e p i t h e l i a ,  _in v i t r o  a preliminary  different-  experiment  t h a t c e l l s o f t h e e p i t h e l i a would undergo  ably greater  elongation  consider-  than t h a t p r e v i o u s l y reported  (Phil-  p o t t a n d C o u l o m b r e , 1 9 6 5 , 1 9 6 8 ) . I ^ s u b s e q u e n t l y became ent  that the e p i t h e l i a l conformation  elongation, ation  and t h a t b y m a n i p u l a t i n g  i n c e r t a i n ways e o t h e r  elongation would r e s u l t . ultrastructural  f o r this  the e p i t h e l i a l conform-  limited  or very great  I t was t h e n o f i n t e r e s t  studies with respect  ia differentiating o g e n e s i s and  very  was i m p o r t a n t  appar-  to anterior  cell  to undertake lens  epithel-  i n v i t r o f r o m t h e p o i n t s o f v i e w o f morph-  cytodifferentiation.  MATERIALS & METHODS General  i n vitro  procedures.  F e r t i l i z e d W h i t e L e g h o r n eggs o f s i x d a y s 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 w e r e r e m o v e d a n d t r a n s f e r r e d t o a p e t r i d i s h  cont-  a i n i n g s t e r i l e P u c k ' 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 bodies w i t h adhering  l e n s e s were p l u c k e d  watchmaker's f o r c e p s and t h a a d h e r i n g  from t h e eye w i t h  iris  and v i t r e o u s  a t e d f r o m t h e l e n s e s . The l e n s e p i t h e l i a w e r e s e p a r a t e d  separfrom 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 , b y one o f t h r e e methods, to e i t h e r a Nuclepore  (Winley-Morris) or M i l l i p o r e  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  ( t y p e TH)  by e i t h e r a P l e x i g l a s s or 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 c u l t u r e medium  (Ham's F-10, N o r t h A m e r i c a n B i o l o g i c a l  1 ml. of 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.) a n d a n t i b i o t i c s  (lOOu/ml. p e n i c i l l i n ,  z o n e , 100 meg./ml. s t r e p t o m y c i n , an o r g a n c u l t u r e d i s h  (Falcon  0.25 m e g . / m l . f u n g i -  Grand I s l a n d B i o l o g i c a l  Co.) i n  Plastics).  T r a n s f e r m e t h o d 1. T h i s m e t h o d o f t r a n s f e r r i n g  epith-  e l i a was o n e 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 the e p i t h e l i a having  a variety of  conformations.  T r a n s f e r m e t h o d 2. T h i s m e t h o d 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 with the lens capsule  inside.  T r a n s f e r m e t h o d 3. T h i s m e t h o d o f e x p l a n t a t i o n was s u c h as t o m a i n t a i n t h e n o r m a l c u r v a t u r e o f t h e e p i t h e l i a s o 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 w e r e i n c u b a t e d a t 37°C. i n a n  atmosphere  o f 9 6 % a i r a n d 4% CC^ ••, a n d a h u m i d i t y a p p r o a c h i n g 100%, d i f f e r e n t p e r i o d s o f t i m e b e t w e e n 1-6  days,  for  a f t e r which  they  were processed f o r m i c r o s c o p i c examination o r f o r e l e c t r o p h o r esis. Microscopy. Lenses i n 0.1N  sodium  in buffer  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  c a c o d y l a t e (pH 7.4)  ( p l u s 1 0 % s u c r o s e ) and p o s t - f i x e d  ( 1 % i n _ p h o s p h a t e b u f f e r , pH were washed, d e h y d r a t e d  7.2).  in cold  Subsequently the  washed  OsO^ tissues  and embedded i n Epon.  S e c t i o n i n g was sections  a t room t e m p e r a t u r e ,  p e r f o r m e d w i t h a LKB  ( a p p r o x i m a t e l y lum)  ultratome. Thick  f o r l i g h t microscope  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  examination  (Fisher S c i e n t i f i c )  in  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  lead  citrate. T h i c k s e c t i o n s w e r e e x a m i n e d and p h o t o g r a p h e d a Zeiss photomicroscope.From  t h e b l o c k s o f embedded  tissue  examined i n t h i s manner, a p p r o p r i a t e ones w e r e c h o s e n thin sectioning. HU  7S, HU  E l e c t r o n m i c r o s c o p y was  1 1 A , a n d A E I 801  electron  with  for  performed w i t h  the  microscopes.  Polyacrylamide g e l electrophoresis. 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 ed as o u t l i n e d b y Truman  (1968). Lenses  h o m o g e n i z e d i n lOmM p h o s p h a t e uged  (10,000 g.  f o r 10 m i n . )  (pH 7 . 2 ) ,  perform-  o r e x p l a n t s were t h e homogenate  and t h e s u p e r n a t a n t  centrif-  (mixed i n 4 0 %  8. sucrose,  1:1), i n appropriate  quantity,  layered  on  the.stack-  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 w e r e s u b s e q u e n t l y f i x e d ( i n 1 2 % T C A ) , stained  ( i na mixture containing  B r i l l i a n t Blue ethanol,  glacial  (Mann R e s e a r c h L a b o r a t o r i e s ) ,  10 m l . g l a c i a l  containing  4 5 m l . o f 0.2% C o o m a s s i e  acetic acid), destained  6 5 m l . w a t e r , 25 m l . a b s o l u t e  acetic acid), stored  scanned w i t h  45ml.  ethanol,  absolute  ( i na mixture 10 m l .  ( i n 1 0 % a c e t i c a c i d ) , and  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- Organization  u r e s 1-4.  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  The  developing  cells  enclosed  sule.  The  by  cells  lens i s a population  of  fig-  epithelial  a m o d i f i e d b a s e m e n t membrane, t h e  of the a n t e r i o r p o r t i o n  in  lens  (anterior lens  cap-  epith-  elium) are 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 the p o s t e r i o r p a r t  (facing the neural  erentiated fibre cells. 1,  4), c e l l s  r e t i n a ) are h i g h l y  In the region of the  d i f f e r e n t stages Figures  can  i l l u s t r a t e how  various  and  observe c e l l s  i n t h e i r remarkable transformation 1-4  diff-  (figures  of the a n t e r i o r lens e p i t h e l i u m elongate  c o n t r i b u t e t o t h e l e n s b o d y . H e r e , one  fibres.  equator  regions  of  at  i n t o lens of the  lens have been d e f i n e d ,  f o r p u r p o s e s o f d i s c u s s i o n , as  enting d i f f e r e n t stages  i n what, i s a c o n t i n u o u s p r o c e s s  6-day  represof  differentiation. I I - Morphology of 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 The  central anterior epithelium  (region I, f i g u r e  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  cuboidal  The  between c e l l s  t h e numerous i n t e r c e l l u l a r  ( f i g u r e s 2,  7)  and b y  a l complexes are each w i t h  lexes  evident  at the  ( f i g u r e s 5,8).  a p i c a l and  The  spaces memb-  the contours of which  ( 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  associated microfilaments  between j u n c t i o n s  association  the r e l a t i o n s h i p between a d j a c e n t  ranes w h i c h are not c l o s e l y apposed, b u t g e n e r a l l y conform  of the  2)  layer  adherent to the lens capsule. i s r e f l e c t e d by  nature  (region I ) .  basal  t r a v e r s i n g the  junction-  boundries, cytoplasm  a p i c a l j u n c t i o n a l comp-  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 o n e s , and  involve  10. regions of t i g h t junction basal  ones i n v o l v e The  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  only the l a t t e r .  mitochondria, although of general 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 are  often  observed t o be a s s o c i a t e d  ure  5) w h i c h a r e c o n s i s t e n t l y  with  lipid  found, u s u a l l y  inclusions  i s present  appear f i l l e d w i t h those associated  (figures  5,6), and i t s elements  dense m a t e r i a l .  with  (fig-  i n the apical  cytoplasm. A moderately d e v e l o p e d . p e r i n u c l e a r rough reticulum  5,7).They  endoplasmic always  Ribosomes, i n a d d i t i o n t o  the endoplasmic reticulum,  appear i n t h e  cytoplasm, though polysomes a r e n o t prominent. A moderately developed Golgi cytoplasm in  this  apparatus i s c o n s i s t e n t l y  (figure 6). Centrioles  and c i l i a c a n a l s o be o b s e r v e d  region. Microtubules are present  sparse  found i n t h e a p i c a l  (except i n m i t o t i c  (figure 8), but r e l a t i v e l y  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 a n d t h e i r d e g r e e o f o r i e n t ation increase  towrd the l a t e r a l  elium. Microfibres  regions of the anterior  ( 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 ,  are  consistently  are  largely restricted i n their d i s t r i b u t i o n to the basal  found i n t h e c e n t r a l a n t e r i o r  l a s m a n d show no p a r t i c u l a r Ill-  Morphology o f region The  epith-  epithelium.  They cytop-  orientation.  IT.  lateral anterior  epithelium  i s multi-layered  (fig-  u r e s 3 - 4 ) . The i n t e r c e l l u l a r s p a c e s f i r s t become r e s t r i c t e d t o the b a s a l II  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 ,  shows t h e i n i t i a l f e a t u r e s  of d i f f e r e n t i a t i o n that w i l l  region be  11. progressively elaborated  i n regions  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 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 ium. lipid  Cilia  of differentiation  of the central epithel-  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 ,  i n c l u s i o n s a r e f e w e r . Some d e g r e e o f c e l l  accompanied by a •reduction i n i n t e r c e l l u l a r Microtubules  though  elongation,  s p a c e s , i-s a p p a r e n t .  a r e more a b u n d a n t a n d a r e o r i e n t e d w i t h t h e l o n g ,  axes o f t h e c e l l s . p e r i p h e r a l aspects  Since these  s t r u c t u r e s appear t o occupy t h e  o f the cytoplasm,  t h e i r abundance c a n b e  appreciated  i n sections c u t t a n g e n t i a l l y through the long  of adjacent  cells  axes  (figure 10).  IV- Morphology o f r e g i o n I I I . C e l l s o f region I I I (figure 4 ) , immediately to  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  oriented microtubules, nucleus  and t h e a p i c a l s u r f a c e .  