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UBC Theses and Dissertations

A developmental analysis of behavioural mutations in Drosophila Melanogaster Wong, David T. L. 1981

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A DEVELOPMENTAL ANALYSIS OF BEHAVIOURAL MUTATIONS IN DROSOPHILA MELANOGASTER  by D a v i d T.L. Wong B.Sc., Simon F r a s e r U n i v e r s i t y , " 1 9 7 6  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE i n Genetics in the Department o f Zoology (FACULTY OF GRADUATE STUDIES) We a c c e p t t h i s t h e s i s as conforming r e q u i r e d standard  to the  THE UNIVERSITY OF BRITISH COLUMBIA June, 1980 © D a v i d T.L. Wong, 1980  In p r e s e n t i n g  this  thesis in partial  r e q u i r e m e n t s f o r an  advanced degree at the  the L i b r a r y s h a l l  it  and  freely  available for reference for extensive  f o r s c h o l a r l y p u r p o s e s may  be  d e p a r t m e n t o r by h i s o r h e r understood that for  financial  copying  gain  granted  s h a l l not  be  allowed  &BrN^rri C£  Columbia  make  further  of this  thesis  the head of  or publication of this  The U n i v e r s i t y o f B r i t i s h 2075 W e s b r o o k P l a c e Vancouver, Canada V6T 1W5  (2/79)  by  I  representatives.  permission.  Department of  study.  copying  the  University  of B r i t i s h Columbia, I agree that agree that permission  DE-6  f u l f i l m e n t of  my  It is thesis  w i t h o u t my  written  ABSTRACT Two types o f s e x - l i n k e d r e c e s s i v e mutations i n D r o s o p h i l a melanogaster have been i n v e s t i g a t e d i n the p r e s e n t study.  The f i r s t  type i n c l u d e s 5 d i f f e r e n t mutations which e x h i b i t a s t r e s s - s e n s i t i v e (ses) phenotype.  F l i e s o f a l l f i v e mutant s t o c k s become p a r a l y z e d  when t h e i r c o n t a i n e r s a r e l i g h t l y by the same treatment. groups  (cistrons).  tapped; w i l d type f l i e s a r e u n a f f e c t e d  The f i v e mutations form t h r e e  complementation  F l i e s mosaic f o r mutant and non-mutant t i s s u e were  s t u d i e d to determine the f o c i o f t h e i r a c t i o n i n embryos by f a t e mapping. 2 These s t u d i e s suggest t h a t the mutation ses D sumptive nervous system o f the b l a s t o d e r m .  has 6 f o c i i n the p r e -  Each focus corresponds  to a s i t e i n the t h o r a c i c g a n g l i o n which c o n t r o l s the movement o f one leg.  The focus f o r ses E"* mutation i s r a t h e r d i f f u s e and o c c u p i e s a -  l a r g e r a r e a i n the t h o r a c i c nervous system o f t h e b l a s t o d e r m f a t e map. Focus mapping s t u d i e s w i t h the ses B"* m u t a t i o n were i n c o n c l u s i v e -  because  of the h i g h l y v a r i a b l e e x p r e s s i o n s o f the a d u l t b e h a v i o u r a l phenotype i n mosaic i n d i v i d u a l s . shifts  Developmental  from 22°C to 29°C  revealed  studies, involving  ( p e r m i s s i v e to r e s t r i c t i v e  temperature  temperatures)  that the ses E^~ mutation has 2 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 s  (TSPs) f o r l e t h a l i t y d u r i n g i t s development,  one i n the l a t e 2nd  l a r v a l i n s t a r s t a g e and the o t h e r i n the l a t e p u p a l s t a g e . In a d d i t i o n 2 to developmental TSPs o f ses B a t the embryonic, 1 s t l a r v a l i n s t a r and p r e - p u p a l stage, t e m p e r a t u r e - s h i f t s t u d i e s a l s o r e v e a l e d a t s m a t e r n a l effect  2  l e t h a l f o r ses B .  iii  The second type o f m u t a t i o n s t u d i e d has a t e m p e r a t u r e - s e n s i t i v e ( t s ) phenotype  of a d u l t death induced by s h i f t i n g  up to the r e s t r i c t i v e  tsl temperature.  The add A  f l i e s have normal b e h a v i o u r and  at 22°C but d i e w i t h i n 24 hours a f t e r s h i f t - u p to 29°C.  longevity  In c o n t r a s t ,  the ses E"* f l i e s are l e s s a c t i v e and r e q u i r e 168 hours a t 29°C to induce -  death.  Both mutations s t u d i e d have a 3rd l a r v a l i n s t a r - p u p a l TSP w i t h  tsl  1  add A and ses E a l s o h a v i n g an a d d i t i o n a l TSP a t the embryonic and 1 s t l a r v a l i n s t a r - 2 n d l a r v a l i n s t a r stage r e s p e c t i v e l y . F a t e mapping tsl s t u d i e s suggest t h a t the a d u l t l e t h a l phenotype  of add A  i s caused  by a l e s o o n i n t i s s u e s d e r i v e d from the mesodermal c e l l s of the b l a s t o d e r m , and f o r ses E\  the l e s i o n i s i n the n e u r a l  cells.  iv  TABLE OF CONTENTS  ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  v i i  ACKNOWLEDGEMENT  *  CHAPTER 1  GENERAL INTRODUCTION  CHAPTER 2  STRESS-SENSITIVE MUTATIONS  I. II.  1  Introduction  17  M a t e r i a l s and Methods Developmental S t u d i e s Genetic Mosaic Studies N e u r o p h y s i o l o g i c a l Studies;  III.  IV. CHAPTER 3 I. II.  19 21 22  Results ses B: C h a r a c t e r i z a t i o n o f TSPs G e n e t i c Mosaic S t u d i e s Drug S t u i d e s ses D: G e n e t i c Mosaic S t u d i e s Drug S t u d i e s ses E: C h a r a c t e r i z a t i o n o f TSPs G e n e t i c Mosaic S t u d i e s Drug S t u d i e s  25 32 32 35 36 36 41 50  Discussion  53  '  DROP-DEAD MUTATIONS Introduction  62  M a t e r i a l s and Methods Examination o f a d u l t drop-dead b e h a v i o u r Examination o f Embryonic Development  III.  ix  65 65  Results add A  tsl  ; C h a r a c t e r i z a t i o n of a d u l t drop-dead ..behaviour  6.7  V  CHAPTER 3, c o n t i n u e d . add A; C h a r a c t e r i z a t i o n o f TSPs G e n e t i c Mosaic S t u d i e s Drug S t u d i e s Photomicrographic Studies ses E: C h a r a c t e r i z a t i o n o f a d u l t drop-dead Behaviour C h a r a c t e r i z a t i o n o f TSPs Genetic Mosaic Studies Drug S t u d i e s IV.  Discussion  67 86 90 90  •  115 115 118 118 121  LITERATURE CITED  128  APPENDIX  135  LIST OF TABLES TABLE 1.  Behavioural c h a r a c t e r i - t i c s of wild-type Ore-R and mutant f l i e s .  2.  L i s t of drugs, t h e i r putative a c t i o n and the Drosophila l a r v a l LD^Q-  3.  Maternal-effects of ses B .  4.  Temperature-sensitive  5.  Temperature-shift  6.  C o r r e l a t i o n of the three body segments of ses B"^ mosaics with t h e i r behaviour.  7.  L i s t of drugs which have a more pronounced e f f e c t on the v i a b i l i t y of d i f f e r e n t s t r e s s - s e n s i t i v e mutations.  8.  Ranked distances of ses E'*" focus with respect to various body landmarks.  9.  L i s t of drugs which have a more pronounced e f f e c t on the v i a b i l i t y of two drop-dead mutations.  2 2  maternal-effects of ses B . 2  studies of ses B .  LIST OF FIGURES  FIGURE 1.  Formation of a mosaic chromosome.  2.  R i g h t h a l f of the b l a s t o d e r m f a t e map body p a r t s .  3.  P r i n c i p l e of mosaic  4.  R i g h t h a l f o f the b l a s t o d e r m f a t e map f o r a d u l t e x t e r n a l body landmarks, v e n t r a l nervous system and mesoderm.  5.  6.  f l y through the l o s s of a r i n g - X  f o r adult  external  f a t e mapping.  2 Percentages o f ses B a d u l t s e c l o s e d from hatched eggs a f t e r s h i f t - u p s and s h i f t - d o w n s a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development. 2 F a t e map of ses D .  7.  Percentages of ses E^" pupae formed a f t e r s h i f t - u p s and shift-downs a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development.  8.  Percentages of ses E"*' a d u l t s e c l o s e d a f t e r s h i f t - u p s and s h i f t - d o w n s a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development.  9.  Percentages of ses E^ a d u l t s and Ore-R a d u l t s e c l o s e d a f t e r 12-hour heat p u l s e s a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development.  10.  F a t e map  of ses E^.  11.  S u r v i v a l of 1-day a d u l t s a t 29°C.  12.  S u r v i v a l of 1-5  13.  Percentages of add A and Ore-R eggs t h a t h a t c h a f t e r 6-hour heat p u l s e s a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development. tsl Percentages of add A eggs which y i e l d a d u l t s a f t e r 6-hour heat p u l s e s a d m i n i s t e r e d at d i f f e r e n t times d u r i n g development.  tsl o l d , 5-day o l d and  10-day o l d add A  day o l d Ore-R a d u l t s a t 29°C.  tsl  14.  Percentages of add A eggs which h a t c h a f t e r s u c c e s s i v e s h i f t - u p s a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development. tsl Percentages of add A eggs which y i e l d a d u l t s a f t e r s u c c e s s i v e s h i f t - u p s a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development. tsl Percentages of add A white pre-pupae which y i e l d a d u l t s a f t e r s u c c e s s i v e s h i f t - u p s and s h i f t - d o w n s a d m i n i s t e r e d p r i o r to and a f t e r w h i t e pre-pupae formation. tsl Percentages of add A w h i t e pre-pupae which y i e l d a d u l t s a f t e r 12-hour heat p u l s e s and 12-hour c o l d p u l s e s a d m i n i s t e r e d p r i o r to and a f t e r white p r e pupae f o r m a t i o n . Percentages of add A ^ t S  mosaic s u r v i v o r s a t 29°C.  tsl F a t e map  of add A  tsl Photomicrographs of add A and Ore-R embryonic development a t 29 C taken a t 2-hour i n t e r v a l s . S u r v i v a l o f 1-day o l d , 5-day o l d and 10-day o l d ses E a d u l t s a t 29°C. Percentages of ses E^ mosaic s u r v i v o r s a t 29°C. tsl Photomicrographs o f add A at 29°C.  and Ore-R c e l l  cultures  ACKNOWLEGEMENT  I am v e r y g r a t e f u l to Dr. David T. Suzuki f o r p r o v i d i n g me w i t h a most i n t e r e s t i n g r e s e a r c h problem, and I am  e s p e c i a l l y i n d e b t e d to Dr. Theodore Homyk, J r . , who  so g r a c i o u s l y and research.  f o r h i s f a i t h and h e l p .  u n s e l f i s h l y of h i s time i n the completion o f  H i s e n t h u s i a s t i c encouragement, p a t i e n t guidance,  this  and  to s i m p l i f y a problem on more than one o c c a s i o n , p r o v i d e d the  gave  ability  necessary  impetus to c o n t i n u e . A s p e c i a l thanks  i s extended  to Dr. Thomas G r i g l i a t t i f o r  h i s h e l p f u l s u g g e s t i o n s on m a t e r n a l - e f f e c t s t u d i e s and h i s r e a d i n g of t h i s  thesis.  I would l i k e culture  critical  to thank M i s s C h r i s t i n e Beard  f o r her  tissue  photographs. My  p a r e n t s and b r o t h e r , and a number of f r i e n d s , have p r o v i d e d  tremendous support to thank them.  throughout  my  y e a r s a t UBC  and  I would l i k e  A l s o , they have been most t o l e r a n t of my  I wish  to thank a l l members o f Dr.  numerous to mention, whose h e l p and both, p o s s i b l e and  enjoyable.  particular  "preoccupation".  Suzuki's l a b o r a t o r y , too  encouragement have made t h i s work  CHAPTER 1 GENERAL. INTRODUCTION  The c o n t r o l of gene e x p r e s s i o n i n b a c t e r i a and t h e i r i s now  understood i n d e t a i l .  In a d d i t i o n to p r o v i d i n g us w i t h i n f o r m a t i o n  on r e g u l a t i o n of gene a c t i v i t y , of  viruses  these s t u d i e s emphasize the v i t a l  each gene product i n s u s t a i n i n g  the l i f e of an organism.  A  role  specific  enzyme i n a m e t a b o l i c pathway can be removed by a mutation i n the s t r u c t u r a l gene.  T h i s a l l o w s the d e t e r m i n a t i o n of the c e l l u l a r e f f e c t s o f the  m i s s i n g enzyme and o f t e n ,  the m e t a b o l i c pathway.  Such p o i n t  mutations  have a l s o proven u s e f u l i n the study of complex developmental pathways such, as b a c t e r i o p h a g e assembly  (Edgar and Wood, 1966;  1971), and the f u n c t i o n of o t h e r macromolecular b a c t e r i a l membranes ( S i l b e r t , 19J5), ribosomes 1977),  flagella  ( A l d e r , 1975). organisms  ( S i l v e r m a n and Simon, 1977)  Meezan and Wood,  complexes  including  (Nomura and Morgan,  and chemotactic r e c e p t o r s  However, phenomena that o n l y occur i n m u l t i c e l l u l a r  such. as. c e l l  to c e l l i n t e r a c t i o n and communication,  d i f f e r e n t i a t i o n , dosage compensation  tissue  and c o - o r d i n a t e d b e h a v i o u r are  l e s s w e l l understood, and hence must be s t u d i e d i n them. One organisms  approach  to study c o - o r d i n a t e d b e h a v i o u r i n m u l t i c e l l u l a r  i s to use mutations which  development  and f u n c t i o n .  in  that both assembly  the f a c t  under  systematically disrupt neural  The r a t i o n a l e b e h i n d t h i s approach  the d i r e c t i o n of genes.  and f u n c t i o n of the nervous Proper performance  lies  system i s  of a g i v e n b e h a v i o u r  2  r e q u i r e s the c o m p l e t i o n of s e v e r a l s t e p s i n a pathway. the b e h a v i o u r a l pathway i s a p h y s i o l o g i c a l p r o c e s s p e p t i d e s coded by genes.  Each step  in  dependent on p o l y -  T h e r e f o r e , an a l t e r a t i o n i n the s t r u c t u r e  o f a p o l y p e p t i d e would a l l o w the s t u d y i n g of a l t e r e d b e h a v i o u r w i t h r e s p e c t to the m a l f u n c t i o n i n g o f a component i n the b e h a v i o u r a l The use of s i n g l e gene m u t a t i o n s . t h u s  pathway.  a l l o w s sequence d e t e r m i n a t i o n of  the pathway by l o o k i n g a t these m u t a t i o n s one at a t i m e . The g e n e t i c approach, to study b e h a v i o u r v i a the d i s r u p t i o n o f n e u r a l f u n c t i o n has s e v e r a l advantages  over the o t h e r w i s e  c h e m i c a l and d e v e l o p m e n t a l m a n i p u l a t i o n s because inheritable,  surgical,  (1). m u t a t i o n s  (.2) t h e i r e f f e c t s a r e v e r y s p e c i f i c , (3) g e n e t i c m a n i -  p u l a t i o n s are m i n i m a l when compared to o t h e r approaches  and (4)  study of b e h a v i o u r is. p o s s i b l e t h r o u g h the use of a c l o n e of  statistical  mutants.  To study the b e h a v i o u r o f m u l t i c e l l u l a r organisms, necessary  are  it  to f i n d an e x p e r i m e n t a l a n i m a l p o s s e s s i n g a complex  is  nervous  system as w e l l as e x h i b i t i n g a r i c h , r e p e r t o i r e of endogenous p a t t e r n e d behaviour.  I n s e c t s axe I d e a l c a n d i d a t e s f o r t h i s purpose of  the f u n c t i o n o f the nervous  system.  studying  Among them, D r o s o p h i l a i s  chosen  f o r e x t e n s i v e b e h a v i o u r a l study because o f i t s complex nervous  system  ( a p p r o x i m a t e l y 10^ neurons v s .  1 0 ^ - 1 0 ' ' ' ^ neurons  i t s well studied behavioural patterns.  i n vertebrates)  F o r example, mating  and  behaviour  i s o f t e n c h a r a c t e r i z e d by the male f o l l o w i n g the f e m a l e , t a p p i n g the f e m a l e ' s abdomen, e x t e n d i n g and v i b r a t i n g a wing i n a s p e c i f i c t o produce a s p e c i e s - s p e c i f i c c o u r t s h i p song ( H o t t a and Benzer,  grequency 1976).  The  stereotyped  demonstrated by  b e h a v i o u r a l s o i n c l u d e s a p o s i t i v e optomotor response f l i e s quickly adjusting  d i r e c t i o n of a t u r n i n g stripes  c y l i n d e r l i n e d w i t h a l t e r n a t e b l a c k and  (Heisenberg and  Drosophila  t h e i r movement to changes i n  Gotz, 1975).  i s a complicated  Moreover, f l i g h t b e h a v i o u r i n  process Involving  box  by  and  T r a c e y , 1973).  and  p o s i t i v e p h o t o t a c t i c behaviour  resonating  the  thoracic  a l t e r n a t e l y s t r e t c h i n g and r e l a x i n g d i f f e r e n t muscles In a d d i t i o n , D r o s o p h i l a  The w e l l known g e n e t i c s i n d u c t i o n , i s o l a t i o n and another r e a s o n why  genetic  e x h i b i t negative  (McEwen, 1918; i n Drosophila  mutagens ( f o r example, e t h y l methanesulfonate  (Homyk, u n p u b l i s h e d ) and,  i s o l a t e d and  studies.  ( T a l k and  v a r i a t i o n s i n t h e i r electroretinograms i s an e x t r a c e l l u l a r r e c o r d i n g from the eye.  of  on  Behavioural  steps have been  Benzer, 1976).  p h o t o t a c t i c responses have  Benzer, 1967).  mutants r e c o v e r e d  chemical  Nash-, 1974), macromolecular  more r e c e n t l y , b e h a v i o u r .  Mutants t h a t have abnormal g e o t a c t i c and  many n o n - p h o t o t a c t i c  Potent  of a wide a r r a y  or a l l of the c o u r t s h i p  (McEwen, 1918;  the  (EMS)) w h i c h induce p o i n t  c h a r a c t e r i z e d s u c c e s s f u l l y (Hotta and  a l s o been s t u d i e d  ,  which f a c i l i t a t e  an enormous amount of I n f o r m a t i o n  p r o c e s s e s such, as b i o s y n t h e t i c s t e p s  mutants unable to perform one  geotactic  Benzer, 1967).  mutations have allowed the d e t e c t i o n and r e c o v e r y  assembly  (Levine  c h a r a c t e r i z a t i o n of mutations i s  i t i s chosen f o r b e h a v i o u r a l  m u t a t i o n s which- ahye p r o v i d e d  white  In the l a t t e r  studies,  were c h a r a c t e r i z a b l e by  abnormal  (ERGs).  An  electroretinogram  of a l i g h t induced e l e c t r i c a l  In f a c t , mutations have been i d e n t i f i e d  that  response specifically  cause a b n o r m a l i t i e s i n l a m i n a l c e l l s and r e t i n u l a c e l l s t h a t are i n d i c a t e d by an abnormal 1978).  optomotor  response and ERG  respectively  (Pak and G r a b o z s k i ,  Recent work has a l s o f o c u s e d on the f l i g h t b e h a v i o u r o f D r o s o p h i l a  v i a the use of s e v e r a l f l i g h t l e s s mutants  (Homyk, u n p u b l i s h e d ) .  