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Mutational analysis of cell development in Paramecium tetraurelia Jones, Donald 1977

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MUTATIONAL ANALYSIS of CELL DEVELOPMENT in PARAMECIUM TETRAURELIA by DONALD JONES B.Sc,  U n i v e r s i t y o f B r i t i s h Columbia, 1973  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology)  We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE WlVERSITY OF BRITISH VoLUMBIA October, ©  1977  Donald J o n e s ,  1977  In  presenting  an  advanced degree  the I  Library  further  for  this  shall  agree  scholarly  by  his  of  this  written  in  at  University  make  that  it  partial  freely  permission  thesis  for  It  financial  is  for  gain  Zoology  University  of  British  Deo.  6.  1977  of  of  Columbia,  British  Columbia  for  extensive by  the  understood  permission.  of  fulfilment  available  p u r p o s e s may be g r a n t e d  2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  the  representatives.  Department  The  thesis  shall  reguirements  reference copying  Head o f  that  not  the  of  I  agree  and this  be a l l o w e d  or  that  study. thesis  my D e p a r t m e n t  copying  for  or  publication  w i t h o u t my  ii  ABSTRACT Temperature-sensitive mutants have been used i n t h i s study t o examine development i n Paramecium t e t r a u r e l i a . f e c t i n g development were described.  Ten mutations a f -  Since two o f the mutants were  a l l e l i c , the e f f e c t s o f nine genes on Paramecium development were studied.  Of these, two a f f e c t formation o f the fission-zone ( c a l l e d  dfz o r defective fission-zone mutants), f i v e a f f e c t c o n s t r i c t i o n o f the  fission-furrow ( c a l l e d dc o r defective c o n s t r i c t i o n mutants),  and two produce a reduction i n c e l l s i z e ( c a l l e d sm o r small mutants). Morphometric measurements were made on inter-fission and d i v i d i n g wild-type and mutant c e l l s t o examine two aspects o f Paramecium d e v e l opment:  changes i n c e l l shape and s i z e which preceed and accompany  c e l l d i v i s i o n and p o s i t i o n i n g o f new structures on the c e l l surface during c e l l d i v i s i o n .  This a n a l y s i s suggested the following hypo-  theses: 1.  The shape and s i z e changes which preceed and accompany c e l l s i o n i n Paramecium are c a u s a l l y r e l a t e d t o c e l l d i v i s i o n .  diviDefec-  t i v e c o n s t r i c t i o n mutants undergo exaggerated c o n t r a c t i o n p r i o r to fission-furrow formation.  This contraction appears t o i n t e r -  f e r e with the decrease i n c e l l width which o r d i n a r i l y occurs during d i v i s i o n . Although the mutant c e l l s are able t o make a normal amount o f furrow surface, the abnormal c e l l width prevents furrow completion.  Premature contractions seen i n d f z mutants s i m i l a r l y  i n t e r f e r e with furrow formation. 2.  Surface growth i n Paramecium i s dependent on p r i o r basal body  iii  proliferation.  Basal bodies appear to act as organizing centres  for surface growth. Reduced basal body proliferation i n mutant c e l l s was always associated with reduced surface growth: There was a consistent relationship between the number of new basal bodies produced proceeding c e l l division and the amount of surface growth which occurred.  The order of the causal relationship  was suggested by the observation that basal body proliferation was completed prior to the beginning of surface growth. 3.  The positioning of new structures during c e l l division i n Paramecium can be affected by the pre-existing c e l l shape, size, or structure. This model, called mechanical guidance, was based on observations of the movement and positioning of the vestibule (the opening leading to the mouth) i n wild-type and mutant c e l l s . This model was discussed i n relation to other developmental mechanisms proposed to account for protozoan morphogenesis.  iv TABLE OF CONTENTS ABSTRACT  i i  LIST OF TABLES  viii  LIST OF FIGURES  x  LIST OF PLATES  xvii  INTRODUCTION  1  MATERIALS AND METHODS  10  RESULTS Section A: Genetics  21  Section B: Morphometric A n a l y s i s o f C e l l D i v i s i o n  28  1.  INTRODUCTION  2.  THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS  32  i.  Growth Phase  32  ii.  Contraction Phase  34  iii.  Fission-Furrow Phase  36  iv.  C e l l Width  37  v.  Basal Body P r o l i f e r a t i o n  38  vi.  Contour Drawings  39  vii.  Growth o f the Dorsal Surface  39  v i i i . Growth o f the Ventral Surface 3.  .28  40  THE MORPHOGENETIC PERIOD IN MUTANT CELLS  42  (a) Mutant sm2  42  i.  Growth Phase  42  ii.  Contraction Phase  44  iii.  Fission-Furrow Phase  44  iv.  Basal Body P r o l i f e r a t i o n  46  V  v.  Width  .47  vi.  Stomatogenesis  48  vii.  Juvenile Cells  49  v i i i . Contour Drawings  50  ix.  Growth of the Dorsal Surface  50  x.  Growth of the Ventral Surface  50  (b) Mutant dc4  51  i.  Growth Phase  53  ii.  Contraction Phase  53  iii.  Fission-Furrow Phase  54  iv.  Basal Body Proliferation  55  v.  Width  56  vi.  Stomatogenesis  57  vii.  Contour Drawings  57  v i i i . Growth of the Dorsal Surface  57  ix.  58  Growth of the Ventral Surface  (c) Mutant dfz2  58  i.  Growth Phase  60  ii.  Contraction Phase  60  iii.  Fission-Furrow Phase  61  iv.  Stomatogenesis  63  v.  Basal Body Proliferation  63  vi.  Width  63  vi  vii.  Contour Drawings and Surface Growth ... 64  (d) Mutant dc2  a  64  i.  Growth Phase  64  ii.  Contraction Phase  66  iii.  Fission-Furrow Phase  66  iv.  Stomatogenesis  67  v.  Basal Body Proliferation  67  vi.  Width  67  vii.  Contour Drawings  68  v i i i . Growth of the Dorsal Surface  68  ix.  68  Growth of the Ventral Surface  Section C: The Pattern of Temperature-Sensitivity During the Cell Cycle  70  1. INTRODUCTION  70  2.  71  EXPERIMENTAL DESIGN  3.. TEMPERATORE-SENSITIVITY OF WILD-TYPE AND MUTANT CELLS Section D: Cortical Patterning  72 76  1. INTRODUCTION  76  2.  77  EXPERIMENTAL DESIGN  3. MEASUREMENTS 4. PROBIT ANALYSIS 5.  77 ..77  TEMPERATURE EFFECT ON sm2 CELL LENGTH  80  THE LENGTHS OF PARTS OF THE-CELL AS A FUNCTION OF CELL LENGTH 7. CORRELATION ANALYSIS  82 86  6.  vii  8.  BIYARIATE SCATTERGRAMS  Section E: G u l l e t Defects and Feeding Discussion A.  Genetics and Fhenotypes o f the Mutants  B.  Cell Division  94 100 105 I l l  (a) Shape and S i z e Changes During the C e l l Cycle  C.  SUMMARY  112  (b) Surface Growth  118  C o r t i c a l Pattern  128  (a) Cytotaxis i n Paramecium  131  (b) P o s i t i o n a l Control i n Paramecium  139 150  viii  LIST OF TABLES TABLE: I.  PAGE: a.  C o r t i c a l A b n o r m a l i t i e s Found i n Morphol o g i c a l Variants 14-16  b.  C o r t i c a l Abnormalities i n Selected Variant Lines  c. II.  t s V a r i a n t Stocks used i n the Study  17-18 . . . 19-20  Exautogamous S e g r e g a t i o n o f t s Heterorygotes  III. IV. V. VI. VII. VIII. IX.  23  _ Complimentation M a t r i x  26  Age-Dependent Changes i n Wild-Type Size  33  Contour Lengths o f V e n t r a l Regions Age-Dependent Changes i n sm2 C e l l S i z e  4-1 . . . . 43  Age-Dependent Changes i n d c 4 C e l l S i z e  . . . . 52  Age-Dependent Changes i n d f z 2 C e l l S i z e  . . . 59  Age-Dependent Changes i n d c 2  . . . 65  C e l l Size  X.  Size-Dependent Changes i n sm2 C e l l Dimensions  XI.  D i s t r i b u t i o n s Obtained from P r o b i t  XII. XIII.  XIV. XV.  Cell  78  Analysis  • 81  Morphometric Measurements as a F r a c t i o n o f C e l l Length  L i n e a r R e g r e s s i o n s o f C e l l Dimensions A g a i n s t Time a t t h e R e s t r i c t i v e Temperature  84  85  Matrix of C o r r e l a t i o n C o e f f i c i e n t s I  88-89  Matrix of C o r r e l a t i o n C o e f f i c i e n t s  90-91  II  LIST OF TABLES -  (Cont'd.)  TABLE: XVI.  PAGE Parametric  Correlation Coefficients  for  Sets o f Homogeneous C o r r e l a t i o n s XVII. XVIII. XIX. XX.  92  P r i n c i p a l Axes o f B i v a r i a t e Scattergrams Morphometric Parameters Experiment  . . 96  - Food Vacuole  Matrix of Correlation C o e f f i c i e n t s  101 for  Data from the Food Vacuole Experiment  P r e d i c t e d Growth of the Furrow S u r f a c e Wild-Type and Mutant C e l l s  . . . . 103  on  123  LIST OF FIGURES  NUMBER: 1.  2.  PAGE: A Schematic Diagram o f the K i n e t i e s on the D o r s a l and V e n t r a l S u r f a c e s o f an I n t e r f i s s i o n Paramecium  160  A Schematic Diagram o f a P o r t i o n o f the Paramecium Cortex  162  3.  Structure  o f the Paramecium G u l l e t  4.  A Schematic Diagram o f O r a l Anlage Development  164 •  166  5.  Camera L uPore c i d a Drawings actilC e e l l s . 168 Vacuole A b n o r m a l iot fi e sC oi n nt rtx-0  6.  Measurements Made on C e l l s o f Known A g e . . . 170  7-12.  Sample S t a t i s t i c s f o r Wild-Type and sm2 C e l l s of Known Age a f t e r a S h i f t t o the R e s t r i c t i v e Temperature  7a.  Length  172  7b.  Length of the Anterior Suture and Vestibule Length.  172  8a.  Length of the P o s t e r i o r Suture  174  8b.  Width  174  9a.  CVA  176  9b.  CVP  176  10a.  CVA'  178  10b.  CVP'  178  11a.  Length-to-Width Ratio  180  lib.  CVINT  *  180  xi  LIST OF FIGURES -  (Cont'd.)  NUMBER:  PAGE:  12a.  Number o f B a s a l Bodies  182  12b.  Spacing Between B a s a l Bodies  182  13.  O u t l i n e Drawings o f Wild-Type and sm2 Dividing Cells  14-.  Growth o f V a r i o u s Regions on the D o r s a l  15.  B a s a l Body P r o l i f e r a t i o n i n sm2 C e l l s D u r i n g the F i r s t C e l l C y c l e at the R e s t r i c t i v e Temperature  16. 17-22.  .Surface  of D i v i d i n g C e l l s  184 186  188  A b n o r m a l i t i e s i n G u l l e t Anlage Development i n sm2 C e l l s  191  Sample S t a t i s t i c s o f Wild-Type and dc4 C e l l s of Known Ages A f t e r a S h i f t t o the R e s t r i c t i v e Temperature  17a.  Length  17b.  Length of the A n t e r i o r Suture V e s t i b u l e Length  193 and 193  18a.  Length o f the P o s t e r i o r Suture  195  18b.  Width  195  19a.  CVA  197  19b.  CVP  197  20a.  CVA'  199  20b.  CVP'  199  21a. 21b.  Length-to-Width R a t i o CVINT  201 201  xii  LIST OF FIGURES -  (Cont'd.)  NUMBER:  PAGE:  22a.  Number of B a s a l Bodies  203  22b.  Spacing Between B a s a l Bodies  203  Camera L u c i d a Drawing o f a dc4 C e l l A f t e r 0 . 9 3 C e l l C y c l e s at the R e s t r i c t i v e Temperature  205  23.  24. 25.  O u t l i n e Drawings o f Wild-Type and dc4 Dividing C e l l s . . . .  207  Growth of Regions o f the D o r s a l S u r f a c e of D i v i d i n g C e l l s .  26-31.  209  Samples S t a t i s t i c s o f Wild-Type and d f z 2 C e l l s o f Known Age A f t e r a S h i f t t o the R e s t r i c t i v e Temperature  26a.  Length  *  26b.  Length o f the A n t e r i o r Suture and the Vestibule...  211  211  27a.  Length o f the P o s t e r i o r S u t u r e . . . .  213  27b.  Width  213  28a.  CVA  215  28b.  CVP  215  29a.  CVA'  217  29b.  CVP'  30a.  Length-to-Width R a t i o  219  30b.  CVINT  219  31a.  Number o f B a s a l Bodies  221  31b.  Spacing Between B a s a l Bodies  221  •  217  xLii  LIST OF FIGURES -  (Cont'd.)  NUMBER; 32.  33.  PAGE: Camera L u c i d a Drawings of d f z 2 C e l l s Which Were A r r e s t e d D u r i n g D i v i s i o n a t the R e s t r i c t i v e Temperature O u t l i n e Drawings o f d f z 2 and d c 2  223  a  Dividing Cells  34-39.  225  Sample S t a t i s t i c s f o r Wild-Type and d c 2 C e l l s A f t e r a S h i f t t o the R e s t r i c t i v e Temperature  34a.  Length  34b.  Length o f the A n t e r i o r Suture  a  227 and the  Vestibule  227  35a.  Length o f the P o s t e r i o r S u t u r e  229  35b.  Width  229  36a.  CVA  231  36b.  cvp..  231  37a.  CVA'  233  37b.  CVP'  233  38a.  Length-to-Width R a t i o  235  38b.  CVINT  235  39a.  Number o f B a s a l Bodies  237  39b.  Spacing Between B a s a l Bodies  237  40.  An Example o f the P l o t s Used t o Obtain  41.  Synchronous P o p u l a t i o n of C e l l s The P a t t e r n o f T e m p e r a t u r e - S e n s i t i v i t y  the Mean C e l l C y c l e Length o f a  During the Paramecium C e l l C y c l e  239 24l  xlv  LIST OF FIGURES -  (Cont'd.)  NUMBER: 42. 43. 44.  45.  46.  47.  48.  PAGE: The P a t t e r n o f T e m p e r a t u r e - S e n s i t i v i t y During the Paramecium C e l l C y c l e  243  The P a t t e r n o f T e m p e r a t u r e - S e n s i t i v i t y During the Paramecium C e l l C y c l e .  245  A Schematic Diagram Showing the Measurements made on sm2 C e l l s o f Various S i z e s . . .  247 -  P r o b i t P l o t s o f sm2 C e l l Length f o r Asynchronous Samples Taken at V a r i o u s Times A f t e r a S h i f t to the R e s t r i c t i v e Temperature  249  P r o b i t P l o t s o f C e l l Width f o r Asychronous Samples o f sm2 C e l l s Taken at V a r i o u s Times A f t e r a S h i f t t o the R e s t r i c t i v e Temperature  251  The Length and Width o f sm2 C e l l s A g a i n s t Time at the R e s t r i c t i v e Temperature  253  Plotted  Sample S t a t i s t i c s o f Asynchronous sm2 C e l l s a t V a r i o u s Times A f t e r a S h i f t t o the R e s t r i c t i v e Temperature  48a.  A n t e r i o r and P o s t e r i o r Suture Lengths  48b.  A n t e r i o r and P o s t e r i o r Suture Lengths as a F r a c t i o n o f C e l l Length  255  255  48c.  CVINT, CVA, and C V P . . .  48d.  CVINT, CVA, and CVP as a F r a c t i o n o f C e l l Length 255 Sample S t a t i s t i c s o f Asychronous sm2 C e l l s a t V a r i o u s Times A f t e r a S h i f t t o the R e s t r i c t i v e T e m p e r a t u r e . . .  49.  255  XV  LIST OF FIGURES -  (Cont'd.)  NUMBER;  PAGE:  49a.  Portions of C e l l L e n g t h . . . .  49b.  P o r t i o n s o f C e l l Length as a F r a c t i o n o f  257  C e l l Length  257  49c.  C y t o p r o c t Length  49d.  C y t o p r o c t Length as a F r a c t i o n of C e l l Length  50-55.  Bivariate Scatter Statistics  50a.  2  Plots  o f sm2 C e l l s  o f Sample  The Length o f the A n t e r i o r  The Length of the P o s t e r i o r  P l o t t e d a g a i n s t C e l l Length  51a.  The C y t o p r o c t Length P l o t t e d C e l l Length  51b.  The Length o f the V e s t i b u l e a g a i n s t C e l l Length  52a.  52b.  53a.  259 Suture 259 against 26l Plotted 261  The D i s t a n c e Between the A n t e r i o r CVP and the A n t e r i o r of the C e l l (CVA) P l o t t e d a g a i n s t C e l l Length  263  The D i s t a n c e Between the P o s t e r i o r CVP and the P o s t e r i o r o f the C e l l (CVP) P l o t t e d a g a i n s t C e l l Length  263  The D i s t a n c e Between the CVPs (CVINT) P l o t t e d a g a i n s t C e l l Length  53b.  265  The Number o f B a s a l Bodies Between the CVPs P l o t t e d a g a i n s t C e l l L e n g t h .  54a.  The S p a c i n g Between B a s a l Bodies a g a i n s t C e l l Length  54b.  . 257  Suture  P l o t t e d a g a i n s t C e l l Length  50b.  57  The T o t a l Number o f K i n e t i e s A g a i n s t C e l l Width  265  Plotted 267  Plotted 267  LIST OF FIGURES -  (Cont'd.)  NUMBER: 55a.  55b.  56.  PAGE The T o t a l Number o f K i n e t i e s P e r C e l l P l o t t e d a g a i n s t the Number o f K i n e t i e s Between the L e f t V e s t i b u l a r W a l l and the F i r s t CVP t o the C e l l ' s L e f t (KTCV)  269  The Value KTCV as a Percentage o f the T o t a l Number of K i n e t i e s P l o t t e d a g a i n s t C e l l Width  269  P r o b i t P l o t s of C e l l Length Measurements from the Feeding Experiment  57.  Bivariate  S c a t t e r P l o t s o f Sample  Statistics  58.  59.  o f sm2 C e l l s  A Schematic Diagram I l l u s t r a t i n g P o s s i b l e Mechanisms f o r the G a i n and Loss of Kineties  273  275  The Fate o f CVPs i n a C e l l Lineage Diagram  60.  271  The R e l a t i v e  277 P o s i t i o n s o f CVPs i n  Dividing Cells  279  xvii  LIST OF PLATES  NUMBER;  PAGE;  1.  Gullet Structure  2.  Somatic Cortex-Normal and Abnormal C e l l s  3.  Gullet Abnormalities  285  4-.  Cytoproct Structure  28?  5.  C e l l Shapes  289  6.  C e l l Shapes  291  ?.  C e l l Shapes  293  8.  C e l l D i v i s i o n Stages  295  9.  B a s a l Body P r o l i f e r a t i o n  10.  Cells  281  in  D i v i s i o n Stages of sm2 C e l l s  Wild-Type  ..  283  297 299  xvlii  ACKNOWLEDGEMENT  I  thank D r . James D. Berger  for his thoughtful  advice  and encouragement d u r i n g the course o f t h i s t h e s i s . h e l p f u l c o n v e r s a t i o n s and c r i t i c i s m s I P h y l l i d a Riseborough, C a r o l P o l l o c k , Connie Boogaard.  I am e s p e c i a l l y  am i n d e b t e d to  Glenn M o r t o n , and  g r a t e f u l to E r i c  and Adele Hunter f o r p r o v i d i n g me w i t h the mutant s t o c k s used i n t h i s s t u d y .  Financial  For  Peterson Paramecium  support f o r the  thesis  work was p r o v i d e d by the N a t i o n a l Research C o u n c i l o f Canada through grants t o Dr. J . D .  Berger.  1  INTRODUCTION  M u t a t i o n a l a n a l y s i s i s a p o t e n t i a l l y p o w e r f u l approach t o the study of developmental b i o l o g y .  T h i s approach  e n t a i l s the use o f chemical or p h y s i c a l agents to produce mutations which a l t e r  some aspect o f development.  Ideally,  the mutant phenotype i s produced by a s i n g l e mutated gene. The a c t i o n of i n d i v i d u a l genes i n development can be examined.  therefore  The i d e n t i f i c a t i o n o f these genes a l s o l a y s  open the p o s s i b i l i t y t h a t t h e i r gene products can be i d e n t i f i e d and the r o l e of these products i n development a n a l y s e d . T h i s approach has been used s u c c e s s f u l l y i n analysis  o f numerous aspects o f development.  These i n c l u d e  f l a g e l l a r development i n Chlamydomonas ( M c V i t t i e , t r i c h o c y s t development i n Paramecium ( P o l l o c k ,  the  1972),  1974), and  development of the f e e d i n g apparatus i n Tetrahymena and P o l l o c k , 1975).  C e l l d i v i s i o n has been examined by  mutational analysis i n b a c t e r i a Schechter,  1975)» y e a s t  (reviewed by S l a t e r and  ( H a r t w e l l , 1974), Tetrahymena  ( F r a n k e l et a l . , 1976), and Chinese Hamster c e l l s and B u t t i n ,  1975).  (Orias  Mutants such as t h e s e , where  (Hatzfeld cell  d i v i s i o n or other e s s e n t i a l c e l l f u n c t i o n s are i m p a i r e d , can o n l y be maintained i f t h e i r e x p r e s s i o n i s c o n d i t i o n a l . These are most commonly t e m p e r a t u r e - s e n s i t i v e t i o n a l mutants.  (ts)  condi-  2  The two h o l o t r i c h o u s c i l i a t e s  Paramecium and  Tetrahymena a r e w e l l s u i t e d f o r s t u d i e s i n v o l v i n g m u t a t i o n a l analysis.  In Paramecium the g e n e t i c p r o c e s s o f autogamy  r e s u l t s i n homozygosis o f a l l gene l o c i i n a s i n g l e g e n e r a t i o n (Sonneborn, 1974).  R e c e s s i v e mutations may  be brought t o e x p r e s s i o n r a p i d l y . not occur i n Tetrahymena, a l t e r n a t e to b r i n g r e c e s s i v e Frankel  alleles  therefore  Although autogamy does methods are a v a i l a b l e  into expression ( A l l e n ,  ( F r a n k e l et a l . ,  196?)•  1976, a , b ) has i s o l a t e d s i x  gene l o c i i n v o l v e d i n c e l l d i v i s i o n i n Tetrahymena pyriformis.  Both heat and c o l d - s e n s i t i v e mutant  of these l o c i have been found.  alleles  They produce d i v i s i o n  a r r e s t a t v a r i o u s c e l l ages r a n g i n g from the i n i t i a t i o n o f f u r r o w i n g t o completion o f c y t o k i n e s i s . N o n - c o n d i t i o n a l c e l l d i v i s i o n mutants o f Paramecium tetraurelia  have been r e p o r t e d by Maly (1958),  (1974b), and W h i t t l e and Chen-Shan (1972).  Sonneborn  W h i t t l e and  Chen-Shan (1972) a l s o i s o l a t e d a t s c e l l d i v i s i o n mutant. The purpose o f the p r e s e n t study i s t o use s e v e r a l new c o n d i t i o n a l c e l l d i v i s i o n and c e l l growth mutants t o analyse the mechanisms u n d e r l y i n g c y t o d i f f e r e n t i a t i o n and d i v i s i o n i n Paramecium. metric analysis  cell  The study w i l l focus on a morpho-  o f growth, s t r u c t u r e ,  and morphogenesis o f  the Paramecium c e l l and w i l l compare the changes i n c e l l dimensions p r o c e e d i n g and accompanying c e l l d i v i s i o n i n  3  wild-type P.  c e l l s with those i n mutant  tetraurelia  i s w e l l s u i t e d to morphometric  . o f c y t o d i f f e r e n t i a t i o n due to i t s c e l l architecture. and  43 pm  cells.  cell  shape and complex  The i n t e r f i s s i o n c e l l i s 100  wide (Kaneda and Hanson,  v a r y w i t h temperature  (Whitson,  analysis  1974).  pm l o n g  These dimensions  and n u t r i t i o n a l  1964)  state.  The c e l l has an enigmatic shape; names such as s l i p p e r shaped, pear-shaped, or p r o l a t e s p h e r o i d are o n l y r o u g h l y applicable.  The c e l l i s wider i n the p o s t e r i o r than the  a n t e r i o r r e g i o n and the c e l l i s d i s t o r t e d by a one-sided groove which extends from the a n t e r i o r t i p to a d e p r e s s i o n (the v e s t i b u l e )  which l e a d s i n t o the f e e d i n g a p p a r a t u s .  Two components of the c i l i a t e analysed i n developmental s t u d i e s . c o r t e x and the f e e d i n g apparatus o f the f i r s t o f the l a t t e r  c e l l are  typically  These are the  (the  gullet).  as stomatogenesis.  Both components can be Silver-  impregnation of paramecia by the Chatton-Lwoff (Chatton and Lwoff,  As a l l  The i n t e r i o r of the c e l l  remains  s i d e s o f the c e l l are impregnated  i s p o s s i b l e to examine a l l  (1962)  technique  r e s u l t s i n impregnation of only  1930)  the c o r t e x and g u l l e t .  Dippell  Development  i s r e f e r r e d to as c o r t i c a l development and t h a t  r e v e a l e d by s i l v e r - i m p r e g n a t i o n t e c h n i q u e s .  transparent.  somatic  o f the c e l l ' s  it  exterior.  has shown t h a t the s i t e s  of  silver  d e p o s i t i o n are at the j u n c t u r e of each c i l i u m with i t s  basal  4  body, at the j u n c t u r e of each t r i c h o c y s t with the s u r f a c e , i n the parasomal s a c s , i n the . vacuole p o r e s , and i n the c y t o p r o c t .  cell  contractile The l o c a t i o n of  a l l of these s t r u c t u r e s can be found i n f i g u r e s 1 and 2 and t h e i r u l t r a s t r u c t u r e w i l l be c o n s i d e r e d i n more d e t a i l later. C i l i a r y b a s a l bodies are arranged on the c e l l i n l o n g i t u d i n a l rows c a l l e d k i n e t i e s . k i n e t i e s Is  shown i n f i g u r e 1.  surface  The arrangement of  On the d o r s a l s u r f a c e the  k i n e t i e s are continuous from p o l e to p o l e .  On the  ventral  s u r f a c e the k i n e t y p a t t e r n i s i n t e r r u p t e d by the v e s t i b u l e . K i n e t i e s to the l e f t s h a r p l y around i t  (cell's left)  of the v e s t i b u l e bend  and meet k i n e t i e s on the r i g h t o f the  v e s t i b u l e along two " s u t u r e l i n e s " . contains within i t  The p o s t e r i o r suture  the c y t o p r o c t which appears as a wavy  black l i n e i n s i l v e r e d preparations ( f i g . l ) .  The c y t o p r o c t  i s the s i t e of e g e s t i o n o f f o o d v a c u o l e s . K i n e t i e s are composed of an a l t e r n a t i n g a r r a y  of  c i l i a r y b a s a l b o d i e s , parasomal s a c s , and t r i c h o c y s t s (fig.l).  The space between t r i c h o c y s t s c o n s t i t u t e s a  cortical unit.  In a d d i t i o n t o e i t h e r one or two b a s a l  bodies and a parasomal sac each c o r t i c a l u n i t c o n t a i n s a complex o f membranes and a v a r i e t y m i c r o f i b e r s ( E h r e t and Powers,  of m i c r o t u b u l e s and  1959;  Hufnagel,  1969).  The anatomy of a c o r t i c a l u n i t i s shown i n f i g u r e  2.  5  Of  the complex a r r a y  only  certain  (see above).  o f s t r u c t u r e s shown i n t h i s  ones are e v i d e n t  in silvered  organelles i s  preparations  As t h i s study w i l l r e l y h e a v i l y  m a t e r i a l a c l o s e r examination o f these  figure  on s i l v e r e d  argentophilic  warranted.  Paramecium c i l i a r y  b a s a l bodies and t h e i r  development  have been e x t e n s i v e l y s t u d i e d by D i p p e l l ( 1 9 6 8 , 1 9 7 6 ) . The b a s a l body i s  composed o f nine groups of  m i c r o t u b u l e s arranged to form a c y l i n d e r i n n e r m i c r o t u b u l e s of each t r i p l e t  (fig.2).  The two  are continuous w i t h the  outer d o u b l e t m i c r o t u b u l e s o f the c i l i a . tubule o f each t r i p l e t  triplet  The o u t e r m i c r o -  ends at a b a s a l p l a t e which  the b a s a l body from the c i l i u m above  separates  it.  Development of new b a s a l b o d i e s i s r e s t r i c t e d t o later  s t a g e s of the c e l l c y c l e  (Dippell,  the  1 9 6 8 ; Kaneda and  Hanson, 1 9 7 4 ) . New b a s a l b o d i e s appear a t r i g h t angles and a n t e r i o r to o l d ones ( D i p p e l l , opment they t i l t Details  1 9 6 8 ) .  During t h e i r  and move upwards t o the c e l l  devel-  surface.  o f t h e i r attachment t o s u r f a c e membranes and the  subsequent growth of c i l i a from the b a s a l bodies have not y e t been determined. T r i c h o c y s t s are l a r g e p r o j e c t i l e c a r r o t - s h a p e d body and a p o i n t e d t i p the t r i c h o c y s t with i t s fine thread.  o r g a n e l l e s with a (fig.2).  The body o f  a t t a c h e d t i p can be e x p e l l e d as a  T r i c h o c y s t s a r i s e deep w i t h i n the  cytoplasm  6  and undergo a complex s e r i e s of movements which b r i n g the mature o r g a n e l l e s t o the c e l l s u r f a c e (Jurand and Selman, 1 9 7 0 ) .  The t r i c h o c y s t attachment s i t e  on the c e l l membrane  c o n s i s t s of a " r o s e t t e " or c i r c u l a r arrangement o f g r a n u l e s . Mutants l a c k i n g the r o s e t t e s have t r i c h o c y s t s i n c a p a b l e of a t t a c h i n g to the c e l l membrane ( B e i s s o n et a l . ,  197&a)•  The parasomal sac i s an i n v a g i n a t i o n of the o u t e r membrane ( H u f n a g e l , 1 9 & 9 ; f i g - 2 ) .  These sacs are  cell  located  t o t h e . r i g h t and a n t e r i o r t o the b a s a l body i n each c o r t i c a l unit.  If  two b a s a l bodies occur w i t h i n a u n i t the parasomal  sac i s a s s o c i a t e d w i t h the p o s t e r i o r one ( H u f n a g e l , 1 9 & 9 ) . The o r i g i n of new parasomal sacs d u r i n g development has not been determined. On the d o r s a l s u r f a c e of the c e l l s i l v e r impregnation reveals tractile  the pores of the c o n t r a c t i l e v a c u o l e s .  The c o n -  v a c u o l e s are the f a m i l i a r w a t e r - e x p u l s i o n  o r g a n e l l e s o f paramecia.  The v a c u o l e s themselves are not  r e v e a l e d by s i l v e r i m p r e g n a t i o n .  The p o r e s of the v a c u o l e s  are 1 . 2 pm l o n g and 1 pm wide i n v a g i n a t i o n s of the  outer  membrane and are surrounded by a h e l i c a l m i c r o t u b u l e complex (McKanna, 1 9 7 3 ) . The g u l l e t can a l s o be seen i n s i l v e r e d p r e p a r a t i o n s . B a s a l bodies i n the g u l l e t are arranged i n twelve k i n e t i e s . The f o u r k i n e t i e s on the d o r s a l g u l l e t w a l l are c a l l e d the quadrulus.  The remaining e i g h t k i n e t i e s comprise the  7  p e n i c u l u s which i s a r b i t r a r i l y  s e p a r a t e d i n t o the d o r s a l  and v e n t r a l p e n i c u l i , each c o n t a i n i n g f o u r (fig.3)«  kineties  B a s a l b o d i e s i n the p e n i c u l u s are spaced more  c l o s e l y than those i n the quadrulus ( f i g . 3 ) .  An a d d i t i o n a l  g u l l e t k i n e t y o c c u r s a t the j u n c t u r e between the r i g h t v e s t i b u l a r w a l l and t h e g u l l e t .  This short kinety i s  c a l l e d the e n d o r a l membrane. Stomatogenesis i n I__ t e t r a u r e l i a has been d e s c r i b e d by Roque (1956),  Kaneda and Hanson (197*0, and Jones  (1976).  F i g u r e 4 i s an o u t l i n e o f stomatogenesis as p r e s e n t e d by these a u t h o r s .  The f o l l o w i n g account o f stomatogenesis  i s given by Jones  (1976).  In s i l v e r - i m p r e g n a t e d m a t e r i a l the o r a l anlage appears at age 0.75 c e l l c y c l e s above the e n d o r a l k i n e t y on the right vestibular wall (fig.4).  At t h i s time the anlage  c o n s i s t s o f only a few i r r e g u l a r l y spaced a r g e n t o p h i l i c loci.  Up t o age 0.84 c e l l c y c l e s a d d i t i o n a l l o c i appear  i n the anlage and the e n d o r a l k i n e t y i s r e s o r b e d .  The  anlage e v e n t u a l l y becomes o r g a n i z e d i n t o t h r e e , and l a t e r six, kineties (fig.4).  By age O.85 c e l l c y c l e s t h e anlage  has i n v a g i n a t e d and taken a p o s i t i o n t o the r i g h t o f the vestibule.  The anlage now l o o k s l i k e an i n v e r t e d "J" when  viewed from above ( p l a t e 1 ) .  The number o f k i n e t i e s i n  the anlage doubles a f t e r age O.85 c e l l c y c l e s and the anlage s e p a r a t e s from the a n t e r i o r end o f the v e s t i b u l e  (plate 1).  8  It  then moves p o s t e r i o r l y t o occupy a p o s i t i o n under the  opisthe's vestibule  (plate  l).  A d d i t i o n a l d e t a i l s o f stomatogenesis are g i v e n by Jones (1976).  The e n d o r a l k i n e t y ,  which i s r e s o r b e d d u r i n g  stomatogenesis, reappears on the r i g h t v e s t i b u l a r w a l l of both p r o t e r and o p i s t h e p r i o r to s e p a r a t i o n of the daughter cells.  In a d d i t i o n , the anlage has been shown to be p r e s e n t  throughout the c e l l c y c l e  ( J o n e s , 1976).  n a t i o n of i n t e r f i s s i o n c e l l s r e v e a l s  P r o t a r g o l impreg-  b a s a l bodies l o c a t e d  beneath the c e l l s u r f a c e i n the r e g i o n of the r i g h t bular wall.  vesti-  At age 0.75 c e l l c y c l e s these l o c i move to  c e l l s u r f a c e where they are s u s c e p t i b l e to silver-impregnation.  the  Chatton-Lwoff  The new g u l l e t anlage a c t u a l l y  forms  around age O.87 c e l l c y c l e s a t which time two new anlagen form, one d e s t i n e d f o r the o p i s t h e and the other f o r proter  the  (fig.4).  The complexity o f the Paramecium c e l l and i t s morphog e n e s i s should be e v i d e n t from the f o r e g o i n g d e s c r i p t i o n s . It  i s hoped, however,  t h a t i n s p i t e o f the  specializations  which a l l o w Paramecium to e x i s t as a f r e e - l i v i n g  single-  c e l l e d organism t h e r e are fundamental developmental p r o c e s s e s which paramecia share with o t h e r e u k a r y o t i c c e l l s .  These  p r o c e s s e s may i n c l u d e c e l l growth, f o r m a t i o n and c o n s t r i c t i o n o f the f i s s i o n - f u r r o w , and deployment o f c e l l such as b a s a l b o d i e s .  In a d d i t i o n , i t  organelles  i s p o s s i b l e that  at  9  the m o l e c u l a r l e v e l the p r o c e s s e s g o v e r n i n g development i n Paramecium w i l l prove to be common t o c y t o d i f f e r e n t i a t i o n i n many c e l l types.  10  MATERIALS AND METHODS:  1.  CULTURE TECHNIQUE Paramecium t e t r a u r e l i a s t o c k 5 1  was grown i n  g r a s s medium (pH 6 . 8 ) i n n o c u l a t e d w i t h K l e b s i e l l a aerogenes as d e s c r i b e d by Sonneborn 2.  (1970).  SYNCHRONIZATION OF CULTURES P o p u l a t i o n s o f synchronous  c e l l s were o b t a i n e d  by manual s e l e c t i o n o f d i v i d i n g c e l l s from log-phase cultures. 3-  TEMPERATURE - SHOCK EXPERIMENTS The procedure f o r temperature-shock was m o d i f i e d from Natchway and Cameron Individual  experiments  (1967).  c e l l s from a synchronous p o p u l a t i o n were  i s o l a t e d i n s m a l l drops o f c u l t u r e plastic petri-dish  (#3001).  f l u i d i n a Falcon  The drops c o n t a i n i n g the  c e l l s were then covered with a l a y e r o f m i n e r a l o i l . To heat-shock the c e l l s , the p e t r i - d i s h was submerged for  30 minutes i n a water bath s e t a t 3 4 . 5 ° C . The median c e l l c y c l e  l e n g t h was determined by  11  3.  TEMPERATURE - SHOCK EXPERIMENTS  (Cont'd.)  t a b u l a t i n g ( e v e r y 10 minutes) the number o f c e l l s i n the  p o p u l a t i o n t h a t had completed d i v i s i o n .  The  cumulative percentage o f d i v i d e d c e l l s was p l o t t e d a g a i n s t time on p r o b a b i l i t y paper.  The i n t e r s e c t o f a  s t r a i g h t l i n e drawn through the p o i n t s a t the 5°^ l e v e l gave the median c e l l c y c l e l e n g t h .  Natchway and  Cameron (1967) p r e s e n t a d e s c r i p t i o n o f t h i s t e c h n i q u e and a d i s c u s s i o n o f i t s v a l i d i t y . 4.  CHATT0N-LW0FF SILVER-IMPREGNATION. S i l v e r - i m p r e g n a t i o n was performed as o u t l i n e d by F r a n k e l and Heckmann (1968).  5.  