associated w i t h basal bodies  Cilia  cells  cells,  are f r e q u e n t l y observed  ( f i g u r e 16) a n d s a t e l l i t e s ,  ( f i g u r e 17) a p p e a r t o b e i n v o l v e d  the elaboration of microtubules.  these  and c o n t a i n  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  at the a p i c a l border o f these  in  adjacent  I n t h e more b a s a l r e g i o n s o f  o r i e n t e d m i c r o f i b r e s a r e remarkably abundant  (fig-  u r e 1 2 ) . The i n t e r c e l l u l a r s p a c e s o f r e g i o n I I I a r e f e w a n d b a s a l . Adjacent ures  12, 1 5 . 18) a n d e n g a g e i n j u n c t i o n s o n l y t o w a r d  a p i c a l and b a s a l The ures  c e l l membranes r u n more o r l e s s p a r a l l e l  (fig-  their  surfaces.  G o l g i apparatus appears t o be very  1 5 , 27) a n d e x t e n d s t h r o u g h o u t t h e a p i c a l  active  (fig-  cytoplasm,  12. o c c a s i o n a l l y f r o m 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 Golgi origin, of c e l l s  are p a r t i c u l a r l y evident  i n this  region  i n the apical  cytoplasm  ( f i g u r e s 14-16). These v e s i c l e s appear  empty a n d 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 p l a s m a membrane, o c c a s i o n a l l y appearing  t o fuse with  i t ( f i g u r e 1 4 ) . Elements o f  the rough endoplasmic r e t i c u l u m , although  tending  t r a t e d j u s t above and j u s t b e l o w t h e n u c l e u s , uted  along  plasmic  the length of the c e l l .  reticulum  than i n c e l l s  an e l l i p t i c a l all  distri-  The q u a n t i t y o f r o u g h  endo-  o f r e g i o n I . H o w e v e r , numerous f r e e p o l y s o m e s ( f i g u r e s 15, 1 8 ) .  n u c l e i of t h i s / r e g i o n of the developing shape, prominent n u c l e o l i ,  f e a t u r e s w h i c h become more f u l l y  cells  are often  ( f i g u r e s 1 3 , 15) d o e s n o t a p p e a r t o b e g r e a t e r  pervade t h e cytoplasm The  t o be concen-  lens have  a n d many n u c l e a r  elaborated  i n young  pores, fibre  ( f i g u r e s 22, 2 3 ) .  V- M o r p h o l o g y o f r e g i o n s The  cells  young f i b r e c e l l s ably p l e n t i f u l  I V & V.  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  ( 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  elongated a r e remark-  ( f i g u r e 1 9 ) . However, i n more m a t u r e f i b r e  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 b o d y ,  cells,  micro-  tubules  a r e much l e s s a b u n d a n t  fibres,  s o a b u n d a n t i n r e g i o n I I I , a r e much l e s s s o i n d i f f e r -  entiating  fibre cells  ( f i g u r e s 21,25,26,28,29).  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 are observed While the adjacent III  Micro-  (figure 25).  p l a s m a membranes o f c e l l s  i n region  a r e n o t 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 adjacent  cell  membranes o f  13. Young f i b r e c e l l s is  the  i s d i f f e r e n t i n two  w a y s . One  a s s o c i a t i o n of plaques o f dense m a t e r i a l w i t h  branes of adjacent c e l l s  at various  points  along  ( f i g u r e 20)..The i n t e r c e l l u l a r space o f these contains  CPannese, *1"9'68). "These j u n c t i o n s the c e l l s  differentiate,  their  appear t o be  i n the  maintained  evident  spaces of these j u n c t i o n s  ing  be  points  derived  and  i l l u s t r a t e d by ions  fused  (figure 24)}suggesting  from the G o l g i . Another  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 , a p p e a r s more and  c e l l membranes i n r e g i o n V,  are q u i t e extensive, While the  initiating  not  ically  develelabor-  and  o f r e g i o n V,  ( f i g u r e s 21,28,29).  many G o l g i d e r i v e d  a very  vesicles  further  cells  are  ( f i g u r e 20), differentiation.  reduced G o l g i body i s  a p i c a l cytoplasm  as  where t i g h t j u n c t -  a p i c a l cytoplasm of these c e l l s  found i n the  strik-  more e x t e n s i v e ,  continuous  G o l g i a c t i v i t y appears to d e c l i n e w i t h In f i b r e c e l l s  the  with  that  G o l g i apparatus i s w e l l developed i n  elongation,  observed i n the  but  the  Coated  m o d i f i c a t i o n o f y o u n g f i b r e c e l l membranes i s t h e  a t i o n of t i g h t junctions  s  amorphous m a t e r i a l r a t h e r t h a n  c e l l membranes a t v a r i o u s t h e s e j u n c t i o n s may  refer-  intercellular  often observed i n a s s o c i a t i o n w i t h  V  junct-  and  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 . are  as  i n region  embryo l e n s  t o t h e m as desmosomes. However, i n t h e  vesicles  chick  e t _ a l . (1968) o b s e r v e d t h e s e  ions between f i b r e c e l l s of the c h i c k  lengths  'adhesion plaques'  since they are  ( f i g u r e s 21,28,29). Porte  t h e mem-  junctions  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  embryo n e r v o u s s y s t e m h a v e b e e n t e r m e d  red  modification  ( f i g u r e 26).  The  typ-  rough  14. endoplasmic  r e t i c u l u m a l s o becomes r e d u c e d ,  the 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 are observed. 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 a l s o become r e d u c e d  mitochondria  .29). Some m i t o c h o n d r i a  can be o b s e r v e d  v e r y abundant i n young f i b r e c e l l s , persed  amidst  an a c c u m u l a t i n g  u r e s 2 1 , 2 6 , 2 8 , 2 9 ) and ies  28,  (figure  Polysomes, which  7  are  become p r o g r e s s i v e l y d i s -  matrix with differentiation  d e c l i n e i n number. C i l i a  are o f t e n seen i n the a p i c a l  I V and  (figures  undergo e x t e n s i v e e l o n g a t i o n  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 .  in  cytoplasm  (fig-  and b a s a l b o d -  of c e l l s i n regions  V. I n summary, i t may  be  considered that the  t u r a l d i f f e r e n c e s between the c e l l s o f r e g i o n s  ultrastruc-  I , I I , and  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 lens c e l l  differentiation.  become m o d e r a t e l y inated.  B e t w e e n r e g i o n s I and  elongated,  Oriented microtubules  and  III  stages  I I I the  of.  cells  i n t e r c e l l u l a r spaces are e l i m -  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 o c c u p y t h e b a s a l c y t o p l a s m . G o l g i  activity  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 nucleoli,  and many n u c l e a r p o r e s .  Marked e l o n g a t i o n o f c e l l s presence  erous.  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  j u n c t i o n s , f i r s t adhesion  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 Polysomes pervade the cytoplasm.  be g r e a t l y reduced  i n number. C e l l s  that with fibre c e l l o f o r g a n e l l e s and  prominent  P o l y s o m e s i n c r e a s e i n number.  o f numerous o r i e n t e d m i c r o t u b u l e s  of i n t e r c e l l u l a r tight  i n shape, develop  differentiation  and  the  plaques  the  development and  then  n u c l e a r pores  num-  M i c r o f i b r e s appear  to  i n region V i l l u s t r a t e there i s a progressive  the d e p o s i t i o n of cytoplasmic  matrix.  loss  15.  F i g u r e .1.- D i a g r a m 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 regions of the lens are i l l u s t r a t e d i n figures 2-4.  ANTERIOR ANT. LENS E P I T H E L I U M  POSTERIOR  16.  F i g u r e s 2-4. - S i x d a y c h i c k embryo i l e n s i n m i d 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 s h o w i n g t h e c e n t r a l a n t e r i o r lens e p i t h e l i u m (region I ) . Note t h e i n t e r c e l l u l a r spaces.  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 r e g i o n I I . N o t e t h a t t h e i n t e r c e l l u l a r s p a c e s 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 . N o t e 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 are 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 prominent.  as  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 are evident w i t h associated mitochondria, 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 s e e n a d j a c e n t t o t h e plasmalemma. N o t e 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. N o t e t h e r o u g h e n d o p l a s m i c reticulum f i l l e d w i t h d e n s e m a t e r i a l and t h e e l e m e n t s 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 h a n d 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 a p p o s e d 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 . N o t e t h e i n t e r c e l l u l a r s p a c e s 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 r o u g h e n d o p l a s m i c r e t i c u l u m . The l e n s c a p s u l e c a n b e s e e n on t h e l e f t and a p p o s e d f i b r e c e l l s o n 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 . N o t e 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 4 4 , 0 0 0 .  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 . N o t e 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 a p p o s e d 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 c a n be s e e n 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 . A r r o w s i n t h e l o w e r r i g h t h a n d 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 a s s o c i a t e d v e s i c l e s . - x 32,000  9&10.  Note the  golgi  and  21a  22.  Figure 12.-Basal aspect o f c e l l o f region 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 4 8 , 0 0 0 .  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 endoplasmic 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 apparent 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 p l a s m a membrane, x 7 0 , 0 0 0 .  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 s h o w i n g g o l g i a n d a s s o c i a t e d v e s i c l e s . N o t e 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.  Figure 16.-Apical portion of c e l l of region I I I i l l u s t r a t i n g c i l i u m . N o t e 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 b o d y and a s s o c i a t e d s a t e l l i t e a n d . 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 I V . N o t e m i c r o t u b u l e s , x 64,000.  