Thus,  the use o f these b e h a v i o u r a l mutations, i t s g e n e t i c a m e n d a b i l i t y , t o g e t h e r w i t h the new  use o f n e u r o p h y s i o l o g y i n D r o s o p h i l a , such as  i n t r a c e l l u l a r r e c o r d i n g techniques (Ikeda and Kaplan, 1970a), outweigh the disadvantages o f i t s s m a l l s i z e .  In f a c t , such techniques have  h e l p e d to r e v e a l the complex neural-motor  circuit  (Kaplan and T r o u t ,  1969;  Ikeda and Kaplan, 1970a; Ikeda and Kaplan, 1974;  1978)  i n Drosophila.  Jan and Jan,  B e h a v i o u r a l a b n o r m a l i t i e s c o u l d be caused by mutations the assembly  o f the nervous system.  system must be s t u d i e d .  Hence, the development  affecting  of the nervous  D r o s o p h i l a has been a f a v o u r i t e organism f o r •  s t u d y i n g the g e n e t i c c o n t r o l o f developmental p r o c e s s e s i n a m u l t i c e l l u l a r eukaryote.  I t s developmental p r o c e s s ( Z a l o k a r , A u d i t and Erk,  1978)  as w e l l as c e l l d i f f e r e n t i a t i o n and d e t e r m i n a t i o n (Lewis, 1978)  have  been s t u d i e d i n d e t a i l .  because  I t i s chosen f o r developmental s t u d i e s  of i t s complex l i f e c y c l e i n v o l v i n g metamorphosis from egg to l a r v a to pupa to a d u l t i n a r e l a t i v e l y s h o r t time (12 days a t 22°C).  Its  development  and  i s most i n t r i g u i n g to both developmental b i o l o g i s t s  g e n e t i c i s t s owing to the v a s t d i f f e r e n c e s i n the a n a t o m i c a l s t r u c t u r e s between the a d u l t and the l a r v a as w e l l as the t r a n s f o r m a t i o n p r o c e s s from the l a r v a to an a d u l t w i t h wings,  l e g s and compound eyes  which  i n i t i a l l y were never  presented.  Among the many t o o l s used i n the s t u d y i n g o f D r o s o p h i l a development, t e m p e r a t u r e - s e n s i t i v e  ( t s ) mutations  have proven to be  a v a l u a b l e a s s e t . They a l l o w e x p r e s s i o n o f a mutant phenotype o n l y at  the r e s t r i c t i v e  temperature is  temperature w h i l e b e i n g normal a t the  (Suzuki, 1970).  permissive  T e m p e r a t u r e - s e n s i t i v i t y i n micro-organisms  a consequence o f a s i n g l e amino a c i d s u b s t i t u t i o n i n a p o l y p e p t i d e  which a l t e r s  the b i o l o g i c a l a c t i v i t y o f a p r o t e i n at d i f f e r e n t  temperatures  (Jockush, 1966) r a t h e r than an e f f e c t on the a c t u a l process o f t r a n s c r i p t i o n or translation.  In D r o s o p h i l a melanogaster, t s l e t h a l mutants  s i m i l a r t o those o f micro-organisms- have been r e c o v e r e d 1967;  B a i l l i e e t ad., 1968).  periods  (Suzuki et^ a]..,  These mutants have t e m p e r a t u r e - s e n s i t i v e  (TSPs) which- a r e o f t e n r e a d i l y d e f i n e d d u r i n g development by  a p p r o p r i a t e s h i f t s o f c u l t u r e s from p e r m i s s i v e to r e s t r i c t i v e The u l t i l i t y engineer  o f t s mutations  s p e c i a l balanced  Is manifold:  temperatures.  they o b v i a t e the need to  stocks of l e t h a l s ,  mutants t h a t c o u l d n o t be d e t e c t e d otherwise  they a l l o w r e c o v e r y o f (such as dominant  Suzuki and P r o c u n i e r , 1969) and most important  lethals,  o f a l l , they permit the  d e l i n e a t i o n o f TSPs to the time o f gene a c t i o n d u r i n g development. One  q u e s t i o n o f t e n asked  where i n the animal to  the mutation  concerning  b e h a v i o u r a l mutations i s  i s exerting i t s effects.  Answers  t h i s q u e s t i o n a r e o f t e n c o m p l i c a t e d by problems e v i d e n t i n the f o l l o w i n g  example.  The d e f e c t i v e t i s s u e i n a n o n - p h o t o t a c t i c mutant may be i n  p a r t s o f the nervous system t h a t a r e not d i r e c t l y r e l e v a n t to the v i s u a l  6  physiology, but to a mere motor defect.  Hence, i t i s necessary to  i n v e s t i g a t e which component i n a behavioural pathway the mutation i s affecting.  In Drosophila, genetic mosaics have been used extensively  to provide answers to these questions (Hotta and Benzer, 1970; 1972; Ikeda and Kaplan, 1970b; Suzuki et a l . , 1971; G r i g l i a t t i et al.*, 1972; Kankel and H a l l , 1976).  Genetic mosaics are i n d i v i d u a l s composed of  g e n o t y p i c a l l y d i s t i n c t types of t i s s u e .  The generation of mosaics  often required manipulations of the f l y ' s genotype through the use of chromosome mechanics.  The more s o p h i s t i c a t e d genetic tools a v a i l a b l e  to Drosophila has allowed i t to become one of the most extensively developed mosaic systems of a l l organisms ( f o r mosaic systems i n other organisms, see review by Pak and P i n t o , 1976).  Other uses of mosaics i n behavioural  a n a l y s i s have involved questions on s p e c i f i c i n t e r a c t i o n between normal and mutant t i s s u e s .  Thus, the problem of whether the phenotype of a  g e n o t y p i c a l l y normal t i s s u e i n a mosaic i s affected by a c i r c u l a t i n g substance produced by a mutant t i s s u e elsewhere can be solved. One powerful technique of analysing genetic mosaics i s to extrapolate the s i t e of the mutation back to the embryonic c e l l s . This method of determining the embryonic t i s s u e w i t h i n which behavioural mutants act has been described i n d e t a i l by Hotta and Benzer (1972). I t i s based on the assumption that In a f e r t i l i z e d Drosophila egg, the p o l a r o r i e n t a t i o n of the spindle i n the f i r s t m i t o t i c d i v i s i o n i s randomly positioned w i t h i n a aphere (Fig. IA). (Parks, 1936).  Following  t h i s , nine synchronous nuclear d i v i s i o n s occur without c e l l d i v i s i o n .  The n u c l e a r d e r i v a t i v e s of the two n u c l e i formed remain c l u s t e r e d w i t h l i t t l e mixing divisions,  ( F i g . IB).  a f t e r the f i r s t  F o l l o w i n g these n i n e  the n u c l e i m i g r a t e to the s u r f a c e " o f the b l a s t u l a where they  d i v i d e t h r e e more times a f t e r which c e l l membranes form around (Fig.  division  IC).  Once the b l a s t o d e r m i s formed,  by a c e l l determines  and Selman, 1961;  the c o r t i c a l p o s i t i o n o c c u p i e d  i t s developmental f a t e ;  blastoderm gives r i s e  them  the a n t e r i o r r e g i o n o f the  to a n t e r i o r s t r u c t u r e s o f the a d u l t f l y (Hathaway  Chan and G e h r i n g , 1971).  I t i s obvious t h a t f o r any  two s i t e s on the b l a s t o d e r m , the f u r t h e r a p a r t they a r e , the more l i k e l y a mosaic boundary would l i e between them ( F i g . ID). of  mosaics,  the f r e q u e n c y w i t h which, any  d i f f e r e n t phenotypes A map  i s related  two  Hence, i n a p o p u l a t i o n  a d u l t landmarks a r e o f  to t h e i r p r o x i m i t y In the  blasoderm.  o f the b l a s t o d e r m p o s i t i o n of s t r u c t u r e s on the e x t e r n a l  s u r f a c e o f a d u l t s can be c o n s t r u c t e d ( F i g . 2) from the d i s t r i b u t i o n of  mutant and w i l d type t i s s u e s i n a p o p u l a t i o n o f mosaics.  w i t h which any said  The  two s u r f a c e landmarks are p h e n o t y p i c a l l y d i f f e r e n t , i s  to be the "map  d i s t a n c e " on an embryonic  f a t e map.  T h i s i s analogous  to mapping two  l i n k e d genes based on the f r e q u e n c i e s o f c r o s s i n g  between them.  A f a t e map  d i f f e r s i n that i t a l l o w s a two  c o n s t r u c t i o n based on the t r i a n g u l a t i o n o f t h r e e d i f f e r e n t (Fig.  frequency  over  dimensional structures  31. The  embryonic  f o c i of b e h a v i o u r a l mutants can a l s o be  by t r i a n g u l a t i n g a b e h a v i o u r a l phenotype r e l a t i v e to s e v e r a l e x t e r n a l c u t i c u l a r markers.  T h i s i s done by measuring  determined  different  the frequency  8  F i g u r e 1.  Formation of a mosaic f l y by l o s s o f one X chromosome. I n i t a l egg c o n t a i n s a r i n g - X chromosome p l u s a rod-X chromosome c a r r y i n g r e c e s s i v e genes f o r marking the body s u r f a c e as w e l l as the b e h a v i o u r t o be s t u d i e d . (A) Loss of r i n g - X a t the f i r s t d i v i s i o n r e s u l t s i n a rod-X/O nucleus (open c i r c l e ) and a ring-X/rod-X nucleus ( s o l i d c i r c l e ) . (B) The n u c l e i devide i n c l u s t e r without mixing then migrate to the egg c o r t e x (C) where c e l l membranes form to produce a one c e l l t h i c k b l a s t o d e r . (D) S u r f a c e view o f the b l a s t o d e r m w i t h the mosaic boundary s e p e r a t i n g the two c e l l types. (from H o t t a and Benzer, 1972)  9  A  B  C  D  10  Figure  2.  Right h a l f of the f a t e map of D r o s o p h i l a melanogaster f o r a d u l t e x t e r n a l body p a r t s , c o n s t r u c t e d by mosaic mapping. A m i r r o r image of the map corresponds to the l e f t h a l f . D o t t e d l i n e s i n d i c a t e d i s t a n c e s to the n e a r e s t m i d l i n e , as o b t a i n e d by h a l v i n g the d i s t a n c e measured between two homologous s i t e s on each s i d e . D i s t a n c e s are i n d i c a t e d i n s t u r t s . One s t u r t i s d e f i n e d as a d i s t a n c e such t h a t the two e x t e r n a l body p a r t s are of d i f f e r e n t genotypes i n 1% of the m o s a i c s . The s i z e o f the c i r c l e i s p r o p o r t i o n a l to the frequency w i t h which the s t r u c t u r e i s i t s e l f s p l i t by the mosaic boundary, (from H o t t a and Benzer, 1972)  DORSAL MIDLINE  12  F i g u r e 3.  P r i n c i p l e of mosaic f a t e mapping. L e f t : Mosaic boundary l i n e s f a l l i n random o r i e n t a t i o n on the b l a s t o d e r m . The f a r t h e r a p a r t two s i t e s are, the more l i k e l y i t i s that the boundary w i l l f a l l between them, and hence the s t r u c t u r e s d e r i v e d from these s i t e s w i l l be o f d i f f e r e n t genotype. The d i s t a n c e between two s i t e s on the f a t e map i s i n d i c a t e d by the frequency w i t h which two c u t i c l e landmarks are o f d i f f e r e n t phenotypes. R i g h t : L o c a t i o n of a t h i r d s i t e by t r i a n g u l a t i o n . S i t e s A and B are 10 s t u r t s a p a r t . If C i s 4 s t u r t s from A and 8 s t u r t s from B, then C can be .. located. A c h o i c e between the two p o s s i b i l i t i e s r e q u i r e s mapping with, r e s p e c t to a d d i t i o n a l s i t e s , (from Hotta and Benzer, 1972).  14  with which mutant behaviour i s found when an e x t e r n a l marker i s w i l d type or v i c e versa.  Using a second set of external landmarks, the distance  between the "behavioural focus" and the second landmark can be determined. Using a number of these surface landmarks, the l o c a l i z a t i o n of the "behavioural focus" responsible f o r the mutant phenotype i s therefore possible.  Emhryological studies have delineated the neural and muscular  tissues i n the embryo (Poulson, 1965) and f a t e mapping of mutations with known n e u r o l o g i c a l defects f i n d s t h e i r mapped embryonic f o c i congruent w i t h the c y t o l o g i c a l p i c t u r e ( P i g . 4). Thus, the genetic and developmental tools developed i n Drosophila provide a means of d i s s e c t i n g the components of behaviour.  This report  involves the use of new mutations and focus mapping i n an attempt to f i n d out where, when and how s p e c i f i c genes c o n t r o l n e u r o b i o l o g i c a l phenomena.  This includes how genes a f f e c t the assembly and f u n c t i o n of  the nervous system throughout the l i f e cycle of the organism.  Also,  an attempt w i l l be made to pinpoint the focus of i t s a c t i o n and determine whether i t i s a f f e c t i n g the muscles or the nervous system. the time and duration of i t s a c t i o n w i l l be i n v e s t i g a t e d .  Moreover,  15  F i g u r e 4.  R i g h t h a l f of the f a t e map of D r o s o p h i l a melanogaster f o r a d u l t e x t e r n a l body landmarks. Dotted l i n e s , according to e m b r y o l o g i c a l s t u d i e s (Poulson, 1965), i n d i c a t e areas of presumptive nervous system and mesoderm, (from Hotta and Benzer, 1972)  16  CHAPTER 2 STRESS-SENSITIVE MUTATIONS  I.  Introduction  A number o f mutations c o n f e r r i n g s e n s i t i v i t y shock have been r e p o r t e d et  (Judd,  Shen and Kaufman, 1972; G r i g l i a t t i  a l . , 1973; F e i t e l s o n and H a l l , 1975).  s e n s i t i v e to s t r e s s .  A light  is  to induce  usually sufficient  to mechanical  Such f l i e s a r e extremely  tap o f the v i a l c o n t a i n i n g these  flies  immediate p a r a l y s i s .  E a r l y s t u d i e s on s t r e s s - i n d u c e d p a r a l y s i s i n the American cockroach, induces  P e r i p l a n e t a americana (Cook, 1967) r e v e a l e d t h a t shock  production  or release of a neuroactive  substance, F a c t o r S,  which causes an i n i t i a l  r i s e i n nervous a c t i v i t y  in  The b e h a v i o u r a l  the nervous system.  parallels  response o f . t h e . i n s e c t to s t r e s s  the above p h y s i o l o g i c a l o b s e r v a t i o n s ;  f o l l o w e d by p a r a l y s i s (Cook and H o l t , 1974). nervous a c t i v i t y  f o l l o w e d by a blockade  an i n i t i a l h y p e r a c t i v i t y  The i n i t i a l  rise i n  i s the f a m i l i a r p h y s i o l o g i c a l phenomenon o f f a c i l i t a t i o n .  F a c i l i t a t i o n i s an enhanced p o s t - s y n a p t i c response caused by pre-synaptic a c t i v i t y . to  repeated  S i n c e f a c i l i t a t i o n i s an i n s e c t ' s normal response  s t r e s s , an abnormal f a c i l i t a t i o n p a t t e r n might be expected among  some s t r e s s - s e n s i t i v e mutants.  Indeed, a b a n g - s e n s i t i v e mutant, bas,  e x h i b i t s an abnormal f a c i l i t a t i o n p a t t e r n a t the neuromuscular j u n c t i o n (Jan and J a n , 1978). and  T h i s f a c i l i t a t i o n p a t t e r n develops much  decays much more s l o w l y  than normal.  faster  Fate mapping a n a l y s i s o f  bas  i n d i c a t e d t h a t i t s primary  ganglion bas  1975).  Thus, taken  to the p r o t h o r a c i c together,  data f o r  i n d i c a t e t h a t t h e mutant d e f e c t i s most l i k e l y p r e - s y n a p t i c i n  origin. of  ( F e i t e l s o n and H a l l ,  focus i s l o c a l i z e d  The s t r e s s - s e n s i t i v i t y phenotype may c h a r a c t e r i z e d a c l a s s  d e f e c t s t h a t w i l l be u s e f u l f o r s t u d y i n g neuromuscular j u n c t i o n s . In the p r e s e n t  study,  the p r o p e r t i e s o f r e c e s s i v e s t r e s s -  s e n s i t i v e mutations i n t h r e e s e x - l i n k e d genes (ses B, ses D, ses E) are i n v e s t i g a t e d . and  Sheppard  The mutants s t u d i e d were a l l r e c o v e r e d by Homyk  (1977) and Homyk, Szidonya  and Suzuki  (1980).  They  were a n a l y z e d f o r t h e i r embryonic f o c i by f a t e mapping, t h e i r to  response  d i f f e r e n t n e u r o t r o p i c drugs and, i n the case o f t e m p e r a t u r e - s e n s i t i v e  alleles,  their  temperature-sensitive  p e r i o d s were d e l i n e a t e d .  II.  M a t e r i a l s and Methods  Except where s p e c i f i e d ,  a l l c u l t u r e s were grown on s t a n d a r d  c o r n m e a l - y e a s t - d e x t r o s e D r o s o p h i l a medium a t  22°C ± 1°C.  The  1  2  isolation  1  p r o c e d u r e s , mapping and b e h a v i o u r a l p r o p e r t i e s of ses B , ses B-,  2 ses D  ses D ,  1 and ses E  have been r e p o r t e d (Homyk and Sheppard,  Szidonya and S u z u k i , 1980).  The map  1977;  Homyk,  p o s i t i o n s o f the genes a r e : ses B,  1-33.0; ses D, 1-20.0; ses E, 1-27.7.  The b e h a v i o u r a l c h a r a c t e r i s t i c s  of the mutants and Oregon—R w i l d type f l i e s  used i n t h i s study are  summarized i n T a b l e 1. Developmental  Studies  The mutations lethals,  ses B^" and ses E^" were found to be  that i s , w h i l e s u r v i v i n g a t  heat-sensitive  22°C, these f l i e s d i e a t 29°C.  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 s (TSPs) f o r h e a t - s e n s i t i v e l e t h a l i t y  are  d e l i n e a t e d by the e a r l i e s t  shift  the  earliest  significant  1970). and  s h i f t up y i e l d i n g A l l shift  at the time o f s h i f t was  The developmental  and  (Suzuki,  22°C ( p e r m i s s i v e )  stage p r e s e n t i n each  culture  determined by i n s p e c t i n g d e v e l o p i n g f l i e s  L a r v a l i n s t a r s were determined by the morphology o f t h e i r  mout p a r t s and a n t e r i o r s p i r a c l e s Flies  l e v e l s of s u r v i v a l  experiments were performed between  29°C ( r e s t r i c t i v e ) .  a t t h a t time.  down which reduces v i a b i l i t y  ( B o d e n s t r i n , 1950).  from which eggs were to be c o l l e c t e d were p l a c e d i n  empty b o t t l e s whose mouths were covered w i t h a watch g l a s s . g l a s s c a r r i e d a s t r i p o f agar  (1% agar i n b o i l i n g water,  The watch  2% e t h a n o l  Table 1 Behavioural characteristics of wild type Oregon-R and mutant f l i e s used i n this study.  