IDENTIFICATION OF CELL CYCLE STAGES The i d e n t i f i c a t i o n o f c e l l  c y c l e stages i n s i l v e r -  impregnated m a t e r i a l was based on photographs and f i g u r e s p r e s e n t e d by Kaneda and Hanson (1974) and Jones (1976).  The age o f c e l l s was l i s t e d as a  decimal f r a c t i o n o f the c e l l c y c l e l e n g t h .  (An age  O.75 c e l l was t h r e e - q u a r t e r s o f the way through the cell  cycle.)  12  6.  MUTAGENESIS AND MUTANT STOCKS The  temperature-sensitive  (ts) morphologically  abnormal v a r i a n t s used i n t h i s study were k i n d l y s u p p l i e d by Adele Hunter and E r i c P e t e r s o n .  These  v a r i a n t s were p a r t o f a group o f s i x t y m o r p h o l o g i c a l l y abnormal v a r i a n t s found  among 1 2 , 0 0 0 s e p a r a t e  i s o l a t e d a f t e r mutagenesis. i s given i n Peterson  clones  The mutagenesis procedure  and Berger  (1976).  v a r i a n t s were t e m p e r a t u r e - s e n s i t i v e .  A l l 60  The r e s t r i c t i v e  and p e r m i s s i v e temperatures were 3 4 . 5 ° C and 17°C respectively. 7.  ANALYSIS OF MUTANT PHENOTYPES To examine the m o r p h o l o g i c a l  abnormalities of  v a r i a n t s , a log-phase p e t r i - d i s h c u l t u r e o f v a r i a n t c e l l s was h e l d a t 3 4 . 5 ° C f o r 20 hours. then f i x e d and s i l v e r - i m p r e g n a t e d .  The c e l l s were  C l a s s i f i c a t i o n of  the a b n o r m a l i t i e s i n the s i l v e r - i m p r e g n a t e d c e l l s was achieved by s e p a r a t i n g the Paramecium c o r t e x i n seven components. found  A number o f d i s t i n c t a b n o r m a l i t i e s were  f o r each o f these components.  Table l a l i s t s  13  7.  ANALYSIS OF MUTANT PHENOTYPES - (Cont'd.) the  seven components of the  and  the  abnormal-  i t i e s noted i n each.  Samples o f v a r i a n t c e l l s were  examined with r e s p e c t  to these seven  components and  cortical  the number of v a r i a n t c e l l s with each  o f the a b n o r m a l i t i e s 8.  cortex  l i s t e d i n t a b l e l a were noted.  SELECTION OF VARIANTS FOR  STUDY  T h i r t y - e i g h t v a r i a n t s were examined by procedure g i v e n above. f o r f u r t h e r study.  Ten v a r i a n t s were s e l e c t e d  These i n c l u d e d  six variants  a defect  i n fission-furrow constriction  2, k, 8,  23,  fission-zone  62 and  63),  two  The  cells  c o r t i c a l abnormalities  with  (variants  v a r i a n t s with a  f o r m a t i o n ( v a r i a n t s 29 and  v a r i a n t s which produce small 26).  the  30),  defect and  ( v a r i a n t s 21  two and  p r e s e n t i n these  ten v a r i a n t s are l i s t e d i n t a b l e l b and d e s c r i p t i o n of t h e i r phenotypes i s g i v e n  a  general i n table l c .  14  TABLE I a C o r t i c a l Abnormalities Cortical Component Somatic Cortex  Gullet  Type  Found i n M o r p h o l o g i c a l  Variants  Description  Normal  K i n e t i e s and b a s a l b o d i e s a r e evenly spaced ( p l a t e 2 ) .  Abnormal, type 1  An o c c a s i o n a l b a s a l body or t r i c h o c y s t i s not a l i g n e d w i t h i n a kinety (plate 2).  Abnormal, type 2  Many b a s a l bodies and t r i c h o c y s t s are n o t a l i g n e d w i t h i n k i n e t i e s , p a r t s o f the c o r t e x may have no k i n e t i e s o r k i n e t i e s may be wavy o r bent a t odd angles (plate 2).  Abnormal, type 3  K i n e t i e s are h i g h l y disorganized, b a s a l b o d i e s are s c a t t e r e d i n a more or l e s s random manner (plate 2).  Abnormal, extra  More than the u s u a l number o f a r g e n t o p h i l i e bases are found on the c o r t e x (plate 2).  Normal  See f i g u r e 3.  Abnormal, type 1  Loss o r d i s o r g a n i z a t i o n o f the innermost p a r t o f the g u l l e t , the d i s o r g a n i z e d r e g i o n i s not more e x t e n s i v e than one-quarter o f the gullet length ( p l a t e 3).  Abnormal type 2  Loss or d i s o r g a n i z a t i o n o f the innermost one-quarter t o o n e - t h i r d o f the g u l l e t ( p l a t e 3).  Abnormal type 3  Loss o r d i s o r g a n i z a t i o n o f more than o n e - t h i r d o f the g u l l e t ( p l a t e 3).  Abnormal, everted  G u l l e t k i n e t i e s are on the c e l l s u r f a c e i n t e r c a l a t e d between somatic k i n e t i e s ( p l a t e 1 ) .  15  TABLE I a  Cortical Component  Type  (con't.)  Description  Gullet  Abnormal, astomatous  No g u l l e t p r e s e n t  Cortical Pattern  Normal  The e n t i r e c e l l s u r f a c e i s covered by c o r t i c a l u n i t s .  Abnormal, patchy  Patches on the c o r t e x are d e v o i d of c o r t i c a l organelles (plates 5 and 6 ) .  Normal  The c y t o p r o c t s i l v e r - i m p r e g n a t e s as a t h i n , wavy b l a c k l i n e about 20 um l o n g ( p l a t e 4 ) .  Abnormal  Abnormalities include short, very wide ( d i s t e n d e d ) , and d i s r u p t e d cytoprocts (plate 4).  Normal  See f i g u r e 1 .  Abnormal, large  C e l l s are abnormally l o n g and wide ( p l a t e s 5 and 6 ) .  Abnormal, small  C e l l s are u n u s u a l l y narrow ( p l a t e 5 ) •  Abnormal, swollen  C e l l s are wide with c o r t i c a l b l i s t e r s or blebs ( p l a t e 6 ) .  Abnormal, round  C e l l s are e q u a l l y wide as l o n g (plate 6 ) .  Abnormal, truncated  C e l l s l a c k e i t h e r an a n t e r i o r o r posterior c e l l region (plate 5)•  Abnormal, division arrest.  C e l l s cannot complete c o n s t r i c t i o n o f the f i s s i o n - f u r r o w ( p l a t e 7 ) «  Cytoproct  Cell Shape  (plate 5 ) '  s h o r t and  16  TABLE l a ( c o n ' t . )  Cortical Component  Cell shape  Contractile vacuole pores  Type  Description  Abnormal, monster  a large, irregularly-shaped c e l l which i s t h e r e s u l t o f c o n t i n u e d c e l l growth i n the absence o f c e l l d i v i s i o n ( p l a t e 7) •  Abnormal, irregular  C e l l has an i r r e g u l a r ( p l a t e s 5 and 6).  Normal  C e l l s have two ( r a r e l y three o r more) pores which are about 1 um i n diameter ( f i g u r e 1 ) .  Abnormal, extra  C e l l s have t h r e e or more c o n t r a c t i l e vacuole pores 6 and 7)•  Abnormal, plate-like  The pores are v e r y broad, with an i r r e g u l a r o u t l i n e ( p l a t e 6 and f i g u r e 5)  outline  (plates  1 7  TABLE l b C o r t i c a l Abnormalities Cortical Character  i n Selected Variant Lines  3 -  V a r i a n t Number CELL SHAPE Normal Normal d i v i d e r Division arrest Monster Irregular Truncation Large Swollen CELL SIZE 0 - 80pm 80-lOOpm 100-120;am >120pm CORTEX Normal Type 1 Type 2 Type 3 Extra mtr'l.  % o f sampled 8 8 0  1 0-  2 6  2 9  30  62  63  92  4 6 4  30  50  4 4 2 6 2 8 1 8  7 7 2 1 1 4  2  3 1 1 2  1 4 2 5 4 8 8 1 4  6-  4  1  -  -  1 6  32  1 1 4  2 4  -  3  -• 6 2 4  1  1 9  7 1  4 4 5 6  1 6 2  1 8  1 4 1 4 1 8 2  1  -  -  29 50 2 6 6  63  1  50  1 4 8 6  4 8  52  15 8 5  90  7 4 2 6  4 8  1 0 mm  2 4 1 1  8 0 6 0  3 3  —  36 1 6 1 4  32 -  2  -.  -  . -  -  -  _ 3 8 7 1 0  5 5 4 5  1 4 8 9 6  9  1 9  _  mm  4 6 4 1 1 2  -  2 2 0 4 2 4  1  —•  —  1 9 1 8  -  -  1 8 8  •—  CORTICAL PATTERN Normal 3 5 Patchy 6 5 CYTOPROCT Normal 7 6 Abnormal 2 2 Absent 2 GULLET Normal Type 1 Type 2 Type 3 On s u r f a c e Astomatous  23  6  3 4 2 3 7 1 8  character  2 1  1  1 4 8 4 1 1  c e l l s with g i v e n  52  3 1 1 3  8 8 0 4  4 7 4 4  1 8  8 2 2 0  30 . 70  9 9 1  25  5 6 4 1  7 9 2 1  3  15 25 13 39 —  8  -  3 3 3 3 9 1 2  9 4  *  51  3 ^ 3  —  8 2 0 1 6  27  —  7 5  6 1 3 7 2  23 3 3 9  27 1 7  *  18  TABLE l b - (Cont'd.) C o r t i c a l Abnormalities Cortical " Character  i n Selected Variant  3  % o f sampled  cells  with g i v e n  character  21  2;  26  29  30  62  63  CONTRACTILE VACUOLE PORES Normal number 85 88 Supernumerary Ik 12 Plate-like 1 -  62 3^ 2  90 10  26  7k  61 39  66 33 1  *  V a r i a n t Number  8  v.  Lines  a.  A f u l l d e s c r i p t i o n o f thes table l a .  *..  No v a l u e s a v a i l a b l e .  b.  Sample s i z e s : v a r i a n t s 8 , , and 62 - 100 c e l l s ;. v a r i a n t s 2 6 - 8 0 c e l l s ; remaining v a r i a n t s - 50 c e l l s .  c h a r a c t e r s may be found i n  19  TABLE l c t s V a r i a n t Stocks Used i n the Study Stock Number  Fission-Zone Formation  Fission-Furrow Formation  Normal  Partial, arrested  Normal  Partial, arrested  Forms v e r y s h o r t d i v i d i n g c e l l s (about 13Cvum long)-division arrest occurs d u r i n g the f i r s t c e l l c y c l e a t 34.5 c c e l l behaviour i s abnormal a t 34.5 C-the c e l l s f r e q u e n t l y swim backwards f o r s h o r t periods.  8  Normal  Partial, arrested  C e l l s w e l l i n g and death o c c u r s a t 34-5 C - g u l l e t d e f e c t s and astomatous c e l l s were commongullets occasionally have supernumerary kineties.  23  Normal  Partial, arrested  C e l l s w e l l i n g and death occurs a t 34.5 C - c e l l s only r a r e l y a r e a r r e s t e d i n fission-supernumerary g u l l e t k i n e t i e s are common.  62  Normal  Partial, arrested  G u l l e t abnormality i s severe with astomatous c e l l s common-has supernumerary c o n t r a c t i l e v a c u o l e pores.  63  Normal  Partial, arrested  Penetrance o f the misd i v i d e r t r a i t ranged from 50 to 95$ i n d i f f e r e n t experiments. Chains o f 3 and 4 c e l l s  Notes  20  TABLE l c - (Cont'd.) t s V a r i a n t Stocks Used i n the Study Stock Number  Fission-Zone Formation  Fission-Furrow Formation  63  Normal  Partial, arrested  Cont'd. as w e l l as h e t e r o p o l a r d o u b l e t s are commong u l l e t and c o r t i c a l abnormalities are severe.  29  Does n o t occur o r partial  Does not occur or partial  Cells arrested prior to f i s s i o n - z o n e format i o n a r e rounded, and are astomatous i n e i t h e r the p r o t e r or opisthe.  30  Does n o t occur or partial  Does not occur o r partial  Same o r s i m i l a r to stock 2 9 .  21  Normal  Normal  C e l l s are small with severe c o r t i c a l abnormality.  26  Normal  Normal  C e l l s are s m a l l w i t h severe c o r t i c a l and g u l l e t abnormalityc e l l s have a t r a n s v e r s e gap a c r o s s g u l l e t kineties (plate l ) .  21  RESULTS;  SECTION A - GENETICS  From a s e r i e s o f 60 m o r p h o l o g i c a l v a r i a n t s following nitrosoguanidine selected f o r genetic  obtained  mutagenesis, t e n v a r i a n t s were  analysis.  These i n c l u d e d  six variants  w i t h a d e f e c t i n f i s s i o n - f u r r o w c o n s t r i c t i o n ( v a r i a n t s 2, 4, 8, 23, 62, and 63), two v a r i a n t s with a d e f e c t i n fission-zone  f o r m a t i o n ( v a r i a n t s 29 and 3°)> and two  v a r i a n t s t h a t produce s m a l l c e l l s Two s e r i e s o f g e n e t i c The  ( v a r i a n t s 21 and 26).  crosses  were made i n the study.  f i r s t was designed t o determine whether the phenotype  of each v a r i a n t was due t o a s i n g l e gene and the second was designed t o determine the a l l e l i c s e l e c t e d mutant  relationship of  lines.  In the f i r s t  series of genetic  crosses,  c e l l s o f each  v a r i a n t were mated with w i l d - t y p e c e l l s c a r r y i n g the b e h a v i o r a l marker gene, pawn (pw).  The heterozygous  progeny o f t h i s cross were induced t o undergo autogamy, which r e s u l t s i n homozygosis a t a l l g e n e t i c 1950).  The exautogamous c e l l  generation. and  loci  (Sonneborn,  l i n e s c o n s t i t u t e d the F^  These l i n e s were Scored f o r the m o r p h o l o g i c a l  pawn phenotypes.  The scored  r e s u l t s were compared with  those that would have been expected on the b a s i s o f the f o l l o w i n g hypotheses: the  (a) a s i n g l e r e c e s s i v e  a b n o r m a l i t y , (b) two r e c e s s i v e  gene caused  genes a c t e d  i n concert  22  to  cause the abnormality, and ( c ) two r e c e s s i v e genes  were present i n the v a r i a n t and each caused a m o r p h o l o g i c a l abnormality.  The f r a c t i o n s o f F  classes morphological  2  c e l l s i n the phenotypic  (m~/m~; pw /pw ), m o r p h o l o g i c a l +  +  pawn (m~/m~; pw~/pw~), and pawn (m /m ; pw~/pw~) which +  were expected to  +  on the b a s i s o f these hypotheses  were 1 t o 1  1, 3 t o 3 t o 1, and 1 t o 1 t o 3 r e s p e c t i v e l y .  The  number o f w i l d - t y p e c e l l s was i g n o r e d as t h i s c l a s s may c o n t a i n heterozygous through autogamy). other phenotypic  cells  (those which had not gone  The observed numbers o f c e l l s i n t h e  c l a s s e s were compared by means o f t h e  G - t e s t ( S o k a l and R o h l f , 1969) with the numbers expected. • In most cases the r e s u l t s i n d i c a t e d t h a t a s i n g l e r e c e s s i v e gene caused  the m o r p h o l o g i c a l a b n o r m a l i t y  s e v e r a l cases  ( t a b l e 2). In  ( v a r i a n t s 26, 3°» n d 63) the mating o f a  v a r i a n t c e l l s with pawn-marked w i l d - t y p e c e l l s was done twice.  In two o f these cases ( v a r i a n t s 26 and 30) t h e  experimental r e s u l t s from one o f the g e n e t i c c r o s s e s d i d n o t s a t i s f y the h y p o t h e s i s t h a t a s i n g l e gene was responsi b l e f o r the m o r p h o l o g i c a l a b n o r m a l i t y .  When the matings  w i t h these two v a r i a n t s were r e p e a t e d , however, the r e s u l t s i n d i c a t e d t h a t a s i n g l e gene was r e s p o n s i b l e f o r the abnormality  ( t a b l e 2).  The a b e r r a n t r e s u l t s i n the f i r s t  c r o s s e s made u s i n g these l i n e s a p p a r e n t l y r e s u l t e d  from  i n c o n s i s t e n c i e s i n s c o r i n g the mutant phenotypes o r from  TABLE 2 EXAUTOGAMOUS SEGREGATION OF t s HETEROZYGOTES F Variant Number 2 4 8 21 23 26(1) 26(2) 2 9  30(1) 30(2) 62 63(1) 63(2)  2 PHENOTYPE  Mutant tt  Pawnmm+  dc5 dc4 dc3 sm3 dc6 sm2(l) sm2(2) dfzl dfz2(l) dfz2(2) dc2 dc2 (l) dc2 (2)  1 4  u  a  a  23 54 4 0  -  37 6 1  38 31 30 38 30 2 8  12 20 49 4 2  -  27 62 57 36 66 58 32 30  VALUE O F "G"  Pawn+ mm+  pw-  Dead ** pw+ ?+  4 6  47  1  8  27 38 69  4 6  —  —  4  80 79 44 29 26 60 35 27  49 88 73  4 0  —  —  74 47 . 83 .  2  4  4 0  —  2  3 10 2  -  -  2  4 8  —  52 70 50  3  3 2 1 —  2  1  *  a d j u s t e d g u s i n g Yate's c o r r e c t i o n .  G  v a l u e s a r e t o be compared t o X  2  73 8 —  _  10 23 2 4  34 3 7 2  R a t i o Tested lilil 3: 1: 3 1: 3: 1 28.13* 1.06 2 . 9 1 4 . 3 . 0 3 1 - 3 2 . 9  6 8 * 2 9 * 6  1 8 . 4 1 9 - 7 8 3 9 . 5 5  83.85 30.99 1 8 . 4 4  36.70 4.04 ' 5 9 . 3 6 3 1 . 7 2 1 0 6 . 2 0  . 7 9 2 2 . 3 9 *  6.00*  55-04  •3 9 . 1 9  23.41  (.05) 2= 5 - 9 9 1 and X  2  2 1 . 9 3  75.32 28.63 3 9 - 3 7 3 7 . 3 4  32.42 1 1 8 . 9 8  '73.23 2 1 . 5 9 1 9 . 8 2  2.09 3 7 . 9 6 2 9 . 1 8  21.13  (.05) 1 = 3 . 8 4 1  + - l i n e s which d i e d b e f o r e b e i n g t e s t e d f o r phenotype **-  t h e pw~ and pw  +  l i n e s were t e s t e d f o r t h i s phenotype before they  died.  ++- t h e b r a c k e t e d numbers r e f e r t o experiment numbers where any one mutant was c r o s s e d t o pawn c e l l s i n more than one experiment.  24 incomplete  penetrance.  The r e s u l t s from c r o s s e s o f s e v e r a l o f the v a r i a n t s w i t h pawn-marked c e l l s d i d not conform t o what was expected i f a s i n g l e r e c e s s i v e gene.caused t h e abnormality. These l i n e s were: (a)  V a r i a n t 2 - The r e s u l t s d i d not agree with any o f the hypotheses  tested.  The number o f m~/m~  and m /m  c e l l s were e q u a l ,  however, i n both the pw~/pw~ and pw /pw +  T h i s suggested  classes.  +  t h a t a s i n g l e gene was r e s p o n s i b l e  f o r the a b n o r m a l i t y b u t t h a t many o f the pw~/pw~ l i n e s d i e d b e f o r e they were scored. (b)  V a r i a n t 21 - The number o f mutant c e l l s may have been underestimated  due t o d i f f i c u l t y i n s c o r i n g  the phenotype o f t h i s l i n e . Although  the c e l l s  were s m a l l , they were o f normal shape and t h e r e f o r e abnormal clones may have been (c)  overlooked.  V a r i a n t 62 - The r e s u l t s d i f f e r only s l i g h t l y from what was expected the  i f a s i n g l e gene  caused  abnormality.  The nine mutant genes were g i v e n names r e l a t e d t o t h e i r phenotypes. These names are l i s t e d i n t a b l e 2. Mutants with d e f e c t s i n furrow c o n s t r i c t i o n were c a l l e d d e f e c t i v e c o n s t r i c t i o n (dc) mutants, those w i t h d e f e c t i v e f i s s i o n zone f o r m a t i o n were c a l l e d f i s s i o n zone ( d f z ) mutants, and those  producing  s m a l l c e l l s i z e were c a l l e d s m a l l (sm) mutants. Each gene  25  was numbered (sml, sm2, superscript  e t c . ) and each a l l e l e was g i v e n a  (dc2 , dc2 , e t c . ) . a  b  The second s e r i e s o f g e n e t i c c r o s s e s were made to determine i f a l l e l i s m o c c u r r e d among any o f the t e n genes examined. To do t h i s , c e l l s o f a mutant l i n e , which had been marked with the pawn gene, were c r o s s e d w i t h c e l l s o f every o t h e r mutant  l i n e . The heterozygous progeny were  t e s t e d f o r the presence o f m o r p h o l o g i c a l a b n o r m a l i t y . (Permanent disappearance o f the pawn t r a i t ensured t h a t r e c i p r o c a l f e r t i l i z a t i o n had taken p l a c e . ) I f the h e t e r o zygous progeny were m o r p h o l o g i c a l l y abnormal then the two parent l i n e s were assumed t o be a l l e l i c .  The complimentation  assay ( t a b l e 3) r e v e a l e d only one a l l e l i c p a i r : dc2  b  dc2  a  and  ( v a r i a n t s 63 and 62). Four mutants were s e l e c t e d f o r f u r t h e r study. These  i n c l u d e d t h r e e genes (dc2 , dc4, and dfz2) which a f f e c t a  c e l l d i v i s i o n and one (sm2)  which a f f e c t s c e l l  growth.  These mutants p r o v i d e the b a s i c m a t e r i a l f o r an a n a l y s i s o f c e l l growth and d i v i s i o n i n Paramecium.  In a d d i t i o n ,  the r e d u c t i o n i n c e l l s i z e produced by the sm2  gene makes  p o s s i b l e an a n a l y s i s o f the e f f e c t s o f a change i n c e l l s i z e on morphogenesis. Morphometric a n a l y s i s was  the main technique used  i n examining the phenotypes o f the mutant  cells.  Two  26  TABLE 3 COMPLIMENTATION MATRIX  dc2 dc2  a  dc2  b  a  dc2  b  dc3  dc4  dc5  dc6  dfzl  +  +  +  +  +  *  +  +  +  *  * +  dc3 dc4 dc5 dc6 dfzl dfz2 sm2  sm2  sm3  +  +  +  +  a  +  a  *  +  +  +  a  a'  +  +  +  a  +  +  +  +  a  +  +  +  a  +  +  +  +  + +  sm3  a - repeatedly  dfz2  refused  t o mate  * - mating t h a t was not t e s t e d  27  types of morphometric data were c o l l e c t e d :  data on  of known age and d a t a on p o p u l a t i o n s of c e l l s age. The  cells  o f unknown  f i r s t type o f morphometric d a t a w i l l be used t o  examine the age-dependent changes i n s i z e and morphology o f w i l d - t y p e and mutant c e l l s .  The second type o f data  w i l l be used to examine size-dependent morphogenesis and s u r f a c e  structure.  aspects of c e l l  28  SECTION B;  MORPHOMETRIC ANALYSIS OF CELL  INTRODUCTION:  DIVISION  ' '  Kaneda and Hanson  (197*0 s e p a r a t e d the Paramecium  c e l l cycle i n t o three periods.  The f i r s t p e r i o d  (post-  f i s s i o n p e r i o d ) spans ages 0 t o 0.1 c e l l c y c l e s and i s a time o f r a p i d c e l l growth.  The second, o r morpho-  s t a t i c , p e r i o d extends up t o age 0-75 c e l l  cycles.  D u r i n g t h i s time t h e c e l l does n o t change i n l e n g t h . The t h i r d , or morphogenetic, p e r i o d begins w i t h the appearance o f the o r a l anlage a t age 0.75 and culminates i n the c o n s t r i c t i o n o f the f i s s i o n furrow.  During t h i s  p e r i o d the c e l l undergoes a s e r i e s o f s i z e and shape changes whose o r i g i n s and f u n c t i o n s a r e p o o r l y understood.  The i s o l a t i o n o f mutants d e f e c t i v e i n c e l l  d i v i s i o n and c e l l growth, however, p r o v i d e s a means o f a s s e s s i n g the importance o f these s i z e and shape changes i n normal c e l l morphogenesis.  I n o r d e r t o use the  mutants f o r t h i s type o f a n a l y s i s , i t was n e c e s s a r y t o compare  t h e s i z e and shape changes d u r i n g the morpho-  g e n e t i c p e r i o d i n w i l d - t y p e c e l l s w i t h the c o r r e s p o n d i n g changes i n mutant c e l l s .  Four mutants were used, one  with d e f e c t i v e f i s s i o n zone f o r m a t i o n ( d f z 2 ) , two with d e f e c t i v e c o n s t r i c t i o n o f the f i s s i o n furrow ( d c 2 and a  dc*0, and one which produced s m a l l c e l l s  (sm2).  29  EXPERIMENTAL DESIGN: The  purpose o f the morphometric study o f w i l d  type and mutant c e l l s was t o c o r r e l a t e d i f f e r e n c e s i n the temporal p a t t e r n  o f s i z e and shape changes between  these two types o f c e l l s with the d e f e c t s i n c e l l d i v i s i o n or development i n the mutants. mutant c e l l s express t h e i r d e f e c t s  S i n c e the  only a t the r e s t r i c -  t i v e temperature, a l l experiments were done a t 34.5°C. To p r o v i d e a s u i t a b l e c o n t r o l , w i l d - t y p e c e l l s were a l s o c u l t u r e d a t 34.'5°C. was  (Kaneda and Hanson's study  done on c e l l s grown a t room temperature and t h e r e -  f o r e does not c o n s t i t u t e a s u i t a b l e c o n t r o l . )  Two  types of experiments were done: (a)  EXPERIMENTS WITH SYNCHRONOUS CULTURES Synchronous c e l l samples were p l a c e d i n 35 x 100 m.m. p l a s t i c p e t r i d i s h e s  containing  about 2 ml. o f c u l t u r e f l u i d w i t h b a c t e r i a .  The  dishes  were immediately immersed i n a 34.5°C water  bath.  The c u l t u r e was p e r i o d i c a l l y examined u n t i l  d i v i d i n g c e l l s were noted, and the c u l t u r e was then f i x e d and (b)  silver-impregnated.  EXPERIMENTS WITH ASYNCHRONOUS CULTURES Asynchronous log-phase c e l l samples were p l a c e d i n culture flasks containing  culture f l u i d  with  b a c t e r i a such t h a t t h e depth o f f l u i d i n t h e f l a s k  30  EXPERIMENTAL DESIGN; - (Cont'd.) (b)  EXPERIMENTS WITH ASYNCHRONOUS CULTURES - (Cont'd.) was no. g r e a t e r than 1 . 5 cm.  A synchronous  sample  of c e l l s was removed from the asynchronous c u l t u r e and prepared as i n d i c a t e d above. the  Both  asynchronous and synchronous c u l t u r e s were  then heated t o 34.5°C i n a water bath.  When  d i v i d i n g c e l l s were seen i n the synchronous the  sample,  asynchronous c u l t u r e was f i x e d and s i l v e r -  • impregnated. The r e s u l t s r e p o r t e d below f o r sm2 and d c 2  a  cells  were based e n t i r e l y , and f o r dfz2 and dc4 c e l l s partially,  on synchronous m a t e r i a l , w h i l e the  r e s u l t s r e p o r t e d f o r w i l d - t y p e c e l l s were based e n t i r e l y on asychronous m a t e r i a l . MORPHOMETRIC ANALYSIS OF SILVER-IMPREGNATED CELLS; The age"*" o f s i l v e r - i m p r e g n a t e d c e l l s was determined by comparing t h e morphology  of the c e l l with data  p r e s e n t e d by Kaneda and Hanson (1974) and Jones (1976), (see  plate 8).  A v a r i e t y o f measurements, i n d i c a t e d i n  f i g u r e 6, were then made on each c e l l  o f known age.  These measurements were made with an o c u l a r micrometer and r e p r e s e n t the l i n e a r l e n g t h o f the c e l l the  cell.  or p a r t s o f  As the c e l l s a r e not f l a t , however, l i n e a r  l e n g t h measurements do n o t r e p r e s e n t the a c t u a l l e n g t h ages a r e i n d i c a t e d as a d e c i m a l f r a c t i o n o f the l e n g t h o f the c e l l c y c l e .  31  EXPERIMENTAL DESIGN: - (Cont'd.) MORPHOMETRIC ANALYSIS OF SILVER-IMPREGNATED CELLS: -  (Cont'd.)  o f the c e l l s u r f a c e .  The a c t u a l , or contour,  surface  l e n g t h s were obtained by c o n s t r u c t i n g o u t l i n e drawings o f d i v i d i n g c e l l s u s i n g the l i n e a r l e n g t h measurements. Separate o u t l i n e drawings were made f o r c e l l s of ages 0.94,  0.95.  0.96  To o b t a i n contour  - 0.97.  then was  - 0.99  shaped t o conform to the  s t r a i g h t e n e d and measured.  and u s i n g the o u t l i n e drawings, two made:  c e l l cycles.  l e n g t h s from the c e l l o u t l i n e s , a  p i e c e of t h i n wire was and  and 0.98  contour  In c o n s t r u c t i n g  assumptions were -  t h a t the r e f e r e n c e p o i n t s used .in the measure-  ments ( f o r example the CVPs) d i d not m i g r a t e or move d u r i n g c e l l d i v i s i o n and  t h a t the  c o n s t r i c t s at a constant  r a t e t h a t i s equal i n both  mutant and w i l d - t y p e  cells.  The  fission  furrow  l a t t e r assumption i s  a t l e a s t q u a l i t a t i v e l y t r u e based on o b s e r v a t i o n s l i v e and  fixed material.  Observations presented Unless  of  on contour  l e n g t h changes w i l l  be  s e p a r a t e l y from l i n e a r l e n g t h changes.  s t a t e d otherwise,  l i n e a r length.  a l l measurements r e f e r t o  32  2.  THE  MORPHOGENETIC PERIOD IN WILD-TYPE CELLS; The  in  data c o l l e c t e d on w i l d - t y p e c e l l s are  t a b l e 4.  against  Morphometric parameters have been p l o t t e d  c e l l age  Figure  7a  i n f i g u r e s 7 through  shows the  type c e l l s t h a t 34.5°C.  listed  12.  changes i n length  occurred d u r i n g one  of  wild-  c e l l cycle  at  Based on t h i s f i g u r e , the morphogenetic  p e r i o d was  s e p a r a t e d i n t o three phases:  (a)  a growth phase from age  0.75  t o O.83  (b)  a c o n t r a c t i o n phase from age  O.83  cell  cycles;  t o 0.95  cell  cycles; (c) . a f i s s i o n - f u r r o w of i.  phase from age  0.95  to  completion  cytokinesis. Growth Phase: Between ages 0.75 the  c e l l length  to 130  /am).  increased  of the  p o s t e r i o r s u t u r e (GP)  cell  by 7 um  cycles  (from  c e l l , with both and  Other p a r t s  change i n l e n g t h middle of the  O.83  123  Growth o c c u r r e d p r i m a r i l y i n  posterior part  increasing.  and  region of the  with the  CVP  the (fig.9b)  c e l l did  e x c e p t i o n of  a n t e r i o r h a l f of the  the  cell  not  the (CVA")  which decreased i n l e n g t h . During the  growth phase two  appeared ( p l a t e 8),  one  new  CVP's  a n t e r i o r to the  old  TABLE 4 AGE-DEPENDENT CHANGE IN WILD-TYPE CELL SIZE DIMENSION ( i n urn) WITH STANDARD ERRORS VARIABLE •75  CELL AGE  .8-.84  .87-.89  .9-.91  .92-.93  Length 123+3.0 130 2.5 124 2.2 124-1.1 121,2.4 Width 41.1.6 44.1.0 43,0.4 44T0.7 41+0.5 L/Wc 3.0^.15 3-1+.04 2.9 .06 2.9+.05 2.8-.06 CVA 43-0.6 24+1.1 25+0.7 24,1.1 24,1.0 CVA* 18.1.0 16,1.0 19+0.5 23*1.2 CVINT 40-1.1 39+1.4 38 1.2 41.1.3 CVP' ----19+1.0 19+1.0 17+1.8 18-0.9 CVP 24J1.7 25+1.4 27+1.5 24-0.9 23+1.3 56+2.1 54x1.0 • 53+1.0 GA 54jl.6 50+1.5 17+0.4 G 17+0.7 17+0.7 22T1.6 19+0.4 53+1.9 GP 52.1.3 57+1.4 49*1.4 51+0.4 34^2.4 #b.b. 365O.6 47+2.3 26^2.4 43J1.4 C V I N T / b . b . l . 6 - . 0 4 1.5-.05 1.2-.08 I.O-.06 O.9-.O3 PROTER LENGTH OPISTHE LENGTH PG OG PGP GAO N 5 8 7 a-see f i g u r e 6 f o r e x p l a n a t i o n o f a b b r e v i a t i o n s used b-in fractions ofa c e l l cycle c - l e n g t h t o width r a t i o ( a r b i t r a r y u n i t s ) d-the number o f b a s a l b o d i e s i n r e g i o n CVINT x  +  Z  ?  d  d  o  f  .94  •95  .96-.97  .98-.99  123+3-5 47J0.8 2.6-.10 24T2.2 19x1.1 37+2.2 20-1.1 23+1.6 49+2.7 27+1.6 48J2.3 49^2.2 O.8-.O3  126.2.9 42x0.5 3.0-.05 20T1.1 22rl.7 43- 1.2 20,0.9 21+0.5 44- 1.1  153+3.3 40- 1.2 3.9+.18 25.O.8 32,2.2 45x2.9 27^2.3 25+4.5 35-2.1  43Jo.7 47^2.4 0.9-.06  138J3.5 42Jo.6 3-5+.08 24Jo.8 25+1.1 44,1.7 25+0.5 20- 1.6 35-1-1 43+0.7 46.1.2 I.O-.03  45+1.0 41- 3-3 1.1-.04  65-1.9  71-2.2  79-1.9  59.1.0 16^0.4 13+0.4 7-1.5  67+1.9. 75+1.7 15+0.3 15+0.5 15+0.3 14-0.4 29-1.4 21- 1.8 8 e-the spacing between b a s a l bodies f-sample s i z e  3*  2.  THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS; i.  - (Cont'd.)  Growth Phase: - (Cont'd.) a n t e r i o r CVP and one a n t e r i o r t o the o l d p o s t e r i o r CVP. Kaneda and Hanson ( 1 9 7 ^ ) d e s c r i b e d a s i m i l a r growth phase and a l s o found t h a t growth was r e s t r i c t e d t o the ends o f the cell.  They s t a t e d t h a t the maximum l e n g t h  a t t a i n e d d u r i n g t h i s phase (ages 0.75 t o  0.89) was 132 pm a t age 0.89ii.  Contraction  Phase:  Between ages O.83 and 0.95 the c e l l l e n g t h decreased by 10 pm.  This  contraction  was accompanied by a change i n c e l l  shape,  which became f l a t t e n e d on the s i d e s  ( p l a t e 7).  The c o n t r a c t i o n o c c u r r e d i n two r e g i o n s o f the cell,  the p o s t e r i o r end (CVP) and the m i d - r e g i o n  of the a n t e r i o r h a l f o f the c e l l CVA' was t h e r e f o r e  (CVA').  Region  continuing a contraction  which began i n the growth phase. D u r i n g the c o n t r a c t i o n phase r e g i o n CVINT ( f i g . 6 ) began an expansion t h a t would continue i n t o the f i n a l  phase o f morphogenesis.  Near age 0 . 9 the f i s s i o n l i n e a c l e a r space devoid of c o r t i c a l  ( o r zone),  organelles,  35  2.  THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS; - (Cont'd.) ii.  Contraction  Phase; - (Cont'd.)  appeared o n ' e i t h e r (plate 8). and  s i d e o f the v e s t i b u l e  T h i s l i n e spread around the c e l l  was complete by age 0.93'  Between ages  0.93 and 0.95 t h e r e was an apparent  twisting  of the p o s t e r i o r h a l f o f the c e l l i n r e l a t i o n to the a n t e r i o r h a l f . .  c and d) t h a t  Note i n P l a t e 9 ( f i g s .  the k i n e t i e s bend a t the  f i s s i o n l i n e as i f the p o s t e r i o r r e g i o n has twisted  c l o c k w i s e i n r e l a t i o n t o the a n t e r i o r  region.  The t w i s t i n g was a l s o seen on the  v e n t r a l surface opisthe's  as the l e f t w a l l  v e s t i b u l e was a l i g n e d  o f the with the  r i g h t w a l l o f the p r o t e r ' s v e s t i b u l e  ( p l a t e 8,  fig.e). The  two separate v e s t i b u l e s i n the  daughter c e l l s are produced by the b i s e c t i o n of the o r i g i n a l v e s t i b u l e by the f i s s i o n and  subsequently the f i s s i o n furrow.  line  The  a n t e r i o r v e s t i b u l e remains a t t a c h e d t o the existing gullet.  The p o s t e r i o r  vestibule  subsequently a t t a c h e s t o the d e v e l o p i n g g u l l e t anlage.  E x t e n s i v e development o f the anlage  occurs a t t h i s time, as w i l l be d e s c r i b e d  below.  36  2.  THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS: - (Cont'd.) ii.  C o n t r a c t i o n Phase: - (Cont'd.) P r o l i f e r a t i o n o f b a s a l bodies  a l s o began  d u r i n g t h i s phase, the d e t a i l s o f which w i l l be presented  later.  The end o f the c o n t r a c t i o n  phase was marked by t h e appearance o f a cons t r i c t i o n i n the r e g i o n o f the f i s s i o n iii.  Fission-Furrow  line,  Phase:  With the c o n s t r i c t i o n o f the f i s s i o n furrow the c e l l underwent a r a p i d i n c r e a s e i n l e n g t h from 123 um (age 0.95) t o 154 pm (age 0.98).  T h i s growth o c c u r r e d p r i m a r i l y i n the  mid-regions o f the p r o t e r and o p i s t h e fig.10a;  CVP', fig.10b).  i n the p o s t e r i o r r e g i o n region  (CVINT).  Minor growth  (CVA', occurred  (CVP) and the c e l l mid-  ( I t should be noted here that  once c o n s t r i c t i o n begins the c e l l becomes h i g h l y curved  i n the furrow r e g i o n and t h e r e -  f o r e l i n e a r measurements do not a c c u r a t e l y gauge growth i n t h i s  area.)  A dramatic s h o r t e n i n g o f the a n t e r i o r and p o s t e r i o r sutures  (GA and GP, f i g s .  7b and 8a)  accompanies the i n c r e a s e i n c e l l l e n g t h . r e s u l t e d from m i g r a t i o n  This  o f the v e s t i b u l e s t o  c e n t r a l l o c a t i o n s on the p r o t e r and o p i s t h e .  37  2.  THE MORPHOGENETIC iii.  PERIOD IN WILD-TYPE CELLS: - (Cont'd.)  Fission-Furrow The  Phase: - (Cont'd.)  forward  vestibule  movement o f the p r o t e r ' s  ( b e g i n n i n g a t age 0.94) l e f t i n  i t s wake a p r o t e r p o s t e r i o r suture.  The  i n c r e a s e i n l e n g t h o f t h i s suture exceeded by 5 pm the decrease i n l e n g t h o f the a n t e r i o r s u t u r e , which i n d i c a t e d t h a t the p o s t e r i o r r e g i o n o f the p r o t e r was growing, a change a l s o seen on the d o r s a l s u r f a c e ( r e g i o n CVINT was i n c r e a s i n g ) . vestibule  M i g r a t i o n o f the o p i s t h e ' s  (also beginning  l e s s dramatic.  a t age 0.94) was  As a r e s u l t , a t f i s s i o n the  o p i s t h e had i t s v e s t i b u l e l o c a t e d i n the anterior third The  o f the c e l l  ( p l a t e 8,  fig.h).  o p i s t h e was t h e r e f o r e shaped r a t h e r d i f -  f e r e n t l y from the p r o t e r .  The former was  p o i n t e d a t the a n t e r i o r end and b l u n t a t the p o s t e r i o r while the l a t t e r was e l l i p s o i d a l (plate iv.  7).  C e l l Width: Width was f a i r l y  constant  growth and c o n t r a c t i o n phases. slight  d u r i n g the There was a  i n c r e a s e i n width j u s t before  of the f i s s i o n furrow ( f i g . 