of region  25a  F i g u r e .20.-. Zone o f t r a n s i t i o n b e t w e e n r e g i o n s I I I a n d I V 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 p o l y s o m e s 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 ) . N o t e a l s o t h e a p i c a l j u n c t i o n a l c o m p l e x e s and. t h e n u m e r o u s cytoplasmic v e s i c l e s i n the a p i c a l aspects of the 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 b e t w e e n r e g i o n s I V and V d e m o n s t r a t i n g numerous p o l y s o m e s a n d 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 & 2 3 . - Y o u n g f i b r e c e l l s o f r e g i o n I V s h o w i n g p r o m i n e n t 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 apparent 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 6 4 , 0 0 0 .  F i g u r e 2 5.- A more m a t u r e f i b r e c e l l . N o t e 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 a n d t h e d i s p e r s i o n o f p o l y s o m e s 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 & 2 7 . - 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 reduced g o l g i apparatus c o m p a r e d 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 s h o w i n g e l o n g a t i o n of nucleus, extensive regions of t i g h t junction 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 . N o t e 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  31. VI- D i f f e r e n t i a t i o n of anterior lens e p i t h e l i a Examination  i n vitro.  o f t h e 6-day a n t e r i o r l e n s  epithelia  e x p l a n t e d b y p r o c e d u r e 1 a n d c u l t u r e d f o r 3,4,5, o r 6 d a y s d e m o n s t r a t e d t h a t i n 8 7 % (105/121) o f t h e c a s e s 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 . were h i g h l y organized  differentiated.  Twenty-six  ( f i g u r e s 34-39). S i x t e e n were  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 extent i l l u s t r a t e d  a significant explants different-  elongated  tothe  i n f i g u r e s 4 3 - 4 4 . A n o t h e r 16 w e r e p o o r l y  I n 11 o f t h e r e m a i n i n g  79 c a s e s ,  a portion of  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 h a d e l o n g a t e d to a l i m i t e d degree. I n a l l , 47  26 e x p l a n t s w e r e h i g h l y  organized,  ( i n c l u d i n g t h e 16 w i t h p o o r d i f f e r e n t i a t i o n ) p o o r l y  ized,  and t h e r e m a i n i n g  48 o r g a n i z e d  only  t o an i n t e r m e d i a t e  organdegree.  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 h a d a common f e a t u r e : t h e e p i t h e l i u m was s o o r i e n t ed t h a t i t s c u r v a t u r e t e n d e d t o e n c l o s e capsule.  Similarly,  i n those  the cells within the  explants with less organization,  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 b e i n a r e a s more o r l e s s e n c l o s e d b y t h e l e n s c a p s u l e On t h e o t h e r h a n d , i n t h o s e  explants or regions of explants  with limited differentiation, epithelial enclosed  folding,  cells  t h e a r r a n g e m e n t was o n e o f l i t t l e  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  the capsule, The  (see f i g u r e s 33, 3 7 ) .  as s e e n i n f i g u r e s 43 a n d 4 4 .  o f the e p i t h e l i a explanted by procedure  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 a n d t h e c a p s u l e consistently  f o l d e d on i t s e l f ,  (15 c a 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  extent  .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 w e r e e x p l a n t e d b y e d u r e 3, w i t h t h e n o r m a l c u r v a t u r e m a i n t a i n e d , c o n s i s t e n t l y elongated  their  to a great extent(8.cases)  d a y s i n c u l t u r e ( f i g u r e s 4 5 - 4 7 ) . E v e n a f t e r 1-1% ure in  (7 c a s e s ) a h i g h d e g r e e o f c e l l e p i t h e l i a explanted Examination  intervals  (figures  of c u l t u r e d e p i t h e l i a  cells  during  three  days i n c u l t -  e l o n g a t i o n was  i n t h i s manner  proc-  apparent  40-42).  fixed at  different  (1-6 d a y s ) i n d i c a t e d t h a t t h e i r c e l l s  elongate  prog-  r e s s i v e l y f o r a p e r i o d o f a b o u t t h r e e d a y s . The  explants  cult-  ured  f o r longer periods  cell  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 c u l a r to the r a f t , by  the medium-air In  will  as  ( s e e f i g u r e s 3 8 - 3 9 ) d i d n o t show g r e a t e r  though r e f l e c t i n g  the p o l a r i t y  summary, 6-day c h i c k embryo l e n s a n t e r i o r  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  the lens capsule  extensive  appears t o be  an e p i t h e l i a l c u r v a t u r e w h e r e b y t h e c e l l s  c u r v a t u r e and  perpendimposed  interface.  elongation involved i n t h i s process by  i n a direction  cell  form the  the g r e a t e r curvature.  elongation.  cell  encouraged  of e p i t h e l i a l curvature, or curvature opposite to the seemi t o d i s c o u r a g e  epithelia  lesser Lack above  F i g u r e 30 & 3 1 . - 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 d a y jLn v i t r o . E x p l a n t e d b y m e t h o d 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 d a y s i n v i t r o . E x p l a n t e d b y m e t h o d 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 d a y s . ..Explanted b y .method 1. B a r r e p r e s e n t s .10.0 micra,.  33a  34-  F i g u r e s 3 6 & 3 7 . - 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 b y m e t h o d 1 a n d c u l t u r e d f o r 3% d a y s . 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 d a y s . 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 3 9 . - Same as f i g u r e 38, b u t c u l t u r e d f o r 6 d a y s .  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 a n d c u l t u r e d f o r 1^ d a y s . 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 4 1 . - E n l a r g e m e n t o f f i g u r e 40. B a r 50 m i c r a .  represents  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 micra.  50  F i g u r e 43.- A n t e r i o r lens e p i t h e l i u m explanted 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 d a y s . E x p l a n t e d b y m e t h o d 2. 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 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 b y 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. VII-  Fine structure of anterior  '  lens e p i t h e l i a  differentiating  in vitro. Those e p i t h e l i a  appeared t o be w e l l w i t h the  cultured for periods  organized  and  o f 1-6  differentiated  e l e c t r o n microscope i n order  explants  illustrated  to evaluate  d i f f e r e n t i a t i o n which, i n general, in  normal lens f i b r e c e l l  shape, w i t h  cells. and  As  their  long  nuclei  closely  central  their  become m o r e  and  numerous n u c l e a r  ellipthe elongated fibre  pores  (fig-^  throughout the cytoplasm , days  t o s u b s e q u e n t l y become somewhat l e s s the c e l l s accumulate a f i n e  "matrix t h a t appears s i m i l a r ure  to  come t o assume an  a r e m o s t 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  With d i f f e r e n t i a t i o n ,  the  these  those of d i f f e r e n t i a t i n g  ures 48,60). Polysomes, d i s p e r s e d  appearing  of  correspond to those observed  their nuclei  c e l l s , w i t h prominent n u c l e o l i  58),  regions  axes a l i g n e d w i t h t h o s e o f  the c e l l s elongate,  narrow, resembling  fine  differentiation.  With d i f f e r e n t i a t i o n , tical  their  examination of  a number o f f e a t u r e s  that  were examined  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 normal embryonic l e n s . U l t r a s t r u c t u r a l  days  to that of lens  (figures  50,  numerous. cytoplasmic  fibre cells  (fig-  63). Cell  elongation  i s accompanied by  numerous o r i e n t e d m i c r o t u b u l e s  (figures  the presence  of  57-58) w h i c h t e n d  to  o c c u p y t h e . p e r i p h e r a l c y t o p l a s m . T h e y a r e t y p i c a l l y more a b u n d ant a p i c a l l y stages  than b a s a l l y ,  of elongation.  o r a t e d by  As  particularly i n the  during  the  initial  l e n s , t h e y a p p e a r t o be  s a t e l l i t e s associated with basal bodies  (figure  elab53).  38. Microtubules  generally 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 explants,  even a f t e r s i x days i n v i t r o , extent  t h e y do i n l e n s Oriented  n o t d e c l i n i n g i n number t o t h e  fibre cells  o f r e g i o n V in v i v o .  100 A m i c r o f i b r e s  are evident  i n a l l the  c u l t u r e d e p i t h e l i a e x a m i n e d , b u t d i f f e r i n number a n d 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 d e v e l o p m e n t . The a p p e a r ance o f c y t o p l a s m i c illustrated cells day  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 n f i g u r e s 4 8 - 4 9 . T h e y a r e much more n u m e r o u s i n  of explants  i n culture  differentiating  during  ( f i g u r e s 50, 5 9 ) . The r e l a t i v e l y h i g h  ration of microfibres the cytoplasm b a s a l  not  t o the nucleus.  Examination o f w e l l  t o t h e same d e g r e e as i n f i b r e c e l l s  q u i t e long  i s indeterminate,  although  i n v i v o , a n d assume a the length  i n some i t i s e v i d e n t  t h e y may  (figure 64).  A relative increase initial  differ-  indicated  this period,  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  of microfibres be  o r more d a y s  t o d e c l i n e i n number d u r i n g  more g e n e r a l  concent-  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  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 microfibres  t h e i r second and t h i r d  stages o f c e l l  i n G o l g i a c t i v i t y accompanies t h e  elongation  ( f i g u r e 5 5 ) . 