Mutants  Total number of flies observed  20  ses  2  n  Behaviour before • tapping of vial  active  II  Behaviour after 1 tap to make flies f a l l to the bottom of the v i a l Climb up the walls of the v i a l quickly II  ses D ses E  II  ses B^  2 ses B  Ore-R  it  n  II  Behaviour after 5 - 10 sec. of tapping ; Flies climb up slowly and occasionally f a l l back. Require 90 sees, for f u l l recovery. 4 flies paralyzed, require 1-2 min. to recover. II  Climb, up the walls of the v i a l quickly  A l l flies begin to climb up the walls of the vials after they were paralyzed for 5 to 10 sees..  Climb up the walls of the vial slowly  After tumbling and falling on their backs for 30 sees., 10 flies recovered. The other 10 remained paralyzed and required more than 3 min. to recover.  moderately active  Climb up the walls of the v i a l slowly  After tumbling and falling on their backs for 30 sees., 16 flies recovered. The other 4 remained paralyzed and required more than 3 min. to recover.  active  Climb up the walls of the v i a l immediately and very quickly  None paralyzed. Flies climb up the walls of the vial immediately and very quickly.  tt  inactive  ro o  and 1% a c e t i c a c i d ) covered w i t h y e a s t p a s t e (yeast and tap water Since females can r e t a i n f e r t i l i z e d two-hour egg c o l l e c t i o n was was  eggs b e f o r e d e p o s i t i o n , the  discarded.  mixed).  first  The second two-hour c o l l e c t i o n  saved and the eggs removed from the agar s t r i p by washing.  These  d e v e l o p m e n t l a l y s y n c h r o n i z e d eggs were c o l l e c t e d on a f i n e wire*mesh and s u b s e q u e n t l y t r a n s f e r r e d to a 20% s u c r o s e s o l u t i o n which a l l o w s the d e b r i s  (yeast p a s t e and agar) to s i n k to the bottom  w h i l e the eggs f l o a t on top of the s o l u t i o n .  of the beaker  The eggs were then r e -  c o l l e c t e d on the w i r e mesh and washed t h o r o u g h l y w i t h d i s t i l l e d The f i n a l washing  o f the eggs i s e s s e n t i a l to ensure t h a t a l l t r a c e s  of s u c r o s e are c o m p l e t e l y removed to prevent mould growth. washing,  water.  After  the  the eggs were counted and l a i d on s t r i p s o f h e a t - s t e r i l i z e d  moistened b l o t t e r paper which, were then p l a c e d i n f r e s h v i a l s D r o s o p h i l a medium.  F o r t y - e i g h t hours l a t e r ,  remoyed and the number o f unhatched  the paper s t r i p s were  eggs noted.  Three s e t s o f c u l t u r e s were e s t a b l i s h e d f o r s h i f t F o l l o w i n g egg c o l l e c t i o n , one s e t was immediately  t r a n s f e r r e d to 29°C.  containing  experiments.  l e f t a t 22°C and another  was  At s u c c e s s i v e i n t e r v a l s of 12 hours,  c u l t u r e s were s h i f t e d up o r down a c c o r d i n g l y .  24-hour heat p u l s e s  were a l s o a d m i n i s t e r e d to the t h i r d s e t o f c u l t u r e s at d i f f e r e n t of development.  A l l c u l t u r e s were s c o r e d f o r the number o f pupae  and the number o f a d u l t s  times formed  eclosed.  G e n e t i c Mosaic S t u d i e s Since a l l o f the mutations were l o c a t e d away from the t i p  22  of the X chromosome, the d i s t a l l y  l o c a t e d e x t e r n a l c u t i c u l a r markers,  y e l l o w and w h i t e were r e a d i l y combined w i t h them by c r o s s i n g o v e r . G e n e t i c mosaics were generated  through  the use of the s o m a t i c a l l y u n s t a b l e  r i n g - X chromosome, I n ( l ) w " (Hinton, 1955). V(  Females c a r r y i n g the  I n ( l ) w ^ chromosome were mated to males c a r r y i n g the s p e c i f i c b e h a v i o u r a l V  mutations  (ses) l i n k e d  males are ;y_ w ses/Y.  to the c u t i c u l a r markers y e l l o w and w h i t e . By c r o s s i n g I n ( l ) w / y w s p l females v C  males, ' I n ( l ) w ^ / y w ses female zygotes V  can be r e c o v e r e d .  These  to y_ w ses/Y Loss of  the  I n ( l ) w ^ r i n g - X chromosome i n the e a r l y cleavage stage ( G a r c i a - B e l l i d o V  and Merriam, 1969)  of I n ( l ) w ^ / y w ses female zygote r e s u l t s i n patches V  o f y w ses/O male t i s s u e i n the a d u l t f l y . the mutant phenotype can be expressed those c e l l s .  The  S i n c e t h i s t i s s u e i s hemizygous,  provided  the gene i s a c t i v e i n  l o c a t i o n and e x t e n t of mutant t i s s u e are  determined  e x t e r n a l l y by e x p r e s s i o n of the y e l l o w and w h i t e phenotype on a background o f w i l d type t i s s u e . mosaic and  The  areas of mutant t i s s u e were r e c o r d e d f o r each  then i t s b e h a v i o u r  shock treatment.  a t 29°C was  I f the mosaic f l y became completely  a shock, the time f o r i t s r e c o v e r y was was  n o t e d b e f o r e and a f t e r each  recorded.  paralyzed  Partial  paralysis  a l s o r e c o r d e d i n those cases where the f l y dragged i t s e l f  u s i n g one  o r more p a i r s of l e g s .  along  In t h i s manner, the mosaics were  examined on a d a i l y b a s i s f o r a t l e a s t Neurophysiological  after  two weeks.  Studies  In o r d e r to t e s t whether s y n a p t i c f u n c t i o n s are i n v o l v e d i n the ses mutant d e f e c t s , the mutants were exposed to v a r y i n g con—  c e n t r a t i o n s of a v a r i e t y o f drugs known to be p s y c h o t r o p i c and neurot r o p i c i n mammals.  The number o f drugs used, t h e i r l a r v a l l e t h a l  and t h e i r known p h y s i o l o g i c a l e f f e c t s a r e l i s t e d Each mutant s t r a i n was c o n c e n t r a t i o n s o f each drug.  subjected  dose  i n T a b l e 2.  to a t l e a s t 5 d i f f e r e n t  The c o n c e n t r a t i o n s were  — , 1, 2 and  4 times the l a r v a l l e t h a l dose as r e p o r t e d by Howard, Merriam and Meshul (1975) o r determined i n the p r e s e n t s t u d y . o r e x t r a r e s i s t a n c e was lower  Where e x t r a  sensitivity  noted, the experiments were r e p e a t e d w i t h  (— times the l a r v a l l e t h a l dose) or h i g h e r (8 times the l a r v a l o  l e t h a l dose) c o n c e n t r a t i o n s ,  respectively.  5 ml. of each drug o f a p p r o p r i a t e c o n c e n t r a t i o n s were added  to 1.4  6 ml. o f medium. was  (see above)  gram o f i n s t a n t D r o s o p h i l a medium ( B o r e a l ) to make Where p o s s i b l e , d i s t i l l e d water at room temperature  used to d i s s o l v e the drug.  In o r d e r to p r e v e n t any heat  inactivation  when the s o l u b i l i t y o f the drug a t room temperature p r e s e n t e d a problem, the water  temperature was  p r e v e n t b a c t e r i a l growth,  r a i s e d j u s t enough to d i s s o l v e the drug. two a n t i b i o t i c s , s t r e p t o m y c i n and  To  tetracycline  (10 m g . / l i t r e ) were added t o t h e d i s t i l l e d water w h i l e mould growth was  i n h i b i t e d w i t h tegosept (100 g m . / l i t r e  ethanol).  Newly d e p o s i t e d eggs were c o l l e c t e d and counted on  strips  o f moistened b l o t t e r paper as o u t l i n e d above and then p l a c e d i n t o c o n t a i n i n g drugs of d i f f e r e n t  concentrations.  These were s u b s e q u e n t l y  s c o r e d f o r the number o f pupae formed and the number o f a d u l t s both, a t  22°C and 29°C.  vials  eclosed  Table 2 L i s t o f drugs, t h e i r p u t a t i v e a c t i o n and the D r o s o p h i l a l a r v a l LD Taken i n p a r t from Howard, Merriam and Meshul, 1975.  _ ~Putative action  T>  nDiT/-  DRUG  Carbamylcholine c h l o r i d e Pilocarpine nitrate Succinylcholine chloride Nicotine Atropine sulfate Hexamethonium bromide d-tubocurare Eserine sulfate Reserpine Nialamide T  •  • J  Iproniazid Caffeine Theophylline Strychnine sulfate Picrotoxin Allylglycine Amino o x y a c e t i c a c i d Methionine s u l f o x i m i n e Sodium b a r b i t a l Urethane Dopamine dl-Norepinephrine Tyramine dl-Octopamine Histamine GABA Glutamic a c i d Catechol Yohimbine  Cholinonimetic " " " Cholinergic blocker " " M M Inhibits acetylcholinesterase Reduces t r a n s m i t t e r s t o r a g e I n h i b i t s monoamine oxidase II  II  II  I n h i b i t s c y c l i c nucleotide phosphodiesterase " " " " Glycine antagonist GABA a n t a g o n i s t Convulsant I n h i b i t s GABA d e g r a d a t i o n Convulsant Nerve, muscle depressant Muscle depressant Neurotransmitter " False transmitter " " ' Neurotransmitter " " Adrenergic blocker " "  Larval lethal dose, mg/ml o f . ' ' i n s t a n t Dros. medium ( B o r e a l ) 6  3 0.8 2.5 06 6 2 4 .025 .2 1 o  1  J0.5 0.5 2 .01 5 .8 .5 .5 1 5 7 3 10 7.5 25 25 4 2 .  III.  Results  ses B C h a r a c t e r i z a t i o n of TSPs:  The  t e m p e r a t u r e - s e n s i t i v e phenotype  of the ses B mutation enabled a n a l y s i s o f gene a c t i v i t y by shift  experiments.  A ses B  2  The ses B  2  temperature-  mutant e x h i b i t a t s m a t e r n a l - e f f e c t .  2 s t o c k m a i n t a i n e d i n the male l i n e as ses B /Y males X ^ f_:=/Y  attached-X females at 29°C y i e l d s a 1:2  r a t i o o f males to females i n  each, g e n e r a t i o n whereas the s t o c k i n which, homozygous ses B have been kept a t 29°C y i e l d s no a d u l t progeny e f f e c t was  f u r t h e r i n v e s t i g a t e d by mating  Oregon-R males a t 2 9 ° C  ses B  at a l l . 2  2  females  This ts maternal-  homozygous females to  Only 4 males were r e c o v e r e d w i t h 291  females  2 In a d d i t i o n , males from the c r o s s ses B /Y X f_: = /Y 2 2 2 attached-X females and ses B /Y X ses B /ses B females s u r v i v e w e l l survived  at 22°C  (Table 3 ) .  (90% and 73% v i a b l e progeny  e f f e c t was  respectively).  a l s o i n v e s t i g a t e d ny s h i f t  studies.  This ts maternal-  A d u l t females from the  homozygous s t o c k were s h i f t e d from 22°C to 29-°C and eggs c o l l e c t e d counted w h i l e t h e i r subsequent  development  at 29°C f o r more than 3 days, l a i d Before the 3rd day, about  was  scored.  Females,  eggs unable to g i v e r i s e  10% of the eggs produced a d u l t s .  and  kept  to a d u l t s . However,  eggs c o l l e c t e d from females which had been h e l d a t 29°C f o r more than 3 days and s h i f t e d down immediately to 22°C y i e l d e d 50% a d u l t These  eclosion.  females, which had been kept at 29°C f o r some time, were then  t r a n s f e r r e d back to 22°C and i n subsequent  c o l l e c t i o n s , eggs were monitored  Table 3  M a t e r n a l - e f f e c t s of s e s B .  The progeny were r e a r e d a t the same temperature t h e c r o s s was made. 2 ses B  Cross  2 females X Ore-R males  ses B  females X Ore-R males  at 29°C  a t 22°C  eggs c o l l e c t e d  930  750  pupae formed  327  726  males 4  Progeny recovered  females 291  2 Cross  females 403  2  ses B /Y males X y f:=/Y females  ses B /Y males X y f:=/Y females  at 29°C  at 22°C  males  females  males  females  135  236  186  205  Progeny r e c o v e r e d  2 2 2 ses B /Y males X ses B /ses B females Cross  Progeny r e c o v e r e d  males 296  2 2 2 ses B /Y males X ses B /ses B females  at 29°C  at 22°C  males  females  males  females  0  0  152  175  f o r f u r t h e r development.  More than 4 days a t 22° C were r e q u i r e d  s u r v i v a l reached the normal v a l u e (Table 4 ) .  (more than 80%) o f a d u l t  before  eclosion  Eggs o f c o n t r o l Oregon-R females h e l d a t 29°C f o r any p e r i o d  of time always had more than 75% a d u l t  eclosion. 2  Shift p u p a l TSP.  s t u d i e s a l s o i n d i c a t e d that ses B  has a 3rd i n s t a r -  A s h i f t - u p a f t e r 240 hours a t 22°C g i v e s  of s u r v i v o r s , thus marking t h e end o f t h e TSP. of the TSP c o u l d n o t be w e l l d e f i n e d  a significant  However, the b e g i n n i n g  s i n c e the d u r a t i o n  l a r v a l p e r i o d c o u l d not be determined.  level  o f the 3rd i n s t a r -  When r a i s e d a t 29°C, a l a r g e  f r a c t i o n o f the 3rd i n s t a r l a r v a e can s u r v i v e without p u p a t i n g f o r as long as t h r e e weeks b e f o r e down experiments I n d i c a t e 2nd  they b e g i n to d i e .  Nevertheless,  that a f t e r 19-2 hours a t 29°C  shift-  (the end o f  i n s t a r - l a r v a l p e r i o d i s 144 hours a t 29°C) the l e v e l o f s u r v i v a l  to a d u l t h o o d I s reduced  ( F i g . 5, T a b l e 5).  T h i s may r e f l e c t  either  the b e g i n n i n g o f the TSP o r a l t e r n a t i v e l y the time a f t e r which s u r v i v a l at 29°C i s not p o s s i b l e .  Therefore,  the TSP i s e s t i m a t e d to be between  the 3 r d i n s t a r l a r v a and b e g i n n i n g o f p u p a t i o n . I t should  be noted t h a t I n s u c c e s s i v e  s h i f t - u p experiments,  most o f the l a r v a e were a b l e to pupate but f a i l e d In c o n t r a s t ,  i n the s h i f t - d o w n experiments, a l a r g e number o f the l a r v a e  s h i f t e d during 5).  l a t e r stages o f development were unable to pupate  2 When ses B embryos or 1 s t i n s t a r l a r v a e were s u b j e c t e d  heat shocks, t h e i r a b i l i t y it  to e c l o s e as a d u l t s .  to pupate was g r e a t l y reduced.  2 seems t h a t ses B c o u l d have another TSP d u r i n g  (Table  to 29°C Therefore,  the embryonic and  Table 4 T e m p e r a t u r e - s e n s i t i v e m a t e r n a l - e f f e c t o f ses B .  ses B /ses B  females was c r o s s e d to ses B /Y males. number of adults  adults eggs  92  250  90  135  66  21  10  275  209  76  37  13  29  100  68  68  10  10  1  29  124  59  48  9  7  29  2  29  142  59  42  8  5  6  29  1  29  70  21  30  0  0  7  29  1  29  134  54  40  3  2  8  29  1 '•  29  100  22  22  1  1  9  29  1  29  62  12  19  0  0  10  29  1  22  52  27  52  26  50  2  22  2  22  200  127  64  124  62  3  22  1  22  52  33  63  33  63  4  22  1  22  112  100  89  98  88  5  22  1  22  77  56  73  55  71  eggs l a i d at °C  for days  eggs developed at °C  Number o f eggs collected  2  22.  1  22  279  258  3  22  1  29  205  1  29  1  29  2  29  1  3  29  5  females l i v e d at 22°C f o r days  females l i v e d at 29°C f o r days  number of pupae  pupae eggs  a/  c/  N3 00  29-  F i g u r e 5.  Percentage o f ses B*" a d u l t s e c l o s e d from hatched eggs a f t e r , • , s h i f t - u p s and T, s h i f t - d o w n s a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development. The developmental stage 22°C a t the time o f s h i f t i s shown at the bottom. The d u r a t i o n . o f 3rd i n s t a r stage cannot be determined due to non-pupating c h a r a c t e r i s t i c s o f t h i s mutant a t 29°C, hence, the curve f o r the s h i f t - d o w n cannot be drawn.  100  j embryo |  1st  24  72  I  48  2nd 84  i —  i  I  114 r  hours at 29°  144 i  —  "D  CU CO  _o o cu  CO *->  50  ~3 T3  CO  24 embryo |  ±_.  i  48  72 1st  i  96  120 2nd  144  i  i  168 192 hours at 22° 3rd  216  i  i  i  i  i  i  240  264  288  312  336  360  pupa  adult LO  O  -31 Table 5 2  T e m p e r a t u r e - s K i f t s t u d i e s o f ses B . Eggs were c o l l e c t e d a t 0 hour. F o r s h i f t up experiments, eggs were c o l l e c t e d a t 22°C over a 2-hour p e r i o d and l e f t a t 22°C u n t i l subsequent s h i f t s . F o r s h i f t - d o w n experiments, eggs' were c o l l e c t e d a t 22°C over a 2-hour p e r i o d , Immediately t r a n s f e r r e d to 29°C and l e f t a t t h a t temperature u n t i l subsequent s h i f t - d o w n s . hours  number of eggs  hatched e  g  g  S  number q  {  pupae  number q  £  adults  pupae_ l  a  r  v  a  e  %  adults P  %  u p a e  adults l  a  r  v  a  %  e  SHIFT-UP'EXPERIMENTS  0  egg  collection  24  78  74  54  1  73  2  1  48  79  73  55  3  75  6  4  72  74  70  59  5  84  8  7  96  78  76  64  13  84  20  17  120  77  69  62  10  90  16  14  144  76  70  65  27  93  42  39  168  77  72  67  24  93  36  33  192  80  74  63  23  98  37  31  216  79  73 '  60  33  82  55  45  240  77  71  59  47  83  80  66  264  74  68  65  48  96  74  71  288  77  67  61  50  91  82  75  312  77  72  64  58  89  91  81  336  38  35  35  25  100  71  71  SHIFT--DOWN EXPERIMENTS  0  egg  collection  45  79  74  58  57  78  98  77  48  70  78  66  63  85  95  81  72  74  71  65  64  92  98  90  96  77  72  58  53  79  91  74  120  77  71  59  53  83  90  75  144  76  70  61  44  87  72  63  168  75  74  65  53  88  82  72  192  75  71  57  35  80  61  49  216  75  69  52  28  75  54  41  240  80  79  39  16  49  41  20  264  78  73  31  12  42  39  16  288  80  79  48  13  61  27  16  312  79  75 ...  43  11  57  26  15  336  76  73  24  1  33  4  1  1st i n s t a r stages of development. Genetic Mosaic Studies:  ses B"^ i s extremely s e n s i t i v e  s t r e s s and u s u a l l y r e q u i r e s more than 3 minutes paralysis.  to r e c o v e r a f t e r  271 mosaics were c o l l e c t e d and the b e h a v i o u r of each were  examined i n s e v e r a l t r i a l s .  Mutant o r normal b e h a v i o u r c o u l d be  e q u i v o c a l l y a s s i g n e d to o n l y 109 mosaics. classified  to  Of the 109 mosaics,  as mutant and 59- were c l a s s i f i e d as normal.  The  un-  50 were  remaining  162 mosaics were l e f t u n c l a s s i f i e d due t o the i n c o n s i s t e n c y o f t h e i r behaviour i n seperate t r i a l s . s e n s i t i v e w h i l e on another day, inability  Thus on one day they might be v e r y bangthey would be completely normal.  to a s s i g n a b e h a v i o u r a l phenotype t o these mosaics  the a p p l i c a t i o n o f a c o n v e n t i o n a l f a t e mapping a n a l y s i s .  