8 b ) .  formation  D u r i n g the  38  THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS; - (Cont'd.) iv.  C e l l Width: - (Cont'd.) f i s s i o n - f u r r o w phase the c e l l width decreased steadily age  v.  from 48 pm a t age 0.95 t o 36 pm a t  0.98.  B a s a l Body P r o l i f e r a t i o n : The number o f b a s a l bodies  between the  innermost CVPs on the d o r s a l s u r f a c e began t o i n c r e a s e a t age 0.82 ( f i g . 1 2 a ) .  Between ages  0.82 and 0.95 the number o f b a s a l bodies i n t h i s r e g i o n doubled (from 25 t o 50). age  0.95 the mean number o f b a s a l bodies i n  CVINT decreased by n i n e .  Although t h i s  decrease was not s t a t i s t i c a l l y it  After  significant  c o n s t i t u t e d a c o n s i s t e n t t r e n d f o r decrease  i n b a s a l body numbers and was seen i n some mutant l i n e s as w e l l as i n w i l d - t y p e  cells.  B a s a l body p r o l i f e r a t i o n d u r i n g c e l l  division  i s i l l u s t r a t e d i n P l a t e 9. The  spacing  ( i n um) between b a s a l  i s shown i n f i g u r e 12b.  This spacing  f o r both the number o f b a s a l bodies  The  accounts  i n the  r e g i o n CVINT and the l e n g t h o f CVINT. 0.75 b a s a l b o d i e s were 1.6 pm a p a r t  bodies  A t age  (fig.12b).  s p a c i n g decreased t o a minimum o f 0.8 jura  39  THE  MORPHOGENETIC PERIOD IN WILD-TYPE CELLS: - (Cont'd.) v.  B a s a l Body  Proliferation:  by age  0.94  at which time b a s a l body p r o l i f -  e r a t i o n was  at a maximum (Kaneda and Hanson,  1974).  By age 0.98  1.1  Lun.  was  s t i l l l e s s than t h a t i n i n t e r f i s s i o n  After fission,  ( p l a t e 9» vi.  the s p a c i n g i n c r e a s e d  to  the b a s a l body s p a c i n g cells  fig.e).  Contour Drawings: Contour drawings o f w i l d - t y p e 13)  i n d i c a t e a l l of the l e n g t h and  d i s c u s s e d above.  cells (fig. width changes  Note t h a t the p o s i t i o n s o f  the CVPs and v e s t i b u l e s changed i n a very r e g u l a r manner with i n c r e a s i n g c e l l age.  There  i s a l s o a marked d i f f e r e n c e i n the extent  of  v e s t i b u l e m i g r a t i o n i n the p r o t e r and vii:  Growth of the D o r s a l  Surface:  Between ages 0.93 linear  and 0.98  t h e r e was  a  i n c r e a s e i n the contour l e n g t h of the  mid-regions o f the p r o t e r and CVP *; f i g . 1 4 ) .  The  (CVA'  and  then began to i n c r e a s e  i n contour l e n g t h ( f i g . l 4 ) . and CVP)  opisthe  c e l l m i d - r e g i o n (CVINT)  i n i t i a l l y c o n t r a c t e d and  (CVA  opisthe.  The  polar  regions  showed no net growth d u r i n g  p a r t of the c e l l c y c l e .  this  40  THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS: - (Cont'd.) viii.  Growth o f the V e n t r a l Contour l e n g t h s  Surface: of regions  s u r f a c e are l i s t e d i n t a b l e 5-  on the v e n t r a l Between ages  0.94 and 0.95 the a n t e r i o r s u t u r e decreased i n contour l e n g t h by 7 tun while the p r o t e r ' s p o s t e r i o r suture  i n c r e a s e d by 7 pm>  In the  o p i s t h e , the decrease i n l e n g t h o f the p o s t e r i o r suture was s i m i l a r l y equal t o the i n c r e a s e i n l e n g t h o f the o p i s t h e ' s  a n t e r i o r suture.  3 p.m i n c r e a s e i n the t o t a l  The  contour l e n g t h of  the v e n t r a l s u r f a c e o c c u r r i n g a t t h i s time appeared as an i n c r e a s e i n the l e n g t h o f the p r o t e r ' s v e s t i b u l e , (from  13 t o 16 jum).  Between ages 0.95 and O.97 the growth o f the p o s t e r i o r suture i n the p r o t e r exceeded the decrease i n the l e n g t h Surface  o f the a n t e r i o r s u t u r e .  growth behind the v e s t i b u l e was t h e r e -  f o r e accounting  f o r some o f the apparent  v e s t i b u l e movement.  I n the o p i s t h e , the l e n g t h  of the a n t e r i o r suture  continues  to increase  a f t e r age 0.95 without any f u r t h e r change i n the p o s t e r i o r suture  length.  TABLE 5 CONTOUR L E N G T H S OF V E N T R A L R E G I O N S MORPHOMETRIC  Sample  Age  GAp  Wild-Type  .94 .95 .96-.97 .98-.99  59 52 43 42  sm2  .91-.94 •95-.96 .97-.98 .99  •  dc4  dc2  a  dfz2  Gp  PARAMETER*  GPp  GAo  13 16 15 15  0 7 23 35  0 9 14 22  13 13 14 15  60 52 52 53  56 38 38 32  11 16 15 15  0 11 20 31  0 5 10 22  11 12 13 14  53 47 43 41  .91-.93 •94-.95 .96-.97 .98-.99  52 40 28 31  11 14 15 15  0 6 25 35  0 5 10 12  11 12 14 14  52 42 45 45  .92-.94 • 95 .96 •97-.99  55 47 37 35  13 15 15 15  0 5 21 33  0 2 10 20  13 12 14 15  57 55 44 50  •93 •94-.95 .96-.97 .98-.99  63 44 35 36  14 17 15 15  0 10 21 35  0 5 14 21  14 14 14 15  55 50 46 44  Go  GPo  * i n pm. abbreviations: GAp, a n t e r i o r s u t u r e o f p r o t e r ; Gp, p r o t e r v e s t i b u l e ; GPp, p r o t e r p o s t e r i o r s u t u r e ; GAo, o p i s t h e a n t e r i o r s u t u r e ; Go, o p i s t h e v e s t i b u l e ; GPo, opisthe p o s t e r i o r suture.  42  THE MORPHOGENETIC PERIOD IN MUTANT LINES: a.  Mutant  sm2  At the r e s t r i c t i v e temperature  sm2 c e l l s were  s m a l l , with severe c o r t i c a l and g u l l e t a b n o r m a l i t i e s ( p l a t e 5)'  C e l l s c o u l d complete two t o f o u r c e l l  c y c l e s a t 34-5°C b e f o r e d i v i s i o n a r r e s t  occurred.  D i v i s i o n a r r e s t was n o t accompanied by m i s d i v i s i o n . Data c o l l e c t e d on sm2 c e l l s are g i v e n i n t a b l e 6.  The parameters a r e p l o t t e d a g a i n s t c e l l age i n  f i g u r e s 7 t o 12.  D i v i s i o n stages o f sm2 c e l l s are  i l l u s t r a t e d i n p l a t e 10.  With the e x c e p t i o n o f  width, the standard e r r o r s o f measurement parameters were s i m i l a r t o those recorded f o r w i l d - t y p e  cells.  Width i s more v a r i a b l e i n sm2 than i n w i l d type, i.  Growth Phase: Between ages 0.75 and 0.83 c e l l c y c l e s there was no s i g n i f i c a n t i n c r e a s e i n l e n g t h (fig.7a).  The o n l y p a r t o f t h e c e l l  changed i n l e n g t h was the mid-region which c o n t r a c t e d ( f i g .  lib).  which (CVINT)  Region CVA' d i d  not change i n l e n g t h (fig.10a) whereas the same region contracted i n wild-type c e l l s during t h i s phase. I n t e r f i s s i o n and growth phase sm2 had an unusual c e l l o u t l i n e with a  cells  conspicuous  TABLE 6 AGE-DEPENDENT CHANGE IN sm2 CELL SIZE DIMENSION ( i n um) WITH STANDARD ERRORS VARIABLE " 3  CELL A G E Length Width L/W CVA CVA' CVINT CVP' CVP GA G c  b  0.75  0.8  123+1.2 55-1.1  120Jl.7 52-1.0  41-1.0  39-0.9  +  57-1.4 _ — — —  25+0.8 52-0.9  18-0.3 53+0.9 35+0.7 I.6-.03  54-1.2 4.  26-1.0 51+1.2 18^0.4 51+0.9 34jl.4 1.6-.05  0.82  117+5 .1 53-3 .2  O.85-.86 0.91-.94 0.95-.96 0.97--98 122^2.2 52-1.5  22±2 .1 25+1.3 20-1 .6 18-1.8 . 40±2 -7 34^2.6 20-1 .4 20^1.1 18^0 .6 25+L3 49^4 .0 51+1.9 17+0 .6 18-1.0 53+2 • 7 52-1.8 23x1 .8 20-1.3 1.7-.07 1.7-.05  lllj2.4 48-1.3  a  114x2.9 44-1.4  1.05  128^3-7 42-1.3  81^4.5 50-2.9  22^1.0 25Jo.9 28-2.5 19^1.5 19+0.9 17+0.8 19+1.0 19J0.6 . 24-1.3 32^0.8 36T2.0 34x2.2 35-3-8 31+1.1 18-1.0 22jl.O 18-2.3 - 25+1-2 22-1.0 20x1.2 18^1.8 17+0.5 23+L7 29-2.1 47+1.7 25-1.1 31+2.5 31-L5 23 0.6 17+0.4 *4* 35+0.8 33+L3 33+1.5 32-3.1 5 25+0.9 27+1.4 30+3.2 22x2.7 29x1-8 1-3-.04 1.2-.07 1.3-.05 1.2-.12 1.6-.12 x  2 d #b.b. CVINT/b.b. PROTER LENGTH OPISTHE LENGTH PG OG PGP GAO n f 16 4 6 16 23 a-see f i g u r e 6 f o r e x p l a n a t i o n o f a b b r e v i a t i o n s used b-in fractions of a c e l l cycle c - l e n g t h t o width r a t i o ( a r b i t r a r y u n i t s ) d-the number o f b a s a l "bodies i n r e g i o n CVINT P  108,2.9 46-1.5  .99  56-2.4  61-1.2  68-1.8  50,1.0 16-1.0 12 1.0 10-2.4 3-1.0  51+-.2 15+1.0 13+1.0 18 2.0 6-1.0 7  60^2.3 15+1.0 I4jl.0 27+1.3 13-1.0 7  x  X  —  —  '  - —  .  —  8 6 e-the spacing between b a s a l b o d i e s f-sample s i z e _p_ u>  44  THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) i.  Growth Phase: - (Cont'd.) bulge on the c e l l ' s l e f t  s i d e ( p l a t e 10). The  mean c e l l width a t age 0.75 was 55 pm  (fig.8b)  which i s s i g n i f i c a n t l y g r e a t e r than the width of w i l d - t y p e ii.  Contraction The at  c e l l s o f t h e same age (42 pm). Phase:  c o n t r a c t i o n phase i n sm2 c e l l s began  age 0.85 and continued  t o age 0.95-  decreased i n mean l e n g t h by 14 pm d u r i n g  Cells this  phase compared t o a 9 pm c o n t r a c t i o n i n w i l d type c e l l s .  At the end o f the c o n t r a c t i o n  phase sm2 c e l l s were s i g n i f i c a n t l y s h o r t e r  than  w i l d type ( f i g . 7 a ) . Most o f the c o n t r a c t i o n i n sm2 c e l l s was l o c a l i z e d i n the c e l l  (CVA and CVP)  and  the mid-region (CVINT).  not  show the c o n t r a c t i o n i t d i d i n wild-type  cells iii.  extremities  Region CVA' d i d  (fig.10a).  Fission-Furrow  Phase:  T h i s phase begins i n sm2 c e l l s a t the same age  as i n w i l d - t y p e  cells  (0.95).  Increase i n  l e n g t h d u r i n g t h i s phase occurred  at a similar  r a t e i n both wild-type  and sm2 c e l l s but sm2  c e l l s were s h o r t e r than w i l d type when they  45  THE  MORPHOGENETIC PERIOD IN MUTANT LINES; - (Cont'd.)  iii.  F i s s i o n - F u r r o w Phase; - (Cont'd.) entered t h i s phase.  By age 0.98 the mean  length  o f sm2 c e l l s reached 128 pm, 6 pm  longer  than i n t e r f i s s i o n c e l l s  (fig.7a).  D u r i n g the f i r s t c e l l c y c l e a t the r e s t r i c t i v e temperature f u r r o w i n g was normal i n most sm2 c e l l s , but a few cases o f m i s d i v i s i o n  (arrested  at about age 0.99) were encountered. Increase i n l e n g t h occurred  (CVA' and CVP', f i g s . 1 0 a and  The e x t r e m i t i e s  also increased The  t h i s phase  p r i m a r i l y i n the mid-regions of t h e  p r o t e r and o p i s t h e b).  during  start  o f the c e l l  i n length  during  (CVA and CVP) t h i s phase.  o f the f i s s i o n - f u r r o w phase i n  w i l d - t y p e c e l l s was marked by a dramatic shortening (GP)  o f the a n t e r i o r (GA) and p o s t e r i o r  sutures ( f i g s . 7 b  and 8a).  I n sm2 c e l l s  the a n t e r i o r suture r a p i d l y c o n t r a c t e d ages 0.94 and 0.96 ( f i g . 7 b ) .  The p o s t e r i o r  s u t u r e showed no dramatic s h o r t e n i n g creased s t e a d i l y i n length 0.97 ( f i g . 8 a ) .  between  but de-  from age O.85 t o age  The p o s t e r i o r suture i n sm2  c e l l s a t age 0.95 was s i g n i f i c a n t l y  shorter  (35 pm) than i n w i l d - t y p e c e l l s (43 jum).  This  46  THE  MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.)  iii.  Fission-Furrow  Phase: - (Cont'd.)  d i f f e r e n c e arose overall cell  from the d i f f e r e n c e i n the  l e n g t h and  not a d i f f e r e n c e i n  v e s t i b u l e movement, iv.  B a s a l Body P r o l i f e r a t i o n : Mutant sm2  c e l l s had  a different  pattern  of b a s a l body p r o l i f e r a t i o n from t h a t seen i n wild-type bodies 0.85  cells  (fig.12a).  The  number of b a s a l  i n CVINT i n c r e a s e d s t e a d i l y  to age  0.99.  from  age  U n l i k e the case i n w i l d - t y p e  c e l l s , t h e r e was  no d o u b l i n g of b a s a l body  numbers.  O.85  At age  b a s a l bodies by age  0.99-  t h e r e was  a mean of  20  i n CVINT and t h i s i n c r e a s e d t o There was  no r e d u c t i o n i n b a s a l  body number d u r i n g the f i s s i o n - f u r r o w phase p r o l i f e r a t i o n continued fission.  Despite  30  and  up to the time of  the d i f f e r e n c e s i n the  pattern  of b a s a l body p r o l i f e r a t i o n between w i l d type and  sm2  c e l l s there was  no s i g n i f i c a n t  ence i n the number of b a s a l bodies of these two The steadily wild-type  c e l l l i n e s a t age  0.99  differ-  i n CVINT (fig.12a).  l e n g t h of the r e g i o n CVINT i n c r e a s e d d u r i n g the morphogenetic p e r i o d c e l l s but decreased s t e a d i l y  in  of sm2  4?  THE  MORPHOGENETIC iv.  PERIOD IN MUTANT LINES; - (Cont'd.)  B a s a l Body P r o l i f e r a t i o n ; cells  ( f i g . lib)'.  - (Cont'd.)  T h i s may have been  related  to the d i f f e r e n c e s noted i n b a s a l body proliferation.  Examination and comparison o f  b a s a l body s p a c i n g e l i m i n a t e s the e f f e c t o f d i f f e r e n c e s i n the l e n g t h o f the r e g i o n i n which the b a s a l bodies  are counted.  Decrease  i n b a s a l body s p a c i n g i n sm2 c e l l s was not as obvious as i n w i l d - t y p e indicating cells.  cells  (fig.12b)  a r e d u c t i o n i n p r o l i f e r a t i o n i n sm2  A t age 0.98 the b a s a l body s p a c i n g on  wild-type  and sm2 c e l l s was i d e n t i c a l .  Although  b a s a l body p r o l i f e r a t i o n was reduced i n sm2 c e l l s t h i s was o f f s e t by a r e d u c t i o n i n c e l l size.  C e l l s which had been a t 34.5°C f o r 1.05  c e l l c y c l e s had the same b a s a l body s p a c i n g as interfission cells  ( t a b l e 3).  B a s a l body p r o l i f e r a t i o n i n sm2 c e l l s o f v a r i o u s ages i s shown i n f i g u r e v.  15•  Width; Age wild-type  0.75 sm2 c e l l s were much wider than c e l l s o f the same age ( f i g . 2 0 b ) .  sm2 c e l l s width decreased s t e a d i l y morphogenetic p e r i o d and reached 40  In  d u r i n g the LUTI  by age  48  THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) v.  Width:  - (Cont'd.)  0.99.  Wild-type c e l l s  o f age 0.99 were a l s o  40 pm wide. The l e n g t h to width r a t i o 2.3 + 0.1 a t age 0.75*  T h i s remained c o n s t a n t  u n t i l the f i s s i o n - f u r r o w phase. it  i n c r e a s e d t o 3.1 +0.2  start  i n sm2 c e l l s was  By age 0.99  (fig.11a).  At t h e  o f the f i s s i o n - f u r r o w phase the l e n g t h t o  width r a t i o  was about 2.5 ± 0.1 i n both w i l d -  type and sm2 c e l l s , vi.  Stomatogenesis: The o r a l of age 0.75-  anlage i s f i r s t seen i n sm2  cells  I t s morphology a t t h a t age was  s i m i l a r t o a n l a g e n i n age 0.75 w i l d - t y p e c e l l s . I n v a g i n a t i o n o f the anlage o c c u r r e d and i t developed t o the three k i n e t y stage ( f i g . l 6 ; a, b, c ) . ent  A b n o r m a l i t i e s i n the anlage were appar-  i n c e l l s age 0.9 o r o l d e r .  Most c e l l s o f  t h i s . a g e had a n l a g e n w i t h i r r e g u l a r l y  spaced  a r g e n t o p h i l i c l o c i but had no k i n e t i e s In  (fig.16).  some cases the anlagen were abnormally  or reduced i n s i z e ( f i g . l 6 ;  shaped  d, g ) .  M i g r a t i o n o f the sm2 anlage began a t age 0.92 or 0.93 and proceeded i n a manner s i m i l a r  49  THE  MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) vi.  Stomatogenesis - (Cont'd.) t o t h a t seen i n w i l d - t y p e  cells.  I n many cases  g r a n u l a r a r g e n t o p h i l i c m a t e r i a l was l o c a t e d between the p o s t e r i o r o f the anlage and i t s original  p o i n t o f c o n t a c t with t h e v e s t i b u l e  (fig.16; g to j ) . The  endoral  k i n e t y , which i s resorbed  d u r i n g the e a r l y stages o f stomatogenesis ( f i g . 4 ) , reappeared i n sm2 c e l l s around age 0.93-  In some c e l l s the new e h d o r a l k i n e t y was  normal (fig.16; f ) while  i n others  i t consisted  o f o n l y a few i r r e g u l a r l y - s p a c e d a r g e n t o p h i l i c bases ( f i g . 16;  e, h ) .  The newanlagen, which  should have formed a t age 0.88, were found i n some c e l l s but n o t i n others vii.  Juvenile  (fig.l6).  Cells:  C e l l s aged 1.05 c e l l c y c l e s c o n s t i t u t e the p r o t e r s and o p i s t h e s r e s u l t i n g from the f i r s t d i v i s i o n a t the r e s t r i c t i v e  temperature.  In  mutant sm2 these c e l l s had a mean l e n g t h o f 81 pm ( t a b l e 6) and were 20 pm s h o r t e r than age 1.05 w i l d - t y p e long).  cells  (these a r e about 100 jum  The j u v e n i l e c e l l s were rounded i n out-  l i n e and had a - t w i s t e d  body ( p l a t e 10). The  50  THE  MORPHOGENETIC PERIOD IN MUTANT LINES; - (Cont'd.)  vii.  Juvenile Cells;  - (Cont'd.)  k i n e t y pattern,- g u l l e t , and o f t e n abnormal on these viii.  cytoproct  were  cells,  Contour Drawings; The  contour drawings of sm2  i l l u s t r a t e the and  t h a t the c o n t r a c t i o n i n sm2  of CVPs and age  cells  s i z e d i f f e r e n c e between these  w i l d type d i v i d i n g c e l l s  longed (to age  dividing  0.95)  a n  d  ( f i g . 13) • c e l l s was  t h a t the  Not e -  more p r o -  displacement  v e s t i b u l e s d i d not change with  i n as r e g u l a r a manner as i n  cell  wild-type  cells. ix.  Growth of the D o r s a l The length  Surface;  greatest r e l a t i v e increase  occurred  i n surface  i n the p o r t i o n o f the  s u r f a c e a n t e r i o r t o the f i s s i o n - l i n e P o s t e r i o r to the f i s s i o n l i n e , the s u r f a c e showed no net growth. c e l l mid-regions was  growth i n the p o l a r x.  Growth of V e n t r a l  furrow the  cells (in  t h e r e was  no  net  regions,  Surface;  Between ages 0.94 a n t e r i o r suture  (fig.l4b).  Growth of  reduced i n sm2  comparison t o w i l d type) and  furrow  and  0.95  the  proter's  decreased by 18 p.m i n l e n g t h  51  THE M O R P H O G E N E T I C P E R I O D I N MUTANT LINES;. x.  Growth  o f Ventral Surface;  (table  5) w h i l e ' i t s p o s t e r i o r s u t u r e  by  11 pm i n l e n g t h .  the  proter's  -  During  (Cont'd.)  A f t e r a g e 0.95  change  i n the length  increased  t h e same  vestibule increased  5 pm.  (Cont'd.)  period  i n length  by  t h e r e , was no f u r t h e r of the proter's  anterior  suture. B e t w e e n a g e s 0.94 the  5 pm  Mutant In type  A f t e r a g e 0.95  continued  asynchronous  cell  strictive Data  i n length,  c u l t u r e a t 35°C t h e dc4  size.  Severe  were n o t e d  after  and g u l l e t  20 h o u r s  at the r e -  temperature. c o l l e c t e d o n dc4  age a r e g i v e n  cells  and c o e f f i c i e n t s  of  cells  t o 22.  of variation  were s i m i l a r  cells.  are given  parameters  i n f i g u r e s 17  errors  wild-type  pheno-  a r r e s t and  cortical  P l o t s o f the morphometric  dc4  by  dc4  abnormalities  cell  increased  the posterior  a slow decrease  was c h a r a c t e r i z e d b y d i v i s i o n  reduced  ?.  the anterior suture  i n length.  suture b.  while  i n the opisthe,  d e c r e a s e d b y 6 pm i n  posterior suture  length  a n d 0.95  i n table  against The  standard  f o r measurements  t o or less than  those f o r  TABLE 7 AGE-DEPENDENT CHANGE IN dc4 CELL SIZE DIMENSION ( i n mn) WITH STANDARD ERROR VARIABLE CELL AGE  b  Length Width L/W CVA CVA ' CVINT CVP • CVP GA ' G c  S d #b.b. P  a  CVINT/b.b. PROTER LENGTH OPISTHE LENGTH PG OG PGP GAO n f  •75 109J2. 1 42^0. 6 2.6 .05 37-1. 0 X  -  —  52-1. 7 —  —  20-1. 3 45^1. 8 16-0. 2 49x0. 6 37x1- 8 1.4-.04  .8- .81  .83-.84  .85-. 87  111 J l . 6 45 J l . 0 2.5 -.06 20 9 8 19 32 I - 5 20 I - 2 1 19 43 h- 5 17 5 +°51 J i - 4 25 l l . 0 1-3 -.04  108jl. 5 46x1. 2  112T"2 .1  1  1  2.4-T.09  19+0. 5 17 Jo. 8 33+1. 0 LSjl. 6 21x1. 8 43 J l . 2 16 Jo. 3 49jl. 5 23x1- 3 1.5-.05  .88-.91  113x3. 1 4 4 j l .2 4741. 6 2.6J. 08 2.4-.12 21x1 •3 22x1. 3 18x0 •5 19+0. 9 33+0 .9 32-1. 0 21.0. 7 .1 19+1 20x0. 4 22x1 •5 45^1 .1 45 J l . 1 18x0 .4 18 Jo. 4 49^1 • 7 51+2. 0 2 6 j l .8 33T1. 3 1.3-. 09 1.0-.04  !  •91-.93  .94-.95  -96-.97  -98-.99  106Jl. 6 48T1. 7 2-3x-10 20 0. 3 16 Jo. 5 33+0. 7 18 J l . 0 19+1. 0 42jl. 6 23x1- 0 4ljl. 7 40T2. 7 0.9-.06  98-3-2 45J1.6 2.2T.13 18Jo.6 17+1.4 30 1.4 18Jo.6 15+1.1 30-1.5  120-1.9 38|l.4 3.2-.13 20x0.8 25+1.1 36Jo.8 25-0.8 15+0.4 23-1.0  128 1.2 38J1.1 3-^+.09 24Jo.7 26jl.2 37+1.2 26-0.9 15+1.2 23-0.9  32J2.4 39+1.3 O.8-.03  35+1.2 36jl.6 1.0-.05  35+1.3 39+2.0  50-1.6  62-1.5  47+1.7 l4 0.3 12+0.5 6jl.l 3-1-0 10  58jl.3 15+0.1 14x0.2 24jl.8 8-I.3 10  x  +  +  8  10  8  a-see f i g u r e 6 f o r e x p l a n a t i o n o f a b b r e v i a t i o n s used b - i n f r a c t i o n s of a c e l l c y c l e c - l e n g t h t o width r a t i o ( a r b i t r a r y u n i t s ) d-the number o f b a s a l bodies i n r e g i o n CVINT  8  10  x  I.O-.05 63-1.0 64jl.l 15+0.2 14x0.2 26jl.O 15-1.4 10  e-the spacing between b a s a l b o d i e s f-sample s i z e ^  53  THE MORPHOGENETIC PERIOD IN MUTANT LINES;. - (Cont'd.) i.  Growth Phase; Age 0.75 dc4 c e l l s had a mean l e n g t h o f 109 pm ( t a b l e 7 ) .  The d i f f e r e n c e i n l e n g t h  between w i l d - t y p e and dc4 c e l l s a t t h i s age (109 pm as compared t o 120 pm f o r w i l d - t y p e c e l l s ) was l a r g e l y due t o d i f f e r e n c e i n t h e l e n g t h o f the a n t e r i o r h a l f o f t h e c e l l .  An  a p p a r e n t d e f e c t i n c e l l growth o c c u r r e d  during  the i n t e r f i s s i o n  period of dc^ c e l l s .  From age 0.75 t o age 0.9 t h e r e was no i n c r e a s e i n dc4 c e l l l e n g t h ( f i g . 1 7 a ) . increase i n p o s t e r i o r suture  The  (GP) l e n g t h n o t e d  i n w i l d - t y p e c e l l s .did n o t o c c u r i n dc4 c e l l s (fig.18a). ii.  C o n t r a c t i o n Phase; C o n t r a c t i o n o f dc4 c e l l s o c c u r r e d between ages 0.9 and 0.95 w i t h a l e n g t h d e c r e a s e o f 16 pm.  A t age 0.95 t h e mean c e l l l e n g t h was 98 jam  as compared t o 126 um i n age 0.95 w i l d - t y p e cells  (fig.17a).  The c o n t r a c t i o n o f dc4 c e l l s  was l o c a l i z e d i n t h e c e l l e x t r e m i t i e s (CVA and CVP, f i g s . 1 9 a and b ) .  I n t h e r e g i o n CVA* , none  of t h e c o n t r a c t i o n seen i n w i l d - t y p e c e l l s was found i n dc4 c e l l s ( f i g . 2 0 a ) .  54  THE  MORPHOGENETIC PERIOD IN MUTANT LINES; - (Cont'd.)  iii.  F i s s i o n - F u r r o w Phase; Between ages 0.95 and 0.98 t h e mean l e n g t h of dc4 c e l l s i n c r e a s e d from 95 t o 128 pm ( f i g . 17a).  The l e n g t h a t age 0.98 was s i g n i f i c a n t l y  l e s s than t h a t o f w i l d - t y p e c e l l s  (153 .um) •  Growth was l o c a l i z e d i n t h e mid-regions  of the  p r o t e r and o p i s t h e (CVA and CVP ; f i g s . 2 0 a and 1  b).  Some growth a l s o o c c u r r e d i n the a n t e r i o r  end o f t h e c e l l (fig.21b). tical  1  (CVA; f i g . 1 9 a ) and i n CVINT  T h i s p a t t e r n o f growth was i d e n -  to that of wild-type  cells.  I t was d i f f i c u l t t o s c o r e dc4 c e l l s o f age 0.98 o r 0.99 s i n c e many o f these c e l l s were misdividers.  Such c e l l s were not used i n the  a n a l y s i s as they c o u l d n o t be a c c u r a t e l y aged. Thus, f o r dc4 c e l l s which appeared as i f they would n o t be a r r e s t e d i n the f i r s t c e l l c y c l e at 3^-5°C there was a s i z e r e d u c t i o n s i m i l a r t o that found i n sm2 c e l l s . Decrease i n l e n g t h o f the a n t e r i o r suture (GA;  fig.17b) i n dc4 c e l l s began i n the c o n t r a c -  t i o n phase a t about age 0.92. reached  The s u t u r e  a minimum l e n g t h o f 23 pm a t age O.96.  The l e n g t h o f t h i s suture i n w i l d - t y p e  cells  55  THE  MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.)  iii.  Fission-Furrow  Phase: - (Cont'd.)  never f e l l below 35 pm  (fig.17b).  In  c e l l s the v e s t i b u l e moved forward a d i s t a n c e of 26 pm  ( t a b l e 4;  dc4  i n the p r o t e r  PGP).  This i s  s i m i l a r to the extent of v e s t i b u l e movement i n wild-type The  cells  (29 pm)  and  i n sm2  p o s t e r i o r suture i n dc4  to decrease i n l e n g t h around age 18a). 0.95  cells  (27  c e l l s began 0.9  (GP; f i g .  I t reached a mean l e n g t h of 35 pm and m a i n t a i n e d t h i s l e n g t h u n t i l  at  s i m i l a r to t h a t found i n w i l d - t y p e  suture  cells.  The minimum suture l e n g t h i n w i l d - t y p e  cells  however, exceeded by 10 pm  cells  t h a t i n dc4  age  fission.  The p a t t e r n of decrease i n l e n g t h of t h i s was  pm).  (fig.18a). Patches appeared on the c o r t e x of c e l l s d u r i n g the f i r s t  c e l l c y c l e a t 34.5°C.  These brown patches had silver-impregnated  dc4  a g r a i n y appearance i n  m a t e r i a l and were i n t e r -  c a l a t e d between k i n e t i e s or were found i n the a n t e r i o r or p o s t e r i o r suture iv.  (fig.23).  B a s a l Body P r o l i f e r a t i o n The number o f b a s a l bodies of dc4  i n r e g i o n CVINT  c e l l s i n c r e a s e d from 22 to 40 between  56  THE  MORPHOGENETIC PERIOD IN MUTANT LINES;. iv.  (Cont'd.)  B a s a l Body P r o l i f e r a t i o n - (Cont'd.) ages O.83  and  0.92  (fig.22a).  b a s a l bodies d i d not  The  number of  change a f t e r age  0.92.  T h i s p a t t e r n of b a s a l body p r o l i f e r a t i o n s i m i l a r to t h a t i n w i l d - t y p e  cells.  was  There i s  however, a s m a l l d i f f e r e n c e i n the number of b a s a l bodies p r e s e n t wild-type  and m4  c e l l s a t age  fewer i n the dc4 The  i n the mid-regions of  cells  0.94  with  (fig.22a).  b a s a l body s p a c i n g i n dc4  d u r i n g the c e l l  ten  cells  c y c l e i n a manner s i m i l a r to  that i n w i l d - t y p e  cells  (fig.22b).  The  slight  r e d u c t i o n i n b a s a l .body p r o l i f e r a t i o n i n c e l l s was in c e l l v.  changed  t h e r e f o r e a s s o c i a t e d with  dc4  a reduction  length,  Width; At age 42 _um.  O.75  the mean width of dc4  T h i s i n c r e a s e d to 48 pm by age  steady decrease i n width occurred f i s s i o n - f u r r o w phase.  The  except t h a t no dc4  cells  O.96.  0.92.  A  the  p a t t e r n of change i n similar  ( f i g s . l 8 b and  decrease i n width was  c e l l s a f t e r age  was  during  width (and l e n g t h to width r a t i o ) was to t h a t i n w i l d - t y p e  cells  21a)  noted i n  57  THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) vi.  Stomatogenesis: Stomatogenesis i n dc4 c e l l s was normal during  vii.  the f i r s t c e l l c y c l e a t 34.5°C.  Contour Drawings: Contour drawings ( f i g . 2 4 ) i n d i c a t e the reduced s i z e o f dc4 d i v i d i n g c e l l s as w e l l as the marked and prolonged c o n t r a c t i o n o f the cell.  T h i s c o n t r a c t i o n was p a r t i c u l a r l y prom-  i n e n t i n the p o s t e r i o r p a r t o f the c e l l , viii.  Growth o f the D o r s a l  Surface:  S u r f a c e growth i n a l l c e l l r e g i o n s was delayed  u n t i l a f t e r age 0.94 ( f i g . 2 5 a ) .  From  age 0.94 to age 0.97 growth o f the c e l l midregions  (CVA' and CVP') i n c r e a s e d the l e n g t h o f  these r e g i o n s by 50% o f t h e i r o r i g i n a l  length.  A f t e r age 0.97 however, growth o f the c e l l midr e g i o n s was attenuated.  Furrow s u r f a c e growth  began a t age 0.95 and continued  t o age 0.98.  There was i n i t i a l l y a s l i g h t c o n t r a c t i o n i n t h e furrow s u r f a c e p o s t e r i o r t o the f i s s i o n No n e t growth occurred  line.  i n the p o l a r r e g i o n s , but  the p o s t e r i o r r e g i o n (CVP) c o n t r a c t e d t o 80$ o f i t s o r i g i n a l l e n g t h and remained c o n t r a c t e d t o age 0.99.  58  THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) ix.  Growth o f the V e n t r a l  Surface:  Between ages 0.93 and 0.95 the decrease i n l e n g t h of the a n t e r i o r suture e q u a l l e d the i n c r e a s e the l e n g t h o f the p r o t e r ' s p o s t e r i o r suture  ( t a b l e 5)•  Surface  growth i n the  p o s t e r i o r suture began a f t e r age 0.95 and continued u n t i l  fission.  Between ages 0.93 and 0.95> c o n t r a c t i o n of the p o s t e r i o r suture i n the o p i s t h e  exceeded  the i n c r e a s e i n l e n g t h o f the o p i s t h e * s a n t e r i o r suture.  A f t e r age 0.95 t h e r e was l i t t l e  further  change i n the l e n g t h o f e i t h e r the a n t e r i o r or p o s t e r i o r suture i n the o p i s t h e . a n t e r i o r suture  The o p i s t h e ' s  a t age 0.98 was considerably-  s h o r t e r (12 pm long) than t h a t found i n e i t h e r wild-type  (22 ;um long) or sm2 (22 jm  long) c e l l s  of the same age. c.  Mutant dfz2 A f t e r 20 hours a t the r e s t r i c t i v e  temperature  the dfz2 phenotype was c h a r a c t e r i z e d by i n a b i l i t y t o form a f i s s i o n - l i n e  o r furrow.  Data c o l l e c t e d on dfz2 c e l l s are g i v e n i n t a b l e 8.  P l o t s o f the morphometric parameters  a g a i n s t c e l l age a r e g i v e n i n f i g u r e s . 2 6  t o 31.  TABLE 8 AGE-DEPENDENT DIMENSION  CHANGE IN dfz2 CELL SIZE  ( i n pm) WITH STANDARD ERROR  VARIABLE CELL AGE Length Width L/W CVA CVA' CVINT CVP' CVP GA G c  .92 .81-.83 .84-.85 .86-.87 .88-.89 .91 •93 .94-.95 .96-.97 .98-.99 120T2.2 1 2 l j l . 9 I30J4.I 123J2 2 13lj4.0 128J2.6 125-I3-3 126J3.O 118J3.4 123J2.2 142J3.5 50jl.2 45+2.3 40x2.3 37+1.3 44JO.8 49x3-1 47x1-4 45x1 3 50x0.8 52x2.7 49Jl.4 2.5J.O8 X-12 2.7+.07 2.5x-20 2.8x-10 2.8T.12 2.6T.10 2.5T.17 2.5 2.7+.13 3.1J.17 3.9J.17 43-2.3 21^0 22 Jo. 7 27J2.5 L S j l . l 24^2 0 26x1-9 26-0 24jl 21J1.1 24jl.9 2 0 j l 1 20jl.8 22±2 21 Jl.8 20jl.9 21Jo.8 25J1.2 33J2.6 23jl 23x1 37x2 39 +1.9 38J2.6 39+3.0 37+1.8 40x2.1 56-2.7 37x1 36j3 2 4lj2.2 36-;i 22^2 19j0 5 22jl.4 20J1 19J2.0 22J0.8 25jl.5 29 J l . 4 25jl 21 Jl.O 18 Jo 20x0 Jl.8 25x2 4 23x1-3 22 21x1.5 23+1.7 17jl.5 I6J1.O 16+1.3 51 2 1 56J2.9 23x1 48 J2.1 53x1-0 48 J l 53J3.0 36jl.8 28-1.8 29-1.6 53x1 55Jl 18 Jo 17.0.4 17x0 22 Jo. 6 28-2.1 20J0 6 20-0.6 21-1 50-1.8 56J2 5 4 j l 5 55x2.4 52Jl 56Jl.6 44Jl.4 39jl.6 37+1.5 38+1.1 57x2 36-1.6 22-1 26-1 37 25-1 -2.3 41-1.4 42-2.5 40-2.5 38-1.8 2 3O-O.7 40-3 b  .75  +  #b.b. CVINT^ b. b. 1.1-.08 1.6-.10 1.5-.07 1.4-.13 1.4-.09 0.9-.07 1.1-.04 0.9-.06 0.9-.04 O.9-.05 1.1-.04 PROTER LENGTH 60-1.4 6I-5.I 74-2.6 OPISTHE LENGTH 57J2.2 6 l j l . 4 67jl.5 PG 19Jo.4 15Jo.3 15Jo.4 OG 14Jo.6 14J0.6 15Jo.3 PGP 8J1.9 19+1.1 3 l j l . l GAO 4-1.6 10-0.8 15-1.6 n f 5 12 8 8 8 9 7 11 9 a-see f i g u r e 6 f o r e x p l a n a t i o n o f a b b r e v i a t i o n s used e-the spacing between b a s a l b o d i e s b - i n f r a c t i o n s of a c e l l c y c l e f-sample s i z e c - l e n g t h t o width r a t i o ( a r b i t r a r y u n i t s ) d-the number o f b a s a l bodies i n r e g i o n CVINT d  VA  60  THE  MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) i.  Growth Phase: In dfz2 c e l l s t h i s phase extended from age  0.75 t o age O.89.  During  c e l l increased i n length  t h i s time the  (from 120 jam to 131  LIUI;  fig.26a) i n a manner s i m i l a r t o t h a t o f w i l d type c e l l s , ii.  C o n t r a c t i o n Phase: The  c o n t r a c t i o n phase o f dfz2 c e l l s ex-  tended from age 0.89 t o age 0.95 with a minimum l e n g t h o f 118 _um a t t a i n e d a t age 0.95'  Wild-  type c e l l s o f the same age had a mean l e n g t h o f 126 jum ( f i g . 26a).  The m a j o r i t y o f t h e dfz2 c e l l  c o n t r a c t i o n was l o c a l i z e d i n the c e l l  extrem-  ities  The  (CVA and CVP; f i g s . 2 8 a and b ) .  standard age  e r r o r o f the mean dfz2 c e l l  length at  0.95 was g r e a t e r than t h a t found i n w i l d -  type c e l l s  ( t a b l e 4) d e s p i t e greater, sample  s i z e i n the dfz2 experiment. lengths 141 jum.  The range o f  o f age 0.95 dfz2 c e l l s was from 101 t o The s m a l l e r c e l l s were very  abnormal.  They were wide (up t o 62 ;um), l a c k e d a f i s s i o n l i n e , and showed no s i g n o f furrowing.  This  group o f c e l l s appeared t o be a r r e s t e d i n the c e l l c y c l e p r i o r to the f o r m a t i o n  o f the  61  THE MORPHOGENETIC PERIOD IN MUTANT LINES: ii.  C o n t r a c t i o n Phase: - (Cont'd.) fission-line.  iii.  - (Cont'd.)  '  F i s s i o n - F u r r o w Phase: Those c e l l s which d i d not have a f i s s i o n line  or furrow were n o t i n c l u d e d i n the morpho-  m e t r i c a n a l y s i s as they had a g r o s s l y abnormal body form (cases of p a r t i a l excluded).  f u r r o w i n g were a l s o  F i g u r e 32 shows the s e r i e s of  changes which occurred i n those c e l l s which underwent d i v i s i o n a r r e s t .  The c e l l s  became  t w i s t e d such t h a t the k i n e t i e s o f the o p i s t h e e v e n t u a l l y l a y a t r i g h t angles t o those of the proter. ision  Monsters.produced by t h i s type of d i v -  a r r e s t were a p p a r e n t l y unable t o continue  i n t o a second d i v i s i o n a t the r e s t r i c t i v e erature.  temp-  Rounded forms with more than two  g u l l e t s were n o t seen even i n samples l e f t a t 35°C f o r 20 hours. The v e s t i b u l e s and g u l l e t s o f d i v i s i o n a r r e s t e d d f z c e l l s were u s u a l l y exposed on the cell  surface (fig.32d).  A b n o r m a l i t i e s i n the  s t r u c t u r e of the g u l l e t k i n e t i e s were however, rare. For  dfz2 c e l l s which were n o t a r r e s t e d  62  T H E MORPHOGENETIC P E R I O D i i i .  Fission-Furrow during  Phase:  the f i r s t  temperature  cell  -  (Cont'd.)  (Cont'd.)  cycle at the restrictive  t h e f i s s i o n - f u r r o w phase  from  a g e 0.95  cell  length  age  I N MUTANT LINES:. -  to fission.  increased  During  extended  this  time t h e  f r o m 1 1 8 t o 1 4 2 pm.  At  0.98 t h e dfz2 c e l l s w e r e a b o u t 1 0 ^jm  shorter  than t h e i r  (fig.26a). ized  w i l d type  The i n c r e a s e  i n the mid-regions  opisthe,  counterparts  i n length  was  local-  o f t h e p r o t e r and  a s was t h e c a s e  i n wild-type  cells  (figs.29a andb ) . The a n t e r i o r a n d p o s t e r i o r s u t u r e s a  r a p i d s h o r t e n i n g . a t a g e 0.92  27a).  r a p i d and continued  (fig.26b). reached length  A minimum  at this  time,  i n wild-type  length 6 pm  35 um a t a g e 0.95> a b o u t length  i n a g e 0.95  The r a t e  until  0.98  o f 29 jum w a s  shorter  reached  than the  wild-type  o f forward  a length of  10 p m s h o r t e r t h a n i t s cells  movement  p r o t e r ' s v e s t i b u l e was i d e n t i c a l  dfz2 c e l l s  age  suture  cells.  The p o s t e r i o r s u t u r e  and  (figs.26b and  The c o n t r a c t i o n o f t h e a n t e r i o r  was v e r y  began  (GF; t a b l e s  (fig.27a). of the  i n wild-type  2 a n d 5).  This  63  THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) iii.  Fission-Furrow  Phase: - (Cont'd.)  i n d i c a t e d t h a t 'the d i f f e r e n c e s i n l e n g t h o f the a n t e r i o r suture between dfz2 and w i l d - t y p e  cells  was not due t o d i f f e r e n c e s i n v e s t i b u l e movement but t o d i f f e r e n c e s i n growth o f the p r o t e r . iv.  Stomatogenesis: Stomatogenesis i n dfz2 c e l l s d u r i n g the first  v.  c e l l c y c l e a t 34.5°C was normal,  B a s a l Body P r o l i f e r a t i o n : The i n c r e a s e i n b a s a l body numbers i n dfz2 c e l l s f o l l o w e d the same p a t t e r n as seen i n wild-type slightly  cells  (fig.31a).  The numbers  lower i n dfz2 c e l l s but so was the  l e n g t h of the r e g i o n CVINT. b a s a l bodies a t a l l c e l l wild-type vi.  were  and dfz2 c e l l s  .The s p a c i n g  between  ages was i d e n t i c a l i n (fig.31b).  Width: Throughout the i n i t i a l growth phase the width o f dfz2 c e l l s was s i m i l a r t o t h a t of w i l d type c e l l s  (fig.27b).  D u r i n g the c o n t r a c t i o n  phase a s i g n i f i c a n t d i f f e r e n c e between the widths of w i l d - t y p e  and dfz2 c e l l s appeared.  T h i s may have been a s s o c i a t e d with i n furrow formation  i n dfz2 c e l l s .  the d e f e c t I n the  64  THE MORPHOGENETIC PERIOD IN MUTANT LINES: vi.  - (Cont'd.)  Width: - (Cont'd.) f i s s i o n f u r r o w phase t h e w i d t h s o f dfz2 c e l l s were i d e n t i c a l t o t h e i r w i l d t y p e c o u n t e r p a r t s ,  vii.  Contour Drawings and S u r f a c e Growth: The i n t e r e s t i n g d e f e c t i n dfz2 c e l l s was a l a c k o f f i s s i o n - z o n e and f u r r o w f o r m a t i o n . A d e t a i l e d a n a l y s i s o f s u r f a c e growth was n o t c a r r i e d out on c e l l s e x p r e s s i n g t h i s d e f e c t . Contour drawings o f d f z c e l l s which formed a p a r t i a l o r complete f u r r o w i n d i c a t e d t h a t t h e r e was a s l i g h t r e d u c t i o n i n s u r f a c e growth.  The  phenotype o f t h e s e d f z c e l l s was s i m i l a r t o t h a t o f dc2  a  c e l l s (compare t h e o u t l i n e drawings  o f dfz2 and d c 2 d.  Mutant  dc2  a  cells,  fig.33).  a  At t h e r e s t r i c t i v e temperature dc2  a  c e l l s have  the m i s d i v i d e r phenotype. D a t a c o l l e c t e d on dc2 9.  a  c e l l s i s given i n table  P l o t s o f the morphometric  parameters a g a i n s t  c e l l age a r e g i v e n i n f i g u r e s 34 t o 39. i.  Growth In  Phase: the dc2  c e l l c y c l e t h i s phase  from age O.75 t o age 0.9.  extended  No i n c r e a s e i n mean  c e l l l e n g t h occurred d u r i n g t h i s time  (fig.34a).  TABLE 9 a CELL SIZE AGE-DEPENDENT CHANGE IN dc2~ DIMENSION  ( i n p i ) WITH STANDARD ERROR  VARIABLE CELL AGE  b  Length Width L/W CVA . CVA' CVINT CVP' CVP GA G GP . #b.b. CVINT/b.b. PROTER LENGTH OPISTHE LENGTH PG c  d  •75  .8-.84 .85-.86  .87  .88-.89  .9-..91 -92-.94  121 2.6 122-T2 120x2.8 120Jr.3 115J2.2 119x2 4 122x2 50^0.9 49^0.8 4 6 j l 50-0 . _ 46Jo.9 48 JL. 2.7-.10 2.3+.10 2.6 .08 2-5 .07 2.5X.05 2.4J.07 2.4J.07 23x0 r, 21x0 22x0 ~,+- ~ 43-1-5 24^1 23+1 23x1 — ~5 22j2 20jo 18 Jo 23x1 19 0 23.1 57-2.8 34j2 4lj7 35x2 38j2 35jl 35jl I6j2 I8j2 21x1 18 J l 19jl -X" 19 1 21 J L 22^2 19j2 20-1.7 27x3 I6j5 23.3 47^2 44jl 47j4 46^2 ^7x1.5 51 2 52JL 18J0 I8J0 19J1 26jl 17j0 17x0.5 17x0 56jl 54jl 44J1 56-1.2 54x1 52x1 56j3 29-> 38J2 34j4 32 2.2 23 l 32x3 4l±l 1.8-.07 1.5-.07 1.4-.17 1.2-.15 1.1-.10 0.9-.09 0.9-.03 T  Z  +  +  x  +  +  x  +  PGP GAO n ** 9 5 4 8 a-see f i g u r e 6 f o r e x p l a n a t i o n o f a b b r e v i a t i o n s used b-in fractions of a c e l l cycle c - l e n g t h t o width r a t i o ( a r b i t r a r y u n i t s ) d-the number o f b a s a l bodies i n r e g i o n CVINT  .95  .96  .97-.99  l l l j l . l 114^3.4 138J3.4 48?!,' '•-+— •'+- . 6 43J1.1 '41Jl.3 2.3J.IO 2.7J.IO 3.4J.18 , 20jl.O 23jl.8 - -._ 23jl.6 29.1.5 - ' 34^1 - 32.1.7 ~ 4lj3.1 20^0 24jl.7 28jl.5 l 6 j l . 2 17jl.6 17+0 29-3.2 30-1.2 38-1 +  +  4ljl 35jl.7 38-2.2 43j2 39J2.6 0.8J.04 0.8J.05 56J1 ~ 60x2.0 '~ ~ - 72Jo.9 54J2.1 66Jo.3 54jo 15x0.4 15x0-5 15x1 14-0.7 15.0.5 12x1 17x1-7 27x0.1 4jl 1-0 5-2.5 12-0.2 8 5 e-the spacing between b a s a l bodies f-sample s i z e * - v a l u e not a v a i l a b l e due t o poor • quality of silver-impregnation. +  ON  66  THE MORPHOGENETIC i.  ii.  Growth  P E R I O D I N MUTANT L I N E S :  Phase:  - (Cont'd.)  At  t h e end o f t h i s  pm  long,  Contraction  phase  dc2  cells  ages  decreased  minimum l e n g t h  0.9  a n d 0.95  b y 10 pm  t h e mean  (fig.34a).  a t t a i n e d w a s 110  0.95)  w h i c h w a s 10 pm s h o r t e r  cells  o f t h e same a g e ( f i g . 3 4 a ) .  contraction the  cell  occurred  The  began  pattern  120  of.the  a t a g e 0.9  o f change  s u t u r e s was s i m i l a r  cell  The  ( a t age wild-type Most  of the  i n t h e p o s t e r i o r end o f  (CVP; fig.36b) w i t h  Contraction  pm  than  no  s e e n i n t h e a n t e r i o r e n d (CVA;  sutures  were  Phase:  Between length  - (Cont'd.)  contraction fig.36a).  a n t e r i o r and p o s t e r i o r (figs.34b and  i n the length  to that  35a).  of the  found i n wild-type  cells. i i i .  Fission-Furrow T h i s phase cell  cycle.  reached occurred b) w i t h This  Phase: began  A mean  b y a g e 0.98. i n regions  a t a g e 0.95  cell  length  Most CVA'  o f 138  pm w a s  o f the growth  a n d CVP'  some g r o w t h i n r e g i o n  pattern  i n t h e dc2 a  (figs.3?a  CVINT  o f g r o w t h was i d e n t i c a l  and  (fig.38b). to that  67  THE  MORPHOGENETIC P E R I O D I N MUTANT L I N E S :  i i i .  F i s s i o n - F u r r o w Phase:  i n wild-type cells.  cells  were about  type  counterparts Patches  iv.  pm  were a l s o  cells  (fig.23).  O.98  A t age  shorter  dc2  than their  a  wild  (fig.34a).  similar  cells  (Cont'd.)  -(Cont'd.)  found  12  -  t o those seen  found  on  the cortex  on  dc4  of  dc2  Stomatogenesis: Stomatogenesis d u r i n g the  first  i n dc2  cell  cycle  c e l l s was at the  normal  restrictive  temperature. v.  B a s a l Body The  change i n numbers and  spacing of  basal  bodies throughout the morphogenetic  period  similar  (figs.39a  and vi.  Proliferation:  to that  i n wild-type cells  was  b).  Width: Throughout tion  phases  dc2  the i n i t i a l  growth  (fig.35b).  At  however, the w i d t h s were i d e n t i c a l rapid  contrac-  c e l l s were c o n s i s t e n t l y  than wild-type c e l l s  A  and  age (47  wider O.95  p.m).  decrease i n width occurred during the  fission-furrow  phase.  68  THE MORPHOGENETIC vii.  Contour  P E R I O D I N MUTANT LINES;. Drawings:  Contour'drawings indicate surface polar viii.  that  regions  i s a slight  and of  reduction i n  prolonged,  Surface: i n dc2 a c e l l s  b y k0%  cells  while  length  the furrow there  follows the  as i n wild-type  of their they  These  original  cells.  i n dc2 a c e l l s  i s no n e t growth  regions  length i n  b y ?0fo  increase  i n .wild-type  surface  cells (fig.  m i d - r e g i o n s (CVA'  CVP') i s n o t i c e a b l y r e d u c e d .  original of  i s  but growth i n the c e l l  increase dc2  trend  cells  the contraction of the  of the c e l l  growth  same g e n e r a l  and  ( f i g . 3 3 ) o f dc2  growth and t h a t  Surface  ix.  there  Growth o f t h e Dorsal  25b)  (Cont'd.)  of  their  The  growth  i s normal,  of the polar  regions  the c e l l ,  Growth of t h e V e n t r a l  Surface:  B e t w e e n a g e s 0.93  a n d 0.95 i n both  vestibule  migration  was e v i d e n t  t h e p r o t e r and  opisthe.  Growth o f t h e p o s t e r i o r suture  p r o t e r and t h e a n t e r i o r suture began a f t e r  a g e O.95  (table  9).  i n the  of the opisthe  69  Continuous heat treatment throughout the c e l l c y c l e mutant and w i l d - t y p e  c e l l s was  necessary f o r consistency  of of  experimental d e s i g n when morphometric s t u d i e s of the mutant phenotypes were c a r r i e d out. the t e m p e r a t u r e - s e n s i t i v i t y d u r i n g the c e l l  cycle.  d e t e r m i n i n g the  of the mutants v a r i e d  temporally  found, i t might have proven  valuable  o r i g i n of the mutant phenotypes.  iments were t h e r e f o r e  c a r r i e d out t o determine the  of t e m p e r a t u r e - s e n s i t i v i t y wild-type  p o s s i b l e however, t h a t  I f a temporal v a r i a t i o n i n temp-  e r a t u r e - s e n s i t i v i t y was in  I t was  and mutant  cells.  throughout the c e l l  Experpattern  cycle  of  70  SECTION C:  1.  THE PATTERN OF TEMPERATURE-SENSITIVITY DURING THE CELL CYCLE:  INTRODUCTION: T h i s s e c t i o n w i l l examine the  e f f e c t o f heat-shocks  on synchronous p o l u l a t i o n s of w i l d - t y p e and cells. two  This p a r t of the study i s designed to answer  questions:  erature  mutant  (a) i s t h e r e  s e n s i t i v i t y during  a temporal p a t t e r n the  cell  does t h i s d i f f e r i n w i l d type and does the p a t t e r n  of temp-  c y c l e , and  i f so,  mutant l i n e s , and  of temperature s e n s i t i v i t y i n the  mutants c o r r e l a t e with the v i s i b l e m o r p h o l o g i c a l in  the mutants?  i s a heat-labile division  p r o t e i n that i s e s s e n t i a l f o r c e l l  review).  1971  or Zeuthen and  d i v i s i o n (see  Rasmussen, 1971  Heat-shocks a p p l i e d d u r i n g  presumably d e s t r o y  t h i s p r o t e i n and  in  The  cell division.  d e s t r o y e d by  defects  S i m i l a r experiments with Tetrahymena  have suggested t h a t t h e r e  Mitchison,  the  some time i n the c e l l  c e l l must r e p l a c e  c y c l e the  p r o t e i n i s s t a b i l i z e d and  i s no  cell  for a cycle  result in a  the heat shock b e f o r e i t can  the  delay  protein  divide.  At  Tetrahymena d i v i s i o n longer  heat-sensitive.  Temperature-shocks a p p l i e d a f t e r t h i s time produce d i v i s i o n delay.  (b)  The  no  time at which the p r o t e i n i s  s t a b i l i z e d i s r e f e r r e d to by M i t c h i s o n  (1971) as  the  71  INTRODUCTION: transition  -  (Cont'd.)  point.  EXPERIMENTAL DESIGN: • Synchronous as  described  •  samples  above  of log-phase c e l l s  (see m a t e r i a l s  w e r e h e a t - s h o c k e d f o r 30  samples  during the c e l l  cycle.  (The  was  determined from a sample  The  division  mined  by  samples was  comparing with  that  d e t e r m i n e d by  1968).  Cameron, cells  The  once  d a t a were  cells  which  plotted  length  given  and  the This  cell  had  cells  cycle  involved  paper  as  time.  40.  as  Thus,  cycle  example  length  The  long  of  deter-  heat-shocked  cell  cycle  length  (Natchway  sample  and of  at ten minute • sample.  the percentage  t e n minute  of  interval  The  median  cell  were r e a d  of a probability  cycle  directly plot  i s  of e x p e r i m e n t a l groups  cell  and  a cumulative percentage  a percentage of the a 120$  length  heat-shocked.)  begun i n t h a t  i t s standard deviation  cell  length  times  c o u n t i n g t h e number  had  a t each  not  method"  synchronous  division  cycle  a h e a t - s h o c k was  "shortcut  against  i n figure  length.  cell  divided  An  calculated  by  The  a t known  cell  was  then transformed i n t o  the graph.  The  control  of the control.  on p r o b a b i l i t y  divided  from  the  p r e s e n t i n each  intervals  of  delay produced  isolated  and methods). minutes  that  were  control  cycle  cell  i s 20$  was  cycle  longer  than  72  2.  EXPERIMENTAL the  DESIGN:  control.  length  (Cont'd.)  I n a l lcases t h e c o n t r o l  was d e t e r m i n e d  heat-shock varied 3.  -  a t 27°C.  cells  34.5°C a n d 35°C. little the  percent  were t e s t e d  AND MUTANT C E L L S :  with heat-shocks o f  of thecell  (fig.4la).  a t a g e 0.6.  a g e 0.7  of the  T h e 34.5°C h e a t - s h o c k s p r o d u c e d  very  age a t t h e time o f  The maximum d e l a y was f i v e  A transition  w h e r e b e t w e e n a g e s 0.6 after  The t e m p e r a t u r e  OF W I L D - T Y P E  delay regardless  heat-shock  cycle  among t h e e x p e r i m e n t s .  TEMPERATURE-SENSITIVITY Wild-type  cell  a n d O.65  point  occurred  and cells  some-  heat-shocked  showed no d e l a y .  Heat-shocks  a t 35°C r e v e a l e d  a definite  pattern of  temperature  sensitivity  i nt h e Paramecium  (fig.4la).  Very  d e l a y o c c u r r e d u p t o a g e 0.2  but in  after  that  little  i tremained  age  O.65  at this  value u n t i l  o f 30 m i n u t e ,  been examined by Whitson 23$ a t a g e 0.9.  later  increase  a g e w a s 20$  a g e O.65. t o about  After 5$-  36°C h e a t - s h o c k s h a s  (1964).  The t r a n s i t i o n  considerably  shocks  The d e l a y a t t h i s  the delay decreased r a p i d l y  The e f f e c t  0.92,  cycle  t i m e t h e r e was a r o u g h l y l i n e a r  d e l a y u p t o a g e 0.5.  and  cell  The maximum d e l a y was p o i n t was f o u n d  than that  found  a tage  f o r 35°C h e a t -  ( a g e O.65).  The p a t t e r n  o f temperature  sensitivity  o f mutant  73  TEMPERATURE-SENSITIVITY OF WILD-TYPE AND  MUTANT CELLS:  ( Cont'd.) dc5 to 34.5°C heat-shocks was for  s i m i l a r to that found  w i l d - t y p e c e l l s at 35°C ( f i g . 4 l b ) .  Both the  maximum delay (120%) and the t r a n s i t i o n p o i n t (age were i d e n t i c a l .  O.65)  In dc5 c e l l s however, heat-shocks  a p p l i e d a f t e r the t r a n s i t i o n p o i n t decreased the c y c l e l e n g t h by as much as 10$.  cell  T h i s was not the case  i n wild-type c e l l s . V a r i a n t 48 c e l l s showed a constant delay o f seven percent up to age 0.3 0.45  (fig.42a).  and  the d e l a y produced by 34.5°C heat-shocks i n c r e a s e d  up to a maximum o f 16%. at  Between ages 0.3  age 0.7  The t r a n s i t i o n p o i n t o c c u r r e d  with c e l l s heat-shocked a f t e r age  showing c e l l  cycle  Mutant dc4 was  0.82  acceleration. the only l i n e examined that had a  c l e a r bimodal p a t t e r n o f t e m p e r a t u r e - s e n s i t i v i t y ( f i g . 42b).  Two  t r a n s i t i o n p o i n t s o c c u r r e d i n the c e l l  The f i r s t was a t age 0.2 Between ages 0.3 did  and 0.45  and the second a t age and a f t e r age 0.7  not produce s i g n i f i c a n t Two  cycle.  0.7.  heat-shocks  delay.  experiments were done with sm2  c e l l s and both  i n d i c a t e d a t e m p e r a t u r e - s e n s i t i v e p e r i o d extending from age O.35  to age O.65  (fig.43).  There a l s o appeared to  be a second h e a t - s e n s i t i v e p e r i o d b e g i n n i n g at age  O.85.  74  3.  TEMPERATURE-SENSITIVITY ( As the be  OF W I L D - T Y P E  AND  MUTANT C E L L S :  Cont'd.) no d a t a extent  are available of this  determined.  for cells  older  temperature-sensitive  than  age  period  O.85  cannot  SECTION CORTICAL  D:  PATTERNING  76  SECTION D;  1.  CORTICAL PATTERNING:  INTRODUCTION: The  Paramecium c e l l r e p l i c a t e s the  highly-ordered  a r r a y of o r g a n e l l e s on i t s c o r t e x at each c e l l  division.  The mechanisms which determine the l o c a t i o n and  orien-  t a t i o n o f newly-forming s t r u c t u r e s c o u l d operate  in  ways:  distance  by p o s i t i o n i n g a new  s t r u c t u r e at a f i x e d  from a r e f e r e n c e p o i n t or by a s s e s s i n g the between two new  r e f e r e n c e p o i n t s and  two  distance  then p o s i t i o n i n g the  s t r u c t u r e at a l o c a t i o n t h a t i s a f i x e d f r a c t i o n o f  t h a t l e n g t h from one  of the r e f e r e n c e p o i n t s .  These  models o f c o r t i c a l p a t t e r n i n g can be c a l l e d f i x e d  two  dis-  tance assessment and p r o p o r t i o n a l d i s t a n c e assessment respectively.  The  f i r s t model operates  to the c e l l l e n g t h , while dependent.  without  regard  the second model i s l e n g t h -  In o r d e r to d i s t i n g u i s h between these  models, t h e r e f o r e , i t i s n e c e s s a r y  to examine the  p o s i t i o n s of c o r t i c a l s t r u c t u r e s i n paramecia o f ferent length.  T h i s study  two  dif-  w i l l attempt to d i s t i n g u i s h  between these models by examining the l o c a t i o n o f c o r t i c a l s t r u c t u r e s i n s m a l l c e l l s produced by the gene.  sm2  The r e s u l t s of t h i s study w i l l be d i s c u s s e d i n  r e l a t i o n t o r e c e n t models f o r p o s i t i o n a l in ciliates  ( F r a n k e l , 197k;  determination  Sonneborn, 1974a, 1975).  77  2.  EXPERIMENTAL  DESIGN;  Asynchronous cultures  o f sm2  cells  were h e a t e d  incubator.  Four  after  1 1 . 5 a n d 24 h o u r s .  6 , 8,  chronous  The  cultures  s a m p l e o f sm2  permissive  3 .  log-phase p e t r i - d i s h ( 1 5 x 1 0 0  fixed  cells  temperature cells  were  was  used  i n an a i r  and were  fixed  An a d d i t i o n a l  from  a culture  f i x e d and u s e d  asyn-  kept  as a  at the  control.  silver-impregnated.  M E A S U R E M E N T S; The  positions  of the c o n t r a c t i l e vacuole  (CVPs) and t h e v e s t i b u l e the  ends o f t h e c e l l  were used was of one  each  was  not.  The  kineties  cytoproct  w e r e made  and 24 h o u r  o f measurements  ( f i g . 4 4 ) .  of basal  sample of  cell  were  on  sample  made,  bodies  The m e a s u r e m e n t  and data  i n table 1 0 .  ANALYSIS:  A probit determine  plots  of the  1 1 . 5 and 24 h o u r  o f t h e numbers  on t h e c e l l s  listed  PROBIT  6 , 8,  series  tallies  length  cells  i n r e l a t i o n t o the ends  For a separate control  including  to  (Only i n t e r f i s s i o n  These measurements  of a control,  a more e x t e n s i v e  are  ( f i g . 4 4 ) .  i n the analysis.)  the c e l l  pores  were measured i n r e l a t i o n t o  measured b u t i t sp o s i t i o n  cells.  4 .  were  to 35°C  m.m.)  analysis  was  performed  on a l l v a r i a b l e s  i f t h e y were n o r m a l l y d i s t r i b u t e d .  of length  and w i d t h a r e shown i n f i g u r e s  Probit 4  5 and  TABLE 10 SIZE-DEPENDENT CHANGE IN sm2 CELL DIMENSIONS VARIABLE  DIMENSION ( i n um) WITH STANDARD ERROR  b  TIME a t 34.5°C LENGTH WIDTH L/W RATIO CVA CVINT CVP GA G GP CYTL n #b.b. £ um/b.b. _ KINETIES KTCV KTCL KTCVV . KTCV/TOTAL C  d  e  g  0 hrs. 123+1.2 50+0.6 2.5+.05 41+0.7 58+0.8 23+0.6 58+0.8 15+0.6 ' 50+O.7 19+0.4 44 35+2.2 1.5+.04 72+1.6 39+2.1 3311.6 1+0.1 5312.6  6 hrs.  8 hrs.  11.5 h r s .  24 h r s . 92+1.1 31+0.6 3.1+.10 32+0.6 36+O.9 23+0.5 46+0.6 ' 14+0.3 . 32+0.7 12+0.4 53  111+1.6 39+0.6 2.9+.07 38+O.7 48+0.9 2510.5 51+0.9 17+0.4 43+0.9 18+0.4 ' 54  112+1.3 45+O.6 2.5+.05 38+O.8 44+0.8 28+0.5 52+0.7 17+0.3 43+O.8 18+0.4 46  101+1.5 34+0.4 3.0+.07 35+0.9 40+0.8 26+0.5 50+0.8 17+0.5 34+0.8 15+0.4 44  •a-  #  *  •8-  *•a*  #  • H -  •*  #  *  #  •*  • H • H -  #  a  24 h r s  ;  80+1.6 40+0.6 2.0+.11 29+0.8 28+1.1 24+0.8 38+1.1 14+0.4 28+1.2  *  47 23+6.0 1.2+.25 72+0.4 41+0.5 30+0.4 1+0.3 58+1.0  CO  TABLE 10 - (Cont'd.) SIZE-DEPENDENT CHANGE IN sm2 CELL DIMENSIONS  * abcdefgh-  n o t measured i n t h i s sample two s e p a r a t e 2k hour samples (from separate experiments) are r e p o r t e d see f i g u r e kk f o r e x p l a n a t i o n o f t h e a b b r e v i a t i o n s used r a t i o o f l e n g t h t o width i n a r b i t r a r y u n i t s sample s i z e number o f b a s a l bodies i n r e g i o n CVINT s p a c i n g between b a s a l bodies the t o t a l number o f k i n e t i e s on the c e l l KTCV expressed as a $ of the t o t a l number of k i n e t i e s .  VO  80  PROBIT ANALYSIS: 46.  (Cont'd.).  Other p r o b i t p l o t s are not shown but i n t h e i r  d e s c r i p t i o n s of the d i s t r i b u t i o n s obtained  place  through  p r o b i t a n a l y s i s are g i v e n i n t a b l e 11. Length was sample and  one  normally  d i s t r i b u t e d i n the c o n t r o l  of the 24 hour samples.  showed a mixture o f two n o r m a l l y In cases where l e n g t h was  A l l others  distributed  bimodally  populations.  distributed  other  v a r i a b l e s f o r t h a t sample were not n e c e s s a r i l y b i modally d i s t r i b u t e d . to  be n o r m a l l y  The  f o l l o w i n g v a r i a b l e s tended  d i s t r i b u t e d i n those  modal l e n g t h d i s t r i b u t i o n :  GA,  samples with a b i -  G, GP,  WIDTH ( a b b r e v i a t i o n s are l i s t e d  CYTL, CVP,  i n f i g u r e 44).  and Most  of the b i m o d a l i t y i n l e n g t h arose from b i m o d a l i t y i n the l e n g t h of r e g i o n s CVA l a t e r , i t was  and  CVINT.  As w i l l  be shown  i n these r e g i o n s where the m a j o r i t y  the l o s s i n c e l l l e n g t h o c c u r r e d d u r i n g the heat TEMPERATURE EFFECT ON  sm2  c e l l s decreased  d u r i n g c u l t u r e at 35°C ( f i g . 4 7 a ) . d u r i n g the  Most of the l e n g t h decrease r e g i o n of the c e l l  (GP;  The  linearly  c e l l length  was  24 hour heat p e r i o d .  o c c u r r e d i n the p o s t e r i o r  f i g . 4 8 a ) , or more s p e c i f i c a l l y ,  i n the p o s t e r i o r p a r t of the c e l l mid-region Region CVP  shock.  CELL LENGTH:  The mean l e n g t h of sm2  reduced by about 35$  of  ( f i g . 4 8 d ) and  (fig.49a).  the v e s t i b u l e ( f i g . 6 )  did  81  TABLE THE  TYPES  11  OF D I S T R I B U T I O N S  OBTAINED  FROM A P R O B I T A N A L Y S I S OF MEASUREMENT  VARIABLES  T I M E A T 34.5 °C MORPHOMETRIC PARAMETER LENGTH GA G GP CYTL CVA CVINT CVP WIDTH KTCV  (HOURS)  a  0 N N SL N N N N N N na  6 BM N N BM N BM N N SR na  8 BM N N N LK N SR SR N na  11.5 BM N N N LK BM BM BM LK na  24 BM BM N N N BM BM SR N na  24  b  N N N N na BM BM N N N  ( A B B R E V I A T I O N S : N = n o r m a l l y d i s t r i b u t e d , BM = b i m o d a l l y d i s t r i b u t e d , S L = s k e w e d l e f t , SR = s k e w e d r i g h t , L K = leptokurtic, na = not applicable) a b  - These measurement v a r i a b l e s a r e d e f i n e d i n f i g u r e - Two i n d e p e n d e n t 24 h o u r s a m p l e s w e r e e x a m i n e d .  44.  82  T E M P E R A T U R E E F F E C T ON not  change  other  regions  During cell  i n length  further ratio  12  increased  from  the heat p e r i o d  50  t o 34 jum a f t e r  t o 3*1  while a l l  ( f i g s . 4 8 and  (fig.47a).  2.5  (Cont'd.)  of the heat p e r i o d  from  occurred  LENGTH:-  i n length  hours  decreased  change  CELL  during  decreased  the f i r s t  width  sm2  The  during  4-9).  t h e mean which  length  no  to  the heat  width  period  (fig.47b). The to  •  cytoproct  i t sp o s i t i o n  regular  l e n g t h was  on t h e c e l l .  decrease  i n length  measured This  without  regard  s t r u c t u r e showed  throughout  the heat  a  period  (fig.49c). The mean n u m b e r o f k i n e t i e s control at  was  t h e same a s t h e n u m b e r f o u n d  35°C ( t a b l e  the  10).  CVPs d e c r e a s e d  (the  distance  body  spacing  f r o m 1.5  sm2  cell  35  t o 23  pm  slightly (table  P A R T S OF  of the reasons  cells  formed  from  decreased  THE L E N G T H S OF LENGTH:  in  The number o f b a s a l during  between t h e CVPs a l s o  /im t o 1.2  One  per c e l l  was  at fixed  THE  24  bodies  during  between  the heat  the heat  hours  period Basal period;  10). C E L L AS A F U N C T I O N OF  to determine  ( t h e CVPs, v e s t i b u l e ,  after  17°C  decreased).  f o r examining  distances  i n the  from  the size  whether c o r t i c a l reference  points  o r ends o f t h e c e l l  CELL  reduction structures on  the  could  be  83  THE LENGTHS OF PARTS OF THE CELL AS A FUNCTION OF CELL LENGTH: - (Cont'd.) used as r e f e r e n c e p o i n t s ) or whether the l o c a t i o n o f these  s t r u c t u r e s was determined with r e f e r e n c e t o the  l e n g t h o f the c e l l .  To d i s t i n g u i s h "between these two  p o s s i b i l i t i e s , the l e n g t h o f each c e l l r e g i o n was c a l c u l a t e d as a f r a c t i o n of the c e l l l e n g t h ( t a b l e 12) and was p l o t t e d a g a i n s t time a t 35°C ( f i g s . 4 8 and 49). The r e g r e s s i o n o f each v a r i a b l e a g a i n s t time was determined ( t a b l e 13)» and s i g n i f i c a n t r e g r e s s i o n  lines  were added to the a p p r o p r i a t e p l o t s . The  a n t e r i o r suture i n i t i a l l y made up 4?$ o f the  c e l l l e n g t h , the v e s t i b u l e , 12$; and the p o s t e r i o r suture, 41$ ( f i g . 4 8 b ) . occupied  The f r a c t i o n s o f the c e l l  length  by these r e g i o n s changed s l i g h t l y d u r i n g the  f i r s t 12 hours o f the heat p e r i o d , with the a n t e r i o r suture i n c r e a s i n g t o 50$ o f the c e l l l e n g t h , the v e s t i bule i n c r e a s i n g to 16$ o f the l e n g t h , and the p o s t e r i o r suture d e c r e a s i n g  t o 34$ o f the c e l l l e n g t h .  f u r t h e r change occurred period (fig.48b).  Little  d u r i n g the remainder o f the heat  There was no s i g n i f i c a n t  regression  of any r e g i o n o f the v e n t r a l s u r f a c e w i t h time a t the r e s t r i c t i v e temperature ( t a b l e 13).  The a n t e r i o r and  p o s t e r i o r sutures t h e r e f o r e occupied  constant f r a c t i o n s  o f the c e l l  length.  84  TABLE  12  MORPHOMETRIC MEASUREMENTS E X P R E S S E D AS  A F R A C T I O N OF THE C E L L L E N G T H - MEANS AND S T A N D A R D ERRORS  T I M E A T 35°C 2L  O  8  6 S x * MEAN S x  G/L GP/L  .01 .01 .01 .01 .12 .01 . 41 .01 .01 .02  •34 .44 .23 .46 .16 •39 .16 .41  (HOURS)  .01 .01 .01 .02 .01 .01 .01 .02  MEAN S x  • 34 .01 .40 .01 .25 .01 .4? .01 .15 .01 •39 .01 .16 .01 .41 .02  11.5 MEAN S x  • 35 .40 .26 .50 .17 • 34 •15 .44  * standard error of proportions + r a t i o s o f morphometric parameters abbreviations).  .02 .02 .01 .02 .01 .02 .02 .03  24  24  MEAN S x  MEAN S x  • 35 • 39 • 25 .50 .16 •34 .13 •39  (seef i g u r e  .01 .02 .01 .02 .02 .01 .02 .03  .36 •35 • 30 .47 .18 • 35  n/a n/a  44 f o r  .02 .02 .02 .02 .01 .02 n/a n/a  85  TABLE 13 LINEAR REGRESSIONS  OF CELL DIMENSIONS  AGAINST TIME AT THE RESTRICTIVE TEMPERATURE _  x  CVA/L CVINT/L CVP/L GA/L G/L GP/L CYTL/L LENGTH CYTL/GP  TIME TIME TIME TIME TIME TIME TIME TIME TIME  *  REGRESSION EQUATION y=33.4+0.97x y-45.2-0.88x y=20.3+0.90x y=46.5+0.56x y=13.6-0.80x y=40.0-0.?9x y=l6.1-0.45x y=83.5-0.92x y=40.3+0.04x  t s < t .05 (3) and the slope does not d i f f e r s i g n i f i c a n t l y from zero.  ts 0.10 -O.38 O.36 0.11 * 0.16 * -0.27* -0.11 -1.05 0.01 *  86  6.  THE LENGTHS LENGTH:  -  OF P A R T S OF THE C E L L AS A F U N C T I O N  of the c e l l  C V P 19$. occurred  Most  38  length,  three  fraction but It  (fig.48d).  a n d 25$  significant  The  t o 39$  length.  appears  that  when c e l l  length  cytoproct  CORRELATION  length  at the r e s t r i c t i v e  a n d CVP i n c r e a s e d  to  length respectively. of the c e l l  length. a  Of  constant  T h e r e was h o w e v e r , a  o f CVA/L w i t h  time  small  (table  13)'  the p o s i t i o n of the anterior size  i s reduced  while  was c a l c u l a t e d a s a  and t h e p o s t e r i o r suture  occupied  suture  ( t a b l e 13 a n d  length  that  a constant  length. of the  but not of the c e l l  length  ANALYSIS: a n a l y s i s was p e r f o r m e d  measurement v a r i a b l e s t o determine varied  fraction  fraction  fig.49d).  A correlation  one  region  CVP p o s i t i o n s  CVA a p p r o a c h e d b e i n g  regression  cytoproct  the c e l l  posterior  7.  R e g i o n s CVA  and  occupied  t h e p o s t e r i o r CVP i s n o t . The  of  only  of the c e l l  CVP i s r e g u l a t e d of  C V I N T 47$,  eight hours  of the c e l l  regions,  therefore  CVA i n i t i a l l y  o f t h e change i n r e l a t i v e  R e g i o n CVINT d e c r e a s e d the  region  region  within the f i r s t  temperature occupy  CELL  (Cont'd.)  On t h e . d o r s a l s u r f a c e 34$  OF  together.  to determine,  This  which  a n a l y s i s would  f o r example,  f o r a l l parameters  therefore  i fa decrease  allow  i n cell  87  7.  CORRELATION ANALYSIS: l e n g t h was  number of  a  decrease  kineties  p r o d u c t moment c o r r e l a t i o n  Rohlf,  1969)  metric  parameters  for  the  tables,  for  a l l samples  which fore  the be  were  r^'s  of  change  the for  groups  parametric  the  and  morpho15.  In  are  given  manner  s h o c k may  in  there-  (Sokal  not  and  change  coefficients Rohlf,  1969).  significantly These  groups  coefficients  (p)  in and  are  listed  16.  correlation  of  correlations the  which  those  were l i k e l y  coefficient)  which  an  In was  correlation,  the  a  were  was  of  the the  magnitude  of  of  r^  appeared  significant which  r^  treat-  value i t  of  period  heat  cases  a more  value  heat  increasing latter  not  difficult.  declining  during  becoming the  which  period  effect  showed an  spurious.  correlation  part-whole  heat  showed  have been i n d i c a t i n g  the  heat  Matrices  of  (r^'s) and  a  (Sokal  and  correlation  correlation  Those c o r r e l a t i o n s  ment but  the  heat period.  homogeneous t h r o u g h o u t  that  of  did  the  Interpretation  may  lk  tables  experiments  homogeneity  during  table  (the  in  during  cell.  comparisons  coefficients  both  correlations  magnitude their  given  the  or  examined.  tested  Certain  are  on  i n width  coefficients  a l l pair-wise  correlation  Some o f  in  (Cont'd.)  a c c o m p a n i e d by  change i n the of  -  would  88  TABLE_14  __  WIDTH  LENGTH  0  6  .442 .598  H-5 24  .322  0 6 8  11.5 24  CVP  0 6 8  11.5 24 CVINT  0 6 8  11.5 -24 CVA  GP  0 6 8  11.5 24 0 6 8  11.5 24 0 6  GA  8 11.5 24 0 6  8 11.5 24  G  *  8  CYTL  GA •375 • 579  •353  •538* •517  •309 • 504  • 05  .606  6  .684** • 920 • 742  • 784* • 774 .812 • 722 .688 #  .480 •359 .429 •554 .829*  .881  • 787 .855 •719  -.361  • 316  • 386  • 758* .831 •734 .808 .648  .333  .300  • 574 • 544 • 542  •730 • 784  GP  .352 .478 .299  .685  .456  .684  .307 .328  .625 -753  .472 .626 — — .498 .524 . 5 6 5 • 719 . 3 4 3 .440 .652 .420 . 5 7 8  •557 -.436 • 436 •339 •375 -.424 .467  • 328 .286  .292 .543  .568  • 509 •758  •529  .508 .38I .327 .374  .690* .632 .487 .694 .717 . 4 6 2 .580 .307  • 768 . 3 2 9 .802 _^ s w \J  • 560 .453 .457  CVA CVINT CVP .461 .413 -374  .403  .665  • 310 • 420  .316 .549 .569  .344 . 4 6 0  .352 .326  ----  CYTL .342  .364  89  TABLE MATRIX  *  OF  14  -  (Cont'd.)  CORRELATION COEFFICIENTS  ( e x c l u s i v e o f t h e two s a m p l e s where body numbers were t a l l i e d . )  kinety  *  and  LEGEND: + = T I M E AT 3 5 ° C ; * = t h e c o r r e l a t i o n s t h e b o x a r e homogeneous; ** = t h e c o r r e l a t i o n s the box a r e n o t homogeneous. Blanks  indicate  non-significant  correlations.  basal  shown i n shown i n  TABLE 15 MATRIX OF CORRELATION COEFFICIENTS T BB  G  GA  0 2 4  KTCVV  LENGTH  GP  • 3 9 3 •5  8  CVA  CVINT  . 8 4 8 . 8 8 9 •5 5 8  . 7 2 8  7  0  •532 .434  2 4  KTCL  0 2 4  KTCV  0 TOTAL KINETIES 2 4 WIDTH  CVINT  0 2 4  GP  0 2 4  G  •6  • 7 1 5  • 5  7  .540 7  •333  KTCV  KTCL  KTCW  . 4 2 0 - . 6 1 8 . 2 8 2  . 7 6 3 - . 3 7 7  . 6 8 4  7 . 3 3 4  _  _  _  _  _  5 . 4 9 1 6 . 4 8 5  •2  7  9  _  _  _  _  _  _  • 577 .361  . 3 2 8  .452  . 4 9 2  _  . 4 2 2  . 4 1 8  . 4 6 1  . 6 2 2  .403  . 7 1 1 •  .852  . 5 7 6 —  . 3 2 6  . 7 3 6 . 7 8 2  .452  • 5 1 3  .530 • 592  • 570  . 4 7 7  . 4 8 1  _  _  _  _  . 6 2 9  . 7 6 3 . 5 5 4  0 2 4  GA  8  .529 .372  0 2 4  CVA  •5  .405  TOTAL KINETIES  —  . 3 6 4  . 3 4 3  0 2 4  WIDTH  . 4 9 6  0 2 4  CVP  . 4 8 6  .303  0 2 4  CVP  . 6 1 9  .650  0 2 4  . 6 2 1  VO o  TABLE 15 - (Cont'd.) MATRIX OF CORRELATION COEFFICIENTS  LEGEND:  Blanks i n d i c a t e n o n - s i g n i f i c a n t  correlations.  *  I n a d d i t i o n t o the c o r r e l a t i o n s l i s t e d , LENGTH and b a s a l body s p a c i n g a r e p o s i t i v e l y c o r r e l a t e d ( r - = .325) f o r the 2k hour sample and WIDTH and KTCV/TOTAL KINETIES a r e n o t s i g n i f i c a n t l y c o r r e l a t e d f o r t h i s same sample.  TABLE 16 PARAMETRIC  CORRELATION COEFFICIENTS  FOR THE HOMOGENEOUS SETS OF CORRELATION COEFFICIENTS FROM THE MATRIX IN TABLE 14.  