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 a p p e a r t o be  involved  i n the establishment  49,50,52,55),and o t h e r t h e c e l l membrane eloped i n c e l l s vitro ity  of adhesion plaques  (figures  v e s i c l e s which are often associated  with  ( f i g u r e s 5 2 , 5 4 ) . The G o l g i a p p e a r s m o s t d e v -  undergoing elongation  during  t h e second day i n  ( f i g u r e 55) a n d a p p e a r s 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 -  (figure 62).  Mitochondria  are,  elongation,  o f at .• . g e n e r a l  elongation,  the mitochondria  i n number, and population adjacent  i n the  : distribution.  the b a s a l cytoplasm  tend  During  p o r t i o n of the  to the lens capsule.  the mitochondria  With i n i t i a l  extent,  elongation  evident.  cell  further  mitochondrial  cell  immediately  elongation,  t o show m o d e r a t e e l o n g a t i o n . W i t h f u r t h e r some m i t o c h o n d r i a  in fibre cell  degeneration,  of  ( f i g u r e 59)  e l o n g a t i o n of the c e l l s , as  stages  a p p e a r t o become somewhat r e d u c e d  a considerable  occupies  initial  in  elongate  vivo.  as c o m p a r e d t o t h o s e in  lens  healthy mitochondria  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  great  Mitochondrial  s i m i l a r to that d e s c r i b e d - i n the  However, a p p a r e n t l y  to a  , i s also  seem t o  fibre cells  be  in vitro  vivo.  A moderate rough endoplasmic r e t i c u l u m i s present the  initial  stages  of i n v i t r o development. W h i l e elements  t h i s o r g a n e l l e tend nuclear  region,  initially  the c e l l s  concentrated  t h e y become d i s p e r s e d  o f t e n a l i g n e d w i t h the long As  t o be  differentiate  r e t i c u l u m appear t o be  i n the  throughout the  axis of the c e l l  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  endoplasmic  i f representing  ( f i g u r e 58).  frag-  Although  disappear  as  the  with  d i f f e r e n t i a t i o n , , some e l e m e n t s g e n e r a l l y p e r s i s t ,  t h a t t h e o r g a n e l l e does n o t  peri-  cytoplasm,  r o u g h e n d o p l a s m i c 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 cell  of  ( f i g u r e s 50,59,60).  , many e l e m e n t s o f t h e  q u i t e s m a l l , as  at  i t does i n t h e  so lens.  With 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 are  modified.  i n c u l t u r e , the  During the  f i r s t day  spaces of the a n t e r i o r lens e p i t h e l i u m  intercellular  are 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 4 0 - 4 2 ) . As  the c e l l s  are formed between a d j a c e n t b y means o f c o a t e d  cells  adhesion  plaques  ( f i g u r e s 48-50), p o s s i b l y  v e s i c l e s which are o f t e n observed  o c i a t i o n w i t h c e l l membranes regions  differentiate,  ( f i g u r e s 49,  of t i g h t j u n c t i o n are elaborated  50,  52,  i n ass-  55).  Also,  (figures^59-60).  However, t h e y do n o t become as e x t e n s i v e as  those  between  /  fibre cells a i n e d and  .  in. v i v o . B a s a l j u n c t i o n a l c o m p l e x e s a r e  apical  j u n c t i o n a l complexes are prominent,  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 (figures  with  junctions  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  i n g jLn v i t r o u n d e r g o m o r p h o l o g i c a l e v i d e n t i n the l e n s . C e l l loss of i n t e r c e l l u l a r the  maint-  spaces,  e x t e n s i v e as  lens. Microtubules,  changes s i m i l a r t o those .  e l o n g a t i o n i s accompanied by  formation of i n t e r c e l l u l a r  n o t become as  differentiat-  the  increased Golgi a c t i v i t y ,  and  junctions. Tight junctions  t h e y do  i n the f i b r e c e l l s  of  do the  apparently e l a b o r a t e d from s a t e l l i t e s  the a p i c a l cytoplasm,  a r e numerous. N u c l e i e l o n g a t e  c o n t a i n numerous p o r e s and  prominent n u c l e o l i .  in  and  Polysomes  become a b u n d a n t . Numerous o r i e n t e d m i c r o f i b r e s a p p e a r i n t h e b a s a l cytoplasm. plasmic  With d i f f e r e n t i a t i o n ,  the l o s s of  o r g a n e l l e s i s n o t n e a r l y as m a r k e d as  cyto-  i t i s i n the  lens. Those e p i t h e l i a elongated  only to a l i m i t e d  e x p l a n t e d b y m e t h o d 2,  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) w e r e e x a m i n e d f i r s t t h e r e w e r e any  which  ultrastruetural  i n order  to determine i f  features that might  account  41. for their not elongating cytoplasmic elongated  differentiation with  to a  examination  f u r t h e r , and second t o compare  great  o f these  that of explants  extent jln v i t r o  contained  that  . Ultrastructural  e x p l a n t s , h o w e v e r , d e m o n s t r a t e d them t o  b e 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 . f r o m t h o s e elongated  their  t h a t had—-,.  t o a g r e a t e r degree  ( s e e f i g u r e s 6 5 - 6 6 ) . The c e l l s . . / numerous m i c r o t u b u l e s , m i c r o f i b r e s a n d p o l y s o m e s .  They had t y p i c a l  j u n c t i o n a l complexes, "with  prominent microfilaments,  associated  and were t y p i c a l l y c i l i a t e d .  Inter-  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 w e r e v e r y much t h e same a s t h o s e which the c e l l s had elongated  i n epithelia i n  t o a much g r e a t e r  extent.  42.  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 . N o t 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 . A r r o w s p o i n t t o nuclear pores of a t a n g e n t i a l l y sectioned nuclear membrane, x 2 0 , 0 0 0 . 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 cultured f o r various periods a f t e r having 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 are of explants 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 a n d 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  43.  F i g u r e 4 9 . - S i m i l a r t o f i g u r e 48 s h o w i n g m i d 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 ( a r r o w s ) t e r m e d 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 d a y s i n v i t r o . N o t e numerous p o l y s o m e s , a n d 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 extending 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 a p p e a r s 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 p l a s m a membranes, x 4 0 , 0 0 0  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 with associated microtubules at apical border of 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 . N o t e a r r a n g e m e n t 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 . Thea r r o w s 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 the cytoplasm toward the l a t e r a l c e l l border, 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 5 6 . - 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 5 7 . - 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 s h o w i n g -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 d a y s 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 p o l y s o m e s , 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 . N o t e 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 e l e m e n t s 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 h a n d c o r n e r , x 20,000  50a  51.  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 . N o t e o f t i g h t j u n c t i o n , x 20,000  region  51a  ''SB.  F i g u r e 61..- C e l l s a f t e r 4 d a y s i n c u l t u r e . N o t e t h e n u m e r o u s m i c r o t u b u l e s a n d m i t o c h o n d r i a . x 26,000.  52a  53,.  F i g u r e 62,- A p i c a l r e g i o n o f c e l l a f t e r 3% days i n c u l t u r e demonstrating the reduced e x t e n t o f the g o l g i . x 30,000.  F i g u r e 63.- C e l l s a f t e r 5 days i n v i t r o . Note the c y t o p l a s m i c matrix, the m i c r o t u b u l e s , and the l a c k o f t i g h t j u n c t i o n s , 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 d a y s . 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  Note  54a  if  55.  F i g u r e 6 5 . - R e g i o n 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 m e t h o d 2. N o t e p o l y s o m e s a n d 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 6 6 . - S i m i l a r t o f i g u r e 65. N o t e t h e numerous p o l y s o m e s and m i c r o t u b u l e s . x 4 0 , 0 0 0 .  56a  •57. VIII-  Polyacrylamide  gel electrophoresis.  E l e c t r o p h o r e s i s of the s o l u b l e p r o t e i n s of and  uncultured  ermine i f c e l l  a n t e r i o r l e n s e p i t h e l i a was elongation  in vitro  uble proteins of c e l l s  elongated  performed to  i s accompanied by  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  pointed  out  feature of lens c e l l  i n the  to a great  the lens  1  extent  the  in  sol-  vitro  degree.  d i f f e r e n t i a t i o n i s the synthesis  crystallins'.  The  unique  of  structural  d i f f e r e n t lens  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 by ility.  a change  i n t r o d u c t i o n , one i n t e r e s t i n g  l a r g e q u a n t i t i e s of what are c o n s i d e r e d proteins,  det-  t o compare the  ' 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 As  cultured  crystal-  e l e c t r o p h o r e t i c mob-  M o s t 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  in  6-day c h i c k embryo l e n s t h e o n l y m a j o r c r y s t a l l i n i s  First  Important Soluble  Y o s h i d a and  Crystallin  K a t o h , 1 9 7 1 a , b ) . The  (FISC)  stage, w i t h respect  the f i b r e c e l l s c e l l s of the eins.  the  Y o s h i d a and  Katoh,  in fibre cells  and  the  additional prot-  1971a,b;Piatigorsky,  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 the eins i s very high  at  i s that  almost e x c l u s i v e l y FISC, w h i l e  anterior epithelium synthesize  ( C l a y t o n , 1970;  fibre cells,  to t h e i r soluble proteins  synthesize  1970;  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 this  (Clayton,  the  r e l a t i v e l y lower i n  protcells  of the 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 of the s o l u b l e p r o t e i n s of e p i t h elia  explanted  b y m e t h o d s 2 and  d e m o n s t r a t e d t h e s e two  3 and  g r o u p s t o be  cultured f o r three t h e same b y  this  days  criter-  58. .ion  ( f i g u r e s 6 8 , 6 9 ) , s i n c e t h e y showed i d e n t i c a l  The  d i f f e r e n c e between  cultured  (figure  67)  cultured  ( f i g u r e s 68,69) and  epithelia with respect  u b l e p r o t e i n s i s t h a t t h e m a j o r peak ed t o i n c l u d e FISC) considerably  of the  profiles.  