The  prevents  However, the  r e s u l t s suggest t h a t the b e h a v i o u r a l phenotype i s most l i k e l y  correlate  to the phenotype of the c u t i c l e markers i n the head and thorax,  and  to a l e s s e r e x t e n t , the phenotype of the abdomen (Table 6). Drug S t u d i e s :  Only those s t r a i n s i n which drug  treatment  r e s u l t e d i n a s i g n i f i c a n t a l t e r a t i o n o f the p e r c e n t p u p a r i a t i o n o r a d u l t e c l o s i o n as compared to the w i l d type s t r a i n , Oregon-R, w i l l discussed.  The r e s u l t s f o r the drugs are shown i n Table 7.  to d i f f e r e n t  be  A l l responses  c o n c e n t r a t i o n o f v a r i o u s drugs which are not shown i n the  t a b l e d i d not d i f f e r from the Oregon-R w i l d can be summarized as  type  (Appendix).  The  results  follows:-  A mutant i s more s e n s i t i v e or l e s s r e s i s t a n t drug i f i t s l a r v a e have a lower v i a b i l i t y  to a p a r t i c u l a r  than Oregon-R at the same  Table 6  C o r r e l a t i o n o f the t h r e e body segments of ses B  1  mosaics w i t h t h e i r  behaviour.  CUTICLE GENOTYPE head mutant  normal  thorax mutant normal  abdomen mutant normal  BEHAVIOUR mutant  23  5  24  2  13  5  inconsistent  39  28  34  16  22  24  6  33  6  20  3  16  normal  Table 7 L i s t of drugs which have a more pronounced e f f e c t on the v i a b i l i t y of v a r i o u s s t r e s s - s e n s i t i v e s t r a i n s . The e f f e c t s of the same drug on Oregon-R a r e a l s o shown. Drugs were d i s s o l v e d i n water to make d i f f e r e n t c o n c e n t r a t i o n s b e f o r e they were added  Ore-R pupae „, larvae " Reserpine  Nicotine  Pilbc&rpine nitrate  Carbamylcholine chloride  H0 control no drugs 2  ses B  adults pupae  pupae „, larvae  2  a d u l t s „, pupae  pupae „ larvae "  adults „ pupae  92.3 80.0 71.4 100.0  20.0 4.8 0.0 0.0 0.0  75w0 100.0  19.0 2.5 0.0 0.0 0.0  62.5 100.0  83.7 94.3 71.4 2.9 0.0  97.2 97.0 88.6 100.0  81.0 64.0 0.0 0.0 0.0  73.0 80.0  88.0 97.0 74.0 34.0 6.0  97.0 94.0 100.0 100.0 100.0  .2 .4 .8 1.6 3.2  72.2 62.5 60.0 0.0 0.0  66.7 100.0 100.0  . 75 1.5 3.0 6.0 12.0  73.8 67.4 33.3 0.0 0.0  96.8 100.0 100.0  .05 .1 .2 .4 .8 .0156 .03125 .0625 .125 .25  33.3 54.1 17.1 2.9 0.0  87.2 72.1 82.5 50.0  .  91.2 90.3 100.0 84.0  pupae „ larvae "  75.0 52.5 35.0 29.3 44.7  94.9 78.4 51.3  94.6 93.1 65.0  75.0 80.0  100.0 96.9  „1 ses E  ses D^"  ses B''"  85.0 94.9 97.4  adults „ pupae  pupae ^ larvae  adults pupae  70.0 76.2 85.7 91.7 29.4  94.1 94.6 100.0  82.1 69.0 89.3 60.7 70.0  91.3 100.0 100.0 70.6 100.0  57.1 96.4 100.0  91.7 92.6 82.5  JO  concentration of drug.  S i m i l a r l y , a mutant i s l e s s s e n s i t i v e or more  r e s i s t a n t to a p a r t i c u l a r drug i f i t s larvae have a greater than Oregon-R at the same concentration of drug. examinied here.  viability  Only m o r t a l i t y i s  The behavioural e f f e c t s of these drugs at concentrations  lower than l e t h a l dose were not studied. The ses B"^" mutation i s more s e n s i t i v e to reserpine catecholamine  (reduce  transmitter storage) and i s more r e s i s t a n t to n i c o t i n e  (an agonist of a c e t y l c h o l i n e ) . 2 The ses B mutation i s more s e n s i t i v e to reserpine (same as the ses B"*" mutation) but u n l i k e the ses B^ mutation, i t i s more s e n s i t i v e to n i c o t i n e as w e l l . ses D 2 Genetic Mosaic Studies: ses D i s not temperature-sensitive and i s moderately s e n s i t i v e to s t r e s s compared to the other mutants 2 1 studied. Paralyzed ses D f l i e s normally recover w i t h i n l - ^ minutes. 2 When homozygous ses D f l i e s are under s t r e s s , a l l s i x legs are often 2 paralyzed.  However, i t was n o t i c e d that when ses D  b i l a t e r a l mosaics  (one side of the f l y having normal c u t i c l e colour while the other side i s t o t a l l y mutant) are stressed, the mutant legs were often paralyzed while the w i l d type legs were never paralyzed.  The other mosaics under  s t r e s s , i n which some of the 6 legs are of d i f f e r e n t phenotypes, often have t h e i r walking w i l d type legs dragging t h e i r paralyzed mutant ones.  Thus the behaviour of each l e g i s independent of the others  with behaviour and c u t i c u l a r genotype having a high degree of concordance,  so the b e h a v i o u r of each l e g o f the mosaic  f l y was  scored independently.  T h i s a l l o w e d the focus mapping of the b e h a v i o u r f o r each l e g . A n a l y s i s o f 1608  l e g s of mosaics  focus of each l e g has i t s own  r e v e a l e d that the b e h a v i o u r a l  s i t e w i t h i n the presumptive  system o f the b l a s t o d e r m f a t e map.  The 6 f o c i occupy  nervous  6 seperate,  n o n - i n t e r a c t i n g s i t e s i n the b l a s t o d e r m a l i g n e d w i t h t h e i r legs  respective  ( F i g . 6). Drug S t u d i e s ;  to p i l o c a r p i n e n i t r a t e  ses D"*" i s more r e s i s t a n t  (less  sensitive)  (an a g o n i s t o f the c h o l i n e r g i c system).  (Table 7)  2 ses D  response d i d not d i f f e r from the Oregon-R w i l d type.  ses E C h a r a c t e r i z a t i o n o f TSPs:  ses E"^ i s the l e a s t  stress-sensitive  as p a r a l y z e d ses E"^ f l i e s u s u a l l y r e c o v e r i n a matter of 5 to 10 The  t e m p e r a t u r e - s e n s i t i v e l e t h a l phenotype  o f ses E mutation enabled  a n a l y s i s of gene a c t i v i t y by t e m p e r a t u r e - s h i f t experiments. single shift  seconds.  Reciprocal  s t u d i e s a t 24-hour I n t e r v a l s from p e r m i s s i v e to r e s t r i c t i v e  temperatures and v i c e v e r s a r e v e a l e d t h a t ses E"^ has a TSP d u r i n g the t r a n s i t i o n of 1st to 2nd I n s t a r l a r v a e (LP) f o r t h i s TSP  Is p u p a r i a t i o n .  ( F i g . 7).  The l e t h a l phase  At the r e s t r i c t i v e  few pupae form; most 3 r d i n s t a r l a r v a e wander around and d i e on the s i d e s w i t h o u t forming a p u p a l case.  temperature, the v i a l o r b o t t l e  T h i s TSP  starts  at a p p r o x i a m t e l y 45 hours a t 22°C a f t e r o y i p o s i t i o n and ends a t  117  hours, a d u r a t i o n of approximately-70 h o u r s . ses E"^ a l s o has another TSP f o r l e t h a l i t y .  To  delineate  37  Figure 6.  Fate map of ses D a, antenna; anp, a n t e r i o r n o t o p l e u r a l b r i s t l e ; hu, humerus; oc, o c e l l a r ; l c , coxa 1; 2c, coxa 2; 3c, coxa 3; 3s, 3rd s t e r n i t e ; I, l e g 1 focus; II, l e g 2 focus; III, l e g 3 focus.  38  $9  Figure 7.  Percentage of ses E pupae formed after, A , shift-ups and T , shift-downs administered at different times during development. The developmental stages at 22°C and 29°C at the i n i t i a t i o n of the s h i f t are also shown at the bottom and at the top respectively.  o  41  the s t a r t of t h i s TSP, f o u r t e e n c u l t u r e s were s y n c h r o n i z e d a t egg  collection,  m a i n t a i n e d a t 22°C f o r 6 days i n o r d e r to pass through the l a r v a l TSP, then s h i f t e d up to 29°C and m a i n t a i n e d t h e r e .  A t 24-hour  intervals,  2 c u l t u r e s were s h i f t e d down to 22°C u n t i l a l l 14 c u l t u r e s a t 29°C had been transferred.  T h i s e s t a b l i s h e d heat p u l s e s of v a r y i n g d u r a t i o n . "  These  heat p u l s e s r e v e a l e d t h a t the second TSP s t a r t s a t a p p r o x i m a t e l y 198 hours a t 22°C.  Standard s h i f t - u p experiments r e v e a l e d t h i s TSP ends a t  288 hours a t 22°C, a time c o r r e s p o n d i n g  to the e a r l y p u p a l s t a g e  (Fig.  Thus, ses E"^ has a 3 r d i n s t a r l a r v a l - p u p a l TSP a p p r o x i m a t e l y 90 hours long. To c o n f i r m the 3 r d i n s t a r l a r v a l - p u p a l TSP and t o examine the s e n s i t i v i t y of the 2 TSPs to h e a t - i n d u c e d l e t h a l i t y , 24-hour p u l s e s were a d m i n i s t e r e d a t v a r i o u s  heat  times d u r i n g development.  24-hour heat p u l s e caused death, o f a l l d e v e l o p i n g f l i e s .  No  The most  s e n s i t i v e time I n t e r v a l d u r i n g the second TSP was a t the 3 r d i n s t a r p u p a l t r a n s i t i o n s t a g e d u r i n g which. 24-hour heat p u l s e s  significantly  reduced the number of a d u l t s : from 85% i n c o n t r o l s t o 15% ( F i g .  9).  There were no p h e n o t y p i c a b n o r m a l i t i e s a s s o c i a t e d w i t h t h e s e 15% survivors.  The 24-hour p u l s e experiments d i d not r e v e a l the l a r v a l  TSP shown by the s h i f t e x p e r i m e n t s , thereby i n d i c a t i n g t h a t a l o n g e r p o r t i o n o f t h i s TSP mustJbe spent a t r e s t r i c t i v e temperatures to i n d u c e death. Genetic Mosaic Studies:  A p a r t from the  phenotype, ses E i s a l s o a t s d r o p - d e a d .  stress-sensitive  The 230 mosaics  recovered  8).  42  F i g u r e 8.  Percentage o f ses E a d u l t s e c l o s e d a f t e r , A , s h i f t - u p s and v, s h i f t - d o w n s a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development. The developmental stages a t 22°C and 29°C at the i n i t i a t i o n of the s h i f t a r e a l s o shown at the bottom and at the top r e s p e c t i v e l y .  44  F i g u r e 9.  Percentage o f , , ses E a d u l t s and , Oregon-R a d u l t s e c l o s e d a f t e r 12 hour heat p u l s e s (29 C) a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development. The developmental stages a t the time of the p u l s e a r e shown a t the bottom. The l e n g t h s of the l i n e s r e p r e s e n t the d u r a t i o n of the p u l s e s a t 29°C.  100  T3  CD CO  _o O  CD  to  50  "D CO  JL  24 emb.  48 1st  72  96 2nd  120 144 168 192 216 240 264 288 312 336 time in hours 3rd  pupa  were observed f o r b e h a v i o u r and v i a b i l i t y a t 29°C. of o b s e r v a t i o n , i t was paralyzed. ways.  noted that i n d i v i d u a l l e g s of mosaic  Hence, the mosaic  be due  o f ses E"*" are a consequence  then b o t h a d u l t l e t h a l i t y and  to e f f e c t s on the same t i s s u e s or on two  By f o c u s mapping both  drop-dead  to high, temperature  to t e s t  stress-sensitivity  different  and s t r e s s - s e n s i t i v e  these a l t e r n a t i v e s might be d i s t i n g u i s h e d . the mosaics  f l i e s became  data of ses E"*" can be examined i n two  Assuming the d i f f e r e n t phenotypes  of a same p o i n t mutation, may  During the course  tissues.  phenotypes,  U n f o r t u n a t e l y , exposing  the p a r a l y t i c b e h a v i o u r  death, i n many f l i e s b e f o r e p a r a l y s i s c o u l d be observed.  caused  Thus, the  occurence of l e t h a l phenotype b e f o r e l e g p a r a l y s i s o b s c u r r e d q u a n t i t a t i v e assessment  of l e g phenotype.  were judged  As a r e s u l t of t h i s , o n l y 27% o f the l e g s  to have become p a r a l y z e d b e f o r e the time of death  c o n v e n t i o n a l focus mapping u s i n g the l e g phenotype was: not  and  possible.  Examination of the s u r v i v a l c u r v e ( F i g . 22, Chapter 3) shows t h a t 73% of the mosaics at 29°C.  d i e d d u r i n g the f i r s t  13 days of i n c u b a t i o n  S p e c i f i c a l l y , most of these d i e d between days 5 and 13,  which time the s u r v i v a l curve l e v e l s o f f . f o r the l e t h a l phenotype was  undertaken,  C o n v e n t i o n a l mosaic  analysis  assuming that t h i s 73%  r e p r e s e n t s mosaics w i t h a l e t h a l focus (or f o c i ) .  after  fraction  I n s p e c t i o n o f the  d a t a showed that 24 of 25 mosaics, which were b i l a t e r a l l y mosaic f o r two of the t h r e e body segments, d i e d p r e m a t u r e l y . data, the ses E^ mutant f o c i a r e judged mathematical  On the b a s i s of these  to be domineering.  U s i n g the  a n a l y s e s d e r i v e d f o r a domineering model (Hotta and  Benzer,  47  1972), the f o c i were l o c a l i z e d r e l a t i v e to c u t i c l e markers. shown i n F i g u r e 10 was places  determined to b e s t  f i t the d a t a .  I t can be  seen, however, t h a t d i s c r e p a n c i e s f o r s e v e r a l of  d i s t a n c e s appear i n t h i s f a t e map. d i s t a n c e between the humeral and  bristle  (24 s t u r t s ) .  ses E f o c i  (17 s t u r t s ) should  area of the b l a s t o d e r m .  that i s , i t o c c u p i e s  which means t h a t only one  of these  b e h a v i o u r a l phenotype to be  order  submissive  The  placed  a relatively  two  f o c i are b i l a t e r a l l y  f u l l y expressed.  This i s i n contrast  the f o c i  i n the v i c i n i t y and  right foci  Using  is diffuse,  (that i s , -|- mutant,  and  the sturts.  f a t e map.  However,  the chance that i t w i l l have a mixed genotype  of mutant t i s s u e i s s u f f i c i e n t  bilateral  coxa 3,  i s found to be about 50  normal) w i l l i n c r e a s e .  of the focus appearing  the same c a l c u l a t i o n  of coxa 2 and  T h i s would p l a c e them i n the middle of the blastoderm the focus  domineering  f o c i i n which. Both, f o c i must be mutant i n  d i s t a n c e between the l e f t  if  large  f o c i need be mutant f o r the mutant  to c o n f e r a mutant b e h a v i o u r a l phenotype.  which had  inconsistancy  I t i s a l s o complex i n that t h e r e a r e 2 f o c i ,  on each s i d e of the b l a s t o d e r m .  to b i l a t e r a l l y  be  anterior notopleural  A possible explanation f o r this  t h a t the focus i s d i f f u s e ,  And  i f a s m a l l amount  to produce a mutant phenotype, the  mutant w i l l i n c r e a s e .  S i n c e ses E"^" f o c i  chances  are  domineering, the p o r t i o n of mosaics e x h i b i t i n g mutant  b e h a v i o u r should be  the  Thus, i t seems i n c o n s i s t e n t - t h a t the  l e s s than the d i s t a n c e between the humeral and  one  This p o s i t i o n  the f o c i near the p o s t e r i o r r e g i o n s o f the presumptive nervous  system.  is  The p o s i t i o n  large.  As Hotta  and  Benzer  (.1972) show, t h i s  *ure 10.  F a t e map of ses E . a, antenna; anp, a n t e r i o r n o t o p l e u r b r i s t l e ; hu, humerus; oc, o c e l l a r ; l c , <joxa 1; 2c, coxa 3c, coxa 3; 3s, 3rd s t e r n i t e ; J | S ^ , ses E focus.  49  would u l t i m a t e l y i n c r e a s e  the d i s t a n c e between 2 b i l a t e r a l l y  domineering  foci. As noted b e f o r e ,  conventional  focus mapping of the  phenotype o f ses E"* c o u l d bot be performed due -  of l e t h a l phenotype.  However, u s i n g  the 27%  the p a r a l y t i c phenotype c o n s i s t e n t l y b e f o r e  leg p a r a l y s i s  to the e a r l y onset  of the l e g s t h a t showed death, i t was  possible  c o r r e l a t e the phenotype of the l e g c u t i c l e w i t h t h e i r p a r a l y s i s . showed t h a t l e g ' p a r a l y s i s d i d not that The  the t h o r a c i c c u t i c l e are  c o r r e l a t i o n a n a l y s i s d i d show, however, t h a t a p a r a l y z e d  implies  separable. prothoracic  most commonly a s s o c i a t e d w i t h mutant t i s s u e i n the a n t e r i o r  notopleural b r i s t l e i n t h a t order  (Table  (ANP), coxae of the f i r s t , 8).  Likewise,  second and  third  p a r a l y s i s of l e g s 2 and  o f t e n a s s o c i a t e d with, mutant c u t i c l e i n the coxae of each leg,  This  always occur i n a mutant l e g and  the f o c i f o r l e g p a r a l y s i s and  l e g was  to  legs  3 were more  respective  than w i t h mutant c u t i c l e i n the r e s p e c t i v e t a r s i or o t h e r  thoracic  parts. These data suggest t h a t the f o c i f o r l e g p a r a l y s i s l i e somewhere below the t h o r a c i c hypoderm on  the b l a s t u l a f a t e map,  i n the  general  area where the a d u l t l e t h a l f o c i were l o c a l i z e d . Therefore, it  i s p o s s i b l e to map  u s i n g both m o d i f i e d out  the same a r e a  of the f l y which i s b e l i e v e d s e n s i t i v e and  to be  and  conventional  mosaic  analyses,  i n the v e n t r a l nervous system  the primary focus  f o r the s t r e s s -  drop-dead phenotypes.  Drug S t u d i e s :  Results  (Table 7) i n d i c a t e d that ses E^ i s  TABLE 8 The d i s t a n c e o f ses E focus w i t h r e s p e c t to v a r i o u s s u r f a c e landmarks. The d i s t a n c e s a r e ranked from 1 to 10 with 1 being c l o s e s t to and 10 b e i n g f u r t h e s t away from the f o c u s . D i s t a n c e from focus to  leg 1 ranking  Antenna  8  10  9  27  9  Humerus  9  9  10  28  10  1  4  4  9  3  Coxa 1  2.5  7  7  16.5.  5'  Coxa 2  2.5  2  3  7.5  2  Coxa 3  4  1  1  6  1  Tarsus 1  6.5  6  6  18.5  7  Tarsus  2  6.5  5  5  16.5  5  Tarsus  3  .5  3  2  10  4  10  8  8  26  8  ANP  Bristle  Sternite 3  leg 2 ranking  leg 3 ranking  Total  combinedranking  tn I— 1  more r e s i s t a n t to an a g o n i s t of c h o l i n e r g i c system, chloride.  carbamylcholine  IV.  Discussion  S i n g l e gene mutations which a f f e c t  the motor a c t i v i t y o f  f l i e s have proven t o be u s e f u l i n p i n p o i n t i n g t h e g e n e t i c r e g u l a t i o n of b e h a v i o u r and i t s neuromuscular  origin.  the d i r e c t e f f e c t o f s t r e s s on normal  While  insects,  the p r e s e n t study has  made use o f mutations which exaggerate s e n s i t i v i t y point of paralysis  to a l l o w us to p i n p o i n t  further physiological  physiologists*study  to s t r e s s to the  the c e l l s a f f e c t e d f o r  studies.  T e m p e r a t u r e - s e n s i t i v i t y i s a u s e f u l p r o p e r t y o f the ses B mutation.  Developmental  s t u d i e s i n d i c a t e t h a t the ses B mutant gene  product i s r e q u i r e d d u r i n g the l a r v a l stage and p o s s i b l y i n the ovary during oogenesis.  