CORRELATED PARAMETERS  p*  L - CVA L - CVP L - GA  . 8 . .821  L  ? 3  .ixlili  - G  L - GP L - WIDTH GP - CYTL  * parametric c o r r e l a t i o n  .730  . 341 ,iei  coefficients.  93  CORRELATION ANALYSIS: naturally those  be  high.  -  (Cont'd.)  For  the  c o r r e l a t i o n s whioh  o r may  not  have  purposes  of a n a l y s i s  were i n i t i a l l y  subsequently  high  deteriorated)  only  (and  may  were  considered. Both  the  significant length  and  the  that  the  decreased after also  low,  24  hours.  correlation  (but  suture  was  not  not  The  length the  CVP).  CVP.  correlated with  but  of  not  CVA  region  cell  length.  constant  f r a c t i o n s of  the  weakly,  but  r^  significant  CVA  and  CVA  nor  was  and  CVINT,  C V I N T was  a  had  Parameters  length, cell  of  correlated with  and  with  posterior  examined as GP  length  anterior suture  a n a l y s i s showed t h a t , were  homo-  The  not  the  was  correlated with  was  was  of  length  were not  width  and  length  p a r a m e t e r s GA, with  cell  magnitude  regions  cell  relationship  the  of the  a  correlated  of  parameters  length,  length.  correlated with  correlation  with  lengths  The  The CVA  but  period  when v a r i o u s  Cell  with  positively  heat  similarly  with  The  was  the  correlated with  had  v e s t i b u l e a l s o showed a  posterior suture,  during  CVINT  but  The  a n t e r i o r suture  of  p o s t e r i o r sutures  homogeneous c o r r e l a t i o n  ( t a b l e 14).  geneous, but of  a n t e r i o r and  as  weakly CVP.  was  seen  fraction a  of  consistent  CVP  did they  and  G  occupy  length. homogeneously,  correlated  9k  7.  CORRELATION ANALYSIS; with  cell  lated The  with  other  cytoproct  length the  length.  The  examined  total  with  suture  (homogeneous). obtained  length,  cell  similarly  KTCV was a l s o  number o f k i n e t i e s ,  width,  on  counterclockwise  the c e l l  from  length.  was  the vestibule to the f i r s t these  suggesting  that  increased  to maintain  t h e CVPs  a fixed  position  The number o f k i n e t i e s  lated  KTCV.  When K T C V w a s s m a l l  corre-  t h e two CVPs  meridian.  SCATTERGRAMS;  clarify  scattergrams variables.  three  when t h e  t h e two CVPs (KTCVV) was n e g a t i v e l y  To  corre-  and t h e a n t e r i o r  separating  BIVARIATE  para-  strongly correlated with the  t e n d e d n o t t o b e i n t h e same 8.  with  when t h e same  correlated with  circumference.  with  cell  ^he number o f k i n e t i e s  number o f k i n e t i e s on t h e c e l l shifted  CVINT.  These  as f r a c t i o n s o f the c e l l  ( t a b l e 15).  (KTCV) was  total  those  counterclockwise  variables.  correlated with  and s t r o n g l y c o r r e l a t e d  length  corre-  notably  number o f k i n e t i e s p e r c e l l  cell  length  counted CVP  l e n g t h was w e a k l y  agreed with  m e t e r s were  was more s t r o n g l y  morphometric parameters,  (heterogeneous)  findings  (Cont'd.)  The w i d t h  p o s t e r i o r suture  lated  -  the correlation  were c o n s t r u c t e d The p r i n c i p a l  axes  analysis, bivariate  f o r selected pairs of of the scattergrams  were  95  B I V A R I A T E SCATTERGRAMS; calculated Equations In  and added  c a s e s where  to the appropriate  are given  i n table  17.  t h e r e was no s i g n i f i c a n t c o r r e l a t i o n  anterior  a b o u t 47$  variables  suture  a line  was d r a w n  was p r e v i o u s l y  of the ventral  surface.  l e v e l on a b i v a r i a t e  through  (fig.50a) p a s s e d  through  points  and had s i m i l a r s l o p e  of the scattergrams.  principal  axes  f o ra scattergram  indicated  a lack  (fig.51b). vestibules often  abnormal The  length  level  of c e l l  t o the of the .  o f GP a n d c e l l  length  but there  length  and v e s t i b u l e  o f c o r r e l a t i o n between these t h a n 65 pm,  t h a n 15 pm  was a  long.  length variables  however, had  These v e s t i b u l e s  were  i n shape.  cytoproct (fig.49d).  CYTL a n d c e l l  of plotted  (fig.50b).  Cells smaller less  cell  The s l o p e  f o r GA a n d l e n g t h ,  intercept  Scatterplots  o f GA a n d  and i n t e r c e p t  axes  different  drawn a t t h e  the cluster  principal  similar to that  shown t o occupy  A line  scattergram  length  was  plots.  mean o f one o f t h e v a r i a b l e s . The  47$  (Cont'd.)  f o r the p r i n c i p a l axes  between two p l o t t e d the  -  occupied  about  P r i n c i p a l axes  length  ran parallel  15$  of the  cell  f o rscatterplots of and close  t o the  15$  (fig.51a). On t h e d o r s a l  surface,  region  CVA o c c u p i e d  about  96  TABLE 17 PRINCIPAL AXES OF BIVARIATE SCATTERGRAMS  Length  Length  Length  Length  Length  Length  EQUATION OF  T  _1 G*  GA**  GP*  CVA*  CVINT**  CVP*  0 6 8 11-5 24M1 24M2  PRINCIPAL AXIS ns  6.1 7.2 4.2 4.0 4.4  17.7 53.7 17.7 9.5 9.3  32.3+1.6 11.5+2.0 2.9+2.1 0.6+2.0 2.2+2.0 61.2+3.5  1-3 1.7 1.7 1.7 1.6 2.7  1.9 2.3 2.7 2.4 2.7 4.9  7.8+2.3 16.2+2.2 32.1+1.8 26.1+2.2 27.4+2.0 31.0+1.7  1.8 1.8 1.5 1.7 1.6 1.1  3-1 2.8 2.3 3.0 2.8 2.8  38.6+2.1 10.9+2.6 31.9+2.1 i 31.7+2.0 i Yt= 9.1+2.6 ± = - 5.0+2.9  1.6 2.2 1.6 1.6 1.9 2.1  2.7 3.2 2.8 2.5 3-7 4.7  1.4 1.7 1.5 1.8 1.1 1.3  2.7 2.2 2.5 3.1 1.6 2.0  2.2 4.3  5.9 11.2  1.8 4.2  2.2 20.3  Y = n  Yt= Y  0 6 8 11.5 24M1 24M2 0 6 8 11.5 24M1 24M2 0 6 8 11.5 24M1 24M2 0 6 8 11.5 24M1 24M2 0 6 8 11.5 24M1 24M2  CONFIDENCE INTERVALS FOR SLOPES OF PRINCIPAL AXES Lower Upper  i=  -46.3+9.1  - 9 8 . 1 + 1 2 . 7 YY:' -12.3+6.8 9-7+5-7 4.6+6.0  I= "  Y  Y  Y  l = " l  =  Y  =  Y  =  Y,  Y  11.8+1.9 Y 17.2+1.9 Y p 27.4+1.9 Y% 9.1+2.3 Y% 43.4+1.3 Y% 33.6+1.6 Y^ 0  Y  i=  l l l Y,= Y  =  Y  =  Y  =  48.0+3.3 Y „ Y^= - 4 4 . 9 + 6 . 3 Y Y, = ns Yt= 48.3+2.0 Y Y|=-l49.2+10.7Y^ Y^= ns * 2  9  97  TABLE 17 - (Cont'd.) PRINCIPAL AXES OF BlVARIATE SCATTERGRAMS  tl Length  CYTL**  CONFIDENCE INTERVALS FOR SLOPES OF PRINCIPAL AXES  EQUATION OF PRINCIPAL AXIS  T  Lower 0 6 8  11.5 24M1  Y, - ns I7.2+7.3 l 1.8+6.5 l 2.6+6.6 l 4.1+7.2 l n/a l= Y  =  Y  =  Y  =  Y  =  Upper  5.5 4.5 4.5 4.4  11.1 11.7 12.1 20.0  2  1.8  4.0  2  9-7  15-0  Y  Length Length  Width  BB  24M2 24M2  Y=  - 3.1+2.5 Y  44.9+11.8Y  1  SPACING BETWEEN BASAL BODIES  24M2  Y=  KTCV TOTAL KINETIES  24M2  Y,=. -46.3+1.0 Y,  ±  *  t h e slopes o f the p r i n c i p a l axes o f these two v a r i a b l e s are homogeneous throughout a l l o f the samples (0-24 h r s . ) .  **  the s l o p e s o f the p r i n c i p a l axes a r e not homogeneous.  n/a  not a p p l i c a b l e  ns  n o t s i g n i f i c a n t (there i s no c o r r e l a t i o n between the v a r i a b l e s , see t a b l e s 14 and 15)•  98  B I V A R I A T E SCATTERGRAMS: 35$  of the c e l l  level  length  plot  changes  other the  cell  in  that by  This  scatterplot 54b.  i n 1.4 o f one  ( r ^ =  against was axes  occupied  about  58$  the total  pm  scatterof  (fig.52b).  The  the remainder o f by t h e  formula;  describing  this  of a  scatter-  number i s g i v e n  w i d t h was  showed  accompanied  kinety. (as a percentage of the width  corre-  The f r a c t i o n o f t h e t o t a l  number  b y KTCV was  (table  10).  indicated  total  no  therefore W h e n KTCV  number o f k i n e t i e s  significant correlation approached  A  pm)  (fig.53a).  i n cell  and c e l l  0.05).  (22  o f the p r i n c i p a l axes  o f KTCV  number o f k i n e t i e s )  equalled  A line  length  The s l o p e  the addition  kineties  occupied  was i n -  a lack  o f width, and k i n e t y  A scatterplot  of  parameters  35$  (fig.52a).  (fig.48C).  c a n be d e f i n e d  and c e l l  an i n c r e a s e  lation  length  length  indicated  (CVINT)  at the  s i m i l a r t o t h e p r i n c i p a l axes  o f CVINT  figure  length  (0.35L + 22 pm).  e q u a t i o n was  A  length  these  region  length.  CVINT = L -  plot  i n cell  between  dorsal  o f CVA.and c e l l  t o be a c o n s t a n t  o f CVP a n d c e l l  correlation  A line  from t h e p r i n c i p a l axes  R e g i o n CVP a p p e a r e d despite  (Cont'd.)  (fig.48d).  on a s c a t t e r p l o t  distinguishable  -  t h e 58$  constant was  (fig.55a)  and  plotted there  ( r ^ = 0.71) a n d t h e p r i n c i p a l  level.  99  8.  B I V A R I A T E SCATTERGRAMS: A scattergram basal  bodies  (r^  scattergram smaller  parameters  between b a s a l  = O.38).  (Cont'd.)  of c e l l  between'the  between these correlation  -  length  CVPs i n d i c a t e d a c o r r e l a t i o n (fig.53b). body  The p r i n c i p a l of these  cells  tended  more c l o s e l y t o g e t h e r  and t h e number o f  T h e r e was a l s o  spacing  axes  and c e l l  their  (fig.54a).  length  f o rthe b i v a r i a t e  two v a r i a b l e s i n d i c a t e d t o have  a  basal  that  bodies  spaced  100  S E C T I O N E:  G U L L E T D E F E C T S AND  Mutant had  severe  sm2  cells  gullet  feeding  ability.  reduced  cell  defect  size might  in  solution  The  of  gullet  The  cells.  mean  paint were  subsequently  vacuoles, (quadrulus  defect  (type  length  o f sm2  on  mutant c e l l s the basis  The  sm2  a  the c e l l s  cells  formed  were  con-  dilute  i n c u l t u r e medium.  After  fixed  i n Champy's s o l u t i o n and  silver  impregnated. f o r t h e number o f length,  o r 3;  cells  plate  after  the length  4).  cells  cells  was  (table 18).  distributed  the wild-type  of  extent  the heat period  f o rwild-type a normally  distribution  cultures  and p e n i c u l u s ) , and t h e  while  was  i n a  1 , 2,  comprised  of length  bimodal length  as  cultures of  a t 34.5°C ( f l a s k  the c e l l  73 pm a s c o m p a r e d t o 1 1 1 pm The  Log-phase  m a t e r i a l was s c o r e d  food  the gullet  such  the f o l l o w i n g experiment  overnight  the c e l l s  parts  lines  their  be r e l a t e d t o s u c h  The n e x t m o r n i n g  silvered  paint-filled the  of phenotypes  therefore  a n d sm2  of red acrylic  samples were  variant  affected  by c e n t r i f u g a t i o n and resuspended  seven minutes the  which might have  problem  were grown  a water bath).  centrated  other  ability.  wild-type  lines  o f many  The a p p e a r a n c e  To e x a m i n e t h i s  both  and c e l l s  defects  i n feeding  done w i t h  FEEDING;  population had a  (fig.56).  fewer  food  vacuoles  than  d i dthe  101  TABLE 18 MORPHOMETRIC PARAMETERS - FOOD VACUOLE EXPERIMENT  LENGTH (pm-s. e. ) Wild-Type Cell  Length  Cells  sm2 C e l l s  111.0-1.6  73-3-1.6  Quadrulus  21.1-0.2  18.8-0.4  Peniculus  20.0-0.2  15.7-0.4  7.6-0.2  1.8-0.2  Number o f Food Vacuoles Number o f Food Vacuoles i n C e l l s with a: (a)  normal g u l l e t  7.6-0.2  3.6-O.3  (b)  type 1 g u l l e t  n/a  2.0-0.4  (c)  type 2 g u l l e t  n/a  O.6-O.3  (d)  type 3 g u l l e t  n/a  0.1-0.1  (na = n o t a p p l i c a b l e ; sample s i z e s - f o r w i l d - t y p e c e l l s 32, f o r . sm2 c e l l s 100).  102  S E C T I O N E; wild-type was  G U L L E T D E F E C T S AND cells  r e l a t e d to  Only  one  of  food  vacuoles  with  types  vacuoles. food  the  the  1  cells  during  the  2 gullets  E v e n sm2  cells  with  length  of  the  wild-type  tended  quadrulus  cells  to have  length  shorter  number o f  of both  c o n t a i n more  of  the  gullet  cells  the  gullets.  fewer  parts  was  food  number o f related,  not  gullet.  to  the  (table  formed  any  numbers  of  formed  food  fewer  correlated with  was  cells,  gullets  but  not  were  80 pm  long  correlated with  cells.  than  due  i n sm2  the  (fig.51a).  with  normal  size  cells  the  sm2  size  cells  with in  of  lengths  (fig.57b).  i n sm2  of  length  i n length  gullet  cells  from  The  numbers  was  d i s o r g a n i z a t i o n of the evident  tended  correlated  vacuole  reduction  the  cells  ones.  weakly  i n food to  Larger  smaller  only  contained  reduced  18).  Cells  less than  than wild-type  f a c t o r not The  cells  reduction  cells  formed  pulse.  gullets  Wild-type  however,  vacuoles  just  sm2  entirely  vacuoles  3 gullet  peniculus  wild-type  E v e n sm2  food  and  vacuoles  was  gullet  reduced  positively  vacuoles  The  not  some o t h e r  of the  food  numbers.  sm2  and  type  number  cells.  gullets  food  sm2  to  vacuole  while  the  normal  ( t a b l e 19).  i n length  The  was  a  a l s o had  The  cell  (Cont'd.)  seven minute p a i n t  than wild-type  constant  to  with  -  a d d i t i o n , the  d i s o r g a n i z a t i o n of  sm2  and  In  vacuoles  lengths in  ( t a b l e 18).  FEEDING:  The therefore  gullet, or  may  had  but  anatomy also  have  TABLE 19 MATRIX OF CORRELATION COEFFICIENTS . for DATA FROM THE FOOD VACUOLE EXPERIMENT  S  Number o f Food Vacuoles  Cell Length  Quadrulus Length  9  .300  .238  .357  .496  .831  PENICULUS LENGTH  .  24 QUADRULUS LENGTH  .9  .264  24  .387  ©  .682  24  .429  .623  CELL LENGTH  S = sample, + = w i l d - t y p e c e l l s , 24 = l e f t a t 35°C f o r 24 hours. Blanks i n d i c a t e  insignificant  sm2  correlations.  cells  104  SECTION Et  GULLET DEFECTS  contributed  t o t h e r e d u c t i o n i n t h e number o f f o o d  per  cell  bility  as s m a l l e r c e l l s  of losing  food  AND  FEEDING:  would have  vacuoles  -  (Cont'd.)  an i n c r e a s e d  at the cytoproct.  vacuoles proba-  105  DISCUSSION  In  this  study  mutants  have  been  organism. of  several  newly-described  used t o examine  The s t u d y  i s based  and s i z e  o f mutant  Morphometric  analysis  has been  size how  an e x a m i n a t i o n  cell  size).  discussed during ining the  A)  division, these  genetics  study  The r e s u l t s  analysis  approached from two v i e w changes  structures  i n cell  pattern (or  are related  of the morphometric study  to cell  and c o r t i c a l  aspects  i n this  cells.  of size-dependent c e l l  of cortical  i n relation  and w i l d - t y p e  of age-dependent  and an e x a m i n a t i o n the positions  morphogenesis  on a m o r p h o m e t r i c  t h e shape  points:  Paramecium  division,  cortical  patterning.  o f Paramecium  to  will  be  growth  Prior  t o exam-  morphogenesis,  however,  and p h e n o t y p e s o f t h e t e n m u t a n t s u s e d i n t h e  w a r r a n t a f e w comments.  G E N E T I C S AND  been  OF THE  MUTANTS:  T e n new m o r p h o l o g i c a l m u t a n t s  o f Paramecium  described  o f these mutants are  alleles of  PHENOTYPES  three  i n this  study.  a t t h e same l o c u s . types  fission-zone  The m u t a n t s  of morphological  formation  Two  defects;  include  have  examples  defects i n  ( d f zmutants, i n c l u d i n g  dfzl  and  dfz2), d e f e c t s i n f i s s i o n - f u r r o w c o n s t r i c t i o n ( d c mutants, and  including  defects  i n cell  dc2 , dc2 , dc3, dc4, dc5, a n d dc6), a  b  growth r e s u l t i n g  i n small  cells  106  G E N E T I C S AND P H E N O T Y P E S OF T H E MUTANTS: ( s m m u t a n t s , i n c l u d i n g sm2 mutants have (Sonneborn,  been p r e v i o u s l y 1974b).  tute  a new t y p e  with  a similar  ten of  described  o f Paramecium mutant  sm a n d d c  i n Paramecium  although  isolation  of only  one p a i r  of alleles that  of  the a l l e l e s  dc2  as  i n d i c a t i n g that for cell  i nthe g e n e t i c  a n d dc2 only  should  a few genes  division, study  were  such  carefully  that  included.  This  studies  were  selected  from  widely  similar  different  allelic  mutants.  on developmental mutants i n Paramecium  similarly Chen-Shan,  revealed  1969).  few cases This  i nTetrahymena, where  determination  of allelism  contrasts  sharply  (Whittle with the  a s u r p r i s i n g number o f t h e  developmental mutants a r ea l l e l i c  products  that  s e l e c t i o n would en-  Other  case  products  variants with  the p r o b a b i l i t y o f i s o l a t i n g  and  produce  as t h ev a r i a n t s  hance  have  Isolation  n o tbe i n t e r p r e t e d  as w e l l as v a r i a n t s with  phenotypes, were  number  b  a g r o u p o f 60 v a r i a n t s phenotypes,  among  a large  genes a f f e c t development i n Paramecium.  used  mutants  e t a l , 1976a).  developmental mutants suggests  essential  consti-  phenotype have been r e p o r t e d i n  a  A  Both  The d f z m u t a n t s , however,  Tetrahymena ( F r a n k e l , The  a n d sm3).  - (Cont'd.)  (Frankel,  e t a l , 1976a).  o f the a c t u a l numbers o f genes  are e s s e n t i a l f o r c e l l  division  whose  or other  107  G E N E T I C S AND  P H E N O T Y P E S OF THE MUTANTS;  developmental awaits  effects  t e n m u t a t i o n s examined on c e l l  arrangements gullet,  influence the  not,  morphology.  of organelles affect  These  similar  types  gradual  deterioration  This  results  affecting  cortex  vacuole were  These  and i n  heat-treatment.  containing  surprisdefects  results  to the c e l l  shape  cause o f t h e a l t e r e d  i n the present  study  a  have  organelles temper-  (presumably by membranes) and  affects  surface  from  phospholipids.  i n abnormal c o r t i c a l  shape p r o b a b l y  are  structure  at elevated  saturated  i n cell  on  L o e t a l (1976)  by g r o w i n g t h e c e l l s  and  t o appear) morpho-  pattern.  and t h e r e f o r e  cortical  The  the attachment of i s the  morphology.  i s abnormal i n a l l o f t h e Paramecium  examined  abnormal  pores  of the normal c o r t i c a l  i n a loss  cell  organelles  shape  (or first  the structure of c o r t i c a l  secondarily  primary  on t h e c e l l  abnormal arrangements o f c o r t i c a l  i n a medium  abnormal  They a l l produce  i n the mutants but r a t h e r r e s u l t  during prolonged  Tetrahymena  Tetrahymena  experiments.  of abnormalities  the primary  defects  ature  and  the structure of the cytoproct,  logical  in  (Cont'd.)  pleiotropic  i n a l l o f the mutants.  however,  produced  have  t h e number o f c o n t r a c t i l e  cell.  ingly  e v e n t s i n b o t h Paramecium  f u r t h e r mutagenesis and g e n e t i c  The  the  -  and one m i g h t  Cell  mutants therefore  108  G E N E T I C S AND  PHENOTYPES  assume t h a t t h i s organelles and  OF THE MUTANTS:  affects  the positioning  on t h e c o r t e x .  cortical  -  In addition,  (Cont'd.) o f new  both  p a t t e r n become i n c r e a s i n g l y  a c a u s a l connection between these  strongly  mutants  These m u t a n t s defective  dc4  not  primary  cycle,  of c e l l  and d f z 2  t o furrow  possible  prior  - a premature  formation.  to correlate  i n sm2 a n d d c 4 c e l l s ,  sensitive.  occur This  to bring growth  when t h e s e  cells  when c e l l  part of the  contraction time  The  occur  primary  at  times  are heatoccurs  grov/th  length.  I n sm2  normally  during  should  occurs during that part of the  when b a s a l b o d y p r o l i f e r a t i o n  i t i s  morphological  to i t s interfission  i s subnormal i n dc4 c e l l s .  -  fission-furrow,  heat-sensitivity  cycle  the c e l l  heat-sensitivity cycle  however,  sm2  prolonged  biochemical lesions.  I n dc4 c e l l s ,  that part of the c e l l  cell  any o f these  defects  cycle  -  At the present  with specific  the c e l l  dc2  i n the early  defects  during  morphometrically.  to formation of a growth  determined  d e f e c t s were:  b a s a l body p r o l i f e r a t i o n ,  - failure  prior  t h a t were examined  and t h e i r  contraction  cell  two d e f e c t s i s  p r i m a r y m o r p h o l o g i c a l d e f e c t s were  those  cell  temperature,  suggested.  The for  shape  abnormal  during continued growth a t the r e s t r i c t i v e and  cell  cells, cell  occurs.  109  A)  G E N E T I C S AND  PHENOTYPES  OF T H E MUTANTS:  B a s a l "body p r o l i f e r a t i o n i s d e f e c t i v e This  correlation  morphological destroys event of  defects suggests  concerned.  While  this  morphogenetic type,  cell  after  division  occurrence  cycle  developmental  does n o t a l l o w t h e n a t u r e  the hypothetical  biochemical  event.  i s always  A l lcell  a t or near  this  related  lines  during  to specific  examined  sensitive  (except i n mutant  of this  peak  division.  (wild  cells,  shocks  o r no d e l a y i n  sm2).  t o some p r o c e s s  i n mutant A peak  Heat  The u n i f o r m  i n heat-sensitivity  i n wild-type cells,  sensitivity  that  suggests  i s heat-  of increased heatand e s s e n t i a l  for cell  i n sensitivity to inhibitors  and  protein  synthesis also  and  Hanson,  1968;  occurs near  Suhama a n d H a n s o n ,  and i t i s p o s s i b l e  metabolic  a g e O.65.  age p r o d u c e l i t t l e  i t corresponds  1967)  treatment  dc4, dc5i sm2, a n d v a r i a n t 48) h a v e a p e a k i n  applied  that  and v i s i b l e  whether h e a t - s e n s i t i v i t y  events.  heat-sensitivity  cell  cells.  the heat  f o rthe  developmental  . I t i s not clear the Paramecium  i n sm2  i n t h e s e m u t a n t s t o be  i t does r e l a t e  to a specific  that  essential  the biochemical lesion  lesion  (Cont'd.)  between h e a t - s e n s i t i v i t y  some p r o d u c t  determined,  -  inhibitors  that  affect  both  a g e O.65  1971;  (Gill  Rasmussen,  heat-shocks  t h e same  o f RNA  and t h e s e  developmental  110  A)  G E N E T I C S AND event. O.65  PHENOTYPES  OF THE MUTANTS;  The o n l y m o r p h o g e n e t i c  i s the p r o l i f e r a t i o n  endoral start that  kinety  of oral oral  related  (Jones,  its  1976).  development.  the onset  of cell  between  documented  possibility O.65  oral  that  to  division.  development  to  suggest  are intimately  processes,  formation. defect  division i s  Tetrahymena, and  Only  such  those  either very  of separate  other  however.  than  mutants which or very  oral  other  cells) of  have  late  w o u l d be  a  i n .the expected  heat-sensitivity.  heat-sensitive periods c y c l e may  of the experimental  at  as p r e p a r a t i o n f o r  early  a n d sm2  The  In mutants,  d e v e l o p m e n t may m a s k  portion of the c e l l  resolution  of rela-  a l s o seems p r o b a b l e .  show m u t a n t - s p e c i f i c p e r i o d s  Detection  type  and c e l l  t o some p r o c e s s  f o r oral  oral  inhibitors  the h e a t - s e n s i t i v e period ending  c y c l e ( a s i n dc4  central  i n the  signals the  This  ciliate,  c a n n o t be e x c l u d e d ,  heat-sensitive  cell  division  or metabolic  i n Paramecium  heat-sensitivity  primary  event  I t i s tempting  i n another  i s related  development  furrow  This  development and c e l l  occurrence  age  o c c u r r i n g a t age  i n Paramecium and t h a t i n t e r f e r e n c e w i t h  tionship well  (Cont'd.)  o f new b a s a l b o d i e s  development by h e a t - s h o c k s delays  event  -  i n the  be beyond t h e  technique.  Ill  CELL  DIVISION: The  of  cell  results  obtained  division  i n wild-type  a number of  issues  Paramecium.  There  shape cell  and  size  division  concerning are  division.  also  be  These  binary  determine whether  cell  defects  and  those  similar  i n diverse  These  i n terms size  compared w i t h  mutants with  mutant  i n the  cell  the  p o s t u l a t e d mechanisms f o r c e l l results  of  an  that premature  or abnormally  ction  the  regions  dc  of  and  dfz  relation  polar  cell  phenotypes.  between b a s a l  g r o w t h i n mutant and study  leads  eration This model  precedes  result of  to the  The  and  is a  is in direct  surface  growth  of  from  process of  (dc  some  of  in study  prolonged  issue  cells.  contra-  examined  agent  of  c o n t r a d i c t i o n to Paramecium.  and  This  that basal  causal  in  cell  obtained  the  body p r o l i f e r a t i o n  conclusion  to  i s associated with  final  wild-type  should  division  indicate  to  control  data  part  few  cells  examination  this  a  changes  division  dfz mutants) then  The  allow  The  cell  relation  m e c h a n i s m s may types.  in  accompany  i n other  and  Paramecium.  and  of t h e i r  found  raise  series of  deserve  shape  analysis  cells  fission  changes which precede  i n Paramecium.  cell  and  a well defined  comments, p a r t i c u l a r l y  division  from a morphometric  i s  the  surface  part  body  of  the  prolif-  surface a  the  growth.  present  112  CELL DIVISION: a)  Shape and  of  the  cell  first  Both  of  o f new  those  not  cortical  changes then  a  Once  the be  soidal  Doerner  attain a  division,  contraction  is  i n the  also  of  1 9 5 8 )  cylindrical this  i n the  polar  evidence  (Doerder,  are Hanson  with  contracts.  polar  the  cell  appearance  These  a  size  stretching  and  i t assumes  which  a  a  shape  cylinder with  facilitates  cells,  including  and  newt  (Sawai,  shape p r i o r to  as  change of  ellip- -  suggested  shape  suggest  et  the  the  units.  centre  Tetrahymena r e s u l t s i n a division  study  available cortical  shape  regions to  only  then  ( 1 9 7 6 ) has  types  although  and  first  as  cylindrical  (Hiramoto,  last  K a n e d a and  organelles.  the  described  Other  than  of  loosely  division.  by  or  contracts,  ends.  the  in this by  associated  cell  of  also  involve  represent  compacting  attainment  urchin  reported  units  therefore  noted  i n length  events  are  Cycle:  fission-furrow formation,  increases  and  Cell  changes d u r i n g  cycle  Preceding  these  regions  size  cell  s i m i l a r to  ( 1 9 7 4 ) .  can  Changes D u r i n g the  shape and  quarter  .  (Cont'd.)  Size  The  very  -  the  the  cell sea1 9 7 6 )  eggs,  cell  i s mediated cell  rather  i n Paramecium. that  that  contraction  There in  shape change p r e c e d i n g  a l . , 1 9 7 5 ) . Although  the  cell means  113  CELL DIVISION: a)  -  (Cont'd.)  Shape and S i z e Changes D u r i n g - (Cont'd.) by  which  the  different for  cell  attaining  functional and  cell  tional and  types this  successful furrow  surface  the  cell  sufficient  to  The  complete  furrow  the  of dc4  occurred  was  i n dc2  change  that a  func-  contraction  and  lesions to  cells.  for  mutants d e f e c t i v e dc4)  cells  the  cell of  amount cycle  the  of was  fission  were not  able  despite normal  Two  such  and  e n h a n c e d and  furrow  noted  or  to  surface  f o r other possible defects  c o n t r a c t i o n i n the  cells  The  shape  cell  constriction  mutants.  contraction prior from  a  that these  search  cortical  same.  that relaxation  I n two  constriction  a  common t o b o t h found:  between  (dc2  these  purpose  contraction i s necessary  division.  fact  in  obscure.  to prove  i t i s evident  complete  l e d to  the  o c c u r r i n g during the  furrow.  growth  exists  constriction  growth  be  between t h i s  i t i s difficult  from  Cycle:  d i v e r s e , the  however, remains  division,  recovery  be  s h a p e may  relationship  cell  Cell  shape i s o b t a i n e d  may  relationship  division  While  in  cylindrical  the  and  defects  formation.  were prolonged  No  posterior polar only  Forces  limited produced  recovery region  recovery by  the  114  CELL DIVISION; a)  -  (Cont'd.)  Shape and S i z e - (Cont'd.) sustained cell  contraction of the polar  must i n t e r f e r e  associated Growth and  with  the furrowing  defect  cause  i n surface  decreases  cell  i n width.  particularly  fore  with  furrow  cell  stages  of cell  sustained cell. the  from  rather  during  i s sharply division.  region  than  which  cleavage  the  i n l e n g t h and  the cell  furrow  impeding  constriction  diameter and  needed  be e x p e c t e d  furrow  an  under-  the width  to indirectly  reduction interfere  I n dc4 c e l l s t h e r e d u c t i o n attenuated T h i s may  constriction,  would  there-  t o complete  i n the l a t t e r be due t o t h e  contraction i n the p o s t e r i o r pole  During  furrow  results  are poorly  as i taids  completion.  width  i n dc c e l l s  The d e c r e a s e I n w i d t h i s  Anything  therefore  furrow.  The m e c h a n i s m s  that  t h e amount o f s u r f a c e  might  in  therefore  increases  reducing  constriction.  i s normal  growth.  important  effectively  of the f i s s i o n  t o move i n w a r d  both  of the  the forces  t o move i n w a r d s  but i ti s obvious  Paramecium  by  defect  Cycle;  regions  or equalize  surface  of the furrow  the furrow  stood,  with  constriction  o f the furrow  inability a  Changes During; t h e C e l l ~~  be f o r c e d  cytoplasm outwards,  of the from towards  115  CELL DIVISION; a)  -  (Cont'd.)  Shape and S i z e - (Cont'd.) the  cell  poles.  regions, furrow  of  Continued  t h e furrow.  reduction  during  cells  throughout  width  decrease  sustained influx  with (Dryl  i n dc2  cycle.  cells cells.  ions,  l e s i o n s produced  detergent  similarly  wild-type  i n relation  dc mutants  t h e increased and by allowing an  a r e known t o s t i m u l a t e  ( K a m a d a a n d K i n o s i t a , 19^5)• by t r e a t i n g paramecia result i ncell  contraction,  a n d M e h r , 1976). The  abnormal behaviour  restrictive  temperature,  o f dc4 c e l l s  with  frequent  of  t h e d i r e c t i o n o f s w i m m i n g , may a l s o  to  thecortical  have  o f width  In addition, the  I n both  which  o f paramecia  than  i sdelayed  contraction, probably  o f calcium  Cortical  areas,  the extent  arewider  l e s i o n s may p r o m o t e  contraction  from the  further constriction  cells  the cell  i nwild-type  cortical  cytoplasm  c o n s t r i c t i o n o f t h e furrow i s  b u t dc2  that  contraction of the polar  blocking  I n dc2  cells  to  Cycle;  from moving i n t o t h e p o l a r  indirectly  normal,  the Cell  however, would prevent  region  thereby  Changes D u r i n g  shown t h a t  lesions.  reversals be r e l a t e d  Kung and E c k e r t  Paramecium b e h a v i o u r a l  at the  (1972)  mutants  have  116  CELL DIVISION; a)  -  (Cont'd.)  Shape and S i z e - (Cont'd.)  Changes D u r i n g the  defects  i n the  ability  conduct  specific  The  cortical  with  ion  flux  across  i n fission-zone also  tion.  At  the  restrictive  do  form  undergo  or  furrow.  different dividing  Their  of  or  the  This  to  membranes.  initiation  furrow.  polar  the  when  contrac  contraction  not  form  form a  cells  and  i t i s possible  of  obstacle  to  fission-  quite  normal  that  the  rounded  furrow  formation. In is  dfz2  associated  while  a polar  cells, with  a polar rounding  contraction  at  contraction of  the  age  cell  0.95  in  is  width.  outline is  cylindrical  at  time  cells rounded  regions  cycles  in cell  do  These  cell  increase  structural  of  0.15  premature  the  a  interfere  cells  from  shape poses  cell  dfz2  a b o u t k-S m i n u t e s ) p r i o r  These h i g h l y - c o n t r a c t e d  the  temperature,  (or  zone  and  to  contrac-  contraction  a marked  of  surface  cycles, approximately  a c c o m p a n i e d by  out  also  fission-zone  occurs.  membranes  cell  0.8  normally  Cycle;  abnormal  age  tion  cell  a  the  r e l a t e d to  either a  or  formation  are  cells  surface  possibly  furrowing  not  the  either into  l e s i o n s may  normal  Defects  ions  of  Cell  at  age  0.8  outline, wild-type  117  B)  CELL DIVISION: a)  -  (Cont'd.)  Shape-and S i z e Changes D u r i n g - (Cont'd.) cells  i s associated with  cylindrical structural furrow that  region  this  prevents  a g e 0.95-  by  age  cell  outward  o f the c o r t e x i nt h e  "between a g e s  cell  that  rounding  0.8  a n d 0.95  b a s a l bodies, l y i n g  bulging of the c e l l a reinforcement  i s complete  Hanson  how  perpendicular to r e s i s t a n c e t o an  i n the furrow parallel  i s required.  and  during contraction  B a s a l body p r o l i f e r a t i o n  of the c e l l  region.  to the long  Sundararaman and  (1976) h a v e r e c e n t l y d e m o n s t r a t e d t h e  presence  of longitudinal  Paramecium  cortex  microtubules division  cortical  microtubules  during  cell  a p p e a r a t a g e 0.9 i s complete  reinforcement  traction.  This  i nthe  division.  tubules  by low temperature  in  rounding  until  a n d may p r o v i d e t h e during cell  conclusion i s supported  that disruption  These  and p e r s i s t  necessary  finding  cell  suggests  s u r f a c e , would provide  Logically,  cell  This  a  0.95» b u t i t i s d i f f i c u l t t o i m a g i n e  additional  axis  shape.  occurs  Cycle:  the attainment.of  reinforcement  at  the  cell  the Cell  by t h e  of the longitudinal or colchicine  con-  micro-  results  (Sundararaman and Hanson,  1976).  118  CELL DIVISION: a)  Shape -  and  -  (Cont'd.)  Size  Changes D u r i n g the  division,  however,  compare the  effects  examine the  during specific  and  i t would  of t h i s  cells  cell  d i v i s i o n , after  contraction  The  contractions  both  with i t s effects  prefission cell  initiation  cytokinesis. the nature the  changes  cell  similar  i n this  of c e l l s ,  contracduring  expansions  has  found  of  for  of  not r e v e a l e d  trigger that  a  or  cause  careful  i s warranted  i s t o be p r o p e r l y  that,  since  shape  i n Paramecium  occur  Paramecium p r o v i d e s a function  of  model  prefurrow  changes.  Growth:  Proliferation portion  on  completed.  