to t h e i r  (which w o u l d be  l a t t e r group comprises  s m a l l e r p r o p o r t i o n of the  unsolexpect-  a  total protein  on  t h e - g e l . i n - c o m p a r i s o n t o t h a t ..of t h e f o r m e r .group. T h a t 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  proportion of t h e i r  u b l e p r o t e i n s i n t h e m a j o r peak r e l a t i v e t o epithelia  s u g g e s t s them t o b e  of f i b r e c e l l  differentiation.  the  sol-  uncultured  demonstrating a  characteristic  .59.  F i g u r e 67.- S c a n 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 m a j o r 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 the cathode.  F i g u r e s 68 & 6 9 . - A s i m i l a r s c a n c o m p a r i n g t h e s o l u b l e proteins of anterior lens e p i t h e l i a cultured f o r 3 days a f t e r h a v i n g b e e n e x p l a n t e d b y m e t h o d 2 ( f i g u r e 68) and b y m e t h o d 3 ( f i g u r e 6 9 ) . E q u i v a l e n t amounts o f s a m p l e , i n t e r m s o f numbers o f e x p l a n t s , w e r e 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 m a j o r 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  o  67  60. DISCUSSION I-  C h i c k embryo l e n s  morphogenesis.  A. P l a s m a membrane. Lens f i b r e d i f f e r e n t i a t i o n cell  elongation.  This  involves  change i n shape r e q u i r e s  o f new s u r f a c e membrane a n d t h e q u e s t i o n  extensive the addition  arises^as  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 ions  suggest t h e G o l g i apparatus t o be i n v o l v e d  e s i s o f new c e l l membrane. The G o l g i in  those c e l l s  ic  vesicles,  cells.  initiating  increases  elongation,  i n the synthi n activity  a n d numerous c y t o p l a s m -  probably of Golgi o r i g i n ,  are evident  i n these  These a r e o f t e n observed i n a s s o c i a t i o n w i t h  cell  membranes w i t h w h i c h t h e y s o m e t i m e s a p p e a r t o f u s e . Cytoplasmic v e s i c l e s have been demonstrated t o c o n t r i b u t e new c e l l membrane d u r i n g (Whaley e t aJL. , 1 9 6 6 ) a n d a n i m a l and  Low, 1 9 7 2 ) c e l l s .  While  mitosis  i n both  (Buck a n d T i s d a l e , 1 9 6 2 ; Hay  Buck and T i s d a l e  t h a t such v e s i c l e s a r e d e r i v e d  (1962) s u g g e s t  f r o m smooth e n d o p l a s m 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 clearly established few  reports  plant  systems i s  ( W h a l e y e t a_l. , 1 9 7 1 ) , a n d t h e r e  i n the l i t e r a t u r e which d i r e c t l y r e l a t e  are a Golgi  activity  t o t h e a d d i t i o n o f new p l a s m a membrane. H i c k s  reported  that i n the r a t t r a n s i t i o n a l epithelium,  specializations  (regions  t h a t s u c h membrane s p e c i a l i z a t i o n s " a p p e a r e d there  from t h e  vesicles.  Falk  were  s u r f a c e , and t o be  transported  G o l g i apparatus i n the form o f f u s i f o r m (1969)  assigned  a  (1966)  membrane  of asymmetrical thickening)  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  retic-  similar  61. o r i g i n and ville  and  '  f a t e to fusiform v e s i c l e s i n c e r t a i n Algae. Weinstock  (1970). d i s c u s s e d  forming v e s i c l e s ' i n the c e l l s  of  what they termed  the  small  apparently  i n the  the  microvilli.  p o l l e n tube extension.  Golgi derived  similar  elongation  new  (1967)  involves the  c e l l membrane w h i c h i s  of very  reported elaborat-  contributed  v e s i c l e s . T h e s e a p p e a r empty, and  are  rather  to those observed i n the present 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  more r e c e n t l y W h a l e y et  (1968) and  G o l g i a p p a r a t u s as iation.  Arnold  iridophore differentiation  i o n of considerable by  and  involved  W h a l e y erf a l . (1971) o b s e r v e d t h e G o l g i t o b e  active during that squid  apical surface  'surface  i n t e s t i n e of  metamorphosing Xenopus. These v e s i c l e s a r e expansion of the  Bonne-  a l . (1971) t o v i e w  a possible centre  I n d e e d , W h a l e y et  led Sjostrand  ='  the  o f membrane d i f f e r e n t -  a l . (1971) s u g g e s t ,  . . . i t seems l i k e l y t h a t much p l a s m a membrane i s , i n one f o r m o r a n o t h e r , t r a n s f e r r e d from the G o l g i apparatus, (p. 28)  described  Wirth r e g a r d  to l e n s development, the  i n sections  I V and  observed i n c e l l s ed w i t h  the  initiating  synthesis  o f new  observations  V suggest t h a t the G o l g i fibre differentiating membrane, and  that  moderately a c t i v e i n the ium,  and  region,  cells  it.  of the  that v e s i c l e s s i m i l a r to the are observations  since these c e l l s  i s concern-  cytoplasmic  v e s i c l e s observed i n these c e l l s c o n t r i b u t e to the 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  activity  That the  plasma-  Golgi i s  a n t e r i o r lens e p i t h e l above are  also consistent with  a r e m i t o t i c a l l y a c t i v e and  seen i n  the must,  stated  this view,  therefore,  62.  elaborate B-  new  Cell  membrane. junctions.  Other v e s i c l e s generated by vesicles.  These have been s t u d i e d  wherein they are considered uptake  (see d i s c u s s i o n by  of i n t e r e s t regarding u r a l observations i o n of the  Hay,  1970)  involved  a l s o termed  (1970) h y p o t h e s i z e d ,  ions by  on  retina,  fusing with  in secretion  involved  (according  and  i n the  to the  and  format-  terminology  S h e f f i e l d and  diminutae' Fischman,  fibre cells.  the b a s i s of s t u d i e s  Sheff-  with  t h a t coated v e s i c l e s form such  t h e p l a s m a membrane. My  are  ultrastruct-  'maculae a d h a e r e n t i a  S h e f f i e l d , 1970;  embryonic n e u r a l  tissues  l e n s m o r p h o g e n e s i s s i n c e ray  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  ield  coated  S h e f f i e l d , 1970). Coated v e s i c l e s  'adhesion plaques'  1968;  G o l g i are  i n a number o f  s u g g e s t them t o b e  o f P a n n e s e , 1968; by  t o be  the  observations  juncton  t fibre cell  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 Junctions  of t h i s  type have been observed i n a  number o f t i s s u e s , b o t h e m b r y o n i c and  F i s c h m a n , 1970;  adult  ( R a v i o l a and  to represent 1 9 6 8 ) , and  points  ( S h e f f i e l d , 1970;  P a n n e s e , 1968; Raviola,  1967;  of strong  Hay,  1968;  Palay,  Aoki,  1967)  t o be  c e l l u l a r relationships during to note t h a t the  formation  development of s y n a p t i c S h e f f i e l d and  one  (Pannese,  ( S h e f f i e l d and  of s t a b i l i z i n g  morphogenesis.  inter-  I t i s of i n t e r e s t  of adhesion plaques precedes  since,  and  1967). They appear  connections i n the  F i s c h m a n 1970)  Sheffield  Aoki,1967)  adhesion between c e l l s  t h e i r function i s considered  F i s c h m a n , 1970;  hypothesis.  i n the  the  embryonic r e t i n a l e n s , the  formation  63.  of adhesion plaques precedes the development of t i g h t  junct-  ions.  to  P o s s i b l y adhesion plaques i n the  stabilize in  extensive  regions The  of t i g h t  h o w e v e r , i t may  pointed  be  i n a n o t h e r way work has  i n a l a t e r s e c t i o n . At  out  that tight  basis  pointed  out  cell  this  point, be  Lowenstein's  j u n c t i o n s are  t o s m a l l e r m o l e c u l e s s o t h a t t o some e x t e n t  c e l l u l a r environment of a f i b r e c e l l  one  of  j u n c t i o n s may  i n lens development.  demonstrated that t i g h t  b o u r s . L o w e n s t e i n has  cells  subsequent development  r o l e these junctions might play i n lens discussed  important  fibre  junction.  e l o n g a t i o n w i l l be  eable  serve  the 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  a manner t h a t f a c i l i t a t e s t h e  ( 1968)  l e n s may  i s shared by  the  permintra-  i t s neigh-  t h a t s u c h j u n c t i o n s may  be  f o r the i n t e r c e l l u l a r communication fundamental  developmental processes,  and  to  i t i s possible that this kind  of  communication f a c i l i t a t e s the degree of u n i f o r m i t y of d i f f e r e n t i a t i o n so v e r y C.  characteristic  of the  Microtubules. This  a reference  c o n s i d e r a t i o n o f l e n s morphogenesis must  to microtubules  oriented microtubules  s i n c e the presence of  them i n t h i s p r o c e s s .  in  their cell  1968;  Microtubules  a p p e a r a n c e and  Porte  distribution  T i l n e y , 1971)  e t a l . , 1968;  elongation  are ubiquitous  s h a p e i n a number o f t i s s u e s  G i b b o n s _et _ a l . , 1969;  include  abundant,  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  the s t a g e a t w h i c h they undergo g r e a t  and  lens.  i n c l u d i n g the  B e y e r s and  implicates organelles  correspond with ( T i l n e y and  at  changes  Gibbons, lens  P o r t e r , 1964). Yet  1969;  (Kuwabara, the  64. circumstances i s not  and  manner i n w h i c h m i c r o t u b u l e s  e n t i r e l y c l e a r , though t h e r e  t h a t t h e y a r e composed o f s u b u n i t s associated  as p o s t u l a t e d b y  are  organized  i s considerable  evidence  t h a t can be r e v e r s i b l y  I n o u e and  Sato  (1967)  (see T i l n e y ,  1971). The  microtubules  differentiating lites  o b s e r v e d b y me  fibre cells  associated with basal bodies.  