T h i s c o n c l u s i o n i s reached a f t e r t e m p e r a t u r e - s h i f t 2  s t u d i e s r e v e a l e d t h a t a t s m a t e r n a l - e f f e c t e x i s t s f o r ses B .  Eggs  m a i n t a i n e d a t 29°C from female p a r e n t s which have been kept a t 29°C f o r more than 3 days do not develop i n t o a d u l t s  ( l e s s than 10%).  a p p r o x i m a t e l y 3 days a r e r e q u i r e d f o r complete 1 to stage 14 oocyte  oogenesis from  Since stage  (see King, 1970 f o r d e f i n i t i o n s o f the s t a g e s )  i n D r o s o p h i l a , the r e s u l t s c o u l d suggest that a t h e r m o l a b i l e p r o d u c t which i s e s s e n t i a l throughout  the l a t e r stages o f development i s  s y n t h e s i z e d by and I n c o r p o r a t e d i n t o the e a r l y o o c y t e . these eggs were s h i f t e d down immediately 50% o f them develop i n t o a d u l t s .  However, i f  a f t e r the egg i s l a i d ,  about  T h i s i n d i c a t e s t h a t e i t h e r the thermo-  l a b i l e product can be r e a c t i v a t e d by a change o f temperature  o r the  embryonic c e l l s make enough product at the p e r m i s s i v e to " r e s c u e "  the embryos.  The  latter possibility  temperature  i s supported by s u r v i v a l  of heterozygous females from homozygous mothers a t 29°C.  I t has  been shown t h a t when ses B embryos or 1st i n s t a r l a r v a e are to 29°C heat p u l s e s , instances,  l a r v a l development i s prolonged and  development i s a r r e s t e d i n the 3rd i n s t a r .  exposure of 2nd  exposed  i n some contrast,  or 3rd i n s t a r l a r v a e to such temperatures r e s u l t s i n  l i t t l e p r o l o n g e d 3rd  instar l a r v a l period.  also i n d i c a t e d a 3rd  i n s t a r - p u p a l TSP  as 2nd  In  also  and  Temperature-shift that s h i f t  I n s t a r are c a p a b l e of forming a d u l t s .  that 2nd  i n s t a r l a r v a e which had  allowing  them to develop throughout the 3rd  These o b s e r v a t i o n s ,  together  (which i n d i c a t e t h a t +/ses B  downs as  studies late  These r e s u l t s i n d i c a t e  been r a i s e d at 29°C are r e s c u a b l e Instar-pupal  TSP  a t 22°C.  w i t h the r e s u l t from m a t e r n a l - e f f e c t 2  by  studies  2 heterozygotes. d e r i v e d from ses B /ses  B  2  2 mothers are capable of s u r v i v i n g ) suggest that ses  B  gene product  i s synthesized  and r e q u i r e d d u r i n g  oogenesis as w e l l as embryonic, 1st  i n s t a r and 3 r d  i n s t a r - p u p a l stage.  These TSPs are r e l a t e d i n such a  way  t h a t the r e d u c e d l a r y a l v i a b i l i t y  TSPs can .be a l l e v i a t e d by TSPs.  due  to exposure to 29°C at  f u r t h e r developing  them at 22°C d u r i n g  Many examples of s e x - l i n k e d m a t e r n a l - e f f e c t  earlier later  mutations e x i s t i n  D r o s o p h i l a i n c l u d i n g sortless (Colaianne and B e l l , 1972), deep orange (Garen and G e h r i n g , 1972)., cinnamon (_Baker, 19.73), f u s e d and r u d i m e n t a r y (Pausto-^Sterling, 1971).,' murkv_ and ( T a r a s o f f and  Suzuki,  1970).  ses  singed B  2  (Mohler, 1977), and  l(l)E25  i s p e c u l i a r i n that i t s ts gene  t S  product i s not o n l y important p r i o r to and a t the embryonic d u r i n g l a t e r s t a g e s as w e l l .  s t a g e , but  A l s o , i t d i f f e r s from o t h e r n o n - c o n d i t i o n a l  mutants i n that i t s gene product i s p a r t i a l l y r e s c u a b l e by a v o i d i n g subsequent  development a t the r e s t r i c t i v e Although mosaic  i n the head and phenotype,  temperature.  a n a l y s i s had suggested t h a t mutant  the thorax w i l l most l i k e l y  c o n f e r a mutant b e h a v i o u r a l  the v a s t amount o f t i s s u e s i n these r e g i o n s make i t almost  i m p o s s i b l e to p i n p o i n t the abnormal b e h a v i o u r to a s p e c i f i c On  tissues  tissue.  the o t h e r hand, the presence o f a l a r g e number of mosaics  i n c o n s i s t e n t behaviour  with  (Table 6) c o u l d suggest that the i n c o n s i s t e n c y  i s a c t u a l l y an i l l u s i o n o f i n t e r m e d i a t e b e h a v i o u r between the extreme phenotypes. and normal  two  Thus they were c l a s s i f i e d as mutant on one  on another.  day  T h i s , t o g e t h e r w i t h the f a c t t h a t t h e r e i s o n l y  a m i n o r i t y o f mosaics with, completely mutant thoraxes showing mutant behaviour  ( f o r example, o f the 64 mutant thoraxes  (Table 6 ) , 24  mutant b e h a v i o u r , 34 had I n c o n s i s t e n t b e h a v i o u r and 6 had normal  had behaviour,  suggest that ses B gene c o u l d a c t non-autonomously, t h a t i s , the mutant gene does n o t - a c t d i r e c t l y on the motor r e g i o n s of the t h o r a c i c but i n d i r e c t l y the f l y .  through a c i r c u l a t i n g substance produced  In f a c t , i n the American  elsewhere i n  cockroach, P e r i p l a n e t a  p h y s i o l o g i c a l s t u d i e s have shown t h a t such a c i r c u l a t i n g  ganglia,  americana, substance  c a l l e d F a c t o r S does e x i s t i n the i n s e c t ' s haemolymph and i s r e l e a s e d i n l a r g e r amounts d u r i n g s t r e s s Sternburg, 1963;  Davey, 1964;  (Beament, 1958;  Helsop and Ray,  Samaranayaka, 1974).  1959;  At low c o n c e n t r a t i o n s ,  F a c t o r S i n i t i a t e s a s t a t e of h y p e r a c t i v i t y i n the nervous system of the organism w h i l e a t h i g h e r  concentrations  the organism i s exposed to prolonged  (which i s the case when  p e r i o d s o f s t r e s s or h i g h e r  o f s t r e s s ) , F a c t o r S causes a n e u r a l b l o c k a d e Cuesta and Pomonis, 1969).  Therefore,  (Cook, 1967;  i t is likely  level  Cook, de l a  that a  circulating  substance analogous to t h a t i n the cockroach, e x i s t s i n D r o s o p h i l a , bearing  i n mind that t h e r e are s t i l l p o s s i b i l i t i e s of mutant  c e l l s being  e x t r a s e n s i t i v e to F a c t o r S and/or mutant c e l l s  a lower t h r e s h o l d f o r r e l e a s e o f F a c t o r  An  easy way  would be  having  S.  These r e s u l t s p o i n t to f u t u r e p u r s u i t s to t e s t autonomy of ses B.  receptor  the gene  to perform r e c i p r o c a l ovary  t r a n s p l a n t s between w i l d type host and mutant o v a r i e s , and v i c e v e r s a . I f the ses B gene does a c t non-autonomously d u r i n g oogenesis, wild  type o v a r i e s . i n ses B host w i l l be p r e d i c t e d to be  s e n s i t i v e , and  to a d u l t s .  see B mutation i s a l s o more s e n s i t i v e to r e s e r p i n e .  pharmacological catecholamine  a c t i o n of r e s e r p i n e i s to r e l e a s e (and (a b i o g e n i c amine) s t o r a g e  and Rosengren, 1956;  Burn and Rand, 1957,  1958,  (Sternburg,  determination concluded  unsuccessful.  t h a t F a c t o r S i s a b i o g e n i c amine.  Carlsson  1959a,b. Burn, 1960). Sternburg  Chang and Kearns, 1959), the exact  o f F a c t o r S was  The  thus reduce)  i n insects (Bertler,  In s p i t e of s e v e r a l e f f o r t s by Cook (1967) and workers  temperature-  ses B o v a r i e s i n w i l d type host w i l l be p r e d i c t e d to  produce eggs at 29°C t h a t w i l l g i v e r i s e The  then  and h i s  co-  biochemical  However, b o t h s t u d i e s B i o g e n i c amines i n  i n s e c t s are a p p a r e n t l y ways:  s t o r e d and  (1) from c o n v e n t i o n a l  r e l e a s e d to a c t i n at l e a s t  neurons where t h e i r e f f e c t s are  to the immediate s y n a p t i c r e g i o n , organs where t h e i r e f f e c t s may  (2) r e l e a s e d from  two localized  neurosecretory  be more g e n e r a l i z e d .  The  extra  sensitivity  of ses B to r e s e r p i n e t h e r e f o r e suggests t h a t s t r e s s p a r a l y s i s i n Drosophila and  c o u l d be mediated by  a b i o g e n i c amine.  Based on  this  the f a c t t h a t the p a r a l y s i s - c a u s i n g agent i s a c i r c u l a t i n g  result  biogenic  amine, the non-autonomous n a t u r e of the ses B gene becomes more Thus, i n a d d i t i o n to i t s s t r e s s - s e n s i t i v i t y e f f e c t s ,  probable. the ses  mutation a l s o a f f e c t s o o g e n e s i s , embryogenesis and p u p a r i a t i o n . represses  p u p a r i a t i o n by p r o l o n g i n g  l o n g as 3 weeks b e f o r e t  n  a  t  s  ^s  The  B c o u l d be an e x c e l l e n t candidate  present  studies  experiments are needed.  any  normal f l i e s ,  fertility,  f u r t h e r s p e c u l a t i o n , more  For example, i f the ses B gene express  non-autonomously, then perhaps q u a n t i t a t i v e a n a l y s i s on by e x t r a c t i o n and  suggest  f o r s t u d y i n g hormones i n  i n s e c t s because hormones are known to p l a y important r o l e s i n Before  It  the 3rd I n s t a r l a r v a l p e r i o d f o r as  death commences.  development and metabolism.  subsequent assay f o r I t s a c t i v i t y by  or a p p l i c a t i o n to i s o l a t e d i n s e c t nerve  itself  the s t r e s s f a c t o r injection into  preparation  (Cook, de l a Cuesta and Pomonis, 1969), or measurement of i t s e f f e c t on s e n s i t i z i n g l a r v a e to a p u p a t i o n One 2 i s ses D .  of the o t h e r  two  Mosaic f o r ses D  2  hormone, ecdysone.  s t r e s s - s e n s i t i v e mutations  Mosaic a n a l y s i s on ses D  B  2  suggested a n e u r a l  studied dysfunction.  e x h i b i t independent b e h a v i o u r of each l e g upon  stress.  This d i f f e r s  from ses B"" mosaics where the e f f e c t i s a l l or 1  none, that i s , e i t h e r a l l 6 l e g s are p a r a l y z e d f a c i f o r ses D (Hotta and  2  map  to the same area of the f a t e map  Benzer, 1972).  sudden movements and anaethesia. l e g s and sites.  The  or they are not.  Hk^  mutants o v e r - r e a c t  Hk  to s t i m u l i such as  l i e near t h e i r  ether  corresponding  T h i s area c l o s e l y corresponds to the r e g i o n of the b l a s t o d e r m to form the v e n t r a l nervous system.  T h i s i n t e r p r e t a t i o n was  f u r t h e r supported by e l e c t r o p h y s i o l o g i c a l s t u d i e s  (Ikeda  and  Kaplan,  which showed t h a t a l l a c t i o n p o t e n t i a l s of a c e r t a i n type of  neuron i n the  thoracic ganglion  are preceded by  slowly r i s i n g  de-  p o l a r i z a t i o n s , hence the rhythmic b u r s t s o f a c t i v i t y i s observed. frequency of t h i s endogenous, a c t i v i t y t h a t of the shaking with  1  each are l o c a t e d at p o s i t i o n s v e n t r a l to t h e i r a d u l t c u t i c u l a r  destined  1974)  as those f o r  o f t e n shake t h e i r l e g s v i g o r o u s l y under  embryonic f o c i f o r Hk^"  The  legs.  i s a l s o found to be  s i m i l a r to  Hence, the congruence of the ses D  those o f Hk""" suggests t h a t ses D p a r a l y s i s c o u l d be  The  due  foci to h y p e r -  a c t i v i t y i n the t h o r a c i c g a n g l i a as w e l l .  2 The  behavioural  phenotype of the ses D  that of another b a n g - s e n s i t i v e i n the nervous system  H a l l , 1975).  t h i s mutant r e v e a l e d  f a c i l i t a t i o n pattern a f t e r mild stimulation. post-synaptic missions.  The  resembles  mutant, bas, whose embryonic focus i s  ( F e i t e l s o n and  p h y s i o l o g i c a l s t u d i e s on  mutant  response caused by  one  Further e l e c t r o -  t h a t there  i s an abnormal  F a c i l i t a t i o n i s an  or more p r e v i o u s  n e u r o n a l f a c i l i t a t i o n observed i n bas  synaptic  enhanced  trans-  develops much  f a s t e r and  decays much slower and  of t r a n s m i t t e r (Jan and  Jan,  at the p r e - s y n a p t i c 1978).  The  to s t r e s s i s known to be 1959;  i s associated  w i t h a prolonged  neuron caused by  insect's hyperactivity  unknown reasons  as a p r e l i m i n a r y  a phenomenon of f a c i l i t a t i o n  S t e r n b u r g , Chang and  Kearns, 1959;  l e v e l s of i t can produce p a r a l y s i s  release  Cook, 1967)  response  (Helsop and where  Ray,  sustain  (Cook, de l a Cuesta and  Pomonis,  1969). 2 T h e r e f o r e , the s i m i l a r l o c a t i o n of f o c i  of ses D  1 , Hk  and  suggest  that  2 bas  and  the  resemblance i n phenotypes o f bas  t h e r e c o u l d be  an  and  ses D  enhanced or a f a s t e r development o f  facilitation  2 and/or a p r o l o n g e d time course of f a c i l i t a t i o n r e l a t i o n s h i p of the p i l o c a r p i n e n i t r a t e resistance unknown.  to the  caused by p r o l o n g e d r e l e a s e then these r e s u l t s c o u l d  conclusion  (an a g o n i s t  i s due  of transmitter  suggest the  The had  of an  i f any,  last  Eldefrawi,  of the  1976;  facilitation  c h o l i n e r g i c system.  Jan  and  i n insects Jan,  This to p r o v i d e  (Hildebrand,  1976).  s t r e s s - s e n s i t i v e mutations s t u d i e d ,  ses  E \  drop-dead pheno-  Although c o n v e n t i o n a l f a t e mapping w i t h mosaics d i d not  p o s i t i o n an embryonic focus f o r the a d u l t  is  inhibitory inter-  p l e i o t r o p i c e f f e c t s i n c l u d i n g s t r e s s - s e n s i t i v e and  types.  acetylcholine)  i s reached a f t e r s e v e r a l independent e f f o r t s f a i l  Osmond, 1979;  The  at the neuromuscular j u n c t i o n ,  existence  the motor neuron v i a the  as w e l l .  to abnormal  evidences of c h o l i n e r g i c neuromuscular j u n c t i o n s H a l l and  of  abnormal response to s t r e s s of ses D,  However, i f the ses D d e f e c t  neuron a c t i n g on  i n ses D  firmly  l e t h a l phenotype, i t d i d  suggest a g e n e r a l  r e g i o n o f the b l a s t o d e r m which i s more n e u r a l  mesodermal i n o r i g i n .  The focus  than  f o r ses E"*" i s q u i t e d i f f u s e and i s  l o c a t e d i n an area o f the b l a s t o d e r m d e s t i n e d  to form the t h o r a c i c  ganglia.  The importance o f the i n t e g r i t y o f the t h o r a c i c g a n g l i a i n  providing  a p r o p e r response to s t r e s s has a l r e a d y been emphasized.  Therefore,  i n a d d i t i o n to i t s e f f e c t on s t r e s s - i n d u c e d  p a r a l y s i s , the  t h o r a c i c g a n g l i a might be i n v o l v e d i n the drop-dead phenotype as w e l l . I t s h o u l d be noted t h a t the t e m p e r a t u r e - s e n s i t i v e c o u l d be e x p l a i n e d  by not o n l y b e i n g  s t r e s s but t o other  l e t h a l i t y o f ses E  extremely s e n s i t i v e t o m e c h a n i c a l  forms o f s t r e s s such as heat as w e l l .  Drug s t u d i e s i n d i c a t e d t h a t ses E^ i s more r e s i s t a n t to another agonist  of a c e t y l c h o l i n e , carbamylcholine c h l o r i d e  f o r e , l i k e ses D, a c h o l i n e r g i c system s u p p r e s s i n g  (carbachol).  There-  the s t r e s s - s e n s i t i v e  phenotype and, i n t h i s case, the drop-dead phenotype as w e l l cannot be r u l out.  A b e t t e r understanding on the n e u r a l  to i s o l a t e the c h o l i n e r g i c r e c e p t o r s study on the b i n d i n g receptors. receptors  d e f e c t o f these mutants i s  ( E l d e f r a w i , 1976) and perform a  properties of d i f f e r e n t neurotransmitters  I f such s t u d i e s i n d i c a t e the b i n d i n g a r e d i f f e r e n t , then d e f e c t s  other  to t h e i r  properties of c h o l i n e r g i c  than a b n o r m a l i t i e s  a t the  neuromuscular j u c n t i o n s must operate i n these s t r e s s - s e n s i t i v e m u t a t i o n s . In summary,  there a r e a t l e a s t two types o f s t r e s s - s e n s i t i v e  m u t a t i o n s : one i s g e n e r a l i z e d ,  a f f e c t i n g a l a r g e amount o f n e u r a l  tissue  w h i l e the other  I s more l o c a l i z e d , a f f e c t i n g a s i n g l e neuron c o n t r o l l i n g  l e g movements.  Hence, one has to be very  s e l e c t i v e i n choosing the  61  mutant f o r f u r t h e r believed  studies  because s t r e s s - s e n s i t i v e mutations  to a c t v i a t h r e e ways ( F e i t e l s o n and  (1) mutant c e l l s are h y p e r s e n s i t i v e to the c e l l s release  normally i n a c t i v a t e d e f e c t s of s e v e r a l and  stress  factor,  Homyk, 1977): (2) mutant  an e x c e s s i v e amount o f s t r e s s f a c t o r d u r i n g s t r e s s ,  (3) mutant c e l l s f a i l  map  H a l l , 1975;  are  to produce an  the  factor.  The  enzyme or enough enzyme which p r e s e n t study has  localized  the  s t r e s s - s e n s i t i v e mutations to t h e i r b l a s t o d e r m  has; d e l i n e a t e d  and  fate  TSPs f o r mutations w i t h ts developmental phenotypes.  More techniques i n anatomy, b i o c h e m i s t r y and n e c e s s a r y to determine the  h i s t o c h e m i s t r y are  s p e c i f i c r o l e of genes i n development  provide i n t e r e s t i n g relationships  between genes, development and  therefore and behaviour.  62  CHAPTER 3 DROP-DEAD MUTATIONS  I.  Introduction  The components of b e h a v i o u r , from the sensory r e c e p t o r s in  the p e r i p h e r a l nervous system to the c e n t r a l nervous system and  back out v i a the e f f e c t o r organs are assembled under the d i r e c t i o n o f genes.  T h e r e f o r e , any a l t e r a t i o n i n these components by mutations  w i l l r e s u l t i n death or abnormal b e h a v i o u r . mutants of  Hence, by s e l e c t i o n of  on the b a s i s o f t h e i r abnormal b e h a v i o u r , s p e c i f i c  the nervous system can be a n a l y z e d .  