i n Paramecium organism  of  interesting  completion  study  indicates  to those  stages  later  and  indicate  f o r a n a l y z i n g the  shape  Surface  processes  effects  therefore critical  this  i t does  I t also  other types  organism  i s  of the mechanisms which  division  understood.  in  Although  of these  cell  cells  o f f u r r o w i n g and  contractions,  study if  regions are  on  be  treatment  ting  the p o l a r  b)  Hanson d i d not  microtubule disruption  cell to  Cycle:  (Cont'd.)  Sundararaman and of  Cell  of the  of b a s a l bodies  Paramecium  cell  i n the  i s completed  central prior  119  CELL DIVISION; b)  Surface to  the  This  - (Cont'd.)  Growth; start  o fsurface  suggests  prerequisite is  - (Cont'd.)  that  basal  f o r surface  s t r o n g l y supported  mutant (sm2  cells  fact,  with  dc4)  and  there  and  sequently bodies  stimulate  by the  occurs. they  the  act  bodies  surface  growth.  and  a s K a n e d a and  fibrillar f o r the  that,  other  o f these this  o fmutant  of Hufnagel  body a s the  i n the  the  here cells,  around units to  kineties, the  cortex suggested,  cortex. comes n o t but  (1969) a n d  central  sub-  which  model  (1974) h a v e  Hanson  l a y e r underneath  examination  basal  t o surface  structures  to  the  when new b a s a l  growth resides  model presented  observations  the  between  growth which  (1970, 1974a) t h a t c o r t i c a l u n i t s a r e with  growth." I n  formed p r i o r  According  for surface  the  surface  elongation  stimulus  the  proliferation  i n a d d i t i o n o f new c o r t i c a l  and  from  body  that  as organizing centres  formation, of  kineties,  Support  basal  i s a  conclusion  observation  I t appears  somatic  the  This  amount o f s u r f a c e  the  in  formation.  i s a consistent relationship  them r e s u l t i n g  not,  furrow  growth.  a l s o have reduced  the  form  and  body p r o l i f e r a t i o n  reduced  number o f new b a s a l growth  growth  only  also  from  Sonneborn organized  component.  The  120  CELL DIVISION; b)  -  (Cont'd.)  S u r f a c e Growth: organizing  -  influence  development  has  based  based  on  Hanson's  urements.  Their  conclusion  that  between  regions bodies only of  This  however,  was  growth  cell  accompanies  temporary,  and  surface  however,  growth. growth  Parts  during  cell  body p r o l i f e r a t i o n  While  i t i s true  from  that  area of the polar  contractions  surface addition  the  growth o f new  i n the  appears basal  was  cortical true  paper  based  and  unit  i n the  was  on  polar  This  reflects  units rather cell  regions  than  i s  actual  show have  surface  temporary  basal  growth  always  preceding  of  a stretching  which  division  growth.  changes  i n the  probably result  sub-cortical fibre  t o be  the  prolif-  proliferation  growth.  of the  basal  surface  dividing wild-  temporal c o r r e l a t i o n  where no  the available c o r t i c a l  stable  no  i s obviously  of the  surface  d a t a on  growth  i n d e p e n d e n t , s e t o f meas-  model, there  surface  eration.  but  by  (1974a).  j u s t as the model i n t h i s  a similar,  cortical  suggested  model f o r surface  on m o r p h o m e t r i c  cells,  b o d i e s on  a l s o p r e v i o u s l y been  Kaneda and  type  of the basal  (1950) a n d S o n n e b o r n  Lwoff  was  (Cont'd.)  initiated  by  lattice,  the  b o d i e s t o k i n e t i e s on  the  121  CELL DIVISION; b)  Surface  -  (Cont'd.)  Growth:  -  (Cont'd.)  cortex. Based surface it a  on  the  growth  by  i s possible cell  could  bodies. found type  furrow  mutant furrow  produce.  This  the  cells, the of  model such  that  an  the  surface  very  to  be  these  surface  not  simple  case  number of b a s a l  region  was  reduced  only  bodies to  lost the  of  results  indicate surface  i n some c a s e s surface  furrow.  bodies furrow  these  test  The  ( w i l d - t y p e , dc2  during  closely  that  compared in  amounts of  fission  the  corresponds very  a  the  could  growth  cells)  basal  cells  of  i n each  number of  such  to predict  t o make m o r e  the  wild-  t h e n be  but  basal  bodies  amount can  ingly,  The  used  observed  capacity  surface  number of  growth presented.  similar,  complete  of  units,  of p r e f i s s i o n  a n a l y s i s ( t a b l e 20)  have the  required  amount  s e r v i n g as  and  stimulate  cortical  given  that  amount  predicted  growth are cells  can  predicted  bodies  number o f b a s a l  region  comparison of  a  the  surface  observed  the  from  cells  amount o f  with  basal  to p r e d i c t the  example,  i n the  that  o r g a n i z i n g new  produce  For  and  model  i n the surface  at  this  the  than  Surpris, and  dfz  furrow growth. time  number t h a t  were  122  CELL DIVISION; b)  Surface  -  (Cont'd.)  Growth:  -  predicted  t o be  to  complete  form  for by  a  surface the  sets  The  obtained  results,  the  the  surface  itself.  The  at  be  effects anism  of  metric  of  furrow basal  the  basal  bodies  the  could  Hanson region  surface  appears  to  extent  growth  Although  the  be  to  of  controlled  the  i t i s  not  proliferation  body p r o l i f e r a t i o n  genes, The  as  this  of  mech-  problem  from of  disappear  resorbed,  as  they the  morpho-  the  control  question  surface  bounded by  of the  proliferation  i s the  (1974), o r  the  solely  which  can.  shown by  nature  extent  furrow  and  surface  body p r o l i f e r a t i o n  bodies  be  supported  growth  determining  determined  during  model  be  between CVPs.  gene.  Related  region  of  of  body p r o l i f e r a t i o n  Kaneda and side  to  c o n t r o l s the  data.  seems t o  of basal  h o w e v e r , be  of b a s a l fate  sm2  The  unexpected  of basal  the  20).  two  i n f l u e n c e d by  which  cannot,  (table  required  however, r a i s e  growth  extent  number  wild-type  what c o n t r o l s the  least  the  and  validity  extent  evident  of  from mutant  measuring distances  amount o f by  furrow  of problems related  challenge by  i n excess  growth therefore  results  cells.  (Cont'd.)  of  from  growth. claimed  CVPs  the These  by  c o u l d move two  the  out-  which  T A B L E 20 P R E D I C T E D GROWTH OF T H E FURROW S U R F A C E  ON W I L D - T Y P E  AND MUTANT C E L L S  +  #•*  A T T A I N A B L E LENGTH  AGE  CONTOUR ' *»» LENGTH OF ++ NO. B A S A L +++ NUMBER OF B A S A L U S I N G NO. FURROW MAX. B O D I E S WHICH A C T U A L CHANGE BODIES/CVINT B A S A L B O D I E S AT SURFACE AT D I F F . CORRESPOND I N NUMBER OF 0.95 MAX.* A G E 0.98 AGE.96 MAX. AGE.98 F I S S I O N ( u m ) ( u m ) TO D I F F E R E N C E B A S A L B O D I E S  WildType  49  49  41  73-5  73.5  61.5  63  -10.5  -7  -8  dc4  39  40  39  58.5  60  58.5  58  - 2.0  -1  -1  sm2  25  30  30  37-5  45  45  53  8.0  5  a  41  43 •  39  61.5  64.5  58.5  60  " M  -3  -4  dfz2  42  42  38  63  63  57  60  - 3-0  -2  -4  dc2  + ++ *** +++  •  0  The maximum mean number o f b a s a l bodies/CVlNT a t any c e l l age A t t a i n a b l e l e n g t h s were c a l c u l a t e d assuming t h a t the a v a i l a b l e b a s a l b o d i e s would move a p a r t t o assume 1.6 um spacing; the v a l u e s l i s t e d correspond t o the number o f b a s a l b o d i e s a t t h e i n d i c a t e d ages. Taken from contour drawings o f d i v i d i n g c e l l s (see t e x t ) The d i f f e r e n c e between t h e maximum a t t a i n a b l e l e n g t h and the mean contour l e n g t h a t fission Assuming a 1.6 pm s p a c i n g The maximum number of b a s a l bodies minus the number a t age 0 . 9 8 - 0 . 9 9 .  124  CELL DIVISION; b)  (Cont'd.)  S u r f a c e Growth; -  (Cont'd.)  were used as r e f e r e n c e p o i n t s i n measuring the furrow s u r f a c e .  I f the l a t t e r a l t e r n a t i v e i s t r u e  then e i t h e r the b a s a l b o d i e s migrate past the CVPs or  t h e CVPs m i g r a t e towards the furrow r e g i o n . I f  the CVPs do migrate towards the furrow (a c o n c l u s i o n reached by K i n g (1954)) then t h e use o f these s t r u c t u r e s as r e f e r e n c e p o i n t s i n measuring s u r f a c e growth becomes unacceptable.  I t would t h e r e f o r e  be p r e f e r a b l e i f the other a l t e r n a t i v e , t h a t the b a s a l bodies move past the CVPs, were t r u e . Evidence t o support t h i s a l t e r n a t i v e i s tenuous, but the a v a i l a b l e r e s u l t s g i v e some i n d i r e c t for  t h i s model.  support  I f the b a s a l bodies move p a s t the  CVPs i n t o the c e l l mid-regions they would c o n t r i b u t e t o s u r f a c e growth.  still  The amount o f b a s a l  body p r o l i f e r a t i o n should t h e r e f o r e c o n t r o l the t o t a l amount o f s u r f a c e growth d u r i n g c e l l  division  and the s i z e o f the r e s u l t i n g daughter c e l l s .  A  d i r e c t demonstration o f the c o n t r o l o f c e l l l e n g t h by b a s a l body p r o l i f e r a t i o n i s p r o v i d e d by t h e data on sm2 c e l l s where reduced b a s a l body p r o l i f e r a t i o n r e s u l t s i n reduced c e l l l e n g t h .  A more i n d i r e c t  demonstration o f t h i s r e l a t i o n s h i p i s p r o v i d e d by  125  CELL DIVISION: b)  -  (Cont'd.)  S u r f a c e Growth: the  with  related  be r e l a t e d  test  of this  34.5°C  about  at  i n this  Hanson  (19?4)  proliferation  this  were  any  found  the only  must direct  was made, i t a p p e a r s used  wild-type cells  A temperature  therefore  that  i n the control  than  i n wild-type cells effect  make Kaneda grown on  seems p l a u s i b l e .  demonstrated,  known  to  A l t h o u g h no  bodies per kinety  conclusively  constitute  example  basal I f  i t would  of this  effect  organism.  The out  study)  temperature.  body  in  body p r o l i f e r a t i o n  relationship  cells  i s related  t o temperature.  1 0 more b a s a l  room  length  (the temperature  experiment  temperature,  producing longer  I f cell  then basal  also  o f paramecia i s  to the c u l t i v a t i o n  1964).  temperature,  and  the length  higher temperatures  (Whitson,  at  (Cont'd.)  observation that  directly  •  -  proposal that  excess  of the furrow region  indirect  basal  therefore  support and i tappears  CVPs a s r e f e r e n c e p o i n t s  bodies  has c o n s i d e r a b l e  that  f o rcell  migrate  the use o f the  measurements i s  valid. Reduction surface  growth  i n basal i n sm2  body p r o l i f e r a t i o n  cells  and  does n o t a f f e c t t h e  126  B)  CELL DIVISION: b)  -  (Cont'd.)  S u r f a c e Growth: ability  -  (Cont'd.)  of these c e l l s  Predictions  based  is  by b a s a l  mediated  that  sm2  cells  than  are required  t o produce  (table  divide  suggests that  the  with  is  occurs only total  furrow  surface  apparently  o f sm2  posterior  this  of the anterior furrow.  Such  cells. furrow  surface growth  growth  i n sm2  The  of the mid-region of  the furrow.  On c o n t o u r  i s indicated  CVP o f t h e o p i s t h e  i n sm2  cells  by a r e d i s t r i b u t i o n  drawings  by movement  towards the  a CVP movement was s e e n  constriction  furrow  t o the fission-zone i s  by growth  growth  that  growth  i s normal.  The n e c e s s a r y f u r r o w s u r f a c e  provided  growth i n  posterior to  such  to the fission-zone  towards  cells  a mechanism  Although furrow, surface  supplied  opisthe  fission  t o the fission-zone  i ti s redistributed  anterior  have  surface  of surface  fission-zone.  growth  the  anterior  indicate  o f sm2 c e l l s t o '  these c e l l s  Furrow  failure  limited,  a complete  The a b i l i t y  growth  bodies per kinety  f o r the reduced  furrow region.  cells  the  20).  surface  body p r o l i f e r a t i o n  have 5 fewer b a s a l  compensates  constriction.  on t h e model t h a t  furrow  which  t o complete  only  i n sm2  t o complete  i s therefore  of surface  growth  and  127  CELL DIVISION; - (Cont'd.) b)  Surface  Growth: - (Cont'd.)  a c o n t r i b u t i o n to furrow s u r f a c e growth of regions  d i s t a l to the furrow.  cell  R e s t r i c t i o n s on  furrow completion are not produced by a l a c k  of  b a s a l body p r o l i f e r a t i o n i n the furrow r e g i o n , suggesting  t h a t the inward d i r e c t e d growth of  furrow i s not due of the d f z and  to s u r f a c e growth alone.  the  All  dc mutants examined have s u f f i c i e n t  c o r t i c a l o r g a n e l l e s to complete growth of the furrow s u r f a c e . t h e r e f o r e not  due  The  d e f e c t i n these mutants i s  to a l a c k of s u f f i c i e n t  to c o n s t r u c t the furrow.  T h i s r e i n f o r c e s the  c l u s i o n s p r e v i o u s l y made about the dfz and  dc phenotypes.  organelles  o r i g i n s of  conthe  128  C)  CORTICAL  PATTERN;  The size the  i s o l a t i o n of a mutation  reduction effects  cortical  of a size  structures  this  problem  cell  size  change  often  proves  regenerative  organism.  away p a r t s  of the c e l l .  Such  as c u t t i n g the  the c e l l s  have  c a p a b i l i t i e s even i f the c u t i s  195^; C h e n - S h a n , 1969» 1970).  The  mutants,  therefore,  means  comparison the  o f such a s i z e  o b s e r v a t i o n s made o n o t h e r  A study positions changes  of certain  the available  vestibule, anisms t h a t  ciliates  size  cell  can  with  m o r p h o l o g y h a s shown t h a t  the  structures  change  i n a manner adequately forciliate  appears  f o rvestibule t o account  predicted  models.  i s therefore  positioning.  The  by mech-  by the a v a i l a b l e  f o r other  with  development.  t o be p o s i t i o n e d  are not predicted  be e x t e n d e d  change  has  cortical  models  however,  t o account  for a  where s i z e  A new m o d e l , m e c h a n i c a l p o s i t i o n i n g , posed  for re-  altered.  o f sm2  i n cell  small  o f p a r a m e c i a and have a l l o w e d  of the results  been s u r g i c a l l y  by  an improved  poor  successful  (Tartar,  ducing the size  ciliates  examined by a l t e r i n g  and since  provide  of  of  In other  are d i f f i c u l t  fatal  examination  on t h e p o s i t i o n i n g  has been f r e q u e n t l y  on Paramecium  resulting i n a  has allowed  i n this  by c u t t i n g  operations cells  i n Paramecium  (sm2)  This  developmental  promodel  129  CORTICAL PATTERN; - (Cont'd.) p r o c e s s e s i n Paramecium. A number of models' have been proposed to account f o r morphogenesis i n the c i l i a t e s .  Many of these have  been adapted from models o r i g i n a l l y designed to development i n m u l t i c e l l u l a r organisms. has  explain  Frankel  (1974)  proposed, f o r example, t h a t Wolpert's h y p o t h e s i s  of p o s i t i o n a l information p l i e d to c i l i a t e s .  The  (Wolpert, 1969) can be ap-  need t o i n t r o d u c e  t h i s hypo-  t h e s i s arose out o f a l a c k o f a s u i t a b l e framework f o r t h i n k i n g about a l a r g e number o f accumulated observations be  on c i l i a t e development t h a t c o u l d not  explained  notably  adequately  by the p r e v i o u s models of development,  Sonneborn's c y t o t a x i s ' t h e o r y  (Sonneborn, 1964).  C y t o t a x i s was d e f i n e d by Sonneborn (1964) as the "ordering  and  arranging  o f new c e l l s t r u c t u r e under  i n f l u e n c e of p r e - e x i s t i n g c e l l s t r u c t u r e " .  the  While t h i s  model can account f o r the growth of k i n e t i e s where developing organelles  are i n c l o s e p r o x i m i t y  to s i m i l a r  p r e - e x i s t i n g s t r u c t u r e s , i t was d i f f i c u l t to apply t h i s model t o s i t u a t i o n s where a new s t r u c t u r e formed at a large distance  from any  structure.  (1974) has  Frankel  similar pre-existing therefore  proposed a  r e t e n t i o n of c y t o t a x i s theory i n a more r e s t r i c t e d sense, renaming i t s t r u c t u r a l guidance, and  suggesting  130  C)  CORTICAL PATTERN; - (Cont'd.) t h a t s t r u c t u r a l guidance i n v o l v e s only c o n t r o l over the o r i e n t a t i o n and arising structures.  He  short-range  p o s i t i o n of newly  suggests t h a t l o n g range  c o n t r o l over development of new  s t r u c t u r e s i s more  adequately  e x p l a i n e d by a model based on Wolpert's  hypothesis  of p o s i t i o n a l information.  T h i s model,  c a l l e d p o s i t i o n a l c o n t r o l , i s proposed to f u n c t i o n through g r a d i e n t f i e l d s which operate  i n accordance  with the r u l e s d e f i n e d i n Wolpert's h y p o t h e s i s .  When  a p p l i e d to c i l i a t e s , the main p o i n t s o f Wolpert's hypothesis  are;  (a)  there are mechanisms i n v o l v e d  i n surface patterning that u t i l i z e reference e i t h e r i n or on the c e l l ,  (b)  points  the main type of s u r f a c e  p a t t e r n i n g mechanism o p e r a t i n g from or between  these  p o i n t s i s a graded d i s t r i b u t i o n o f some substance or q u a l i t y of the s u r f a c e s t r u c t u r e , (c)  the  reference  p o i n t s and g r a d i e n t s are r e g u l a t e d or r e - e s t a b l i s h e d i f they are l o s t or d i s t u r b e d , (d)  r e g i o n s o f the  cell  s u r f a c e along the g r a d i e n t s can i n t e r p r e t the quanr  y^-  t i t a t i v e v a r i a t i o n i n the h y p o t h e t r i c a l f a c t o r s which c o n s t i t u t e the  gradient as a source  r e l a t e d to how  t h a t p a r t o f the c e l l w i l l develop,  (e)  of information and  t h i s i n f o r m a t i o n i s t r a n s l a t e d l o c a l l y i n t o morpho-  g e n e t i c events.  The n o v e l aspect  o f t h i s model i s the  131  CORTICAL PATTERN; - (Cont'd.) s e p a r a t i o n of the i n f o r m a t i o n ( p o s i t i o n a l  information)  f o r morphogenesis from the v i s i b l e morphogenetic events n o r m a l l y  a s s o c i a t e d with p a t t e r n i n g .  t u n a t e l y , as Sonneborn (1974a and t h e r e i s no evidence genesis  to support  of the model.  from any  study  has i n d i c a t e d ,  of c i l i a t e morpho-  the e x i s t e n c e o f t h i s n o v e l  The r e s u l t s on sm2  not p r o v i d e support  1975)  Unfor-  aspect  cells similarly  do  f o r the e x i s t e n c e o f p o s i t i o n a l  information i n c i l i a t e s .  Deprived  of i t s p o s i t i o n a l  i n f o r m a t i o n a s p e c t , F r a n k e l ' s p o s i t i o n a l c o n t r o l becomes a model, based on g r a d i e n t f i e l d s , which i s s t i l l c a b l e and u s e f u l i n t h i n k i n g about c i l i a t e A study  of sm2  appli-  development.  c e l l s i n d i c a t e s t h a t both t h i s model  and  c y t o t a x i s can be a p p l i e d to Paramecium development,  a)  C y t o t a x i s i n Paramecium; In order to a p p l y  the c y t o t a x i s model to some  o f the o b s e r v a t i o n s made on sm2  c e l l s i t i s nec-  essary to r e s t a t e the d e f i n i t i o n of c y t o t a x i s as: the o r g a n i z i n g and a r r a n g i n g of new  c e l l structures  under the i n f l u e n c e o f the p r e - e x i s t i n g c e l l s i z e , or components.  T h i s does not  shape,  significantly  a l t e r the o r i g i n a l d e f i n i t i o n of the model and i s s i m i l a r to other l i b e r a l i n t e r p r e t a t i o n s o f c y t o t a x i s (Grimes, 1976).  I t i s a l s o p o s s i b l e to  132  C)  CORTICAL PATTERN: - (Cont'd.) a)  C y t o t a x i s i n Paramecium: - (Cont'd.) d i s t i n g u i s h two types o f c y t o t a c t i c events: range e f f e c t s and l o n g range e f f e c t s .  short  Cytotactic  events o p e r a t i n g over a s h o r t range, such as the a d d i t i o n o f new b a s a l bodies t o a k i n e t y , can be r e f e r r e d t o by F r a n k e l ' s  term, s t r u c t u r a l guidance.  C y t o t a c t i c events o p e r a t i n g over a l o n g range can be c a l l e d mechanical guidance.  Examples o f t h i s  l a t t e r type o f c y t o t a x i s i n Paramecium i n c l u d e the p o s i t i o n i n g o f the v e s t i b u l e , the d e t e r m i n a t i o n  of  the number o f k i n e t i e s on the c e l l , and c o n t r o l o f the l e n g t h o f k i n e t i e s . The  Paramecium v e s t i b u l e does not develop i n  s i t u a t each b i n a r y f i s s i o n but i s d e r i v e d  from  b i s e c t i o n o f the p a r e n t a l v e s t i b u l e and m i g r a t i o n o f the r e s u l t i n g p a r t s t o a c e n t r a l l o c a t i o n on the v e n t r a l s u r f a c e o f the p r o t e r and o p i s t h e . study on w i l d - t y p e  The  c e l l morphogenesis has shown  t h a t two f a c t o r s c o n t r i b u t e t o v e s t i b u l e movement: v e s t i b u l e m i g r a t i o n and s u r f a c e growth p o s t e r i o r to the d i r e c t i o n of m i g r a t i o n .  Initially,  migration  accounts f o r a l l o f the movement o f the v e s t i b u l e , but l a t e r , s u r f a c e growth p o s t e r i o r t o the m i g r a t i o n adds t o the apparent v e s t i b u l e movement.  Surface  133  CORTICAL PATTERN; - (Cont'd.) a)  C y t o t a x i s i n Paramecium; - (Cont'd.) growth p o s t e r i o r t o the d i r e c t i o n o f v e s t i b u l e movement does not a c t u a l l y c o n t r i b u t e t o the movement, but c o n t r i b u t e s t o v e s t i b u l e p o s i t i o n i n g by c r e a t i n g new c e l l s u r f a c e b e h i n d o r a n t e r i o r t o the v e s t i b u l e .  Both t h i s s u r f a c e growth and the  i n i t i a l vestibule migration for  are therefore necessary  proper p o s i t i o n i n g o f the v e s t i b u l e .  migration  Vestibule  begins when the c e l l width decreases  d u r i n g f i s s i o n furrow f o r m a t i o n .  This r a p i d  decrease i n width r e s u l t s i n a narrowing o f the c e l l circumference  and a decrease i n the s p a c i n g o f  k i n e t i e s around the c e l l circumference.  A decrease  i n k i n e t y s p a c i n g would be expected t o produce tensions  i n the c o r t e x t h a t would p u l l the v e s t i b u l e  i n the p r o t e r forward and the v e s t i b u l e i n the o p i s t h e backward.  These t e n s i o n s a r i s e s i n c e the  k i n e t i e s meet the l e f t  s i d e s o f the a n t e r i o r and  p o s t e r i o r s u t u r e s a t a sharp angle ( f i g . l ) . decrease i n k i n e t y s p a c i n g w i l l t h e r e f o r e the l e n g t h o f the s u t u r e s migration  shorten  and c r e a t e v e s t i b u l e  i n the r e q u i r e d d i r e c t i o n .  of v e s t i b u l e migration  A  The extent  i s t h e r e f o r e c o n t r o l l e d by  the number o f k i n e t i e s on the c e l l  (which i s  134  C)  CORTICAL PATTERN; - (Cont'd.) a)  Cytotaxis  i n Paramecium: - (Cont'd.)  p o s i t i v e l y c o r r e l a t e d with  c e l l width) and  the  amount of width decrease which occurs d u r i n g division.  T h i s model f o r v e s t i b u l e  adequately e x p l a i n s  Porter's  cell  migration  observation  that  the  number o f k i n e t i e s i n the a n t e r i o r suture does not change d u r i n g v e s t i b u l e movement i n the ( P o r t e r , 1962). would not be were due  A change i n the number o f k i n e t i e s  expected i f the v e s t i b u l e movement  t o an i n t e r - k i n e t y c o n t r a c t i o n .  model a l s o p r e d i c t s and observations (a)  proter  explains  made on mutant and  the  The  following  wild-type  cells:  In mutant c e l l s where a normal width decrease occurs d u r i n g first  cell  d i v i s i o n at the  end  c e l l c y c l e at the r e s t r i c t i v e  of  the  tempera-  t u r e the v e s t i b u l e moves forward a constant distance  without regard  length.  (The  to the t o t a l  change i n k i n e t y s p a c i n g  dependent on the width o f the c e l l but  (b)  the  length.)  The  length  cell is not  of the a n t e r i o r suture i s h i g h l y  c o r r e l a t e d with the number of k i n e t i e s on cell.  The  suture i s longer  (and  the  contains  more k i n e t i e s ) on c e l l s with a l a r g e number  135  C)  CORTICAL PATTERN; - (Cont'd.) a)  C y t o t a x i s i n Paramecium; - (Cont'd.) (b)  - (Cont'd.) of k i n e t i e s .  S i n c e the number o f k i n e t i e s  on t h e c e l l i s c o r r e l a t e d w i t h both l e n g t h and width,  cell  t h i s implies that i n long  c e l l s t h e r e w i l l be more k i n e t i e s i n the a n t e r i o r s u t u r e , and v e s t i b u l e movement  will  be more e x t e n s i v e than i n s h o r t c e l l s where t h e r e a r e fewer k i n e t i e s i n the s u t u r e . R e g u l a t i o n o f v e s t i b u l e p o s i t i o n i n g when a change i n c e l l l e n g t h occurs i s t h e r e f o r e mediated through changes i n the number o f k i n e t i e s on the c e l l . (c)  The e x t e n t o f v e s t i b u l e m i g r a t i o n i s g r e a t e r i n the p r o t e r than i n the o p i s t h e .  The  a n t e r i o r suture has more k i n e t i e s a b u t t i n g it  than does the p o s t e r i o r s u t u r e , and the  k i n e t i e s meeting the a n t e r i o r suture a r e a t an angle approaching  90° w h i l e those meeting  the p o s t e r i o r s u t u r e are a t a l e s s angle.  acute  Changes i n k i n e t y s p a c i n g w i l l  there-  f o r e a f f e c t the l e n g t h o f the a n t e r i o r , more than the l e n g t h o f the p o s t e r i o r , s u t u r e .  136 CORTICAL PATTERN; - (Cont'd.) a)  Cytotaxis (c)  i n Paramecium: - (Cont'd.)  - (Cont'd.)• Vestibule  .  p o s i t i o n i n g i s a c y t o t a c t i c phenom-  enon as the  extent of m i g r a t i o n i s determined  by the  c e l l width and  on the  cell  Vestibule  the  (pre-existing  number of form and  p o s i t i o n i n g i s an  responsible  v e s t i b u l e m i g r a t i o n operates over a  distance  Two  structures).  example of mechan-  i c a l guidance s i n c e the mechanism for  kineties  on the  cortex.  o t h e r examples o f mechanical  are the  positioning  c o n t r o l of k i n e t y number and  control  of the number o f b a s a l b o d i e s w i t h i n kinety.  large  each  K i n e t y number i s d i r e c t l y r e l a t e d  c e l l width i n sm2  cells.  induced width r e d u c t i o n ,  to  During a temperaturesm2  c e l l s lose  such t h a t the number of k i n e t i e s per  kineties  unit  of  c e l l width (or c i r c u m f e r e n c e ) remains c o n s t a n t . These k i n e t i e s are p r o b a b l y l o s t by ism proposed by Heckmann and explain  Frankel  (1968)  l o s s of k i n e t i e s i n E u p l o t e s .  model, k i n e t i e s which do not of the  the mechan-  c e l l , but  reach the  In  to  this  poles  which terminate somewhere i n  137  CORTICAL PATTERN; - (Cont'd.) a)  C y t o t a x i s i n Paramecium; - (Cont'd.) (c)  - (Cont'd.) the c e n t r a l p o r t i o n o f the  c o r t e x , are  even-  t u a l l y l o s t d u r i n g c e l l d i v i s i o n when e i t h e r the p r o t e r or o p i s t h e r e c e i v e s no  cortical  o r g a n e l l e s from t h i s incomplete k i n e t y ( f i g . 58).  On the Paramecium c o r t e x i t i s not  p o s s i b l e f o r a l l of the somatic k i n e t i e s to terminate suture  a t the c e l l p o l e s or a l o n g  lines.  two  K i n e t i e s are f r e q u e n t l y seen  t h a t terminate  a t other p o i n t s on the  presumably  to  at  the  due  the c e l l p o l e s .  cortex  overcrowding of k i n e t i e s One  might t h e r e f o r e expect  there to be a tendency f o r paramecia to l o s e k i n e t i e s during c e l l d i v i s i o n .  To  counteract  t h i s e f f e c t the c e l l s must have a mechanism for  i n c r e a s i n g the number o f k i n e t i e s .  c o r r e l a t i o n between k i n e t y numbers and width i n sm2  cell  c e l l s suggests t h a t when the  i n t e r k i n e t y spacing a new  The  exceeds  a c e r t a i n value,  k i n e t y w i l l form on the c e l l s u r f a c e  by  i n s e r t i o n between e x i s t i n g k i n e t i e s ( f i g . 5 8 ) . The  width of the c e l l would t h e r e f o r e  mine whether new  deter-  k i n e t i e s would form, q u a l i f y i n g  138  C)  CORTICAL PATTERN: - (Cont'd.) a)  C y t o t a x i s i n Paramecium: - (Cont'd.) (c)  - (Cont'd.) c o n t r o l over k i n e t y numbers as a c y t o t a c t i c phenomenon.  The p r e c i s e mechanism by which  new k i n e t i e s a r e added t o t h e c o r t e x , however, has n o t been determined.  The  amount o f b a s a l body p r o l i f e r a t i o n  during  c e l l d i v i s i o n i n sm2 c e l l s appears t o i n f l u e n c e the c e l l l e n g t h , v e s t i b u l e l e n g t h , and g u l l e t l e n g t h by i n f l u e n c i n g the l e n g t h o f the c o r - t i c a l kineties. can  T h i s suggests t h a t paramecia  c o n t r o l t h e i r s i z e by c o n t r o l l i n g the  amount o f b a s a l body p r o l i f e r a t i o n at  c e l l division.  Regulation  occurring  of c e l l  size  must be a s s o c i a t e d with r e g u l a t i o n o f b a s a l body p r o l i f e r a t i o n .  When abnormally l a r g e  c e l l s r e t u r n t o normal s i z e , they must do so by l i m i t i n g the amount o f b a s a l body proliferation.  When abnormally s m a l l  cells  r e t u r n t o normal s i z e , they must i n c r e a s e t h e amount o f p r o l i f e r a t i o n .  The extent  l i f e r a t i o n must be r e l a t e d t o c e l l  o f pro-  size  ( e x i s t i n g s i z e ) and would c o n s t i t u t e a  139  CORTICAL PATTERN: - (Cont'd.) a)  Cytotaxis (c)  i n Paramecium: - (Cont'd.)  - (Cont'd.) c y t o t a c t i c phenomenon.  The  t h r e e examples of mechanical guidance  t h a t have been p r e s e n t e d c o n s t i t u t e a new c y t o t a c t i c events. distances  predicted  model o f s t r u c t u r a l guidance.  not  of  They e i t h e r operate over l a r g e  or i n ways not  anisms o p e r a t i n g  class  by  Frankel's  P o s i t i o n a l mech-  by mechanical guidance are  d i r e c t l y under genie c o n t r o l .  also  Genes may  influ-  ence these developmental mechanisms only by l i m i t i n g the a v a i l a b i l i t y o f n e c e s s a r y p r o t e i n s by a l t e r i n g the r a t e c o n s t a n t s of the reactions  involved  i n the mechanisms.  or environmental f a c t o r s may mechanisms by a f f e c t i n g the materials  or  possibly  biochemical Physiological  s i m i l a r l y a f f e c t these availability  of  n e c e s s a r y f o r the normal f u n c t i o n i n g  the mechanisms.  The  of  b a s i c p r o c e s s e s by which  m e c h a n i c a l p o s i t i o n i n g o c c u r s , however, are i n dependent of d i r e c t gene c o n t r o l , b)  P o s i t i o n a l Control The  i n Paramecium:  mechanisms f o r the p o s i t i o n i n g of c e r t a i n  structures  i n Paramecium (the CVPs and  the  cytoproct)  140  CORTICAL PATTERN: - (Cont'd.) b)  P o s i t i o n a l Control remain unknown. therefore  i n Paramecium: - (Cont'd.)  The study o f these mechanisms i s  l i m i t e d t o a d e s c r i p t i o n o f the topo-  g r a p h i c a l r u l e s by which these mechanisms operate. These r u l e s are d i s c u s s e d  as examples o f p o s i t i o n a l  c o n t r o l only because t h i s i s p r e s e n t l y which b e s t e x p l a i n s  the o b s e r v a t i o n s made on the  positioning of certain structures. vations  should not be i n t e r p r e t e d ,  p r o o f o f the e x i s t e n c e ciliates.  the model  These obserhowever, as  of p o s i t i o n a l information i n  What the o b s e r v a t i o n s do i n d i c a t e i s  t h a t the s i z e o r s i t e o f f o r m a t i o n o f c o r t i c a l structures  can be i n t e r p r e t e d as b e i n g due t o a  morphogenetic f i e l d Wolpert (1969).  or gradient  as d e f i n e d by  T h i s i n t e r p r e t a t i o n o f the r e s u l t s  r e l i e s on the demonstration t h a t the s i t e s o f organellogenesis the  are determined w i t h r e f e r e n c e  s i z e o f the c e l l .  CVP forms a t a d i s t a n c e the  In Paramecium the a n t e r i o r from the a n t e r i o r end o f  c e l l t h a t i s a f i x e d f r a c t i o n o f the c e l l  length.  The p o s t e r i o r CVP, however, i s l o c a t e d a t  a f i x e d distance regardless not  to  from the p o s t e r i o r end o f the c e l l ,  of c e l l length.  The a n t e r i o r CVP, but  the p o s t e r i o r CVP, i s t h e r e f o r e  p o s i t i o n e d by  141  CORTICAL PATTERN; - (Cont'd.) b)  P o s i t i o n a l C o n t r o l i n Paramecium; - (Cont'd.) the r u l e s g o v e r n i n g Wolpert's h y p o t h e t i c a l morphogenetic  fields.  Another s t r u c t u r e which conforms  t o these r u l e s i s the c y t o p r o c t , which i s a constant p o s t e r i o r suture The  the l e n g t h  f r a c t i o n of the  of  l e n g t h of  the  i n which i t i s l o c a t e d .  a n t e r i o r CVP  i n paramecia not  only main-  t a i n s a r e l a t i v e p o s i t i o n along the p o l a r a x i s  of  the c e l l , but a l s o m a i n t a i n s a r e l a t i v e p o s i t i o n on the c e l l  circumference,  opposite  the mouth r e g a r d l e s s  k i n e t i e s on the c e l l . two  reference  a n t e r i o r end  being located  approximately  o f the number o f  There are t h e r e f o r e a t l e a s t  p o i n t s f o r CVP o f the c e l l  and  positioning; the mouth.  