structures considered  to serve  merization of microtubular microtubules p l a s m may  e x p l a i n why  help  sites  are  for initiating  Cell  That  a r e more a b u n d a n t  stages  of c e l l  apically  elongation,  are  j u n c t i o n s are  implicated i n c e l l  and and  formed by  cells  suggests these are a l s o important  elongation,  undergoing  in this  process.  T h e r e i s g o o d e v i d e n c e t h a t ,. i n c e r t a i n s y s t e m s a 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 i n m o r p h o g e n e s i s . B e y e r s and  ed m i c r o t u b u l e s and  poly-  elongation.  that i n t e r c e l l u l a r extension  dense  'lens m o r p h o g e n e s i s .  While microtubules  ant  satel-  small  ( T i l n e y , 1971).  and  from a p o i n t i n the a p i c a l c y t o -  microtubules initial  e l a b o r a t e d by  to account 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  orientation in D.  as  presumptive  Satellites  subunits  are elaborated  than b a s a l l y during may  appear t o be  in  i n the c e l l s  s u g g e s t e d them t o be  Porter  of the  responsible  are  (1964) r e p o r t e d  c h i c k embryo l e n s  importorientplacode,  f o r the p a l i s a d i n g of  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 c e and t h a t the a p p l i c a t i o n of colcemid  least,  Zwaan  (1970)  t o t h e c h i c k embryo  reported lens  65.  placode, while  reducing  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 -  ules observed i n the c e l l s ,  d i d not r e s u l t i n the loss of  t h e i r columnar form. These authors suggested m i c r o t u b u l e s be  to  i n v o l v e d i n the i n i t i a t i o n , b u t not t h e maintenance o f c e l l  elongation. Somewhat s i m i l a r c o n c l u s i o n s and  h i s associates  of elegant  were reached by T i l n e y  (see T i l n e y , 1971) on t h e b a s i s  experiments i n v o l v i n g the e f f e c t s o f  sea water, D 2 O , h y d r o s t a t i c pressure  o f a number  calcium-free  and c o l c h i c i n e on s e a  u r c h i n m o r p h o g e n e s i s . The p r i m a r y mesenchyme c e l l s o f t h e s e a u r c h i n embryo u n d e r g o , 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  r i b u t i o n of microtubules.  A l l t h e above t r e a t m e n t s  the morphogenesis o f these c e l l s , tubules  i n their i n i t i a l  the  prevented  £>y s t a b i l i z i n g  d i s t r i b u t i o n and t h e o t h e r 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 s e d o n l y reduction  o f ectodermal c e l l  the dist-  elongation,  by d i s -  partial  treatment with  calcium-  free sea water, which not only disassembles microtubules interferes with  intercellular  junctions  t h e b a s e m e n t membrane a n d h y a l i n e spherulation.  egrity, has  junctions  resulted i n partial  concluded that while  changes i n c e l l ions  and t h e i n t e g r i t y o f  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 ,  intercellular  which  a n d i n t e r f e r e s some-  a n d b a s e m e n t membrane  spherulation  microtubules  of the c e l l s .  are required  shape, a d d i t i o n a l f a c t o r s  intTilney  t o produce  (intercellular  and a t t a c h m e n t s t o e x t r a c e l l u l a r m a t e r i a l )  i n i t s maintenance.  but  layer, resulted i n their  effects the disassembly of microtubules what w i t h  micro-  junct-  are important  66. While microtubules process of c e l l  elongation  a c c o u n t f o r i t . Nor for  a p p e a r t o be i n the  number o f m i c r o t u b u l e s fibre cells.  provide  elongation,  not  any  the  fully  explanation  particularly  i s g r e a t l y reduced i n  since  elongation  f u r t h e r that i t i s important  and  of the l e n s body i n the c o n t e x t  junctions  i t s maintenance,  to recognize  the  energetic  of these  great  reinforces t h a t are  extent  of t i g h t  l e n s body i s maintained  j u n c t i o n s between f i b r e  a n o t h e r . The  i n the absence of the  lens  the lens develops,  g r o w t h o f t h e l e n s , new  tends,  continued  f i b r e c e l l s wrap around the  They p r o c e e d t o e l o n g a t e  l e n s body.  somewhat more t h a n t h o s e c e l l s  down p r e v i o u s l y s i n c e t h e y h a v e a l a r g e r c i r c u m f e r e n c e a t t a c h t o and getically an  over which extend i n order  s t a b l e conformation  integral  new  to maintain  of the lens body  as  p a r t of the s t r u c t u r e . I t i s suggested,  t h a t the l e n s body p l a y s f i b r e c e l l s elongate,  ortant i n lens  the  capsule,  that  toward the s p h e r i c a l . With  a r o l e i n determining  laid to  the  ener-  t h e y become then,  t o what degree  i . e . that p h y s i c a l f a c t o r s are  morphogenesis.  cells  cells  shape of  which suggests i t t o have a s t a b l e conformation as  body  fibre  t h e v i e w t h a t t h e l e n s body i s composed o f  t i g h t l y a d h e r e n t t o one  stab-  lens  i n f l u e n c e s the degree of e l o n g a t i o n of d i f f e r e n t i a t i n g The  and  processes.  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  cells.  the  differentiated  I t i s suggested here t h a t i n t e r c e l l u l a r  play a role both i n c e l l  ility  l e n s , t h e y do  do m i c r o t u b u l e s  the maintenance of c e l l  involved i n  imp-  67. While microtubules  probably  ution to lens c e l l  extension,  probably  to t h i s process  has  important  p o i n t e d out  cell  on  simple  intercellular junctions  t h e o r e t i c a l grounds the  considers  a s h e e t o f c e l l s w i t h b a s a l and  tachments, the .reinforcement of the former w i l l  favour  o f t h e l a t t e r and  differentiate,  lateral  aspects  and  (1967)  influence  initiating  and  intercellular  for fibre cell  maintenance of c e l l  reduction  the young  to the l e n s  i n terms o f the  a d h e s i o n s may  This fibre  their cap-^ latershape  a l s o "serve  to  j u n c t i o n s may  be  considered  elongation. After c e l l  elongation  intercellular  junctions contribute to  shape i n t h e sense t h a t they  the  maintain  as an i n t e g r a l p a r t o f t h e l e n s b o d y . The  lular  the  at-  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  been achieved,  each c e l l  lateral  e l o n g a t i o n become  important  they u n d e r t a k e . These l a t e r a l  important  l e n s , f o r as  l o s e t h e i r attachment  a l l y adherent might w e l l be  microtubules  shape.  t h e y become s t r o n g l y a d h e r e n t a t  s u l e b a s a l l y . That c e l l s  stabilize cell  are  the e l o n g a t i o n of the c e l l s .  p r e c i s e l y what happens i n t h e  cells  has  as w e l l . W o l p e r t  adhesions might have i n the development of c e l l  I f one  is  make a m a j o r c o n t r i b -  e s s e n t i a l suggestion  j u n c t i o n s formed d u r i n g  here i s t h a t the  lens c e l l  intercel-  e l o n g a t i o n are  not  merely a r e f l e c t i o n of the t r a n s f o r m a t i o n of u n d i f f e r e n t i a t ed c e l l s junctions  i n t o d i f f e r e n t i a t e d ones. Rather, formed c o n t r i b u t e t o the process  the  intercellular  of c e l l  extension.  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 lens suggest t h e f o l l o w i n g view o f f i b r e c e l l g a t i o n i n l e n s morphogenesis.New plasmalemma, Golgi activity, tubules,  i s contributed to the c e l l  elaborated  contribute to c e l l  from s a t e l l i t e s elongation.  -through t h e f o r m a t i o n  hments t o : t h e l e n s c a p s u l e extend along  an e x t e n t  from  Micro-  i n t h e a p i c a l .-cytoplasm, elongation  is^assisted with  junctions, while basal  are lost.  These d e v e l o p i n g  This  attaclens  other previously d i f f e r e n t i a t e d c e l l s to  determined by the degree t o which the l a t t e r  elongated.  have  extended s t a t e i s i n t u r n i n f l u e n c e d by t h e  s t a b i l i t y of the o v e r a l l conformation E.  surface.  o f surface adhesions l a t e r a l l y  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  cells  This  derived  elon-  o f t h e l e n s body.  Cilia. That c i l i a have been observed i n t h i s i n v e s t i g a t i o n  in cells respect  of a l l regions to the c e l l  that while  a l l cells  o f t h e l e n s may b e o f i n t e r e s t w i t h  c y c l e . F o n t e ejt aJL. (1971) h a v e  reported  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 b u d ( 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 they conclude that the c e l l s cell  c y c l e may h a v e c i l i a .  i n t h e G^, S, G 2 p h a s e s o f t h e  Similarly,  i n the lens,  c e l l s have been observed i n t h e present ions  i n which a l l the c e l l s  ciliated,  i n v e s t i g a t i o n i n reg-  are reported  ( P e r s o n s a n d Modak,  1970) t o b e 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 of the c e l l  i n something other  c y c l e so t h a t i n t h e c e l l s  than t h e M phase  of regions  II-V c i l i a  69.  w o u l d be  expected i n those c e l l s  the c e l l s  i n m i t o s i s r o u n d up n e a r t h e a p i c a l b o r d e r .  that c i l i a region  examined the a p i c a l b o r d e r s epithelium,  cells  not  cells  of regions  cell  The  fact  I may  be  of c e l l s  the r e s u l t of  of the  having  multi-layered  frequent  observation  of c i l i a  I I I - I V , c e l l s which have entered  (Modak and  the  in terminal  P u r d u e , 1 9 7 0 ) , s u g g e s t s t h a t as  the  c o m p l e t e 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  proceed to d i f f e r e n t i a t e .  Therefore,  t h a t some c e l l s  and  of  the  a n t e r i o r lens epithelium are c i l i a t e d i s c o n s i s t e n t w i t h view t h a t the presence of c i l i a cell  ( F o n t e et  cycle  of  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  i n m i t o s i s . The  cycle  cells  since  a p p e a r e d 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  I I as c o m p a r e d t o r e g i o n  lateral  involved i n elongation,  a l . , 1971)  reflects  the  status of  rather than m i t o t i c  the  the  activity  (Rash e t a l . , 1 9 6 9 ) .  