a stress-sensitive  components  One group o f mutations w i t h  (ses) phenotype has a l r e a d y been d e s c r i b e d .  The  second c l a s s s t u d i e d f o r t h i s t h e s i s i s c h a r a c t e r i z e d by death of a d u l t s (drop-dead) a t r e s t r i c t i v e temperatures. Other t e m p e r a t u r e - s e n s i t i v e a d u l t drop-dead mutations have been r e p o r t e d mutant ts  ( T a r a s o f f and Suzuki, 1968).  r e c o v e r e d i n the p r e s e n t study was  The f i r s t  adult  drop-dead  i s o l a t e d as a r e c e s s i v e  l e t h a l on the X chromosome and o n l y l a t e r ,  the t s a d u l t  drop-dead  tsl (add was  A  ) phenotype was  found among f l i e s  found.  A second t s drop-dead mutant,  recovered i n i t i a l l y  as s t r e s s - s e n s i t i v e .  ses E, Non-  c o n d i t i o n a l mutations w i t h an a d u l t l e t h a l phenotype, i n c l u d i n g d r d (Hotta and Benzer, 1972), dmd 1973) have been d e s c r i b e d . die  (Flanagan, 1977) and 1(4)18 (Hochman,  They a l l develop and e c l o s e n o r m a l l y but  p r e c o c i o u s l y soon a f t e r e c l o s i o n .  A n a l y s i s of g e n e t i c mosaics  showed that the primary l e t h a l focus o f drd l i e s and of dmd  i s i n the t h o r a c i c g a n g l i o n .  i n the b r a i n  H o t t a and Benzer  a b l e to c o n f i r m t h e i r c o n c l u s i o n s based on mosaic  way  Thus, the drop-dead  phenotype may  (1972) were  a n a l y s i s and  h i s t o l o g i c a l work which showed t h a t a l l drd f l i e s have brain tissue.  tissue  by  degenerated  prove to be a u s e f u l  to i d e n t i f y n e u r a l d e f e c t s . tsl The  t e m p e r a t u r e - s e n s i t i v e l e t h a l phenotypes  ses E~^ p r o v i d e advantages l e t h a l phenotypes  First,  the ts nature o f the  to perform s h i f t and p u l s e experiments  the p e r m i s s i v e and n o n - p e r m i s s i v e  A TSP  as the time of s y n t h e s i s and/or (Suzuki,  to  determine  i s a period during  which development at the n o n - p e r m i s s i v e temperature i n t h i s case l e t h a l i t y .  lethal  between  ( r e s t r i c t i v e ) temperatures  the 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 s (TSPs).  mutant phenotype,  and  over s i m i l a r mutations w i t h n o n - c o n d i t i o n a l  ( S u z u k i , 1970).  phenotype a l l o w s one  o f add A  r e s u l t s i n the  TSPs are g e n e r a l l y  regarded  f u n c t i o n of a mutant gene product  1970). In the p r e s e n t case, t e m p e r a t u r e - s h i f t s t u d i e s r e v e a l e d  t h a t the add A mutation has TSPs i n the embryonic e a r l y p u p a l stages as w e l l as i n the a d u l t s t a g e . ses E mutation was  and l a t e l a r v a l to Similarly,  found to have TSPs i n the m i d - f i r s t  the  to mid-second  l a r v a l i n s t a r s , m i d - t h i r d l a r v a l i n s t a r to e a r l y p u p a l stage and o f course, the a d u l t s t a g e .  Furthermore,  examination o f a d u l t mosaics p e r m i s s i v e temperature,  q u a n t i t a t i v e r e c o v e r y and  f o r l e t h a l i t y and b e h a v i o u r at the  non-  a l l o w e d the l o c a l i z a t i o n o f the add A a d u l t  l e t h a l phenotype for  to f o c i i n the mesoderm and suggested that  the ses E phenotype  the f o c i  a r e most l i k e l y i n the v e n t r a l nervous  system.  II.  M a t e r i a l s and Methods  A complete d e s c r i p t i o n of the i s o l a t i o n and p r o p e r t i e s of tsl add A  1 and ses E  mutations used i n t h i s study has been r e p o r t e d  (Homyk and Sheppard,  1977;  Homyk, Szidonya and S u z u k i , 1980).  map  p o s i t i o n s o f the genes a r e : add A, 1-20.9:; ses E, 1-27.7. tsl  for  the a d u l t t s l e t h a l c h a r a c t e r i s t i c , add A  The Except  mutants have develop-  mental r a t e s , b e h a v i o u r a l a c t i v i t y and l o n g e v i t y i d e n t i c a l to Oregon-R at  22°C.  In c o n t r a s t , ses E"^ f l i e s are d e t e c t a b l y l e s s a c t i v e  develop much more s l o w l y than Oregon-R. tsl add A  As w e l l , f l i e s  and  carrying  either  1 or ses E  have t s developmental l e t h a l  phenotypes.  U n l e s s o t h e r w i s e s t a t e d , a l l c u l t u r i n g c o n d i t i o n s , heat p u l s e s , temperature s h i f t s , drug s t u d i e s and g e n e t i c mosaic a n a l y s e s are i d e n t i c a l to those d e s c r i b e d i n Chapter 2. Examination o f a d u l t drop-dead b e h a v i o u r tsl add A ages were s h i f t e d  1 , ses E  to 29°C and each day, t h e i r v i a b i l i t y  examined by r e c o r d i n g vial  and Oregon-R w i l d type f l i e s  the number of s u r v i v o r s l e f t  c o n t a i n e d 10 f l i e s o f one age group.  of d i f f e r e n t a t 29°C  was  i n each v i a l .  Approximately 300  Each  flies  d i v i d e d e q u a l l y i n t o t h r e e age groups were used w i t h the e x c e p t i o n of  Oregon-R where only 100  f l i e s of one age group was  used.  Examination of embryonic development In o r d e r to determine the n a t u r e o f embryonic l e t h a l i t y of tsl add A , embryos were photographed at 2-hour i n t e r v a l s b e g i n n i n g from  66  o v i p o s i t i o n and c o n t i n u e d u n t i l h a t c h i n g .  Eggs were c o l l e c t e d w i t h i n  one-hour p e r i o d s and d e c h o r i o n a t e d i n 3% NaOCl (50% b l e a c h ) .  The embryos  were then l a i d on the s l i d e s which were f i x e d on dry i c e and photographed using b r i g h t f i e l d i l l u m i n a t i o n . polarizing  light  S t a i n s were not used.  However, a  f i l t e r was used to a i d i n o b s e r v a t i o n o f the muscles  which w i l l be b i r e g r i n g e n t under such c o n d i t i o n s . made w i t h Kodak Panatomic-X  film  A l l p i c t u r e s were  i n a C a r l Z e i s s photomicroscope w i t h  a b u i l t - i n camera s e t a t the automatic exposure s e t t i n g .  The exposed  f i l m was developed and p r i n t e d a c c o r d i n g to the manufacturer's  specifications.  III.  Results  ,, t s l add A A  tsl C h a r a c t e r i z a t i o n o f a d u l t drop-dead b e h a v i o u r : f l i e s were s h i f t e d  When add A  to 29°C, almost 95% d i e d w i t h i n 24 hours  whereas o n l y 7% m o r t a l i t y r a t e was  ( F i g . 11)  observed f o r Oregon-R f l i e s  after  20 days a t 29 C ( F i g . 12). C h a r a c t e r i z a t i o n o f TSPs:  TSPs o f t h i s mutant were i n i t i a l l y  determined by s t u d y i n g the consequences  o f 6-hour heat  a d m i n i s t e r e d a t d i f f e r e n t developmental s t a g e s . first  24 hours  (29°C) p u l s e s  Anytime d u r i n g the  (embryo s t a g e ) , such, p u l s e s s i g n i f i c a n t l y reduce  egg  tsl  hatchability hatch  ( F i g . 13).  eggs kept at 22°C  (data from s h i f t s t u d i e s , F i g . 15) whereas as few as 9% of heat  p u l s e d embryos s u r v i v e . is  More than 75% o f add A  In c o n t r a s t , h a t c h a b i l i t y of Oregon-R eggs  u n a f f e c t e d by temperature; more than 75% of the eggs u s u a l l y h a t c h . The e f f e c t of heat p u l s e s on post-embryonic  was  monitored by c a l c u l a t i n g the f r a c t i o n o f hatched eggs which  into adults.  The  data ( f i g ,  development  o r d u r i n g the e a r l y  i n s t a r l a r v a l s t a g e reduces the frequency o f a d u l t  The same data a l s o h i n t  survivors.  that a d d i t i o n a l TSPs e x i s t at the m i d - t h i r d  i n s t a r s t a g e (120 hours at 22°C) and e a r l y pupal s t a g e 22°C).  develop  14) show that a 6-hour heat p u l s e  a d m i n i s t e r e d near the end o f embryonic 1st  development  S h i f t s t u d i e s were performed  (216 hours a t  to i d e n t i f y and d e s c r i b e the  F i g u r e 11.  S u r v i v a l o f A , 1 day o l d ; o, 5 day o l d ; 10 day o l d add A t s l a d u l t s at 29°C and , 1-5 day o l d add A adults at 22°C. t s l  ON VO  70  i  F i g u r e 12.  S u r v i v a l of 1-5  day  o l d Oregon-R a d u l t s at 29°C.  72  Figure 13.  Percentage o f , — — , add A eggs; and, , Oregon-R eggs that hatch after 6-hour heat pulses (29"°C) administered at different times during development. The lengths of the bar indicate the duration of the pulses at 29°C. Approximately 100 eggs were used for each strain. Both strains have identical times for embryonic development. t s l  embryo  1st instar  100  to a>  o o ~o <D JZ  o 50  JC  12  24 time  in  36 hours  74  F i g u r e 14.  Percentage of add A eggs which y i e l d 6 hour heat p u l s e s (29°C) a d m i n i s t e r e d d u r i n g development. The developmental are shown a t the bottom. Approximately  adults after a t d i f f e r e n t times stages a t 22 C 100 eggs were used.  %  adult  survivors  76  F i g u r e 15.  Percentage o f , 0 — O , add A eggs which h a t c h a f t e r s u c c e s s i v e s h i f t - u p s a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development a n d , » — • , c o n t r o l add A ^ eggs which h a t c h a f t e r d e v e l o p i n g at 22°C throughout. A p p r o x i m a t e l y 100 eggs were used f o r each experiment. Developmental r a t e s d i d not d i f f e r i n b o t h experiments. t s l  t s  the TSPs more p r e c i s e l y .  C u l t u r e s s y n c h r o n i z e d to w i t h i n 2 hours o f  d e p o s i t i o n were e s t a b l i s h e d a t 22°C and the v i a l s were s h i f t e d to 29°C a t s u c c e s s i v e 6-hour i n t e r v a l s  (shift-up).  Any s h i f t - u p a t o r  b e f o r e the 24 hour embryonic p e r i o d s i g n i f i c a n t l y reduces the egg hatchability  ( F i g . 1.5).  The r e c i p r o c a l s h i f t - d o w n experiments were  a l s o performed by c o l l e c t i n g eggs a t 22°C, immediately  transferring  the c u l t u r e s to 29°C and then s h i f t i n g back down at 6-hour i n t e r v a l s . The o n l y c u l t u r e i n which, eggs hatched (9%) was In a l l o t h e r c u l t u r e s , no eggs hatched.  s h i f t e d down a t 6 h o u r s .  These experiments  the p u l s e experiments i n d e f i n i n g an embryonic TSP The s h i f t - u p s a l s o r e v e a l e d a s i g n i f i c a n t  corroborate  for lethality. increase i n s u r v i v a l  to a d u l t h o o d i n c u l t u r e s s h i f t e d up a f t e r 150 hours and an even more pronounced  i n c r e a s e i n s h i f t ups a f t e r the w h i t e pre-pupae  at 216 hours  ( F i g . 16).  (wpp) s t a g e  Thus, the d a t a I n d i c a t e the e x i s t e n c e of TSPs  d u r i n g the embryonic, mid-3rd i n s t a r and e a r l y p u p a l s t a g e s . to c h a r a c t e r i z e the p u p a l TSP more f u l l y ,  s h i f t s were made a t 6-hour  i n t e r v a l s on c u l t u r e s s y n c h r o n i z e d by c o l l e c t i o n of wpp  a t 22°C and  S h i f t - u p and s h i f t - d o w n experiments c o v e r i n g the i n t e r v a l p r e c e e d i n g wpp One  s e t was  up to 29-°C.  In o r d e r  60-hour  f o r m a t i o n were performed u s i n g 2 s e t s o f c u l t u r e s .  i n c u b a t e d at 22°C u n t i l wpp  began to appear, then s h i f t e d  White pre-pupae were c o l l e c t e d a t 6 hour i n t e r v a l s  t h e i r i n c u b a t i o n c o n t i n u e d a t 29°C f o r the s h i f t - u p experiment. second s e t was  29°C.  s h i f t e d up to 29°C d u r i n g the 3rd l a r v a l i n s t a r  incubated t h e r e u n t i l wpp  began to appear.  and The and  At t h i s time, the c u l t u r e s  79  Figure  16.  tsl Percentages of add A eggs which y i e l d a d u l t s a f t e r s u c c e s s i v e s h i f t - u p s a d m i n i s t e r e d a t d i f f e r e n t times d u r i n g development. The developmental stages a t 22°C a t the time of s h i f t - u p s are shown a t the bottom.  were s h i f t e d down to 22°C and wpp the s h i f t - d o w n experiments.  For s h i f t - u p s and shift-downs  the 60-hour i n t e r v a l a f t e r wpp at 29°C and  c o l l e c t e d a t 6-hour i n t e r v a l s f o r  f o r m a t i o n , wpp  covering  were c o l l e c t e d from c u l t u r e s  22°C and s h i f t e d down and u p . r e s p e c t i v e l y at 6-hour i n t e r v a l s .  T h i s technique of s y n c h r o n i z i n g the c u l t u r e s a t the wpp  stage y i e l d s  r e s u l t s which i n d i c a t e a s i g n i f i c a n t i n c r e a s e o f a d u l t s u r v i v o r s i n those c u l t u r e s s h i f t e d up at 6 hour or more a f t e r wpp 1.7).  This signals  the end of a TSP.  S i m i l a r l y , decreases  e c l o s i o n b e g i n to appear when shift-downs wpp  formation  ( F i g . 17) and  d e l i n e a t e a TSP  6 hours a f t e r wpp  formation.  (Fig..  i n adult  are made 6 hours o r l e s s b e f o r e  this indicates  These experiments  formation  the b e g i n n i n g o f a  extending  TSP.  from 6 hours b e f o r e to  Twelve-hour heat and c o l d p u l s e s were made s t r a d d l i n g wpp  stage.  survivor  The  data c l e a r l y  the  i n d i c a t e d that the f r e q u e n c i e s of a d u l t  i n c r e a s e when the c u l t u r e s develop  at p e r m i s s i v e  d u r i n g the p e r i o d 12 hours b e f o r e and a f t e r wpp  formation  temperatures ( F i g . 18).  Data f o r c o l d p u l s e s a d m i n i s t e r e d at 42 hours or more a f t e r wpp  formation  are not shown because of too few o b s e r v a t i o n s to be s i g n i f i c a n t , f o r example, a 50% s u r v i v a l i n a c o l d p u l s e 54 to 66 hours a f t e r wpp was  based on 2 s u r v i v o r s out o f 4 wpp.  as e a r l y as 54 hours b e f o r e wpp  formation  Twelve-hour heat p u l s e s b e g i n n i n g  f o r m a t i o n were found  to reduce  the  f r a c t i o n of e c l o s i n g a d u l t s , w h i l e the same heat p u l s e s 12 hours wpp  f o r m a t i o n had no e f f e c t on a d u l t e c l o s i o n  s e v e r e r e d u c t i o n of a d u l t s was  ( F i g . 18).  after  The most  observed when the heat p u l s e s were  82  F i g u r e 17.  Percentage of add A white pre-pupae (wpp) which e c l o s e d as a d u l t s , A - — A , a f t e r s u c c e s s i v e s h i f t - u p s a n d , v — v , a f t e r s u c c e s s i v e shift-downs a d m i n i s t e r e d p r i o r to and a f t e r wpp formation. t s l  JZ  2 50 JZ  o CL Q.  84  72  60 48 36 24 hours before wpp formation  12  •ntr--M^Nt/*Y  0 W  D K  12  D K  24  J  36 48 60 72 hours after wpp formation  L 84  96  84  F i g u r e 18.  Percentage of add A white pre-pupae (wpp) which e c l o s e d as a d u l t s , O O, a f t e r 12 hour heat p u l s e s (29°C) and, > «, a f t e r 12 hour c o l d p u l s e s (22°C), a d m i n i s t e r e d p r i o r to and a f t e r wpp f o r m a t i o n .  85  100 r  oo —  -- -o  -o  - f c — •  -o  o  o.  CO  0  T3  ro if) ro TJ  o  • - -o  0  o  jS 50  o-  _c u lc  ©  ©  -o  o -o  -o  Q.  Q.  cN  o  o  o  •©•  o  12  0 wpp  o- — -o  72  60  48 36 24 hours before wpp formation  J  12  I I I II  24 36 48 hours after wpp formation  60  72  performed i n the i n t e r v a l from 30 hours p r e c e e d i n g wpp f o r m a t i o n to 6 hours f o l l o w i n g wpp f o r m a t i o n . A l t h o u g h t h e r e a r e minor d i s c r e p a n c i e s between the s h i f t and p u l s e experiments d e f i n i n g  the s t a r t of the TSP, these experiments  i n d i c a t e a TSP around the wpp stage which commences a t l e a s t prior  to and ends 6 hours f o l l o w i n g wpp f o r m a t i o n .  6 hours  Thus, we have  tsl shown t h a t the add A  mutant has 3 TSPs; one embryonic TSP which  o c c u p i e s the whole embryonic p e r i o d o f 24 hours, one 12 hour TSP which, b e g i n s a t 6 hours b e f o r e wpp f o r m a t i o n and ends a t 6 hours  after  wpp f o r m a t i o n , and another i n the a d u l t s t a g e . G e n e t i c Mosaic S t u d i e s :  The 292 mosaics used i n t h i s  analysis  were r e c o v e r e d from the c r o s s y_ w add A " "^/Y males X I n ( l ) w ^ / y w s p l t  S  V  females.  The mosaics were developed and mosaic patches were  at 22°C.  A l l mosaics were s h i f t e d  identified  t o 29°C w i t h i n 2 days o f e c l o s i o n  and observed f o r 19 days f o r drop-dead b e h a v i o u r . tsl I t was found t h a t s u r v i v a l mosaics s h i f t e d  (or l e t h a l i t y ) o f the add A  to 29°C do not show any s i g n s o f l e v e l l i n g  the 19 days o f o b s e r v a t i o n  ( F i g . 19).  to a s s i g n a drop-dead phenotype  o f f during  Therefore, i t i s not p o s s i b l e  to these mosaics due t o the p o s s i b i l i t y  that not a l l o f them w i t h a premature  t s l e t h a l phenotype were s c o r e d .  F o r t u n a t e l y , each mosaic was a l s o observed f o r the onset o f b e h a v i o u r a l abnormalities.  I t was noted that some mosaics demonstrated  l e g p a r a l y s i s b e g i n n i n g the f i r s t 19).  individual  day a f t e r the s h i f t up to 29°C ( F i g .  In most c a s e s , the l e g p a r a l y s i s phenotype was observed a t l e a s t  87  F i g u r e 19.  Percentage o f , 292 add A mosaic s u r v i v o r s at 29°C; •» ^5 y w add A"" mosaic s u r v i v o r s a t 29°C ( c o n t r o l ) ; and A , % p l o t of 292 X 6 = 1752 l e g s f o r n o n - p a r a l y s i s . S  1  percent  o  00 00  two  days b e f o r e  lethality.  The major e x c e p t i o n s  dead on day two and a f r a c t i o n of those no o b s e r v a t i o n s were made b e f o r e  were those  dying on day two.  day one.  mosaics Unfortunately,  I t was noted, however, t h a t  a l l s i x l e g s o f many mosaics dying on days two and t h r e e had become severely paralyzed leg  the p r e v i o u s  the f i r s t  the data f o r  t h r e e days, b u t had n o t shown s i g n s o f motor p a r a l y s i s ,  were mutant f o r a l l l e g f o c i . the f o l l o w i n g reasons:  is  Thus, i n p l o t t i n g  p a r a l y s i s ( F i g . 