the Whether  gradients  or f i e l d s operate from o r between these  reference  p o i n t s cannot be  morphometric data. sibility  The  determined from  d a t a i n d i c a t e only the pos-  t h a t such g r a d i e n t s may  occur.  Similar  g r a d i e n t s , w i t h the o r a l apparatus as one gradient  reference  the c o n t r o l of CVP (Nanny, 1972)  and  the  of  p o i n t s , have been proposed f o r p o s i t i o n i n g i n Tetrahymena C h i l o d i n e l l a (Kaczanowska,  1975). The  the  p o s i t i o n of the p o s t e r i o r CVP  on  the  1974;  142  CORTICAL PATTERN: - (Cont'd.) b)  P o s i t i o n a l C o n t r o l i n Paramecium: - (Cont'd.) Paramecium c e l l i s not r e g u l a t e d a c c o r d i n g t o p o s i t i o n a l c o n t r o l model s i n c e t h i s CVP  the  does not  form de novo d u r i n g c e l l d i v i s i o n but i s i n h e r i t e d i n s i t u from the parent on a c e l l w i l l ,  cell.  w i t h i n two  become the p o s t e r i o r CVP descent ( f i g . 5 9 ) . t h i s CVP  cells  CVP  which forms  generations,  of an o p i s t h e l i n e o f •  In sm2  c e l l s the d i s t a n c e  of  from the p o s t e r i o r o f the c e l l i s c o n s t a n t ,  regardless of c e l l s i z e . CVP  Any  forms on the c o r t e x  T h i s means t h a t once a (by p o s i t i o n a l c o n t r o l ) i t s  p o s i t i o n i n the c o r t e x i s f i x e d and s u b j e c t to r e g u l a t i o n .  i s no  longer  T h i s i s p o s s i b l y due  to  the l a c k o f b a s a l body p r o l i f e r a t i o n and  surface  growth p o s t e r i o r t o t h i s CVP  division.  Small  cells will  t a c t and  during c e l l  inherit t h i s c o r t i c a l region i n -  c o u l d only reduce the d i s t a n c e from  p o s t e r i o r CVP  t o the p o s t e r i o r o f the c e l l  r e s o r b i n g c o r t i c a l u n i t s between these two E v i d e n t l y , the c e l l s are not capable During a s i z e r e d u c t i o n i n sm2 of the c y t o p r o c t remains a constant l e n g t h o f the p o s t e r i o r s u t u r e .  the  by points.  o f doing c e l l s the  this. length  f r a c t i o n o f the  It is  difficult,  however, to d e f i n e the r e f e r e n c e p o i n t s f o r  any  143  CORTICAL PATTERN: - (Cont'd.) b)  P o s i t i o n a l C o n t r o l i n Paramecium: - (Cont'd.) g r a d i e n t t h a t would determine the c y t o p r o c t The  length.  c y t o p r o c t forms d u r i n g c e l l d i v i s i o n p r i o r t o  the s e p a r a t i o n o f the daughter c e l l s .  Growth o f  the p o s t e r i o r suture i s n o t y e t complete when the c y t o p r o c t forms.  The h y p o t h e t i c a l g r a d i e n t con-  t r o l l i n g c y t o p r o c t l e n g t h t h e r e f o r e cannot use the l e n g t h o f t h i s suture as a r e f e r e n c e f o r d e t e r m i n i n g the c y t o p r o c t l e n g t h .  Although a  gradient-based  mechanism f o r c o n t r o l o f c y t o p r o c t l e n g t h cannot be r u l e d out, i t i s d i f f i c u l t such a g r a d i e n t would During  t o imagine how  operate.  c e l l d i v i s i o n the o p i s t h e r e t a i n s the  parental cytoproct.  During  a s i z e r e d u c t i o n i n sm2  c e l l s one would t h e r e f o r e expect t o see two k i n d s of c y t o p r o c t s ; the l o n g p a r e n t a l c y t o p r o c t s and s h o r t e r newly-formed c y t o p r o c t s .  No such b i m o d a l i t y  i n c y t o p r o c t l e n g t h was found, s u g g e s t i n g  that  there  i s a mechanism f o r r e d u c i n g the l e n g t h o f i n h e r i t e d c y t o p r o c t s when c e l l s i z e i s decreased. has  Ng (1976)  r e p o r t e d a s i m i l a r o b s e r v a t i o n on paramecia and  a l s o found t h a t the c y t o p r o c t i n the o p i s t h e s i o n a l l y disappears s i o n although  and reappears d u r i n g c e l l  occadivi-  he does not i n d i c a t e how f r e q u e n t l y  144  CORTICAL PATTERN; - (Cont'd.) b)  P o s i t i o n a l C o n t r o l i n Paramecium; - (Cont'd.) t h i s happens.  I f the p a r e n t a l c y t o p r o c t i s r e -  s t r u c t u r e d d u r i n g c e l l d i v i s i o n the c e l l  could  presumably c o n t r o l the l e n g t h o f the newly  forming  cytoproct. The p r e c e d i n g account has demonstrated t h a t the s i t e s o f f o r m a t i o n  o f new  CVPs and  the  length  o f the c y t o p r o c t are determined w i t h r e f e r e n c e the l e n g t h o f the c e l l .  The  s h i p between c e l l l e n g t h and  topographical  r e p o r t e d f o r Tetrahymena (Nanney, 1972) Chilodinella  relation-  s i t e s o f CVP  d e s c r i b e d above are t h e r e f o r e s i m i l a r to  (Kaczanowska, 1974).  to  formation those and  Whether or not  these t o p o g r a p h i c a l r e l a t i o n s h i p s are determined by a g r a d i e n t system, however, i s not  a question  can be answered by morphometric d a t a alone. g a r d l e s s of t h i s , the p r e v a i l i n g o p i n i o n i n the l i t e r a t u r e i s t h a t these types  Re-  expressed  of topo-  g r a p h i c a l r e l a t i o n s h i p s are based on g r a d i e n t tems ( F r a n k e l , 1974; Kaczanowska, 1974; 1975).  Jerka-Dziadosz, Lynn and  that  sys-  1974;  Tucker, 1976;  Sonneborn,  A l t e r n a t e l y , s t r u c t u r e s which form on  c o r t e x a t l o c a t i o n s t h a t are independent of  the  cell  s i z e are thought to be p o s i t i o n e d by mechanisms  145  CORTICAL PATTERN; - (Cont'd.) b)  P o s i t i o n a l C o n t r o l i n Paramecium: - (Cont'd.) t h a t are not based on g r a d i e n t s .  These mechanisms  i n c l u d e c y t o t a x i s (Sonneborn, 1 9 6 4 ) , control (Frankel, 1 9 7 4 ) ,  and  (Lynn and Tucker, 1 9 7 6 ) . no r e a s o n , however, why not  positional  structural positioning  There i s t h e o r e t i c a l l y c y t o t a c t i c mechanisms can-  account f o r size-dependent p o s i t i o n i n g o f  cortical  structures.  The  new  v e s t i b u l e i n Paramecium  appears to be p o s i t i o n e d by a c y t o t a c t i c mechanism, and y e t i t i s not any  reference  located at a f i x e d distance  p o i n t on or i n the c e l l .  remains i n the approximate c e n t e r surface regardless f o r e not  of the  Its position  o f the v e n t r a l  c e l l length.  I t i s there-  e s s e n t i a l t h a t a l l size-dependent mechan-  isms f o r p o s i t i o n i n g c o r t i c a l s t r u c t u r e s be on g r a d i e n t  f o r p o s i t i o n i n g the CVPs or d e t e r m i n i n g  l e n g t h of the  cytoproct  s t r u c t u r e s form before c e l l s i s determined. t o any  i n paramecia i s t h a t  mechanthe these  the s i z e o f the daughter The  CVPs, i n f a c t , form p r i o r  s u r f a c e growth i n the d i v i d i n g c e l l .  o b j e c t i o n to the g r a d i e n t model would not be s e r i o u s one  based  systems.  The main o b j e c t i o n t o a gradient-based ism  from  This a  i f s u r f a c e growth were uniform throughout  146  CORTICAL PATTERN: - (Cont'd.) b)  P o s i t i o n a l C o n t r o l i n Paramecium: - (Cont'd.) the l e n g t h o f the c e l l .  As shown "by the morpho-  m e t r i c study, however, s u r f a c e growth i s d e c i d e d l y non-uniform.  I f g r a d i e n t s account f o r the  size-  dependent p o s i t i o n i n g o f c o r t i c a l s t r u c t u r e s , they must t h e r e f o r e be able to " p r e d i c t " how where s u r f a c e growth w i l l  much  occur d u r i n g c e l l  and division.  To i l l u s t r a t e t h i s p o i n t , c o n s i d e r the l o c a t i o n o f the CVPs on the d o r s a l c o r t e x i n terms of t h e i r r e l a t i v e p o s i t i o n i n r e g a r d t o the a n t e r i o r end the c e l l .  In the i n t e r f i s s i o n c e l l  the two  CVPs are  l o c a t e d at p o s i t i o n s which are 35  and  cell  o f the c e l l ( f i g .  60). will  l e n g t h from the a n t e r i o r end Two  new  80$  of  of  CVPs form d u r i n g c e l l d i v i s i o n which  become the a n t e r i o r CVPs o f the p r o t e r  o p i s t h e and  the  and  they should t h e r e f o r e be l o c a t e d a t  35$  of the c e l l l e n g t h from the a n t e r i o r o f the p r o t e r and  opisthe.  ever, and  They do not  form at t h i s s i t e , how-  only reach i t j u s t p r i o r to the  of c y t o k i n e s i s  (fig.60).  Although a g r a d i e n t - b a s e d formation  completion  cannot be excluded,  mechanism f o r  i t i s also possible  t h a t the c e l l uses c y t o t a c t i c mechanisms to mine the s i t e s of CVP  CVP  formation.  deter-  As p r e v i o u s l y  147  CORTICAL PATTERN; - (Cont'd.) b) P o s i t i o n a l C o n t r o l i n Paramecium;  - (Cont'd.)  shown, any CVP which forms on a c e l l w i l l  become  the p o s t e r i o r CVP o f an o p i s t h e w i t h i n two cycles.  cell  Once i t has reached t h i s p o s i t i o n i t r e -  mains as the p o s t e r i o r CVP o f an o p i s t h e throughout all  future generations  o f the c l o n e .  The p o s t e r i o r  CVP o f the o p i s t h e i s l o c a t e d i n a d i s t i n c t  part  o f the c o r t e x which has the f o l l o w i n g f e a t u r e s ; (a) d u r i n g c e l l d i v i s i o n , t h e r e i s no b a s a l body  pro-  l i f e r a t i o n p o s t e r i o r to the CVP i n t h i s r e g i o n but .there i s e x t e n s i v e p r o l i f e r a t i o n a n t e r i o r to i t and (b) the c e l l s u r f a c e p o s t e r i o r to the CVP i n t h i s r e g i o n c o n t r a c t s p r i o r t o c e l l d i v i s i o n while the s u r f a c e a n t e r i o r t o i t does not.  The p o s t e r i o r CVP  of the o p i s t h e i s t h e r e f o r e l o c a t e d near t o o r i n a boundary zone between r e g i o n s o f the c o r t e x have s i g n i f i c a n t l y d i f f e r e n t p r o p e r t i e s .  that  It is  p o s s i b l e t h a t these p r o p e r t i e s o f the c o r t e x , as w e l l as the p o s i t i o n o f the CVP, are c o r r e l a t e d with some y e t unknown s t r u c t u r a l f e a t u r e s o f the cortex.  Regional  s p e c i a l i z a t i o n s o f the Paramecium  c o r t e x have been d e s c r i b e d by N a i t o h  and E c k e r t  (1969)» and i t i s p o s s i b l e t h a t the CVPs can form only i n c e r t a i n p a r t s o f the c o r t e x .  The  regions  148  CORTICAL PATTERN: - (Cont'd.) b)  P o s i t i o n a l C o n t r o l i n Paramecium: - (Cont'd.) of the c o r t e x i n which the CVPs form may  be  mined l o n g b e f o r e the CVPs appear i n them.  deterThe  o r i g i n a l p o s i t i o n s of these r e g i o n s would t h e r e f o r e be d i f f i c u l t  to p r e d i c t by examining the l o c a t i o n s  where CVPs form.  Although the d i s c u s s i o n of  these  s p e c i a l c o r t i c a l regions i s h i g h l y - s p e c u l a t i v e , i t • has been i n t r o d u c e d to show t h a t an simple  apparently  t o p o g r a p h i c a l r e l a t i o n s h i p between the l o c a -  t i o n of a s t r u c t u r e on the c e l l s u r f a c e and l e n g t h o f the c e l l may  t u r n out to be a complex  r e l a t i o n s h i p not r e a d i l y e x p l a i n a b l e by any model of c i l i a t e Regardless  the  simple  morphogenesis. of whether mechanical guidance (or  other c y t o t a c t i c mechanisms) proves to account f o r CVP  p o s i t i o n i n g or the c o n t r o l of c y t o p r o c t  length,  t h i s model does p r o v i d e  an a l t e r n a t i v e to g r a d i e n t -  based models o f c i l i a t e  morphogenesis and  removes the b i a s imposed by having  therefore  o n l y one  type  of model to a p p l y to size-dependent p o s i t i o n i n g of c o r t i c a l structures. study,  The  adherence, i n the  t o mechanical guidance and  other  present  cytotactic  models should not be i n t e r p r e t e d as i n d i c a t i n g t h a t g r a d i e n t systems i n c i l i a t e s are not p o s s i b l e .  149  C)  CORTICAL PATTERN: - (Cont'd.) b)  P o s i t i o n a l C o n t r o l i n Paramecium: - (Cont'd.) V i s i b l e g r a d i e n t s o f c e l l s t r u c t u r e are common i n ciliates:  f o r example, the s p a c i n g o f k i n e t i e s  around the Paramecium c e l l circumference  shows a  g r a d a t i o n from narrow t o wide (Sonneborn, 1975). Chemical g r a d i e n t s , although demonstrated i n c i l i a t e s ,  they have never been  c o u l d e x i s t i f they were  r e s t r i c t e d t o the c o r t i c a l l a y e r where they not be d i s t u r b e d by c y t o p l a s m i c  flow.  would  Alter-  n a t i v e l y , the g r a d i e n t s c o u l d be " b u i l t i n " t o the c o r t e x by g r a d a t i o n s  i n molecular  proposed by Sonneborn ( 1 9 7 5 )  s t r u c t u r e as  and Roth e t a l . ( 1 9 7 7 ) .  150  SUMMARY  Ten mutants a f f e c t i n g morphogenesis o f Paramecium t e t r a u r e l i a have been d e s c r i b e d i n t h i s study.  The pheno-  type o f each o f the mutants was shown to be due t o a mutated s i n g l e gene.  Since two o f the mutants were a l l e l i c ,  nine genes a f f e c t i n g morphogenesis i n Paramecium have been studied.  Of these, two a f f e c t f o r m a t i o n o f the f i s s i o n  zone ( c a l l e d d e f e c t i v e f i s s i o n zone or d f z mutants), f i v e affect constriction constriction cell size origins  o f the f i s s i o n furrow ( c a l l e d d e f e c t i v e  or dc mutants), and two produce a r e d u c t i o n i n  ( c a l l e d s m a l l or sm mutants).  To a n a l y s e the  o f the d i v i s i o n - a r r e s t phenotypes of the mutants,  w i l d - t y p e c e l l s were examined t o determine how they changed, i n l e n g t h and width p r i o r t o and d u r i n g c e l l d i v i s i o n . was found t h a t the c e l l s f i r s t  It  i n c r e a s e d i n l e n g t h and then  c o n t r a c t e d p r i o r t o forming a f i s s i o n furrow.  Once the  c e l l s c o n t r a c t e d , they assumed  equivalent  a shape roughly  to a c y l i n d e r w i t h e l l i p s o i d a l ends.  Furrowing, which began  when the c e l l s reached t h i s c y l i n d r i c a l shape, was plished  by a r a p i d  accom-  i n c r e a s e i n the c e l l l e n g t h and a r a p i d  decrease i n the c e l l width.  I n the d f z mutant examined  ( d f z 2 ) t h e r e was a premature c e l l c o n t r a c t i o n which produced a rounding o f the c e l l o u t l i n e .  These rounded c e l l s d i d not  form e i t h e r a f i s s i o n zone o r furrow.  I t was suggested t h a t  151  the abnormal shape o f these' c e l l s p r e v e n t s f i s s i o n  furrow  f o r m a t i o n and t h a t the attainment of the normal  cylindrical  p r e - d i v i s i o n shape i s e s s e n t i a l f o r f u r r o w i n g .  The  d e f e c t i v e c o n s t r i c t i o n mutants examined ( d c 2 c o n t r a c t a t the u s u a l time i n the c e l l  and  dc4)  c y c l e , but  this  c o n t r a c t i o n i s e x c e s s i v e and prolonged. the c o n t r a c t i o n was  two  a  The enhancement o f  p o s s i b l y due to the presence o f c o r t i c a l  l e s i o n s on the dc c e l l s t h a t would p e r m i t the e n t r y of c a l c i u m i o n s which s t i m u l a t e c o n t r a c t i o n i n Paramecium. The prolonged c o n t r a c t i o n o f the dc c e l l s p r o b a b l y i n t e r f e r e s w i t h c y t o k i n e s i s by p r e v e n t i n g the width o f the from d e c r e a s i n g d u r i n g c e l l  division.  The width  i s n e c e s s a r y to reduce the diameter o f the c e l l  cell  decrease and hence,  the amount of s u r f a c e needed f o r furrow completion. Although the dc mutants produce  a normal  amount o f furrow  s u r f a c e , without the normal width r e d u c t i o n the furrow cannot complete  c o n s t r i c t i o n o f the c e l l .  mutant examined (sm2), t h e r e was i n s u r f a c e growth d u r i n g c e l l ciliary  In the s m a l l  a significant reduction  division.  b a s a l body r e p l i c a t i o n was  The e x t e n t of  a l s o reduced and i t was  suggested t h a t a c a u s a l r e l a t i o n e x i s t e d between b a s a l body r e p l i c a t i o n and s u r f a c e growth.  This r e l a t i o n s h i p  t e s t e d i n other mutants (dc4, d c 2 , a  was  and dfz2) and was  found  152  t o h o l d t r u e i n a l l o f them. C o r t i c a l p a t t e r n was examined by c r e a t i n g s m a l l u s i n g the sm2 m u t a t i o n ( o r mutant s t r a i n ) . for  This  cells  allowed  an a n a l y s i s o f the p o s i t i o n s o f c o r t i c a l s t r u c t u r e s i n  r e l a t i o n t o the s i z e o f the c e l l T h i s study r e v e a l e d  on which they were found.  t h a t the p o s i t i o n i n g o r number o f  c e r t a i n c o r t i c a l s t r u c t u r e s were determined by the p r e e x i s t i n g shape, s i z e , o r s t r u c t u r e o f the c e l l .  A morpho-  g e n e t i c a l model c a l l e d mechanical guidance was proposed to account f o r the p o s i t i o n i n g o r r e p l i c a t i o n o f these structures bodies).  ( i n c l u d i n g the v e s t i b u l e s , k i n e t i e s , and b a s a l The study a l s o d e s c r i b e d  the  topographical  r e l a t i o n s h i p between c o n t r a c t i l e v a c u o l e p o s i t i o n s and c e l l s i z e and the r e l a t i o n between c y t o p r o c t size.  l e n g t h and  I t seemed u n l i k e l y t h a t these t o p o g r a p h i c a l  cell relations  were due t o g r a d i e n t - b a s e d developmental mechanisms, a l though such mechanisms could n o t be t o t a l l y excluded.  The  study a l s o showed t h a t the l o c a t i o n s o f c e r t a i n s t r u c t u r e s , such as the CVPs and the v e s t i b u l e s were determined by the c e l l with r e f e r e n c e  t o the c e l l l e n g t h .  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Experimental C e l l Research £0: 345-356.  157  P e t e r s o n , E.L. and J.D. Berger (1976) M u t a t i o n a l o f DNA s y n t h e s i s i n Paramecium t e t r a u r e l i a . J o u r n a l o f Zoology j>4: 2089-2097.  blockage Canadian  P o l l o c k , S.L. ( 1 9 7 4 ) Mutations a f f e c t i n g the t r i c h o c y s t s i n Paramecium a u r e l i a . I . Morphology and d e s c r i p t i o n o f the mutants. J o u r n a l o f P r o t o z o o l o g y 21: 352-362. P o r t e r , E.D. (1962) A theory o f morphogenetic m i g r a t i o n i n Paramecium a u r e l i a .  Journal of Protozoology 2  :  27a.  Rasmussen, L. (1967) E f f e c t s o f m e t a b o l i c i n h i b i t o r s on Paramecium a u r e l i a d u r i n g the c e l l c y c l e . Experimental C e l l Research 4 8 : 132-139. Roque, M.' (1956) L ' e v o l u t i o n de l a c i l i a t u r e b u c c a l e pendant l'autogamie e t l a c o n j u g a i s o n chez Paramecium a u r e l i a . Comptes Rendus des Seances de 1'Academic des S c i e n c e s  242: 2592-2595.  Roth, L.E. and D.J. P i h l a j a (1976) G r a d i o n a t i o n : h y p o t h e s i s f o r p o s i t i o n i n g and p a t t e r n i n g . J o u r n a l o f P r o t o z o o l o g y 24:  2-9.  Sawai, T. (1976) Movement o f the c e l l s u r f a c e and change i n s u r f a c e a r e a d u r i n g cleavage i n the newt's egg. J o u r n a l o f C e l l S c i e n c e 21: 537-551. S l a t e r , M. and M. Schaechter ( 1 9 7 4 ) C o n t r o l o f c e l l d i v i s i o n in bacteria. B a c t e r i o l o g i c a l Reviews _3_8: 199-221. S o k a l , R.R. and F . J . R o h l f (1969) Biometry. and Co. San F r a n c i s c o .  W.H.  Freeman  Sonneborn, T.M. (1950) Methods i n the g e n e r a l b i o l o g y and g e n e t i c s o f Paramecium a u r e l i a . Journal of Experimental Zoology 113: 8 7 - 1 4 8 . Sonneborn, T.M. (1964) The d i f f e r e n t i a t i o n o f c e l l s . Proceedings o f the N a t i o n a l Academy o f S c i e n c e £ L :  915-929.  •  Sonneborn, T.M. (1970) Methods i n Paramecium r e s e a r c h . In: Methods i n C e l l P h y s i o l o g y ; v o l . 4. ed. D.M. P r e s c o t t pp. 182-203.  158  Sonneborn, T.M. (1974a) C i l i a t e morphogenesis and i t s b e a r i n g on g e n e r a l c e l l u l a r morphogenesis. Actualites P r o t o z o o l o g i q u e s (proceedings o f the f o u r t h I n t e r n a t i o n a l Conference on P r o t o z o o l o g y , Clermont-Ferrand)  1:  327-355-  Sonneborn, T.M. (1974b) G e n e t i c s of the 14 s p e c i e s of Paramecium a u r e l i a . In: Handbook o f G e n e t i c s , v o l . II,. ed. R.C. King. Plenum P r e s s , New York, N.Y. Sonneborn, T.M. (1975) P o s i t i o n a l i n f o r m a t i o n and n e a r e s t neighbour i n t e r a c t i o n s i n r e l a t i o n t o s p a t i a l p a t t e r n s in ciliates. L'Anne B i o l o g i q u e 14: 5 6 5 - 5 8 4 . Suhama, M. and E.D. Hanson (1971) The r o l e o f p r o t e i n s y n t h e s i s i n p r e f i s s i o n morphogenesis o f Paramecium aurelia. J o u r n a l o f E x p e r i m e n t a l Zoology 177: 4 6 3 -  478:  Sundararaman, V. and E.D. Hanson (1976) L o n g i t u d i n a l microt u b u l e s and t h e i r f u n c t i o n s d u r i n g a s e x u a l r e p r o d u c t i o n i n Paramecium t e t r a u r e l i a . G e n e t i c a l Research 27_: 205211. T a r t a r , V. (1954) Anomalies i n the r e g e n e r a t i o n o f Paramecium aurelia. J o u r n a l of Protozoology 1: 11-17. Whitson, G.L. (1964) Temperature s e n s i t i v i t y and i t s r e l a t i o n t o changes i n growth, c o n t r o l of c e l l d i v i s i o n , and s t a b i l i t y of morphogenesis i n Paramecium a u r e l i a , syngen 4 , s t o c k 51« J o u r n a l o f c e l l u l a r and Comparative P h y s i o l o g y 64: 4 5 5 - 4 6 5 . W h i t t l e , J.R.S. and L. Chen-Shan (1972) C o r t i c a l morphogenesis i n Paramecium a u r e l i a : mutants a f f e c t i n g c e l l shape. G e n e t i c a l Research 1 2 : 2 7 1 - 2 7 9 Wolpert, L. (1969) P o s i t i o n a l i n f o r m a t i o n and the s p a t i a l p a t t e r n of c e l l u l a r d i f f e r e n t i a t i o n . J o u r n a l o f T h e o r e t i c a l B i o l o g y 2$: 1-47. Zeuthen, E. and L. Rasmussen (1971) Synchronized c e l l d i v i s i o n i n Protozoa. In: Research i n Protozoology; v o l . 4. ed. T.T. Chen. Oxford, Pergamon P r e s s .  F i g u r e 1.  A schematic diagram o f the k i n e t i e s on  the  d o r s a l and v e n t r a l s u r f a c e s o f an  interfission  Paramecium c e l l .  represented  by s o l i d l i n e s .  The The  k i n e t i e s are  p a t t e r n of c o r t i c a l  organ-  e l l e s w i t h i n the k i n e t i e s i s shown i n the lower figure.  anterior right field anterior left field contractile vacuole pores  posterior right 'field  vestibule  posterior left field cytoproct  VENTRAL  DORSAL para soma I sac basal body trichocyst  F i g u r e 2.  Schematic diagram of a p o r t i o n o f  the  Paramecium cortex showing the arrangement of v a r i o u s c o r t i c a l s t r u c t u r e s (based observations used ares axp,  of o t h e r a u t h o r s ) .  amr,  a n t e r i o r microtubular  c, c i l i u m ;  fibrils;  iam,  basal  i n n e r a l v e o l a r membrane; k f , oam,  o u t e r a l v e o l a r mem-  brane; ps, parasomal sac; pso,  cyst.  ribbon;  e l , epiplasmic layer; f i b ,  kinetodesmal f i b e r ;  opening; pmr,  published  Abbreviations  a x i a l p l a t e ; bb, b a s a l body; bp,  plate;  tmr,  on  parasomal sac  p o s t e r i o r microtubular  transverse microtubular  ribbon;  ribbon; t r , tricho-  Figure  S t r u c t u r e o f the Paramecium g u l l e t .  s o l i d and dashed l i n e s i n d i c a t e gullet  kineties.  The  the paths of the  164  Figure 4.  A schematic diagram  o f o r a l anlage develop-  ment d u r i n g b i n a r y f i s s i o n based on the p u b l i s h e d o b s e r v a t i o n s o f Kaneda and Hanson ( 1 9 7 4 ) and Jones of  (1976).  the c e l l  C e l l ages a r e g i v e n as f r a c t i o n s  c y c l e l e n g t h . Between ages 0 . 2 and  0 . 6 t h e r e i s a p r o l i f e r a t i o n o f b a s a l bodies i n the e n d o r a l membrane (the row o f l a r g e dots on the r i g h t v e s t i b u l a r w a l l ) . A t age 0 . 7 5 the anlage appears  j u s t above t h e e n d o r a l membrane  and by age O . 8 5 the anlage o r g a n i z e s i n t o three k i n e t i e s w h i l e t h e e n d o r a l membrane v a n i s h e s . By age O . 8 7 the anlage has i n v a g i n a t e d and c o n s i s t s of  s i x k i n e t i e s . A t t h i s time, two a d d i t i o n a l  anlagen appear  ( t h e i r r e g u l a r l y spaced dots i n  the f i g u r e ) , one a d j a c e n t t o the l a r g e , developing  anlage, and one on the r i g h t v e s t i b u l a r w a l l .  These two anlagen develop t o the t h r e e k i n e t y stage and then s i n k below the c e l l s u r f a c e (age 0-98)  and w i l l  sequent  l a t e r reappear d u r i n g the sub-  c e l l c y c l e a t age 0 . 7 5 *  At age 0 . 9 the  l a r g e anlage has developed t o the 12 k i n e t y stage and moved p o s t e r i o r l y t o a p o s i t i o n beneath the opisthe's vestibule. The c i r c l e d numerals i n d i c a t e the stages o f g u l l e t development g i v e n by Kaneda and Hanson  (1974).  F i g u r e 5«  Camera l u c i d a drawings o f c o n t r a c t i l e  vacuole pore a b n o r m a l i t i e s i n t s - 0 c e l l s . shaded areas are presumeably abnormal v a c u o l e pores. The k i n e t i e s  contractile  (solid lines)  abnormal paths around these s t r u c t u r e s . position  of the g u l l e t i s i n d i c a t e d  l i n e s . The v e r t i c a l bar i n d i c a t e s  15  The  by  follow  The solid  pm.  Figure 6 .  Measurements made on c e l l s o f known age.  A- Measurements made on c e l l s with two c o n t r a c t i l e vacuole pores  (CVPs).  B- Measurements made on c e l l s with f o u r CVPs and no f i s s i o n - f u r r o w . C- Measurements made on c e l l s which have a fission-furrow.  GP^ O G P  >  O G  IOGAI  PGP  Figure  7.  Sample s t a t i s t i c s  of c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e  temperature.  The v e r t i c a l bars i n d i c a t e 9 5 % c o n f i d e n c e i n t e r vals . A- Sample s t a t i s t i c s :  length  o f w i l d type (o)  length  o f the a n t e r i o r  and sm2 c e l l s ( © ) . B- Sample s t a t i s t i c s :  s u t u r e (GA) o f w i l d type (o) and sm2 cells; and sm2  v e s t i b u l e length (©) c e l l s .  (•)  (G) of w i l d type (o)  172  —i  •8  —i  •75  1  -8  i -85  1  .9  A G E (cell c y c l e s )  1  -85 AGE (cell  1  -9 cycles)  1  .95  1  -95  r—  to  1 —  1 0  Figure  8.  Sample s t a t i s t i c s  after a shift The  vertical  of c e l l s  o f known age  to the r e s t r i c t i v e  temperature..  bars  a r e 95f° c o n f i d e n c e  intervals  o f t h e mean. A-  The l e n g t h wild  type  B- The w i d t h cells.  of the posterior  suture  (GP) o f  ( o ) a n d sm2 (©) c e l l s . of wild  type  ( o ) and sm2 (©)  I  •75  ^75  1  -8  ^  1  1  1  -85 • 9 A G E ( eel I c y c l e s )  li5  AGE  ^  (cell cycles)  -95  35  1—  1-0  V'o  F i g u r e 9«  Sample s t a t i s t i c s  o f c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e The v e r t i c a l  temperature.  bars a r e 95% c o n f i d e n c e  intervals  the mean. A- CVA (see f i g . 6 ) o f w i l d - t y p e  (o) and sm2  (o) c e l l s . B- CVP (see f i g . 6 ) o f w i l d - t y p e (©)  cells.  (o) and sm2  176  —i •75  1  -8  1  1  1  -85 -9 -95 A G E ( eel I eye les)  1—  1-0  Figure 1 0 .  Sample s t a t i s t i c s  o f c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e The v e r t i c a l  temperature.  bars a r e 9 5 $ c o n f i d e n c e  intervals  the mean. A- CVA' (see f i g . 6 ) o f w i l d - t y p e  (o) and sm2  (•) c e l l s . B- CVP' (see f i g . 6) o f w i l d - t y p e (©)  cells.  (o) and sm2  Figure 1 1 .  Sample s t a t i s t i c s  o f c e l l s o f known age  a f t e r a s h i f t to the r e s t r i c t i v e The v e r t i c a l  temperature.  bars a r e 95% c o n f i d e n c e  intervals  the mean. A- The l e n g t h - t o - w i d t h r a t i o and sm2 (e)  o f w i l d - t y p e (o)  cells.  B- CVINT o f w i l d - t y p e  (o) and sm2 (•) c e l l s .  180  F i g u r e 12.  Sample s t a t i s t i c s  o f c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e The v e r t i c a l  temperature.  bars are 95$ c o n f i d e n c e  intervals  of the mean. A- The number o f b a s a l b o d i e s between the two innermost CVPs (see f i g . 6) of w i l d - t y p e (o) and sm2  (©) c e l l s .  B- The s p a c i n g between b a s a l bodies on w i l d - t y p e (o) and sm2  (e)  cells.  F i g u r e 13. and sm2  Outline  drawings o f w i l d - t y p e  (left)  ( r i g h t ) d i v i d i n g c e l l s o f ages 0.94 ( a ) ,  0.95 ( b ) , 0.96-0.97 ( c ) , and 0.98-0.99 ( d ) . The open c i r c l e s  i n d i c a t e the p o s i t i o n s  o f the CVP's  The s c a l l o p s on the l e f t o f each diagram i n d i c a t the p o s i t i o n and s i z e o f the v e s t i b u l e .  F i g u r e 14.  Growth of v a r i o u s r e g i o n s  on the  s u r f a c e of d i v i d i n g c e l l s o f ages 0.93  dorsal to  0.99-  To produce these p l o t s , the contour l e n g t h s c e l l regions  at the i n d i c a t e d ages were compared to  the contour l e n g t h of t h a t r e g i o n a t age and  the  of  " r e l a t i v e growth" r a t i o was  0.93  calculated.  The graphs show the amount o f i n c r e a s e  or  decrease i n contour l e n g t h as compared to l e n g t h a t age  0.93'  The  contour l e n g t h s  the  indicated  are: (©) -  CVA  (+) -  CVA'  (o) - The  d i s t a n c e from the f i s s i o n - f u r r o w to  the p o s t e r i o r CVP (o) - The  distance  -  CVP'  (v)  -  CVP  f i g u r e A - wild-type f i g u r e B - sm2  proter.  from the f i s s i o n - f u r r o w to  the a n t e r i o r CVP (A)  of the  cells.  of the  cells.  opisthe.  201  I  •93  1  -95 AGE  1  -97 (cell eye les )  T-  -99  Figure  15«  during  B a s a l body p r o l i f e r a t i o n the f i r s t c e l l  i n sm2  c y c l e a t the  cells  restrictive  temperature. The  f i g u r e s show the p o s i t i o n s  b a s a l b o d i e s and  parasomal sacs i n k i n e t i e s i n  the m i d r e g i o n (CVINT) of the The  ages of the c e l l s  below each f i g u r e . The  dorsal  (in c e l l  surface.  c y c l e s ) are  Large dark spots are  extent of b a s a l body p r o l i f e r a t i o n  sm2  given  CVPs. can  compared with t h a t o f the w i l d - t y p e c e l l s similar  of  be of  ages shown i n p l a t e 9« A b n o r m a l i t i e s  c e l l s i n c l u d e reduced p r o l i f e r a t i o n ,  in  cortical  u n i t s l a c k i n g e i t h e r a b a s a l body or parasomal sac, c o r t i c a l u n i t s with two and  parasomal  sacs,  d i s o r g a n i z a t i o n of the k i n e t i e s i n the  region.  The  v e r t i c a l bar r e p r e s e n t s  20  ;um.  furrow  188  F i g u r e 16.  A b n o r m a l i t i e s i n g u l l e t anlage development  i n sm2 c e l l s d u r i n g the f i r s t restrictive  c e l l c y c l e a t the  temperature.  a - A c e l l o f about age 0.85 w i t h a normal containing three k i n e t i e s  (the s o l i d  anlage  lines).  b - A c e l l o f about age 0.87 w i t h an anlage cont a i n i n g three k i n e t i e s . The a n t e r i o r end o f the anlage has moved p o s t e r i o r l y w h i l e the p o s t e r i o r end has n o t , r e s u l t i n g i n an i n v e r t e d "C"-shaped anlage. c - An anlage from a c e l l o f about age 0.9. are  There  t h r e e wide s t r i a t i o n s i n the anlage which  may comprise  s i x k i n e t i e s . Dark g r a n u l a r  m a t e r i a l l i e s between the a n t e r i o r o f the anlage and the v e s t i b u l e  wall.  d, e, f - Anlagen from c e l l s o f age 0.92. I n each case no k i n e t i e s are v i s i b l e i n the anlage. In  f i g u r e f , an anlage and endoral k i n e t y have  appeared on the r i g h t v e s t i b u l a r w a l l . I n f i g u r e d, o n l y t h e e n d o r a l k i n e t y can be seen and i n f i g u r e e the e n d o r a l k i n e t y i s v e r y irregular. g - An anlage from a c e l l o f age 0.93« The anlage i s e x c e e d i n g l y s m a l l and c o n t a i n s no k i n e t i e s .  F i g u r e 16  (con't)  h, i , j - Anlagen from c e l l s o f about age 0.95* In f i g u r e s h and i t h e r e are no k i n e t i e s i n the anlagen.  I n f i g u r e j there are f i v e  k i n e t i e s . I n each case t h e e n d o r a l k i n e t y can be found a l o n g the r i g h t v e s t i b u l a r w a l l and  there i s i r r e g u l a r , g r a i n y m a t e r i a l  between the p o s t e r i o r o f the anlage vestibule.  and the  191  Figure 1 7 -  Sample s t a t i s t i c s  o f c e l l s o f known ages  a f t e r a s h i f t t o t h e r e s t r i c t i v e temperature.  The  v e r t i c a l bars are 95$ c o n f i d e n c e i n t e r v a l s o f t h e mean. A - The l e n g t h o f w i l d - t y p e  (o) and dc4 (©) c e l l s .  B - The l e n g t h o f the a n t e r i o r suture v e s t i b u l e (G) o f w i l d - t y p e cells.  (GA) and  (o) and dck (©)  I  ~—T-  •75  — i  •75  .8  1—  8  1  .85  A G E (cell  1  -85 AGE  (cell  1  .9  cycles)  1  -9 cycles)  p-  .95  — r -  .95  >  F i g u r e 18.  Sample s t a t i s t i c s  of c e l l s  o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature.  The  v e r t i c a l bars are 9 5 $ c o n f i d e n c e i n t e r v a l s o f the mean. A - The l e n g t h o f the p o s t e r i o r suture (GP) o f wild-type B - The width  (o) and dc4 (©) c e l l s . of wild-type  (o) and dc4 (©) c e l l s .  