When c o l u m n a r c e l l s m i t o t i c phase of the c e l l surface,  tubules  c y c l e , t h e y r o u n d up  undergo d i v i s i o n ,  1 9 6 9 ) . The  a p p e a r a n c e and  and  subsequently re-elongate  c o r r e s p o n d t o t h i s c h a n g e i n s h a p e . Thus t h e  microtubules, the l a t e r a l ency t o be  and  the  near the a p i c a l  d i s t r i b u t i o n of cytoplasmic  c y c l e i m p o s e s a c o n s t r a i n t on  ion  of the l e n s placode enter  the o r g a n i z a t i o n of  (Zwann, micro-  cell  cytoplasmic  t h e r e f o r e on m o r p h o g e n e s i s . Many c e l l s  a n t e r i o r lens epithelium somewhat e l o n g a t e d ,  of microtubules,  but  with  (region II) have a an  appropriate  since these c e l l s  undergo  of tend-  distributdivision,  7Q. t h e y undergo  r e c u r r e n t changes  in cell  shape.  i n t e r e s t t o know i f t h e t e n d e n c y o f t h e c e l l s t o b e somewhat e l o n g a t e d ion  is a reflection  o f the growth phase:of t h e i r c e l l  of the c e l l s  of region  those demonstrated ically  inactive.  microtubules in  of a greater  and I V  cycle.  ( P e r s o n s a n d Modak, 1970) t o b e m i t o t -  Thus i t seems f a i r l y  c l e a r that lens  o n l y when i n a n  c y c l e . However, w h e t h e r  may d e p e n d  on o t h e r  or not  cells  of region  r e l a t i v e to the ocular r e t i n a (Coulombre,  environment,,  1969). Y e t , c o n s i d i s the  o f l e n s g r o w t h b y means o f r e g u l a t i n g t h e c e l l  cycles of the c e l l s of the a n t e r i o r lens epithelium, very  tempting to speculate  neural  r e t i n a a f f e c t s the c e l l  c y c l e and t h e r e b y  i t is  that the i n f l u e n c e mediated by the cycle of the c e l l s  such t h a t the l a t t e r are 'locked' cell  organized  seem t o d e r i v e f r o m t h e  t h a t one e s s e n t i a l theme o f l e n s d e v e l o p m e n t  regulation  cytoplasmic  factors i n a d d i t i o n to the c e l l  Such a d d i t i o n a l f a c t o r s would  p a r t i c u l a r l y the neural  cells  appropriate  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  p o s i t i o n of the c e l l s  ering  durat-  I I Icorrespond to  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  III  of region I I  c y c l e s as c o m p a r e d t o t h a t  I. Cells of region  show t h e i r t e n d e n c y t o e l o n g a t e phase o f t h e c e l l  I t w o u l d be o f  permitted  of region I I I  i n t h e growth phase o f t h e to differentiate.  rather than a t t r i b u t i n g the i n a b i l i t y o f the c e l l s 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 ,  That i s , of region I I I i t may b e  more p r o f i t a b l e t o v i e w t h e i r c o m m i t m e n t 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 ,  at least i n part, of c e l l  cycle regulation.  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 w h e t h e r 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 m o r p h o g e n e s i s 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 appeara n c e i n l a r g e numbers i n a n 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 of c e l l  elongation  during  the i n i t i a t i o n  of cell  elongation  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 morphogenetic import, .orientation could be-merely a r e f l e c t i o n o f c e l l abundance o f m i c r o f i b r e s ion.  Porte  et _al.  a specific  i . e .lens  h a n d , t h e 100 A m i c r o f i b r e s microtubules  polarity, the  r e f l e c t i n g cytoplasmic  differentiat-  1  crystallin*  are often  transformation  p r o t e i n . On t h e o t h e r  found together  (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 ) ,  some c i r c u m s t a n t i a l e v i d e n c e  (Wisniewski  inhibitors,  and T e r r y ,  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 as t h o u g h d e m o n s t r a t i n g a common b a s i s  u n i t s . A t t h e moment, t h e q u e s t i o n represent  with  and t h e r e i s  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 i b l y convert  speculated  l e n s p r o t e i n , and t h a t  t h e i r s u b s e q u e n t d i s a p p e a r a n c e m i g h t b e due t o into c e l l matrix,  or the  ( 1 9 6 8 ) , who o b s e r v e d t h e s e f i b r e s ,  t h a t they might represent  that  o f whether these  1968$ W i s may  revers-  mitotic t o these microfibres  t h e 100 A c l a s s o f f i b r e f o u n d i n many c e l l s ,  or  w h e t h e r t h e y a r e i n some way u n i q u e t o l e n s c e l l s ,  i snot  resolved.  further  study.  The p r o b l e m a n d t h e s e s t r u c t u r e s w a r r a n t  72.  II-  Chick  embryo l e n s m o r p h o g e n e s i s 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 of s i x - d a y  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 can ation  than the'limited  p o t t and  elongation'  C o u l o m b r e , 1965;  undergo g r e a t e r  1968). This  i s probably  dish.  out  on  medium, b u t relating  t h a t the degree of e l o n g a t i o n  the o b s e r v a t i o n greater  cell  e p i t h e l i a l conformation, t h a t the  elongation  depends on  view i s a l s o  s i n c e t h e s e end  conformations.  greater  cell  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 than r e p o r t e d by (1965; 1 9 6 8 ) , my  their observation t h r e e d a y s . Of  results  that elongation  further interest  takes  or  explanted  by  the  elongation,  capable  with  for only  i s their observation  about  that  e p i t h e l i a c u l t u r e d i n u n s u p p l e m e n t e d medium p r o g r e s s i v e l y over a p e r i o d o f t h r e e days subsequently placed  lost  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  i n s u p p l e m e n t e d medium. A p p a r e n t l y  isolated  epithelium lost a factor  ion, but  the loss  c o u l d be  of  Philpott  are c o n s i s t e n t place  by  consistent  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 b e  Coulombre  conditions  limited  explanted  and  and  the  a view supported  that those e p i t h e l i a  i n various  cult-  i n supplemented  f i r s t m e t h o d show b o t h l i m i t e d up  my  Petri  to whether i t i s  t h e t h i r d o r second method. T h i s  w i t h the observation  authors  e p i t h e l i u m shows e i t h e r according  to  examination that  a capacity to d i f f e r e n t i a t e  to the  (Phil-  due  the bottom of a  I t w o u l d seem f r o m t h e p r e s e n t  e p i t h e l i u m has  by  flattened  lens elong-  previously reported  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 ured the e p i t h e l i a  anterior  the  necessary for d i f f e r e n t i a t -  compensated f o r , i n t h e i r work, i f  73. the  e p i t h e l i u m were subsequently p l a c e d  ectomized host,  or  in association with  i n the  eye  of a l e n t -  e m b r y o n i c mesoderm i n  culture. U l t r a s t r u c t u r a l observations e r i o r lens  epithelia  on  lens  cells  that d i f f e r e n t i a t e d to a high  v i t r o d e m o n s t r a t e them t o h a v e f e a t u r e s to those of  the  fibre cells  of  ant-  degree i n  essentially similar  d i f f e r e n t i a t i n g _in v i v o .  Cell \  elongation  i s accompanied by  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 of 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 adhesion plaques, i o n a l complexes w i t h aments; the basal  lens  associated  cytoplasm; a r e l a t i v e  a cytoplasmic fibre cells.  the  l i g h t and  the  a n t e r i o r lens  e n t i a t i o n in  the  prominent bands of  increase  and  microfil-  i n the  subsequent  decline polysomes  a t the  l e v e l s of  epithelium  undergo l e n s  fibre cell  of both of  differ-  vitro. d i f f e r e n c e s a t the u l t r a s t r u c t u r a l l e v e l differentiation  i_n v i t r o and  _in v i v o ,  l e s s e r degrees of t i g h t j u n c t i o n formation organelles  from t h i s c o n c l u s i o n . t h a t the c e l l s  emble d i f f e r e n t i a t i n g  i n the  R a t h e r , t h e y may differentiating  f o r m e r , do be  et  not  interpreted  and  of  detract to  i n v i t r o come t o  res-  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  interpretation is consistent with  (Piatigorsky  more  s i m i l a r i n appearance to t h a t  Thus, o b s e r v a t i o n s  loss of cytoplasmic  ones. T h i s  apical junct-  e l a b o r a t i o n o f numerous f r e e  matrix  between f i b r e c e l l  signify  and  e l e c t r o n microscopes i n d i c a t e t h a t the c e l l s  The  involving  tight junctions  appearance of o r i e n t e d m i c r o f i b r e s  in Golgi activity; and  t h e p r e s e n c e o f numerous o r i e n t - \  a l . , 1972)  that  lens  the  epith-  report  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 The  o f s i x - d a y embryonic  c e l l s of explants  a b o u t t h r e e days. These c e l l s ,  elongate  lenses.  i n culture f o r only  at the u l t r a s t r u e t u r a 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  after this  level, time i n  t e r m s 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 a n d a l m o s t completely  losing cytoplasmic  i n the l e n s . This suggests  o r g a n e l l e s , as f i b r e c e l l s  a common b a s i s t o t h e s e  do  observations.  Possibly,  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; 1 9 6 8 ) 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  three  d a y s 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 further elongate, cytoplasmic  but also their a b i l i t y  t o undergo f u r t h e r  d i f f e r e n t i a t i o n that would r e s u l t i n the character-  i s t i c morphology o f t h e mature lens f i b r e c e l l . then,  I t i s suggested,  t h a t whatever i s l o s t during a p e r i o d o f time  a c t u a l l y concerns processes r a t h e r than o n l y those  involved i n c e l l  Considering this, thelium differentiates  fundamental t o c e l l  the reason  i n vitro  i n culture  differentiation  elongation. the a n t e r i o r lens  i s n o t because lens  epi-  develop-  ment i s 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 m e n t ' s i n f l u e n c e , o r b e c a u s e t h e 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 o n m e n t 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 acquired,  through  i t s developmental h i s t o r y ,  capacity to d i f f e r e n t i a t e independently ( c f . McKeehan, 1 9 5 4 ) . A c e r t a i n  some m e a s u r e o f  o f these  influences  period of isolation,  demonstrates a c e r t a i n measure o f dependency.  