19), i t was assumed t h a t those mosaics which d i e d  during  and  day.  T h i s assumption seems r e a s o n a b l e f o r  (1) In those mosaics which d i d show l e g p a r a l y s i s  e a r l y death, a l l s i x l e g s were p a r a l y z e d .  (2) In g e n e r a l ,  there  a good c o r r e l a t i o n between time o f death and the number o f p a r a l y z e d tsl  legs.  (3) Very few y w add A  males which, s u r v i v e d i n c u b a t i o n a t  29°C f o r s e v e r a l days, d i e d b e f o r e  showing any motor  debilitation.  (.4) M o s a i c s dying thorax, Few  e a r l y were g e n e r a l l y mutant f o r c u t i c l e i n the lower tsl t i s s u e n e a r e s t the add A f o c i as determined below. (5) tsl+  c o n t r o l , y_ w add A  , mosaics d i e d d u r i n g  the f i r s t  14 days o f  i n c u b a t i o n a t 29°C. If  the number o f non-paralyzed  the days o f o b s e r v a t i o n off  l e g s were p l o t t e d a g a i n s t  ( F i g . 1 9 ) , i t was found that the curve  a t the 50% mark a t about day 16.  levels  T h i s i s c o n s i s t e n t w i t h the  i n t e r p r e t a t i o n that the b e h a v i o u r o f each l e g i s c o n t r o l l e d by a s i n g l e f o c u s , and the chance that such a focus w i l l be mutant i s , l i k e any c u t i c l e marker, 50%.  Thus, i t seems j u s t i f i a b l e  l e g s that were p a r a l y z e d  to c l a s s i f y  those  on day 0 to day 16 i n c l u s i v e as mutant and  90  the remaining  l e g s normal.  F a t e mapping a n a l y s i s showed t h a t the primary paralysis has coxa  foci  for leg  l i e s i n the mesodermal t i s s u e s of the blastoderm.  i t s own  independent focus  Each l e g  l o c a t e d at about 22 s t u r t s from i t s r e s p e c t i v e  ( F i g . 20). Drug S t u d i e s :  In o r d e r  to t e s t whether s y n a p t i c f u n c t i o n s  are i n v o l v e d i n the drop-dead mutations,  the mutants were exposed to  v a r y i n g c o n c e n t r a t i o n of d i f f e r e n t p s y c h o t r o p i c and n e u r o t r o p i c known to a f f e c t n e u r a l f u n c t i o n s i n mammals. s t u d i e s a r e summarized i n T a b l e 9. are presented  The  drugs  r e s u l t s of the drug  Complete r e s u l t s of the drug s t u d i e s  i n the Appendix, tsl  add A  i s more s e n s i t i v e to Yohimbine, an a d r e n e r g i c b l o c k e r ,  and more r e s i s t a n t  to p i l o c a r p i n e n i t r a t e which i s an a g o n i s t of c h o l i n e r g i c  system. Photomicrographic embryonic TSP, wild  Studies:  tsl Because add A mutants have an tsl  the embryonic development of add A  type were compared and  and  Oregon-R  examined f o r a b n o r m a l i t i e s u s i n g photo-  micrography . The for  r e s u l t s o f photomicrography of embryos kept  v a r y i n g l e n g t h s o f time can be seen i n F i g . 21.  at 29°C  The p i c t u r e s  w i t h a darker background were taken u s i n g a p o l a r i z e r w h i l e  those  w i t h a b r i g h t e r background were taken u s i n g s t a n d a r d b r i g h t f i e l d illumination.  The m a l p i g h i a n  under p o l a r i z e d l i g h t  t u b u l e s and muscles w i l l be  to p r o v i d e an e a s i e r way  birefringent  of i d e n t i f y i n g  these  91  F i g u r e 20.  tsl F a t e map of add A . a, antenna; anp, a n t e r i o r n o t o p l e u r a l b r i s t l e ; hu, humerus; oc, o c e l l a r ; l c , coxa 1; 2c, coxa 2; 3c, coxa 3; 3s, 3rd s t e r n i t e . f ^ , f o c i of add_A ^. ts  Table 9 L i s t of drugs which have a more pronounced e f f e c t on the v i a b i l i t y o f two dropdead s t r a i n s . The e f f e c t s o f the same drug on Oregon-R are a l s o shown. Drugs were d i s s o l v e d i n water to make d i f f e r e n t c o n c e n t r a t i o n s b e f o r e they were added to the i n s t a n t D r o s o p h i l a medium ( 3 o r e a l ) . C u l t u r e s w i t h no drugs d i s s o l v e d i n the water serve as c o n t r o l s . Ore-R pupae „ larvae °  DRUG Pilocarpine nitrate  add  A  ses  adults „ pupae  pupae larvae  72.2 62.5 60.0 0.0 0.0  66.7 100.0 100.0  79.5  54.8  71.1 56.4 63.4  88.9 81.8 65.4  c/  °  adults „ pupae  °f' mg/ml  .2 .4 .8 .1.6 3.2 .75 1.5 3.0 6.0 9.0  73.8 67.4 33.3 0.0 0.0  96.8 • 100.0 100.0  Yohimbine  .5 1.0 2.0 4.0 8.0  88.5 54.1 51.4 0.0 0.0  100.0 100.0 68.4  16.2 11.4  33.3 100.0  87.2 72.1 82.5 50.0  91.2 90.3 100.0 -84.0  58.3 88.2 44.7 51.4  76.2 92.4 94.1 100.0  2  a d u l t s „, pupae  C  Carbamylcholine chloride  H0 control no drugs  pupae „ larvae °  82.1 69.0 89.3 60.7 70.0  100.0 57.1 96.4 91.6  91.3 100.0 100.0 70.0 100.0  '  82.5 91.7 92.6 90.9  94  F i g u r e 21.  Photomicrographs of add A and Oregon-R embryos, developed at 29°C, a t 2 hours p o s t - f e r t i l i z a t i o n : add A embryos under I , p o l a r i z e d l i g h t ; I I , b r i g h t f i e l d i l l u m i n a t i o n ; Oregon-R embryos under I I I , p o l a r i z e d l i g h t ; IV, b r i g h t field illumination. s  9.6  F i g u r e 21 ( c o n t . ) . Photomicrographs of add A and Oregon-R developed at 29 C, a t 4 hours p o s t - f e r t i l i z a t i o n : embryoj^under V, p o l a r i z e d l i g h t ; V I , b r i g h t f i e l d add A embryos under V I I , p o l a r i z e d l i g h t ; V I I I , field illumination.  embryos, Oregon-R illumination; bright  98  F i g u r e 21 ( c o n t . ) . Photomicrographs of add A and Oregon-R embryo^, developed a t 29°C, a t 6 hours p o s t - f e r t i l i z a t i o n : add A embryos under IX, p o l a r i z e d l i g h t ; X, b r i g h t f i e l d i l l u m i n a t i o n ; Oregon-R embryos under X I , p o l a r i z e d l i g h t ; X I I , b r i g h t field illumination.  100  F i g u r e 21 ( c o n t . ) Photomicrographs of add A and Oregon-R embryo developed a t 29 C, a t 8 hours p o s t - f e r t i l i z a t i o n : add A embryos under X I I I , p o l a r i z e d l i g h t ; XIV, b r i g h t f i e l d i l l u m i n a t i o n ; Oregon-R embryos under XV, p o l a r i z e d l i g h t ; XVI, b r i g h t f i e l d i l l u m i n a t i o n . S  102  F i g u r e 21 ( c o n t . ) . Photomicrographs of add A and Oregon-R embryos developed a t 29 C, a t 10 hours p o s t - f e r t i l i z a t i o n : add A embryos under x v i i , p o l a r i z e d l i g h t ; x v i i i , b r i g h t f i e l d i l l u m i n a t i o n ; Oregon-R embryos under x i x , p o l a r i z e d l i g h t ; xx, b r i g h t f i e l d i l l u m i n a t i o n .  o  104  tsl F i g u r e 21 ( c o n t . ) Photomicrographs of add A and Oregon-R embryos. developed a t 29°C, a t 12 hours p o s t - f e r t i l i z a t i o n : add A embryos under XXI, p o l a r i z e d l i g h t ; XXII, b r i g h t f i e l d i l l u m i n a t i o n ; Oregon-R embryos under X X I I I , p o l a r i z e d l i g h t ; XXIV, b r i g h t f i e l d i l l u m i n a t i o n . S  XXJV  Tip  6  F i g u r e 21 ( c o n t . ) . Photomicrographs of add A and Oregon-R embryo.Sj developed a t 29 C, a t 14 hours p o s t - f e r t i l i z a t i o n : "add A embryos under XXV, p o l a r i z e d l i g h t ; XXVI b r i g h t f i e l d i l l u m i n a t i o n Oregon-R embryos under XXVII, p o l a r i z e d l i g h t ; XXVIII, b r i g h t f i e l d i l l u m i n a t i o n , mu, muscles; ma, m a l p i g h i a n t u b u l e s .  X X V I I  X X V I I I  ,108  F i g u r e 21 ( c o n t . ) Photomicrographs of add A and Oregon-R embryoj^ developed at 29 C, a t 16 hours p o s t - f e r t i l i z a t i o n : add A embryos under XXIX, p o l a r i z e d l i g h t ; XXX, b r i g h t f i e l d i l l u m i n a t i o n ; Oregon-R embryos under x x x i , p o l a r i z e d l i g h t ; x x x i i , b r i g h t f i e l d i l l u m i n a t i o n , mu, muscles; ma, m a l p i g h i a n tubules.  •,110  tsl F i g u r e 21 ( c o n t . ) Photomicrographs of add A and Oregon-R embryos developed a t 29°C, a t 17% hours p o s t - f e r t i l i z a t i o n : add' A embryos under x x x i i i , p o l a r i z e d l i g h t ; x x x i v , b r i g h t f i e l d i l l u m i n a t i o n ; Oregon-R embryos under xxxv, p o l a r i z e d l i g h t ; xxxvi, bright f i e l d illumination.  Figure 2 1 (cont.)Photomicrographs of add A embryos, developed a t 29 C: a t 22 hours p o s t - f e r t i l i z a t i o n under x x x v i i , p o l a r i z e d l i g h t ; x x x v i i i , b r i g h t f i e l d i l l u m i n a t i o n ; at 46 hours p o s t - f e r t i l i z a t i o n under x x x i x , p o l a r i z e d l i g h t ; xu, bright f i e l d illumination.  XXXVII  s t r u c t u r e s i n the d e v e l o p i n g embryos. From f e r t i l i z a t i o n  (0 hour) through g a s t r u l a t i o n  (3 - 3y  hour) and head segmentation (.6 hour) to d o r s a l and v e n t r a l segmentation (8 h o u r ) , t h e r e were no d e t e c t a b l e d i f f e r e n c e s between the add A and Oregon-R embryos.  tsl  However, a f t e r 10 hours at 29°C, the m a l p i g h i a n  t u b u l e s and muscles o f add A  tsl  embryos appear to develop a t a f a s t e r  r a t e s i n c e Oregon-R have none a t t h i s time w h i l e the 22°C c o n t r o l s o f both Oregon-R and add A  tsl  embryos developed a t e x a c t l y the same r a t e .  I t s h o u l d be noted that the add A  tsl  embryos may have an o v e r a l l  increase  i n r a t e of development of o t h e r t i s s u e s as w e l l , but o n l y the muscles and m a l p i g h i a n t u b u l e s were e a s i l y d e t e c t e d by the techniques used tsl here.  By 12 hours, the add A  embryos have more prominent but  p o o r l y d e f i n e d muscles and m a l p i g h i a n t u b u l e s and the segmentations once v i s i b l e e a r l i e r have d i s a p p e a r e d . In c o n t r a s t , Oregon-R at t h i s stage have w e l l d e f i n e d muscles and m a l p i g h i a n t u b u l e s . A t 14 hours, Oregon-R embryos s t a r t  to show advanced muscle development and segmentation  tsl w h i l e the add A  embryos completely l a c k segmentation.  A t 16 hours,  tsl the muscles and m a l p i g h i a n t u b u l e s i n the add A  embryos become  l e s s o r g a n i z e d and more d i s p e r s e d w h i l e i n the Oregon-R embryos w e l l d e f i n e d muscles form d i s t i n c t bands along the l o n g i t u d i n a l s i d e s of the embryos. At t h i s stage, the Oregon-R embryos a l s o have t r a c h a e tsl 1 w h i l e add A have none. By 17~2 h o u r s , when most Oregon-R embryos tsl have hatched, the muscles and the m a l p i g h i a n t u b u l e s o f the add A embryos, though d i s o r g a n i z e d , a r e s t i l l p r e s e n t but the embryos  fail  to  hatch.  At 22 hours,  these s t r u c t u r e s s t i l l  A~  p e r s i s t i n add  l  ii±  embryos but no l a r v a e h a t c h . In  summary, i t appears  that the muscles and m a l p i g h i a n t u b u l e s  tsl of  add A  embryos, although formed a t an e a r l i e r s t a g e than Oregon-R,  are never w e l l o r g a n i z e d and are a l s o i l l d e f i n e d when compared* to tsl Oregon-R embryos.  T h e r e f o r e , i t seems t h a t the l e t h a l i t y  at  to a l a c k of muscle or m a l p i g h i a n t u b u l e development  29°C i s not due  but  to a f a i l u r e o f the muscular  system  and the m a l p i g h i a n t u b u l e s to  m a i n t a i n t h e i r i n t e g r i t y and to o r g a n i z e d themselves. the muscular  sysstem  falls  o f add A  to form d i s t i n c t bands.  F o r example,  I t s h o u l d be  noted  t h a t the m a l p i g h i a n t u b u l e s and muscles were used as i n d i c a t o r s of embryo development because of t h e i r ease of i d e n t i f i c a t i o n under p o l a r i z light.  Moreover, these are v e r y g r o s s m o r p h o l o g i c a l d i f f e r e n c e s  observed  through  the l i g h t microscope  and  as  the more s u b t l e d i f f e r e n c e s ,  i f p r e s e n t , are beyond the c a p a b i l i t y of the methodology used  here.  ses E C h a r a c t e r i z a t i o n of a d u l t drop-dead b e h a v i o u r : flies  are s h i f t e d  to 29°C, almost  whereas 95% of Oregon-R f l i e s (Fig.  95% d i e w i t h i n 6 days  When ses E^" ( F i g . 22)  u s r v l v e f o r a t l e a s t 17 days a t 29°C  12). C h a r a c t e r i z a t i o n o f TSPs:  has a l r e a d y been p r e s e n t e d i n Chapter have 2 TSPs - a 70-hour TSP  The d e s c r i p t i o n o f TSPs o f ses E"*" 2.  To r e c a p i t u l a t e , ses E"*" f l i e s  extending from m i d - l s t i n s t a r l a r v a l to  mid-2nd i n s t a r l a r v a l stage and a 90-hour TSP  d u r i n g the  transition  116  F i g u r e 22.  S u r v i v a l of v, 1 day o l d ; o, 5 day o l d ; •, 10 day o l d ses E a d u l t s at- 29°C and , 1-5 day o l d ses E adults at 22°C. 1  1  11'  of  3rd i n s t a r l a r v a l to p u p a l stage G e n e t i c Mosaic S t u d i e s :  ( F i g . 4, F i g . 5 ) . 229  mosaics were r e c o v e r e d among  o f f s p r i n g of the c r o s s y_ w ses E~*"/Y males X I n ( l ) w ^ / y w s p l females. V  The  d e t a i l s o f the mosaic a n a l y s i s have a l r e a d y been mentioned i n the  previous  chapter.  The  suggests  t h a t those mosaics which d i e on o r b e f o r e the 13th  drop-dead i n phenotype. f a t e mapping a n a l y s i s .  s u r v i v a l curve o f the ses  mosaics  T h i s a l l o w s a more simple and  ( F i g . 23) day  straight  are forward  C o n t r o l XO males r e c o v e r e d from the same c r o s s ,  are a r e s u l t of f e r t i l i z a t i o n  o f a n u l l o - X egg by a v_ w ses E"^" sperm.  These XO males a l l d i e b e f o r e day  10.  I t was  found  t h a t the embryonic  focus of ses E~^ i s l o c a t e d i n the a r e a of n e u r o b l a s t s about 9 s t u r t s below coxa 2 and  coxa 3 i n the blastoderm  Drug S t u d i e s : carbamylcholine  ses  f a t e map  i s more r e s i s t a n t  ( F i g . 10,  Chapter  than Oregon-R to  c h l o r i d e , an a g o n i s t o f c h o l i n e r g i c system  (Table 9 ) .  2).  119  Figure  23. Percentage o f ^ o, ses E mosaic s u r v i v o r s a t 29°C; •, y w ses E /0 male s u r v i v o r s a t 29°C and •, y w ses E mosaic s u r v i v o r s a t 29  C.  IV.  Temperature-shift  Discussion  s t u d i e s i n d i c a t e t h a t the add A  mutant  has  s u c c e s s i v e TSPs d u r i n g the embryonic, 3rd i n s t a r l a r v a l - p u p a l  and  adult stages.  2nd  l a r v a l i n s t a r and  stage.  The  The  ses E"^ mutant has  TSPs between the 1st  the 3rd i n s t a r l a r v a l - p u p a l stages  embryonic and  and  and the a d u l t  the 3rd l a r v a l - p u p a l stages r e p r e s e n t  of c o n s i d e r a b l e a c t i v i t y of developmental genes these r e s u l t s are not s u r p r i s i n g .  (Hadorn, 1951)  periods so  The m i c r o s c o p i c o b s e r v a t i o n s made  tsl on embryos of add A  p r o v i d e c y t o l o g i c a l v e r i f i c a t i o n of abnormal  embryonic development. normally  I t was  observed  t h a t the embryos  develop  u n t i l 10 hours a f t e r o v i p o s i t i o n at which time d i f f e r e n c e s tsl  between the Oregon-R and add A l i g h t microscope. studies.  embryos were f i r s t  This coincides with  A f t e r 12 hours,  Notch embryonic l e t h a l s  the TSP  v i s i b l e with  i n f e r r e d from  E a r l i e r s t u d i e s of add A  shift  an amorphous mass o f t i s s u e r e m i n i s c e n t  ( S h e l l e n b e r g e r and Mohler, 1978) tsl  the  of  appears.  embryonic t i s s u e c u l t u r e s r e v e a l e d that  t h e r e i s a poor a s s o c i a t i o n of nerve and muscle c e l l s , as w e l l as a poor agglomeration  of o t h e r d i f f e r e n t e e l l types  communication, F i g . 24). formation,  In a d d i t i o n to poor neuromuscular j u n c t i o n  the number of n e u r a l and muscle c e l l s  to the c o n t r o l s .  These o b s e r v a t i o n s  both! muscle and nerve development. cells  (Buzin _elt a_l., 1978)  (G. Beard, p e r s o n a l  suggest  i s reduced tsl  t h a t add A  relative  could  affect  Reduced numbers of nerve and  muscle  and poor development  (Cross and  Sang,  1978)  122  F i g u r e 24.  C e l l c u l t u r e s o f I , Oregon-R a t 29°C; I I , same as I but a t a h i g h e r m a g n i f i c a t i o n ; I I I , add A l a t 29°C; IV, same as I I I b u t a t a h i g h e r m a g n i f i c a t i o n . t s  12*  have a l s o been observed w i t h  cell  c u l t u r e s o f another embryonic  lethal,  tsl shi  .  I t i s tempting, t h e r e f o r e , to suggest t h a t the developmental tsl  abnormalities facilitate  o f add A  cell  to c e l l  stem from a d e f e c t i n c e l l membranes t h a t connections.  l a c k o f o r reduced p r o d u c t i o n for  Among o t h e r p o s s i b i l i t i e s , the  o f chemical  signals i n cells  responsible  i n t e r c e l l u l a r communication c o u l d r e s u l t i n some o f the observed  phenotypes.  T h i s i n t u r n c o u l d be due to a l a c k o f o r a reduced  level  of enzymes which are r e s p o n s i b l e f o r s y n t h e s i s , r e l e a s e o r r e c e p t i o n of such chemical  signals.  F o r example, L e v i - M o n t a l c i n i e t a l . (1968)  demonstrated that a p r o t e i n , c a l l e d  the nerve growth f a c t o r (NGF),  p l a y s an important r o l e i n the a r c h i t e c t u r e o f the nervous system both i n v i v o and i n v i t r o . Results  from t e m p e r a t u r e - s h i f t  of an a l t e r e d p r o t e i n . diffusible  I t i s p o s s i b l e to t e s t  t s gene product by m i x i n g c e l l  mutant embryos and determining w i l d type c e l l s  s t u d i e s suggest the p o s s i b i l i t y the e x i s t e n c e  c u l t u r e s from normal and  whether there i s any i n f l u e n c e o f the  on the development o f mutant myocytes and  I d e a l l y , a c e l l marker system would be i n c o r p o r a t e d so that the genotype o f i n d i v i d u a l c e l l s Unfortunately, But,  neurocytes.  