Figure 1 9 «  Sample s t a t i s t i c s  o f c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature.  The  v e r t i c a l bars are 9 5 $ c o n f i d e n c e i n t e r v a l s o f the mean. A-  CVA (see f i g . 6) o f w i l d - t y p e  (o) and dc4 (©)  cells. B - CVP (see f i g . 6) o f w i l d - t y p e cells.  (o) and dc4 (•)  10  A  0 J  ,  .75  1 -8  1 1— 1 -85 -9 -95 AGE ( ce 11 eye les )  r— 1 0  F i g u r e 20.  Sample s t a t i s t i c s  of c e l l s  o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature.  The  v e r t i c a l bars are 95$ c o n f i d e n c e i n t e r v a l s o f the mean. A - CVA' (see f i g . 6) o f w i l d - t y p e  (o) and dc4 (•)  cells. B - CVP' (see f i g . 6) o f w i l d - t y p e cells.  (o) and dc4 («)  F i g u r e 21.  Sample s t a t i s t i c s  o f c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e vertical  temperature.  The  bars are 9 5 % c o n f i d e n c e i n t e r v a l s o f the  mean. A - The l e n g t h - t o - w i d t h r a t i o  o f w i l d - t y p e (o)  and dc4 (•) c e l l s . B - CVINT o f w i l d - t y p e  (o) and dck («) c e l l s .  6CH  204  I  • 75  1  1  -8  -85 AGE  (cell  I  -9 cycles)  1  -95  1  -  1-0  F i g u r e 22.  Sample s t a t i s t i c s  f o r c e l l s of known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature.  The  v e r t i c a l bars are 95% c o n f i d e n c e i n t e r v a l s o f the mean. A - The number of b a s a l bodies between the two innermost CVPs o f w i l d - t y p e (o) and dc4  (•)  cells. B - The s p a c i n g between b a s a l b o d i e s on w i l d - t y p e (o) and dc4 (©) c e l l s .  F i g u r e 23.  Camera l u c i d a drawing o f a dc4 c e l l  0.93 c e l l c y c l e s  a t the r e s t r i c t i v e  after  temperature.  Large, i r r e g u l a r patches have, appeared i n the central portion interrupt  of the d o r s a l  s u r f a c e . The patche  t h e paths o f k i n e t i e s  entire c e l l  around them. The  shown i s about 110 pm l o n g .  205  Figure  24.  dc4 (b),  Outline  drawings o f w i l d - t y p e ( l e f t ) and  ( r i g h t ) d i v i d i n g c e l l s o f ages 0.94 ( a ) , 0.95 0.96-0.97 (c),  and' 0.98-0.99 ( d ) . The open  c i r c l e s r e p r e s e n t the p o s i t i o n s  o f the CVPs. The  s c a l l o p s on the l e f t o f the diagrams i n d i c a t e the s i z e and p o s i t i o n o f the v e s t i b u l e s .  2QZ  Figure 2 5 .  Growth o f r e g i o n s  o f the d o r s a l s u r f a c e o f  dividing cells.  The p l o t s were made by comparing the  contour l e n g t h s  o f each d o r s a l r e g i o n a t v a r i o u s  c e l l ages with the l e n g t h o f t h a t r e g i o n a t age 0.92-0.93.  The r a t i o o f l e n g t h s  was then p l o t t e d  a g a i n s t c e l l age. The graphs show the r e l a t i v e amount o f i n c r e a s e o r decrease i n contour l e n g t h at  v a r i o u s c e l l ages. The r e g i o n s  on the d o r s a l  surface are: (•) - CVA (+) - CVA' (o) - The d i s t a n c e from the f i s s i o n - f u r r o w t o the p o s t e r i o r CVP o f the p r o t e r . (0.) - The d i s t a n c e from the f i s s i o n - f u r r o w t o the a n t e r i o r CVP o f the o p i s t h e . (A)  - CVP'  (v) - CVP figure A - dc4 figure B - d c 2  cells, a  cells.  209  F i g u r e 26.  Sample s t a t i s t i c s  f o r c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature.  The  v e r t i c a l bars are 95$ c o n f i d e n c e i n t e r v a l s o f the mean. A - The l e n g t h o f w i l d - t y p e  (o) and dfz2 («) c e l l s .  B - The l e n g t h o f the a n t e r i o r suture (GA) and the v e s t i b u l e (©)  cells.  (G) o f w i l d - t y p e  (o) and dfz2  211  —i •75  1 .8  1 1 -85 9 AGE ( e e l I eye les )  1 95  1— 1 -0  F i g u r e 27.  Sample s t a t i s t i c s  forcells  a f t e r a s h i f t t o the r e s t r i c t i v e  o f known age  temperature.  v e r t i c a l bars a r e 95$ c o n f i d e n c e i n t e r v a l s  The  o f the  mean. A - The l e n g t h o f the p o s t e r i o r s u t u r e (GP) o f wild-type B - The width  (o) and dfz2 (©) c e l l s . of wild-type  (o) and dfz2 ( a )  cells.  213  F i g u r e 28.  Sample s t a t i s t i c s  forcells  a f t e r a s h i f t t o the r e s t r i c t i v e  o f known age  temperature.  v e r t i c a l bars a r e 9 5 $ c o n f i d e n c e i n t e r v a l s  The  o f the  mean. A - CVA o f w i l d - t y p e (o) and d f z 2 (•) c e l l s . B - CVP o f w i l d - t y p e (o) and dfz2 (o) c e l l s .  ure 29.  Sample s t a t i s t i c s  f o r c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature.  Th  v e r t i c a l bars a r e 95$ c o n f i d e n c e i n t e r v a l s o f t h mean. A - CVA' o f w i l d - t y p e  (o) and dfz2 (9) c e l l s .  B - CVP* o f w i l d - t y p e  (o) and dfz2 (®) c e l l s .  217  0  1  1  .75  -8  1  1  -85 AGE  (cell  -9 cycles)  r— -95  1— 1-0  F i g u r e 30.  Sample s t a t i s t i c s  f o r c e l l s o f known age  a f t e r a s h i f t to the r e s t r i c t i v e vertical  temperature.  The  bars are 9 5 $ c o n f i d e n c e i n t e r v a l s o f t h e  mean. A - The l e n g t h - t o - w i d t h r a t i o  o f w i l d - t y p e (o)  and dfz2 (o) c e l l s . B - CVINT o f w i l d - t y p e  a  (o) and dfz2 (©) c e l l s .  5  —i 75  -8  1  1 1 i -85 -9 -95 AGE (cell c y c l e s  i 1 0  F i g u r e yi.  Sample s t a t i s t i c s  f o r c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature.  The  v e r t i c a l bars are 95% c o n f i d e n c e i n t e r v a l s o f the mean. A - The number o f b a s a l bodies between the i n n e r most CVPs on w i l d - t y p e  (o) and d f z 2  (©) c e l l s .  B - The s p a c i n g between b a s a l bodies on w i l d - t y p e (o) and d f z 2  (•) c e l l s .  F i g u r e 32.  Camera l u c i d a drawings o f dfz2 c e l l s  which  were a r r e s t e d d u r i n g d i v i s i o n a t the r e s t r i c t i v e temperature. a and e - Both s i d e s o f a c e l l which had been a t the r e s t r i c t i v e temperature f o r about cell  c y c l e s . There are 4 CVPs but no  0.9 fission  l i n e or furrow. The k i n e t i e s i n the c e n t r a l p o r t i o n of the c e l l are bent, b and f - Both s i d e s o f a c e l l which had been a t the r e s t r i c t i v e temperature f o r about cell but  0.95  c y c l e s . There are 4 CVPs and 2 v e s t i b u l e s o n l y a v e r y s h o r t f i s s i o n - l i n e and no  fission-furrow, c and g - Both s i d e s of a c e l l which had been a t the r e s t r i c t i v e temperature f o r about cell  0.97  c y c l e s . The o p i s t h e p o r t i o n o f the c e l l  has t w i s t e d i n r e l a t i o n t o the p r o t e r . d and h - Both s i d e s o f a c e l l which had been a t the r e s t r i c t i v e temperature f o r about cell  1.0  c y c l e s . The g u l l e t s o f the p r o t e r and  opisthe  are exposed on the c e l l  The o p i s t h e ' s  surface.  g u l l e t l i e s t o the r i g h t o f  the p r o t e r ' s . On the d o r s a l s u r f a c e  ( f i g . h)  the k i n e t i e s o f the o p i s t h e are a t r i g h t a n g l e s t o those o f the p r o t e r .  223 3  F i g u r e 33.  Outline  drawings o f dfz2 ( l e f t ) and d c 2  a  ( r i g h t ) d i v i d i n g c e l l s o f ages 0.94 ( a ) , 0.95 (b),  0.96-0.97 ( c ) , and 0.98-0.99 ( d ) . The open  c i r c l e s i n d i c a t e the p o s i t i o n s s c a l l o p s on the l e f t s i z e and p o s i t i o n s  o f the CVP's. The  o f the diagrams i n d i c a t e t h e  o f the v e s t i b u l e s .  225  F i g u r e 34.  Sample s t a t i s t i c s f o r c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature. v e r t i c a l bars are 95$  confidence i n t e r v a l s  The of the  mean. A - The l e n g t h o f w i l d - t y p e  (o) and d c 2  a  (o)  cells. B - The l e n g t h o f the a n t e r i o r suture v e s t i b u l e (G) o f w i l d - t y p e cells.  (GA) and  (o) and d c 2  a  (©)  227  AGE  (cell  cycles)  F i g u r e 35-  Sample s t a t i s t i c s  f o r c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature.  The  v e r t i c a l bars are 95$ c o n f i d e n c e i n t e r v a l s of the mean. A - The l e n g t h of the p o s t e r i o r suture w i l d - t y p e (o) and d c 2  a  B - The width o f w i l d - t y p e  (GP) o f  (©) c e l l s . (o) and d c 2  a  (•) c e l l s .  F i g u r e 36.  Sample s t a t i s t i c s  f o r c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature.  The  v e r t i c a l bars are 95f° c o n f i d e n c e i n t e r v a l s o f the mean. A - CVA o f w i l d - t y p e  (o) and d c 2  a  (0) cells.  B - CVP o f w i l d - t y p e  (o) and d c 2  a  (•) c e l l s .  F i g u r e 37-  Sample s t a t i s t i c s  f o r c e l l s o f known age  a f t e r a s h i f t to the r e s t r i c t i v e temperature.  Th  v e r t i c a l bars are 95$ c o n f i d e n c e i n t e r v a l s o f t h mean. A - CVA' o f w i l d - t y p e  (o) and d c 2  a  (•) c e l l s .  B - CVP' o f w i l d - t y p e  (o) and d c 2  a  (©) c e l l s .  40  1 30U  20-  10  0 •  r  •75  1 1— •8 -85 -9 -95 AGE ( ce II c y c l e s )  -]  I  1-0  F i g u r e 38 •  Sample s t a t i s t i c s  f o r c e l l s o f known age  a f t e r a s h i f t t o the r e s t r i c t i v e vertical  temperature.  Th  bars are 95$ c o n f i d e n c e i n t e r v a l s of t h  mean. A - The l e n g t h - t o - w i d t h r a t i o and d c 2  a  o f w i l d - t y p e (o)  (e) cells.  B - CVINT o f w i l d - t y p e  (o) and d c 2  a  (•) c e l l s .  ro  F i g u r e 39-  Sample s t a t i s t i c s  f o r c e l l s of known age  a f t e r a s h i f t t o the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95$ c o n f i d e n c e  i n t e r v a l s of the  mean. A - The number o f b a s a l bodies innermost CVPs o f w i l d - t y p e (•)  between the two (o) and d c 2  a  cells.  B - The s p a c i n g between b a s a l bodies (o) and d c 2  a  (o) c e l l s .  on  wild-type  237  Figure  40.  An example o f the p l o t s used t o o b t a i n  the mean c e l l c y c l e l e n g t h  o f a synchronous  population  o f c e l l s . The cumulative percentage  of d i v i d e d  c e l l s i s p l o t t e d on a s e m i - l o g  against  the time e l a p s e d s i n c e the c e l l  scale  popul-  a t i o n was i s o l a t e d . The i n t e r c e p t o f a s t r a i g h t l i n e drawn through the p o i n t s  w i t h the 50$  l e v e l on the X - a x i s g i v e s t h e median c e l l length.  A further discussion  cycle  o f t h i s technique  may be found i n Natchway and Cameron (1972). The two p l o t s shown a r e f o r p o p u l a t i o n s of dc5 c e l l s heat-shocked a t age 0.55  (*) and a t age 0.7 ( o ) .  CUMULATIVE PERCENTAGE DIVIDED CELLS .W  ro I  i  -» i  i  i  O  i i  O  I  U  i  J  l  O I  O  |  O  i  O  O  1  -  W O i L_L_I |_  ro  VJ  Figure  4i.  during  The p a t t e r n  of  temperature-sensitivity  the Paramecium c e l l c y c l e . T h i r t y minute  heat-shocks were a p p l i e d t o synchronous p o p u l a t i o n s of c e l l s of known age. The c e l l of the t r e a t e d samples was a non-heat-shocked  length  compared to t h a t of  c o n t r o l sample and the  r e l a t i v e c e l l c y c l e l e n g t h was The r e l a t i v e c e l l c y c l e l e n g t h s against  cycle  the time d u r i n g  calculated. were p l o t t e d  the c e l l c y c l e a t which  the c e l l s were heat-shocked. The v e r t i c a l  bars  are 95$ c o n f i d e n c e i n t e r v a l s f o r p r o p o r t i o n s . A - Wild-type c e l l s heat-shocked at 3 4 . 5 ° C and a t 35°C ( o ) . B - dc5  c e l l s heat-shocked a t  34.5°C.  (•)  CELL CYCLE  LENGTH  ( ° / of c o n t r o l ) 0  C E L L CYCLE  LENGTH  (•/. of  control)  t\3  Figure 4 2 .  The  pattern of  temperature-sensitivity  d u r i n g the Paramecium c e l l c y c l e . T h i r t y minute heat-shocks were a p p l i e d to synchronous popula t i o n s of c e l l s of known age.  The  l e n g t h of the t r e a t e d samples was  cell  cycle  compared to  t h a t of a non-heat-shocked c o n t r o l and relative  c e l l c y c l e l e n g t h was  relative  c e l l c y c l e lengths  the time d u r i n g the  cell  the  calculated.  were p l o t t e d  c y c l e at which  against the  c e l l s were heat-shocked. The  vertical  95% confidence  proportions.  A  - V a r i a n t 48  B - dc4  intervals for  c e l l s heat-shocked at  The  bars  are  34..5°C.  c e l l s heat-shocked a t 3 4 . 5 ° C . .  -H  c O  o 115  110-  r i r-  O 105UJ  _i  100-  ui  U 95H U 90 0  T" -i 1 1 1 1 r 20 40 60 80 100 TIME OF HEAT SHOCK ( %> of cell c y c l e )  o t_  1 2 0  c O  « 115 110  O 105  z  UJ  o O 95 LU  O 90 —i  1  1  1  1  1  20 40 60 TIME OF HEAT SHOCK (%  T  1—  80 of cell  1  1  100 cycle)  F i g u r e 43. during  The p a t t e r n  of temperature-sensitivity  the Paramecium c e l l c y c l e . T h i r t y minute  heat-shocks were a p p l i e d t o synchronous p o p u l a t i o n s o f c e l l s o f known age. The c e l l  cycle  l e n g t h o f the t r e a t e d samples was compared to t h a t o f non-heat-shocked  c o n t r o l s and the r e l a t i v e  c e l l c y c l e l e n g t h was c a l c u l a t e d . The r e l a t i v e c e l l c y c l e lengths  were p l o t t e d a g a i n s t  the time  d u r i n g t h e c e l l c y c l e a t which the c e l l s were heat-shocked. The v e r t i c a l b a r s are 9 5 $ c o n f i d ence i n t e r v a l s of p r o p o r t i o n s . The graph shown i s f o r sm2 c e l l s heatshocked a t 34.5°C. The r e s u l t s o f two separate experiments are shown.  CELL  §  CYCLE  LENGTH  <•/.  of  control)  F i g u r e kk. A schematic diagram showing the measurements made on sm2 c e l l s  o f v a r i o u s s i z e s . The measure-  ments shown i n B were made on only two c e l l  samples.  Abbreviations include: CYTL - c y t o p r o c t  length.  KTCV - the number o f k i n e t i e s t o the f i r s t  con-  t r a c t i l e v a c u o l e pore (CVP) encountered c o u n t i n g c o u n t e r c l o c k w i s e from t h e v e s t i b ule. KTCL - t h e number o f k i n e t i e s from the f i r s t CVP encountered t o the r i g h t edge o f the vestibule. KTCVV - the number o f k i n e t i e s between the CVPs.  F i g u r e 45.  Probit  asynchronous  plots samples  o f sm2  c e l l length f o r  taken a t v a r i o u s times  a f t e r a s h i f t t o the r e s t r i c t i v e A - sm2  samples:  (x) - non-heat-shocked (.)  control  - sample taken 6 hours a f t e r a s h i f t t o the r e s t r i c t i v e  (•)  temperature.  - sample taken 1 1 . 5  temperature. hours a f t e r a s h i f t  to the r e s t r i c t i v e  temperature,  (o) - sample taken 24 hours a f t e r a s h i f t to the r e s t r i c t i v e B - sm2 (•)  temperature.  samples: - sample taken 8 hours a f t e r a s h i f t t o the r e s t r i c t i v e  temperature,  (o) - sample taken 24 hours a f t e r a s h i f t to the r e s t r i c t i v e  temperature.  ®  8  8  B  7  7 6 ,.' J  5  >o6  h-  \— CO 4 O CL  x  •  CQ  8  68  O Cd CL  3  3 2  50  70  90~ LENGTH  110 (pm)  130  70  90  110 130 £ LENGTH (pm)  F i g u r e 46. samples  Probit of sm2  plots  o f c e l l width f o r asynchronous  c e l l s taken a t v a r i o u s times  a s h i f t to the r e s t r i c t i v e A - sm2  after  temperature.  samples:  (x) - non-heat-shocked (o) and  (o)  control,  - two independant  samples  taken  a f t e r 24 hours a t the r e s t r i c t i v e temperature. B - sm2  samples:  (x) - sample taken a f t e r 8 hours a t the restrictive  temperature.  (••) - sample taken a f t e r 6 hours a t the restrictive  temperature,  (o) - sample taken a f t e r 11.5 hours a t the restrictive  temperature.  ®  8  8-  B  7-  7  o o  6  5  o o o  5  5  5  O 4  Occ A  o  or 0_  \  CD  CD  X  0_  3  3  1 O-  30  WIDTH  40  (pm)  50  60  0  30  40 WIDTH (jjm)  F i g u r e 4?. UPPER - The l e n g t h (o)  and width (•)  o f sm2  c e l l s p l o t t e d a g a i n s t time at restrictive  the  temperature.  LOWER - The length-to-width r a t i o of sm2 c e l l s p l o t t e d a g a i n s t time at the temperature.  restrictive  140  0-J  ,  0  ,  6  ,  12 TIME(hours)  ,  18  r  24  Figure 4 8 .  Sample s t a t i s t i c s  o f asynchronous  sm2  c e l l s a t v a r i o u s times a f t e r a s h i f t t o the r e s t r i c t i v e temperature. The v e r t i c a l b a r s are 95% confidence A - Sample  i n t e r v a l s o f the mean.  statistics:  (o) - a n t e r i o r suture  length.  (©) - p o s t e r i o r suture (o,  length.  lower) - v e s t i b u l e l e n g t h .  B - Sample s t a t i s t i c s shown i n f i g u r e A as a f r a c t i o n o f the c e l l  length:  (o) - GA/length (•) - GP/length (o,  lower) - G/length  C - Sample  statistics:  (o) - CVINT (see f i g . 4 4 ) (©) - CVA  (see f i g . 4 4 )  (x) - CVP (see f i g . 4 4 ) D - Sample s t a t i s t i c s shown i n f i g u r e C as a f r a c t i o n of the c e l l (o) - CVINT/length (•) - CVA/length (x) - CVP/length  length;  L E N G T H (p m)  LENGTH  (pm)  Figure  49 •  Sample s t a t i s t i c s  c e l l s at various restrictive  o f asynchronous sm2  times a f t e r a s h i f t t o the  temperature. The v e r t i c a l  bars a r e  95% c o n f i d e n c e i n t e r v a l s o f the mean. A - Sample s t a t i s t i c s : (o) - the d i s t a n c e  from the p o s t e r i o r CVP  t o the a n t e r i o r o f the v e s t i b u l e . (•) - the d i s t a n c e  from the a n t e r i o r CVP t o  the p o s t e r i o r o f t h e v e s t i b u l e , (x) - the d i s t a n c e  from  the p o s t e r i o r of  the v e s t i b u l e t o t h e p o s t e r i o r CVP. (+) - the d i s t a n c e  from the a n t e r i o r CVP t o  the a n t e r i o r o f t h e v e s t i b u l e . B - Sample s t a t i s t i c s  shown i n f i g u r e A expressed  as a f r a c t i o n o f the c e l l  length.  The symbols  used a r e the same as i n f i g u r e A. C - Cytoproct  length.  D - The c y t o p r o c t c e l l length length  length  (e)  as a f r a c t i o n o f the  and as a f r a c t i o n o f the  o f the p o s t e r i o r s u t u r e ( o ) .  F i g u r e 50-  B i v a r i a t e s c a t t e r p l o t s of sample s t a t -  i s t i c s o f sm2 A - The  cells.  l e n g t h of the a n t e r i o r suture  a g a i n s t the l e n g t h o f sm2 at  (GA)  plotted  c e l l s which had  been  the r e s t r i c t i v e temperature f o r 24 hours.  The  dotted l i n e s  (  are:  ) - The p r i n c i p a l a x i s of the s c a t t e r p l o t of GA  and l e n g t h f o r a sample taken  a f t e r 8 hours a t the  restrictive  temperature. (-.-)  - The p r i n c i p a l a x i s of the s c a t t e r p l o t of GA  and  l e n g t h f o r a non-heat-shocked  sample. The to  s o l i d l i n e i n d i c a t e s values  of GA  equal  47.5$ of the c e l l l e n g t h (see t e x t ) .  The  s l o p e s o f a l l p r i n c i p a l axes are g i v e n i n table B - The  17.  l e n g t h o f the p o s t e r i o r suture  p l o t t e d a g a i n s t the l e n g t h of sm2 had  (GP) c e l l s which  been at the r e s t r i c t i v e temperature f o r  24 hours. The axis.  d o t t e d l i n e i s the  principal  F i g u r e 51 •  B i v a r i a t e s c a t t e r p l o t s o f sample  t i c s o f asynchronous sm2  statis-  cells.  A - The c y t p r o c t l e n g t h (CYTL) p l o t t e d a g a i n s t c e l l l e n g t h f o r a non-heat-shocked sample (o) and a sample taken a f t e r 24 hours a t the r e s t r i c t i v e temperature (•). The p r i n c i p a l axes o f the non-heat-shocked sample and the 24 hour sample •  s o l i d l i n e represents  (  )  (-.-) are shown. The cytoproct  equal t o 15% o f the c e l l  lengths  length.  B - The l e n g t h o f t h e v e s t i b u l e (G) p l o t t e d a g a i n s t cell  length:  (o) - non-heat-shocked sample (©) - sample taken 24 hours a f t e r a s h i f t t o the r e s t r i c t i v e temperature. The p r i n c i p a l a x i s o f the 24 hour sample i s shown (-.-). The l e n g t h o f the v e s t i b u l e i s not c o r r e l a t e d with c e l l heat-shocked sample  (  l e n g t h i n the non).  261  Figure  52.  Bivariate  istics A  o f sm2  scatter  plots  distance  "between t h e . a n t e r i o r  the  anterior  of the c e l l  cell  - non-heat-shocked  (©)  - sample  t a k e n 24  . the r e s t r i c t i v e distance  the  and  plotted  sample. hours a f t e r  of the c e l l  cell  a shift  to  temperature.  between the p o s t e r i o r  posterior  against  (CVA)  CVP  length:  (o)  - The  stat-  cells.  - The  against  B  of sample  (CVP)  CVP  and  plotted  length:  (o)  - non-heat-shocked  (•)  - sample  sample.  taken after  restrictive On  both p l o t s ,  as  follows:  24 h o u r s , a t  the  temperature.  the p r i n c i p a l axes  (-—)  - non-heat-shocked  (-.-)  - 24 h o u r  sample.  sample.  are  indicated  263  Figure 5 3 istics  B i v a r i a t e s c a t t e r p l o t s of sample o f sm2  stat-  cells.  A - The d i s t a n c e between the CVPs (CVINT) p l o t t e d a g a i n s t the c e l l l e n g t h : (o) - non-heat-shocked sample. (•) - sample taken 24 hours a f t e r a s h i f t t o the r e s t r i c t i v e  temperature.  The p r i n c i p a l axes o f the o f the non-heat.  shocked sample (-.-)  (  ) and the 24 hour sample  are i n d i c a t e d . The s o l i d l i n e  describes  the equation CVINT = LENGTH - (-35LENGTH + 22 um)  (see t e x t ) .  B - The number o f b a s a l b o d i e s between  the CVPs  p l o t t e d a g a i n s t c e l l l e n g t h f o r a sample of c e l l s taken 24 hours a f t e r a s h i f t to the restrictive  temperature.  265  Figure 5 ^ . istics  Bivariate  scatter  of asynchronous  A - The  plots  sm2  cell  length  bodies  f o r a sample  plotted af  t a k e n 2k h o u r s a f t e r a s h i f t  to the  tive  line  t e m p e r a t u r e . The  principal B - The  dashed  against  restric-  i s the  cell  width f o r a  cell sample  t a k e n 2k h o u r s a f t e r a s h i f t  to the  tive  line  t e m p e r a t u r e . The  principal  cells  axis.  t o t a l number o f k i n e t i e s p e r  plotted  stat-  cells.  s p a c i n g between b a s a l  against  o f sample  axis.  dashed  restric-  i s the  104 /  •8H O  /  O  /  EV ' 6  O O  o CD  <  3  oo  qfo Q  /  o  o o of  0  •  -  •4J  O  /  /  Q  o  /  o  O  0  ° . 0  o  o/'  o  60 80 100 C E L L LENGTH (pm)  40  e 80  B  E  o  o  /  2 76 o  /  o o O O  O/Q  o CD  ooo CD O  /  * 68-I  is  _i  AD  O  o o  O  o  o  <t>/  <  0 64-j 604 34  /O  s  /  38  o  o o  42  46 WIDTH  50 (pm)  54  120  Figure 5 5 - Bivariate  s c a t t e r p l o t s o f sample s t a t -  i s t i c s o f asynchronous  sm2 c e l l s .  A - The t o t a l number o f k i n e t i e s p e r c e l l against  the number o f k i n e t i e s between the  l e f t v e s t i b u l a r w a l l and the f i r s t the  plotted  cell's left  CVP t o  (KTCV) f o r a sample o f c e l l s  taken 24 hours a f t e r a s h i f t t o the r e s t r i c tive  temperature. The dashed l i n e  i s the  p r i n c i p a l a x i s . The s o l i d l i n e d e s c r i b e s the equation: KTCV = .58TOTAL KINETIES (see t e x t ) . B - The v a l u e KTCV expressed as a percentage o f the  t o t a l number o f k i n e t i e s p l o t t e d  c e l l width. There i s no c o r r e l a t i o n .  against  KTCV C/o of k i n e t i e s )  TOTAL K I N E T I E S ( n u m b e r )  i  ro ON  NO  F i g u r e 56.  P r o b i t p l o t s of c e l l l e n g t h measurements  from the f e e d i n g (•) -  experiment.  non-heat-shocked'sample.  (o) - sample taken a f t e r 24 hours a t the r e s t r i c tive  temperature.  8  40  60  CELL  80 ' LENGTH (pm)  100  120  F i g u r e 57 • istics  B i v a r i a t e s c a t t e r p l o t s o f sample s t a t of sm2  cells.  A - The l e n g t h o f the quadrulus p l o t t e d cell  against  length:  (o) - non-heat-shocked sample. (•) - sample taken a f t e r 24 hours at the r e s t r i c t i v e temperature. B - .The number o f p a i n t - f i l l e d food v a c u o l e s p l o t t e d a g a i n s t the l e n g t h of the quadrulus: (o) - non-heat-shocked  sample.  (•) - sample taken a f t e r 24 hours a t the r e s t r i c t i v e temperature.  ©  273 oa  9 S3  E  »  X  r-  9  LU  i/) Z> j  16-  e e». o 0  o  GC  vn  Q  0  0  0  oo o oo o  •  O  OO  OO  O  g C O O O o  eas eo o es o o oo o • oeooedo e ©e> e  20-  o z _1  0  e» o  «D <D <ID  a  O&>  o  e 0  0  121  35  55  1  1  1—  75 95 115 CELL LENGTH (um)  135  o  9 0  fe 8  0  8  S  8  S8  • 8  -i  0  1  4  1  1 — — — '  o  0  o  o  S o .  8  %  o  o  • S  • •  1  8 12 16 20 OUADRULUS LENGTH (pm)  I  •  • ••  77  24  F i g u r e 58.  A schematic diagram i l l u s t r a t i n g p o s s i b l e  mechanisms f o r the gain and l o s s o f k i n e t i e s (from Heckmann and F r a n k e l , 1968). The zone o f b a s a l body p r o l i f e r a t i o n on d i v i d i n g c e l l s i s represented  by the dotted  which are o u t s i d e lost  ( a ) . Short  l i n e s . Short k i n e t i e s  o f t h i s zone are e v e n t u a l l y  k i n e t i e s which extend i n t o t h i s  zone (b) e v e n t u a l l y i n c r e a s e i n l e n g t h t o extend pole-to-pole.  GAIN  AND  LOSS OF  KINETIES  Figure  59.  The f a t e  diagram.  The 4 CVPs  shown. E a c h new by  o f CVPs i n a c e l l  a separate  explanation.  lineage  of dividing cells  generation  symbol.  are  o f CVPs i s i n d i c a t e d  See t h e t e x t  f o r further  Figure  60.  The r e l a t i v e p o s i t i o n s  dividing  cells.  relative  positions  cell  The u p p e r  o f a g e 0.8.  figure  The CVP p o s i t i o n s  of the c e l l  the  end o f t h e c e l l .  positions division cortex  that  i fgrowth  are given  as  measured from  The r e l a t i v e assume  were u n i f o r m  figure  after  cell  throughout the  shows t h e r e l a t i v e  o f t h e 4 CVPs i n d i v i d i n g  treated  ages.  In this figure,  as i f t h e y  (the proter  fission-furrow position linear  and o p i s t h e )  may  posi-  of the  dividing cells o f two  l i n e s a t 35$  a n d 80$  o f t h e 2 CVPs a l o n g  interfission  cells.  75$  indicate  the positions  the  contour  were  separate  even though  not have been present.  (©) a n d c o n t o u r  length  cells  were c o m p r i s e d  o f t h e CVPs i s g i v e n  length  hatched tions  dividing  a r e shown t o t h e r i g h t .  indicated  cells  length,  t h e CVPs w o u l d  The l o w e r tions  shows t h e  o f t h e k CVPs on a  a proportion anterior  o f CVPs i n  a The  i n terms of both length indicate  ( o ) . The the posi-  the linear length  The h a t c h e d  l i n e s a t 35$  o f t h e 2 CVPs  of i n t e r f i s s i o n cells.  of and  along  279  100-85 .9 -95 A G E ( celJ c y c l e s )  .85 AGE  -9 -95 ( cell c y c l e s )  1-0  P l a t e 1.  Gullet  structure.  a - I n v e r t e d " J " anlage from a c e l l a t age 0.9b - I n t e r f i s s i o n age 0.92. The a n t e r i o r end o f the anlage i s p u l l e d away from the a n t e r i o r of the v e s t i b u l e , c - I n t e r f i s s i o n age 0.95* The anlage p o s t e r i o r t o the f i s s i o n  lies  line,  d - A g u l l e t e v e r t e d on the s u r f a c e o f a dfz2 cell. e and f - Transverse gap a c r o s s a l l g u l l e t k i n e t i e s i n sm2  cells.  231  P l a t e 2.  Somatic c o r t e x - normal and abnormal c e l l s .  a - A normal cortex i n a w i l d - t y p e c e l l . l a r g e r b l a c k spots' ( s i l v e r  The  deposits) indicate  the p o s i t i o n s o f c i l i a r y b a s a l b o d i e s  and  parasomal s a c s , the s m a l l e r spots i n d i c a t e the p o s i t i o n s of t r i c h o c y s t s . b - Supernumerary t r i c h o c y s t s i n a t s O - l  a  cell.  ( T h i s mutant, a l o n g with s e v e r a l o t h e r s , although not c o n s i d e r e d i n d e t a i l i n the present  study, w i l l be used i n t h i s  other p l a t e s f o r i l l u s t r a t i v e  and  purposes.)  c - Supernumerary components on a t s O - l  a  cell.  d, e, f - C o r t i c a l a b n o r m a l i t i e s of types 1, and  2,  3 r e s p e c t i v e l y , ( f i g . d - v a r i a n t 46,  figs,  e and  f -  sm2)  g, h - C o r t i c a l p a t c h e s (p) a p p a r e n t l y no c o r t i c a l  containing  o r g a n e l l e s , ( v a r i a n t s 42 and  46)  283  »••••.»«.  * ' * • • > • • * * »  CT) A . 4 A M * * * * * * * * ' ' * * .»>r 0  • 11  *  P l a t e 3'  Gullet abnormalities.  a, b - Type 1 abnormality. are i r r e g u l a r ,  The q u a d r u l a r  (variant  53  kineties  cells)  c, d - Type 2 abnormality. The innermost o n e - t h i r d of the g u l l e t  i s m i s s i n g , the k i n e t i e s  very i r r e g u l a r .  (sm2  cells)  e, f - Type 3 abnormality. The innermost of the g u l l e t  are  i s m i s s i n g . (sm2  one-half  cells)  285  P l a t e 4.  Cytoproct s t r u c t u r e ,  a - normal  cytoproct.  b - disrupted cytoproct - d f z l  cell,  c - disrupted cytoproct - dc2  cell,  b  d - p l a t e - l i k e cytoproct - t s O - l e - distended  cytoproct - t s O - l  a  a  cell, cell.  287  Plate 5'  Cell  shapes.  a - normal shape - w i l d - t y p e b - l a r g e , pear-shaped' c e l l c - large c e l l  - tsO-1  d - irregularly-shaped  cell. - variant  27-  cell. cell  e - t h i n , astomatous, c e l l  - variant - sml  18.  cell.  f - a n t e r i o r t r u n c a t i o n - the specimen has no a n t e r i o r suture or v e s t i b u l e - sm2 g  p o s t e r i o r t r u n c a t i o n - sm2  cell,  cell,  h - small c e l l  - sm2  i,  c e l l s - note t h a t the k i n e t i e s  j - twisted  cell.  s p i r a l around the c e l l  surface  - dc6 c e l l s .  Plate 6.  C e l l shapes.  a - l a r g e tsO-1  cell.  b - l a r g e , round, and d e e p l y - a r g e n t o p h i l i c  tsO-3  cell. c - swollen, irregularly-shaped d - l a r g e tsO-1  dc6  cell.  c e l l with abnormal c o n t r a c t i l e  vacuole pores, e - i r r e g u l a r l y shaped dfz2 c e l l with  patchy  cortex. f - dc6 c e l l with supernumerary c o n t r a c t i l e v a c u o l e pores  (most are s l i g h t l y  out of f o c u s ) .  29/  P l a t e 7.  C e l l shapes.  a - normal d i v i d i n g  cell.  b and d - blockage o f c e l l d i v i s i o n l a t e i n the cell  cycle  - mutants dc4 ( f i g . b) and  dfzl.  c and e - blockage o f c e l l d i v i s i o n d u r i n g the early  stages o f f u r r o w i n g - mutant  f - monster c e l l  (mutant dc2^) r e s u l t i n g  continued c e l l normal c e l l  dfz2  from  growth i n the absence o f  division.  cells,  273  Plate  8. a  Cell  division  - interfission  stages.  cell  with  no  obvious signs  of  morphogenesis, b  - appearance of  c  of the f i s s i o n - l i n e  ( f l ) on a  cell  0.92.  age  - a g e 0.94  cell  with  around  the c e l l  a fission-line  and w i t h  complete  the sides  of the  cell  flattened. d  - four 0.94  CVPs on t h e d o r s a l  surface  cell  form around  e - a cell and  a g e d O.95  a large  ibule f  ( t h e new CVPs with  o f a n age age 0 . 8 ) .  a slight constriction  c l e a r space  posterior to the vest-  i n the proter.  - a n a g e 0.99  cell  with  a nearly  complete  fission-furrow. g - proter  from  anterior  of the c e l l  posterior h  blunt  i s pointed  from  a recently  and p o s t e r i o r ends  and the v e s t i b u l e  located.  divided  cellwhile  the the  i s blunt.  - an o p i s t h e anterior  a recently  divided  cell-  of the c e l l  i s not c e n t r a l l y  the are  295"  P l a t e 9-  B a s a l body p r o l i f e r a t i o n i n w i l d - t y p e  a - c e l l age has  0.75  occurred  b - c e l l age  - no  cells.  b a s a l body p r o l i f e r a t i o n  on the d o r s a l s u r f a c e .  0.9  - b a s a l body p r o l i f e r a t i o n  a few c o r t i c a l u n i t s p o s t e r i o r t o the  extends  fission-  line. c - c e l l age  0.93  - b a s a l body p r o l i f e r a t i o n extends  p o s t e r i o r l y beyond the a n t e r i o r C V P of o p i s t h e and  the  a n t e r i o r l y to the p o s t e r i o r C V P  of the p r o t e r . d - c e l l age  0.96  - b a s a l body p r o l i f e r a t i o n  . a n t e r i o r l y past the p o s t e r i o r C V P of the and  the k i n e t i e s appear bent i n the  extends proter  furrow  region. e - c e l l age  0.99  - s u r f a c e growth i s i n c r e a s i n g  the s p a c i n g o f p r e v i o u s l y c l o s e l y packed (compare with f i g u r e d) b a s a l  bodies.  297  r*»r% • > *  - * *  T  • * • • * • t « • .•  2• *% «  j) ••2-5!  3  •3  •  ftps  i l  W W W  • * •ill*  1 lit  Plate  10.  Cell  division  restrictive cell  stages  temperature.  cycle  o f sm2  Cells  at the r e s t r i c t i v e  a  - age  b  - a g e 0.8  - The  c  - a g e 0.93  - Fission-line  d  - a g e 0.94  - The  cells  at the  were g r o w n f o r one temperature,  0.75  anterior in  cell  i s abnormally  cell  wide.  formation  i s normal.  i s contracted  at the  and p o s t e r i o r ends.  the oral  groove r e g i o n  Contraction  i s particularly  noticeable. e - a g e O.96  - The  furrow end  of the c e l l  the  due  i s normal.  to continued of the  - An a b n o r m a l l y  a very  disorganized  and o p i s t h e  cell  division  short  The  i s abnormally  anterior pole  - a g e 0.99  g - proter  i s abnormally  formation  possibly  f  cell  but  anterior  pointed,  contraction of cell.  short  divider with  cortex.  ( a g e 1.05)  resulting  at the r e s t r i c t i v e  from  temper-  ature . h  - wild-type size  cell  a g e 0.99  o f sm2  The v e r t i c a l other  - Compare  d i v i d e r s shown  with  the  i n figure f.  b a r i n d i c a t e s 20  pm. A l l  f i g u r e s a r e t o t h e same s c a l e .  299  

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