however,  T h i s i s f u r t h e r i l l u s t r a t e d by t h e o b s e r v a t i o n (Coulombre and Coulombre, 1971) t h a t when the a n t e r i o r l e n s e p i t h e l i u m i s p l a c e d i n t h e o c u l a r environment, i t does n o t immediately  undertake t o 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 a c t i v e and forms a . v e s i c l e t h a t develops  i n t o a l e n s , w i t h f i b r e c e l l s p o s t e r i o r l y and  -a g e r m i n a t i v e .epithelium . a n t e r i o r l y . -Thus t h e o c u l a r .environment i s e s s e n t i a l f o r i t s i n f l u e n c e on t h e l e n s w i t h e c t t o m i t o t i c a c t i v i t y , which permits  growth, and t o t h e  encouragement o f c o n t i n u e d d i f f e r e n t i a t i o n 1951,  resp-  ( c f . McKeehan,  1954; Muthukkaruppan, 1965).  The  u l t r a s t r u c t u r a l observations regarding  those  e x p l a n t s i n which t h e c e l l s had elongated o n l y t o a l i m i t e d degree were made t o determine i f t h e r e were any m o r p h o l o g i c a l f e a t u r e s t h a t might account 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 . However, i n terms o f t h e u l t r a s t r u c t u r e o f these moderately elongating c e l l s ,  t h e r e proved t o be n o t h i n g remarkably  e r e n t from t h a t o f those e l o n g a t i n g t o a g r e a t e x t e n t .  diffNeither  was there any d i f f e r e n c e apparent between these groups w i t h r e s p e c t t o the p o l y a c r y l a m i d e g e l l e l e c t r o p h o r e s i s p r o f i l e s o f t h e i r s o l u b l e p r o t e i n s . These o b s e r v a t i o n s  are consistent  w i t h the view t h a t an e x p l a n a t i o n o f l i m i t e d c e l l  elongation  i s probably r e l a t e d t o p h y s i c a l f a c t o r s r e s u l t i n g from the conformation  o f the e p i t h e l i u m . I t i s necessary,  then, t o  c o n s i d e r how t o i n t e r p r e t t h e r e l a t i o n s h i p between an e p i t h elium's  conformation  and the degree t o which i t s c e l l s  extend.  76.  In order  t o elongate,  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 a d h e r e t o and o v e r w h i c h t o e x t e n d For lens c e l l s ,  the substrate i s other c e l l s ,  pointed out previously i n this  themselves.  and i t has been  discussion that i n the lens  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  previously laid  down, w r a p -  p i n g a r o u n d t h e l e n s body s o t h a t t h e newer f i b r e c e l l s dep•osi-ted - d u r i n g -growth u l t i m a t e l y b e c o m e l o n g e r ones, h a v i n g  than the o l d e r  t o extend over a greater circumference.  d u r i n g g r o w t h 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 exists,  Since  added,  there  a t a g i v e n time 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  fibre cells with a  r e s s i o n o f l e n g t h s . Each c e l l ,  i s somewhat s h o r t e r  t h e one p r e c e d i n g ing. al  This  then,  suggests t h a t elongation takes place  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 ,  One c a n c o n c e i v e  c e l l s which a t a given neighbours,  i n an  increment-  the next  and so on.  that c e l l  w e l l depend on t h e e s t a b l i s h m e n t  than t h e i r  than  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 -  younger c e l l can a l s o extend,  few  prog-  elongation i n vitro  may  o f a s u b s t r a t e o f one o r a  t i m e a r e m a r g i n a l l y more  elongated  so t h a t t h e c e l l s can elongate  e a c h o t h e r , y e t more o r l e s s t o g e t h e r .  along  In 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 b e t w e e n s e r v i n g as s u b s t r a t a and  elongating  i n a s s o c i a t i o n with neighbouring  all  the c e l l s could elongate  group o f c e l l s proceeding  more o r l e s s t o g e t h e r .  i n t h i s way c o u l d s e r v e  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 of centres  c e l l s so t h a t Also, a continuously  p e r i p h e r y , g i v i n g t h e appearance  of r e l a t i v e l y greater elongation surrounded by  77.  areas of r e l a t i v e l y It  less  elongation..  i s p o s s i b l e t h a t my  e p i t h e l i a resulted i n a high the e p i t h e l i a l and  conformation  was  and  elium c e l l s  are not  curved,with  the capsule  may  c e l l s would tend  cell  elongation  such t h a t the c e l l s the o t h e r hand,  h a v e done so b e c a u s e w i t h c o n v e r g e n t , and when t h e  t e n s i o n on  encourage c e l l This  forming  to diverge.  elongation, while  formation  because  converged  explanting  the  a flat  cell  epith-  epithelium  the l e s s e r curvature,  is  the  I n o t h e r w o r d s , a c e r t a i n amount  sheet o f c e l l s would work  a reduction i n l a t e r a l  against  t e n s i o n would  elongation. investigation indicates that epithelial  i s important  suggests t h a t the  for fibre  tendency  c e l l morphogenesis  con-  and  of the lens primordium t o form  v e s i c l e d u r i n g normal development i s important, for  the c o n s t r u c t i o n of a g e r m i n a t i v e  but  a l s o f o r the morphogenesis i f f i b r e  part.  explanting  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  elongation,  of l a t e r a l  method o f  degree of c e l l  s u b s t r a t a w e r e g e n e r a t e d . On  e p i t h e l i a by  third  not  a  only  anterior epithelium, cells  i n the  posterior  78. SUMMARY  1.  A study  with  the  l i g h t and  e l e c t r o n microscopes of  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 ior  e p i t h e l i a was  e n t i a t i o n and cell  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  revealed  (in vivo) _in v i t r o  6-day c h i c k embryo l e n s  lens c e l l  that i n i t i a l  suggested that t h i s  stages  of lens c e l l  increased  activity  elongation  i n the  cells  in  '\  (adhesion  along  their lengths.  intercellular  Golgi activity.  elongation.  and b y  It is  synthesis  the  of  Extensive presence  the development  of  t i g h t j u n c t i o n s ) between  I t i s suggested that  j u n c t i o n s may  study  differentiation  l e n s Is accompanied by  p l a q u e s and  properly  This  i s r e l a t e d to the  o f numerous o r i e n t e d m i c r o t u b u l e s junctions  to  differentiation.  s u r f a c e membrane r e q u i r e d f o r c e l l  cell  and  involved ' '  v i v o are accompanied by  new  differ-  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  the normal evaluate  anter-  lens c e l l  elongation.  2... I t was  in  undertaken to evaluate  the  be  important  microtubules  for c e l l  elong-  ation. 3.  In v i t r o s t u d i e s demonstrated t h a t c e l l s  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 ation,  d e p e n d i n g on  conformation.  elong-  epithelial  Experiments were performed t h a t i n v o l v e d  ed o r r e v e r s e d . extensive  anterior  or l i m i t e d  f a c t o r s d e r i v i n g from the  lanting epithelia with  in  of the  The  cell  t h e i r normal curvature  exp-  either retain-  former procedure c o n s i s t e n t l y r e s u l t e d  elongation while  the l a t t e r  always  result-  79. ed  in limited cell  elongation,  indicating  a t i o n s encouraged or discouraged ination  of explants  extensively during elongation 4.  elongation.  i n w h i c h the c e l l s had 1-6  Exam-  elongated  days i n v i t r o i n d i c a t e d t h a t  progresses  Fine s t r u c t u r a l  cell  these conform-  f o r o n l y about three  s t u d i e s on  cell  days.  lens e p i t h e l i a  that  elongated  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 w e r e , , in general,  ultrastrueturally  differentiating  and  they d i d not iation, 5.  the  Ultrastructural  6.  of t i g h t  junction  organelles.  Indeed,  different-  c e l l s of the  only to a l i m i t e d  d e m o n s t r a t e d them t o h a v e f e a t u r e s  anterior degree  similar  to  e x t e n s i v e l y _in v i t r o .  Anterior lens e p i t h e l i a  normal c u r v a t u r e were the p r o t e i n s as  differentiat-  t h r e e days i n v i t r o .  studies regarding  those that elongated  \  come t o  a p p e a r t o show f u r t h e r c y t o p l a s m i c  i n these terms, a f t e r  vitro  extent  l o s s of cytoplasmic  lens epithelium that elongated in  fibre cells  s i x days _in v i t r o , f u l l y  f i b r e c e l l s i n terms o f the  formation  to lens  i n v i v o . However, t h e y d i d n o t  resemble, even a f t e r ed  similar  i n d i c a t e d by  explanted  with  reversed  same i n t e r m s o f t h e i r polyacrylamide  gel  or  soluble  electrophor-  esis. 7.  I t was  ends on  concluded that continued as y e t u n d i s c l o s e d  onment, b u t t o be  l e n s d e v e l o p m e n t dep-  i n f l u e n c e s of the o c u l a r  t h a t the degree of lens c e l l  elongation  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  e p i t h e l i a l conformation  and  enhanced by  in  envirappears  the  microtubules  and  80.  intercellular curvature  junctions.  I t i s suggested that  o f the _in v i v o l e n s  the  i s important i n this  normal regard.  81. BIBLIOGRAPHY A o k i , A. Arnold,  Temporary c e l l j u n c t i o n s i n the d e v e l o p i n g r e n a l glomerulus. Developmental B i o l . , 1_5: 1 5 6 -  ( 1 9 6 7 ) .  J.  6 4 .  Organellogenesis o f the 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 .  ( 1 9 6 7 ) .  B e y e r s , B. and K. P o r t e r . 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