i n t o such an experiment  can be u n e q u i v o c a l l y  such a system i s n o t a v a i l a b l e a t the p r e s e n t  i t i s p o s s i b l e to compare r e l a t i v e c e l l  mixing w i t h w i l d type c e l l s Altogether,  of a  counts b e f o r e  determined. time.  and a f t e r  (Cross and Sang, 1978).  t i s s u e c u l t u r e and t e m p e r a t u r e - s h i f t  studies  suggest a t s product which i s e s s e n t i a l f o r the development o f nerve  and muscle c e l l s most l i k e l y o p e r a t e s i n add A  tsl  .  More c o n c r e t e  tsl c o n c l u s i o n s on the add A  d e f e c t came from f a t e mapping a n a l y s i s which  i n d i c a t e t h a t i t s a b n o r m a l i t i e s l i e i n the muscular system. the  l a c k o f c o o r d i n a t i o n between the nerve and muscle c e l l s i n d e v e l o p i n g  cell are  Therefore,  c u l t u r e s c o u l d be a t t r i b u t e d to a membrane d e f e c t because membranes known t o be important i n c e l l  t o c e l l communications  such as neuro-  muscular j u n c t i o n f o r m a t i o n s d u r i n g development. tsl I f add A does a f f e c t s k e l e t a l muscles, then drugs a f f e c t i n g s k e l e t a l muscle n e u r o - t r a n s m i s s i o n s would be expected to have an e f f e c t on add A  tsl  .  The f a c t  tsl that add A does not respond to any of these  drugs o t h e r than drugs that a f f e c t  the CNS  and smooth muscles  argues  tsl a g a i n s t the somatic muscle o r i g i n o f t h i s m u t a t i o n . s e n s i t i v e to Yohimbine, to  p i l o c a r p i n e n i t r a t e , an a g o n i s t o f c h o l i n e r g i c system.  and smooth muscles  (Pichon, 1 9 7 4 ) . tsl  r e l a t i o n s h i p between add A doubtful.  i s more  an a d r e n e r g i c b l o c k e r , and i s more r e s i s t a n t  and c h o l i n e r g i c system have an e x c i t a t o r y e f f e c t  is  add A  Both a d r e n e r g i c  on the i n s e c t ' s  CNS  T h e r e f o r e , the e x i s t e n c e o f a  drug responses and i t s muscular  defect  However, these r e s u l t s p o i n t to f u t u r e p u r s u i t s i n g e n e t i c  mapping s t u d i e s o f the a l t e r e d drug r e s i s t a n c e phenotype.  Unless  mapping o f t h i s l o c u s c o i n c i d e s w i t h the drop-dead l o c u s , the a l t e r e d tsl drug r e s i s t a n c e c o u l d w e l l be due to another l o c u s a f f e c t e d by add A M o s a i c a n a l y s i s c l e a r l y i n d i c a t e d t h a t the b e h a v i o u r o f each tsl leg  o f add A  mosaic was  independent o f the o t h e r s .  these l e g s o f t e n remained so d u r i n g subsequent t r i a l s .  A l s o , once These  paralyzed,  observations  suggest t h a t the b e h a v i o u r o f each l e g must has i t s own somatic muscular system r e s p o n s i b l e f o r c o n t r o l l i n g  focus i n the  the movement of  each l e g . L e t h a l mutations a f f e c t i n g muscle d i f f e r e n t i a t i o n i n D r o s o p h i l a t i s s u e c u l t u r e s has been documented due to muscular d e f e c t i s s t i l l seemingly u n r e s o l v e d r e s u l t s  (Wright, 1 9 6 0 ) , but a d u l t  unheard o f .  lethality  In the p r e s e n t case, the  from drug s t u d i e s and g e n e t i c mosaic  analysis  tsl demand the need f o r c o n f i r m a t i o n o f the muscular d e f e c t i n add A T h i s can be done by using.enzyme of mosaicism i n t e r n a l l y .  markers  that w i l l allow  Indeed, such t e c h n i q u e s have been used to  i d e n t i f y mosaicism i n the nervous system o f D r o s o p h i l a Hall, 1976).  identification  (Kankel and  These i n t e r n a l markers are u s u a l l y n u l l enzyme markers  on the rod-X chromosome.  Somatic l o s s of an u n s t a b l e r i n g - X chromosome  from the r i n g / r o d h e t e r o z y g o t e would l e a d to XO  t i s s u e s which would not  s t a i n f o r the enzyme i f these mosaics a r e s c o r e d i n t e r n a l l y by and h i s t o c h e m i s t r y .  sectioning  I t would be p r e d i c t e d t h a t the absence o f a c e r t a i n  type o f mutant muscle c e l l s would be found among s u r v i v o r s at 29°C. U n f o r t u n a t e l y , an enzyme marker to be d i s c o v e r e d .  that w i l l  s t a i n f o r muscles has y e t  The o t h e r drop-dead mutation s t u d i e d , ses E \  i s different  tsl from add A nature.  i n t h a t i t s d e f e c t i s n e u r a l r a t h e r than mesodermal i n  F a t e mapping a n a l y s i s i n d i c a t e s that the d e f e c t o f ses  m u t a t i o n i s i n the nervous system.  The d e m o n s t r a t i o n o f the n e u r a l  b a s i s f o r drop-dead mutations i s not new.  The drd l e t h a l i t y  was  demonstrated both by h i s t o l o g i c a l and to a l e s i o n i n the b r a i n t i s s u e s dmd  l e t h a l i t y was  1977).  due  (Hotta and  to a d e f e c t i n the  Drug s t u d i e s showed t h a t the ses  carbamylcholine c h l o r i d e  (an a g o n i s t  a c t i o n i n the i n s e c t s ' CNS cell  f a t e mapping s t u d i e s  (Pichon,  1974).  Benzer, 1972)  to be while  thoracic ganglion  the  (Flanagan,  E"^ mutant i s more r e s i s t a n t to  of the c h o l i n e r g i c system) whose  i s p r i m a r i l y e x c i t a t o r y on the  This,  due  post-synaptic  together w i t h t e m p e r a t u r e - s h i f t  studies,  would suggest a c h o l i n e r g i c system p l a y i n g a r o l e i n t r i g g e r i n g c e r t a i n s i g n a l s t h a t cause the r e l e a s e o f , or s y n t h e s i s which are e s s e n t i a l both d u r i n g  and  a f t e r development.  n a t u r e of t h i s mutant d i r e c t s f u t u r e s t u d i e s s t r u c t u r e s using  of c e r t a i n p r o t e i n s  to f a t e map  The  neural  internal  the aforementioned enzyme marker t e c h n i q u e i n  order  to l o c a l i z e i t s d e f e c t more p r e c i s e l y . tsl Therefore,  the add A  gene product c o u l d be  o f the muscular system which f a i l s The  ses E"^ product seems to be  r e l e a s e of a t h e r m o l a b i l e  to f u n c t i o n a t a h i g h e r  i n v o l v e d i n the s y n t h e s i s  the  p r o t e i n important i n the f u n c t i o n i n g  and  To  fully  characterize  mutations, more experiments are needed such as s t u d i e s on a f f e c t e d organs as suggested by  using  of a t e m p e r a t u r e - s e n s i t i v e  s t u d i e s of n e u r o t r a n s m i t t e r s  ( E l d e f r a w i , 1976).  these drop-dead  electron-microscopical  f a t e mapping a n a l y s i s ,  muscle p r o t e i n  c u l t u r e s of embryonic mutant c e l l s  binding  temperature.  of o r  i n t e g r i t y of the nervous system.  tification  a component  iden-  biochemically  ( S t o r t i et a l . , 1978)  to the i s o l a t e d r e c e p t o r  and sites  LITERATURE CITED  (1975),. 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F a t e mapping o f nervous system and o t h e r i n t e r n a l t i s s u e s i n g e n e t i c mosaics o f D r o s o p h i l a melanogaster. D e v e l . B i o l . 48: 1-24. Kaplan,  W. D. and T r o u t , W. E. (1969). The behaviour o f f o u r neurol o g i c a l mutants o f D r o s o p h i l a . G e n e t i c s 61j 399-409.  K i n g , R. C. (1970). O v a r i a n development i n D r o s o p h i l a melanogaster. Academic P r e s s . New York. L e v i - M o n t a l c i n i , R. and A n g e l e t t i , P. U. P h y s i o l . Rev. .48(3): 534-569.  (1968).  Nerve Growth F a c t o r .  L e v i n e , J . D. and Tracey, D. (.1973)."' S t r u c t u r e and f u n c t i o n o f the g i a n t motorneuron o f D r o s o p h i l a melanogaster. J . Comp. P h y s i o l .  87: 213-235.  Lewis,  (1978). A gene complex c o n t r o l l i n g segmentation Nature 276(5688): 565-570.  E. B.  McEwen, R. S. (1918). The r e a c t i o n s t o l i g h t and g r a v i t y and i t s mutants. J . Exp. Z o o l . _25_: 49-105.  i n Drosophila.  i n Drosophila  Meezan, E. and Wood, W. B. (1971). Sequence o f gene product i n t e r a c t i o n i n b a c t e r i o p h a g e T4 t a i l core assembly. J . M o l . B i o l . 58(3):  685-692.  Mohler, J . D. (1977). Developmental g e n e t i c s o f the D r o s o p h i l a egg, I . I d e n t i f i c a t i o n o f 59 s e x - l i n k e d c i s t r o n s w i t h m a t e r n a l e f f e c t s on embryonic development. G e n e t i c s £>5: 259-272. Nomura, M. and Morgan, E. A. (1977). G e n e t i c s o f b a c t e r i a l Ann. Rev. o f G e n e t i c s '11: 297-347. Pak,  W. L. and Grabowski, S. R.  (1978).  ribosomes.  I n "The G e n e t i c s and B i o l o g y  of D r o s o p h i l a " Y o l . 2a, p.553-604. Pak,  W. L. and P i n t o , L. H. (1976). G e n e t i c approach to the study o f the nervous system. Ann. Rev. B i o p h y s i c s and B i o e n g i n e e r i n g  5: 39-7-448.  Parks, H. B. (1936). Cleavage p a t t e r n s i n D r o s o p h i l a and mosaic Ann. Entomol. Soc. Amer. 29: 350-392.  formation.  133  Pichon, Y.  Poulson,  (1974).  The pharmacology of the i n s e c t nervous system. In "The p h y s i o l o g y of I n s e c t a " V o l . TV. ed. M. R o c k s t e i n . Academic P r e s s , New York and London.  D. F. (1965). H i s t o g e n e s i s , organogenesis and d i f f e r e n t i a t i o n i n the embryo of D r o s o p h i l a melanogaster. In " B i o l o g y of D r o s o p h i l a " ed. M. Demerec. p.246. Hafner, New York.  Samaranayaka, M. (1974). I n s e c t i c i d e - i n d u c e d r e l e a s e of hyperglycaemic and a d i p o k i n e t i c hormones of S c h i s t o c e r c a g r e g a r i a . Gen.  Comp. Endoc. 24* 424-436. S h e l l e n b a r g e r , D. L. and Mohler, J . D. (1978). Temperature-sensitive p e r i o d s and autonomy of p l e i o t r o p i c e f f e c t s of l ( l ) N l , a c o n d i t i o n a l Notch l e t h a l i n D r o s o p h i l a . Develop. B i o l . t s  62: 432-446. S i l b e r t , D.  F.  (1975).  G e n e t i c m o d i f i c a t i o n of membrane l i p i d .  Ann.  Rev. Bioch. 44: 315-339. Silverman, M. and Simon, M. I. (1977). M i c r o b i o l . 31: 397-419. Sternburg,  J . G.  (1963).  Bacterial flagella.  A u t o i n t o x i c a t i o n and  Ann.  Rev.  some s t r e s s phenomena.  Ann. Rev. Entomol. 8: 19-38. Sternburg,  J . , Chang, S. C. and Kearns, C. W. (1959). The r e l e a s e of a n e u r o a c t i v e agent by the American cockroach a f t e r exposure to DDT or e l e c t r i c a l s t i m u l a t i o n . J . Econ. Ent. 5_2: 10701076.  S t o r t i , R. V., H o r o v i t c h , S. J . , S c o t t , M. P., R i c h , A. and Pardue, M. L. (1978). Myogenesis i n primary c e l l c u l t u r e s from D r o s o p h i l a melanogaster: P r o t e i n s y n t h e s i s and a c t i n h e t e r o g e n e i t y d u r i n g development. C e l l 13: 589-598. Suzuki, D.  T. (1970). T e m p e r a t u r e - s e n s i t i v e mutations i n D r o s o p h i l a melanogaster. S c i e n c e 170: 695-706.  S u z u k i , D.  T., G r i g l i a t t i , T. amd W i l l i a m s o n , R. (1971). Temperatures e n s i t i v e mutations i n D r o s o p h i l a melanogaster, V I I . A mutation ( p a r a ) causing r e v e r s i b l e adult p a r a l y s i s . Proc. N a t l . Acad. S c i . (U.S.A.) 68: 890-893. t s  Suzuki, D.  T., P i t e r n i c k , L. K., Hayashi, S., T a r a s o f f , M., B a i l l i e , D. and. Erasmus, U. (19.67). T e m p e r a t u r e - s e n s i t i v e mutations i n D r o s o p h i l a melanogaster, I. R e l a t i v e f r e q u e n c i e s among gamma r a y and c h e m i c a l l y induced s e x - l i n k e d r e c e s s i v e l e t h a l s and semi-lethals. P r o c . N a t l . Acad. S c i . (U.S.A.) 57: 907=912.  13*  Suzuki, D. T. and P r o c u n i e r , J . D. (1969). T e m p e r a t u r e - s e n s i t i v e mutations i n D r o s o p h i l a melanogaster, I I I . Dominant l e t h a l s and semil e t h a l s on chromosome 2. P r o c . N a t l . Acad. S c i . (U.S.A.) 62: 369-376. T a r a s o f f , M. and Suzuki, D. T. (1970).. T e m p e r a t u r e - s e n s i t i v e mutations i n D r o s o p h i l a melanogaster, V I . Temperature e f f e c t s on development of s e x - l i n k e d r e c e s s i v e l e t h a l s . Develop. B i o l . 23(3): 492-509. Wright, T. R. P. (I960).. The phenogenetics o f the embryonic mutant l e t h a l myospherbid i n D r o s o p h i l a melanogaster. L. Exp. Z o o l . 143: 77-99. Z a l o k a r , M., A u d i t , C. and Erk, I . (1975). Developmental d e f e c t s of f e m a l e - s t e r i l e mutants of D r o s o p h i l a melanogaster. Develop. B i o l . 47: 419,-432.  APPENDIX % pupae f o r m a t i o n  from hatched  eggs developed  under v a r i o u s c o n c e n t r a t i o n s  o f dr  STRAINS DRUGS  CONC.  Ore-R  ses B""  47* 50 83 87 72  75 84 85  .75 1.5 3.0 6.0 12.0  74 67 33 0 0  .2 .4 .8 1.6 3.2 .625 1.25 2.5 5.0 10.0  Control no drugs  Carbamylcholine chloride  Pilocarpine nitrate  Succinylcholine chloride  2 ses B  ses D""  add A  85 95 97 56 -  68 80 78 -  100 57 96 92 31  86 68 64 16 0  77 89 50 0  92 68 47 0  92 100 87 29 0  82 69 89 61 70  72 62 60 0 0  80 82 21 -  51 3 0 0  75 53 35 29 45  -  32 70 49 78 22  60 80 92 93 80  90 94 85 86 17  100 100 90 67  68 3 50 36  63 83 65 85 . 25  25 70 67 46 . 24  81 100 67 0 0  81 71 69 9 0  63 60 68 8 3  83 77 60 18 0  88 61 78 10 0  63 56 28 34 -  90 95 93 43 0  70 69 11 0  -  —  .0156 .03125 .0625 .125 .25  84 94 71 3 .0  88 97 74 34 6  81 64 0 0 0  97 75 58 13 . 0  Atropine sulfate  1.5 3.0 6.0 12.0 24.0  57 31 6 0 0  63 30 21 4 0  56 74 24 0 0  95 87 46  Hexamethonium bromide  .5 1.0 2.0 4.0 8.0  84 88 21* "0 0  57 40 13 7* 0  _  83 45 15 0  90 83 80 0  dTubocurare  1.0  80 34 86 50  64 79 100 33  79 75 50. 0  69 81 69 52  4.0 8.0  ses E^"  51 95 78 -  Nicotine  2.0  ses D  —  —  — 0  —  —  _  —  — — — 80 84 ?0 14  97  — — -  92 88 84 41  —  58 51 45 88  -  .  65 44 36 0 0  —  80  71 56 63  73 74 74 0 -  STRAINS DRUGS  CONC.  ses  B^  2 ses  B  ses  D^ .  0 0 0 0 0  83 75 82 3  85 55 76 63 82  33 54 17 3 0  19 3 0 0 0  20 5 0  26  91 21 67  79 92 58  0  87 92 66 79 75  100 88 95 51 87  60 40 34* 0 0  83 68 21 27 0  82 47 40 23 5  68 64 38 23 3  33* 60 73 18* 79  53* 13 51* 0 0  68 0 90 0 0  66 20 80 67 -  .125 .25  73  88  78  .5 • 1.0 2.0  50 13* 0  75 0 0  46* 0 0  70 70* 59* 16 0  62 29* 0 "0 0  83 83 35 27  71 92 0 0 46  88 98 54  36 58 50 67 41  56 75 78 69 11  95 86 82 42 24  88 95 82 76 15  Eserine  .00625  sulfate  .0125  .025 .05 .1 Reserpine  .05  .1 .2 .4 .8 Nialamide  .25  .5 1.0 2.0 4.0 I p r o n i a z i d .25  .5 1.0 2.0 4.0 Caffeine  .125  .25 .5 1.0. 2.0 Theophylline  S t r y c h n i n e .5 sulfate 1.0  2.0 4.0 8.0  Picrotoxin  Ore-R  '  .0025 .005  .01 .02 .04  -  -  -  —  76 53  -  3 0  -  -  D  72 67 90 34 0  _  62 13  --  21  2 ses  -  '  -  43  ses E"^"  82 90 45 3 0 60 100  JJ  A  add A  100 59 63 13 5*  62 0  0 78 0 00  50  80 97 77 28 62  54 61 58 71 32  85 33 29* 53 5*  80 63 39 26 11*  55* 68 46 54 18  — —  87 88 95 83 2  33 48 28 13 5  87  43  _  55* 0 0  — — _  90 100  -  -  — •— _  — — 100 75 69 57 21  _  _  0 61*  _  68 80 83 38 45  100 28 42 36 0  90 87 55 90 50  43 44 64 26 23  t  S  l  STRAINS DRUGS Ally1- glycine  CONC. 1.25 2.5  .5.0 10.0 Amino oxyacetic acid  .2 .4 .8  1.6 3.2 M e t h i o n i n e .125 s u l f o x i m i n e .25  .5 1.0 2.0 Sodium barbital  .125 .25  .5 1.0 2.0 Urethane  .25  .5 1.0 2.0 4.0 Dopamine  1.25  2.5 5.0 10.0 20.0 dl-Nor1.75 e p i n e p h r i n e 3.5  7.0 14.0 Tyramine  .75  1.5 3.0 6.0 12.0  2  2  JJ  A  Ore-R  ses B''"  58* 72* 2* 0*  71* 10* 0 0  80* 49* 16* 0  25* 0 0 0  38* 0 0 0  64* 50* 0 0  52 0 0 11* -  88 35 35 0 0  66 37 30 0 0  36 86 0 0 0  46 73 0 0 0  84 90 92 0 0  33 0 0 0 0  42 54 66 16* 28  89 58 81 58 68  56 36 73 81 55  85 30 75 28 53*  59 37 80 47 7  53 58  35 5  76 66 ' 62  11  87 78 71* 0  65* 41* 0 0  95 93 63* 81* 0  .-  77 64 61 13 .0  92 95 92 8 0  73 50 53 0  97 83 57 0 0  57 77 58 8  72 9.0 66 76 7  85 55 76 63 82  92 85 90 58 0  31  53 42 4 23-  72 65 57 21  60 63 66 46  87 85 56 30  74 100 95 51  83 86 56 42  45 34 25 8  90 82 36 0 0  90 58 45 3* 0  -  55* 40 0 0  63 54 63 3 0  -  —  100 26 0 0  ses B  o.  ses D"*"  ses D  —  46 73  <  add A  5* 0 0 0  42  5 0  59 48 36* 0 0  73 76 95 0 0  66 84 73 5 0  26 40 38 0 0  90 85 79  82 55  43 55 44 15 0  .  —  —  t  66 77 72 0  — —  22 0 0  ses E^"  •— — —  —  54 ... .0  S  l  138  STRAINS DRUGS  CONC.  dlOctopamine  ses B"^  64 61 31 0  77 44 56  1.8 3.7 7.5 15.0 30.0  54  89 84  62  62 11 0  38 0  38  6.0 12.0 25.0 50.0  _  92  91 88 56 0  2.5 5.0  10.0 20.0  Histamine  Gama aminobutyric acid  Glutamic acid  Catechol  Yohimbine  2  Ore-R  -  -  13  -  11  -  — —  61  ses B  92 78 70 9  -  -  ses D^"  97 95 18* 0  -  0 0  80  —  2 ses D  ses E^"  73 100 76 0  87 89 39 0  54 0 10 0  0 90 0 52 0  87 15 5 0  65 83 22 0 0  70 59  _ _ 42  79 10  97 85 96 8  33 63 60 27  .0  .0  -  -  A  AA  t  S  -  55 61 37 37 11  81 47 56 33 0  66 69 61 22 0  100  80 33 13  87 72 92 28 0  2.0 4.0 8.0 16.0  90 82 - 31 0 0  78 92 0 0 0  78 49 0 0 0  49 72 21 0 0  73 95 20 0 0  80 81 30* 0 0  38 18 o • 0 0  .5 1.0 2.0 4.0 8.0  89 54 51 0 0  66 72 45 0 0  39 0 16 0 0  61 42 26 0 0  93 86 62 0 0  88 53 24 0  16* 11 0 0  6.25 12.5 25.0 50.0 100.0 1,0  —  0.  l  add A  . . . .0  A l l experiments have over 80% adult/pupae with, the e x c e p t i o n o f *, which have l e s s than 50% adult/pupae. —, data not a v a i l a b l e due to e i t h e r e x t e n s i v e mould growth I n c u l t u r e or experiments not performed. A l l experiments were performed a t 22°C. A l l mutant s t r a i n s f a i l to develop a t 29°C.  

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