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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 David T.L. Wong B.Sc., Simon Fraser University,"1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE i n Genetics i n the Department of Zoology (FACULTY OF GRADUATE STUDIES) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1980 © D a v i d T.L. Wong, 1980 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f &BrN^rri C£  T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6 T 1W5 DE-6 (2/79) ABSTRACT Two types of sex-linked recessive mutations i n Drosophila melanogaster have been investigated i n the present study. The f i r s t type includes 5 d i f f e r e n t mutations which exhibit a s t r e s s - s e n s i t i v e (ses) phenotype. F l i e s of a l l f i v e mutant stocks become paralyzed when t h e i r containers are l i g h t l y tapped; w i l d type f l i e s are unaffected by the same treatment. The f i v e mutations form three complementation groups ( c i s t r o n s ) . F l i e s mosaic f o r mutant and non-mutant tissue were studied to determine the f o c i of t h e i r action i n embryos by fate mapping. 2 These studies suggest that the mutation ses D has 6 f o c i i n the pre-sumptive nervous system of the blastoderm. Each focus corresponds to a s i t e i n the thoracic ganglion which controls the movement of one leg. The focus f o r ses E"*- mutation i s rather d i f f u s e and occupies a larger area i n the thoracic nervous system of the blastoderm fate map. Focus mapping studies with the ses B"*- mutation were inconclusive because of the highly v a r i a b l e expressions of the adult behavioural phenotype i n mosaic i n d i v i d u a l s . Developmental studies, involving temperature s h i f t s from 22°C to 29°C (permissive to r e s t r i c t i v e temperatures) revealed that the ses E^ ~ mutation has 2 temperature-sensitive periods (TSPs) f o r l e t h a l i t y during 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 stage and the other i n the l a t e pupal stage. In addition 2 to developmental TSPs of ses B at the embryonic, 1st l a r v a l i n s t a r and pre-pupal stage, temperature-shift studies also revealed a ts maternal-2 e f f e c t l e t h a l f o r ses B . i i i The second type of mutation studied has a temperature-sensitive (ts) phenotype of adult 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 t s l temperature. The add A f l i e s have normal behaviour and longevity at 22°C but die within 24 hours a f t e r shift-up to 29°C. In contrast, the ses E"*- f l i e s are less active and require 168 hours at 29°C to induce death. Both mutations studied have a 3rd l a r v a l instar-pupal TSP with t s l 1 add A and ses E also having an a d d i t i o n a l TSP at the embryonic and 1st l a r v a l instar-2nd 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 . Fate mapping t s l studies suggest that the adult l e t h a l phenotype of add A i s caused by a lesoon i n tissues derived from the mesodermal c e l l s of the blastoderm, and for ses E\ the l e s i o n i s i n the neural c e l l s . i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENT * i x CHAPTER 1 GENERAL INTRODUCTION 1 CHAPTER 2 STRESS-SENSITIVE MUTATIONS I. Introduction 17 I I . Materials and Methods Developmental Studies 19 Genetic Mosaic Studies 21 Neurophysiological Studies; 22 I I I . Results ses B: Characterization of TSPs 25 Genetic Mosaic Studies 32 Drug Stuides 32 ses D: Genetic Mosaic Studies 35 Drug Studies 36 ses E: Characterization of TSPs 36 Genetic Mosaic Studies 41 Drug Studies 50 IV. Discussion ' 53 CHAPTER 3 DROP-DEAD MUTATIONS I. Introduction 62 I I . Materials and Methods Examination of adult drop-dead behaviour 65 Examination of Embryonic Development 65 I I I . Results t s l add A ; Characterization of adult drop-dead 6.7 ..behaviour V CHAPTER 3, continued. add A; Characterization of TSPs 67 Genetic Mosaic Studies 86 Drug Studies 90 Photomicrographic Studies 90 ses E: Characterization of adult drop-dead Behaviour 115 Characterization of TSPs • 115 Genetic Mosaic Studies 118 Drug Studies 118 IV. Discussion 121 LITERATURE CITED 128 APPENDIX 135 LIST OF TABLES TABLE 1. Behavioural characteri-tics of wild-type Ore-R and mutant f l i e s . 2. L i s t of drugs, their putative action and the Drosophila l a r v a l LD^Q-2 3. Maternal-effects of ses B . 2 4. Temperature-sensitive maternal-effects of ses B . 2 5. Temperature-shift studies of ses B . 6. Correlation of the three body segments of ses B"^  mosaics with their behaviour. 7. L i s t of drugs which have a more pronounced effect on the v i a b i l i t y of different stress-sensitive 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 effect on the v i a b i l i t y of two drop-dead mutations. LIST OF FIGURES FIGURE 1. Formation of a mosaic f l y through the loss of a ring-X chromosome. 2. Right ha l f of the blastoderm fate map for adult external body parts. 3. P r i n c i p l e of mosaic fate mapping. 4. Right h a l f of the blastoderm fate map for adult external body landmarks, v e n t r a l nervous system and mesoderm. 2 5. Percentages of ses B adults eclosed from hatched eggs a f t e r shift-ups and shift-downs administered at d i f f e r e n t times during development. 2 6. Fate map of ses D . 7. Percentages of ses E^ " pupae formed a f t e r shift-ups and shift-downs administered at d i f f e r e n t times during development. 8. Percentages of ses E"*' adults eclosed a f t e r shift-ups and shift-downs administered at d i f f e r e n t times during development. 9. Percentages of ses E^ adults and Ore-R adults eclosed a f t e r 12-hour heat pulses administered at d i f f e r e n t times during development. 10. Fate map of ses E^. t s l 11. S u r v i v a l of 1-day o l d , 5-day old and 10-day old add A adults at 29°C. 12. Sur v i v a l of 1-5 day old Ore-R adults at 29°C. t s l 13. Percentages of add A and Ore-R eggs that hatch a f t e r 6-hour heat pulses administered at d i f f e r e n t times during development. t s l 14. Percentages of add A eggs which y i e l d adults a f t e r 6-hour heat pulses administered at d i f f e r e n t times during development. Percentages of add A eggs which hatch a f t e r successive shift-ups administered at d i f f e r e n t times during development. t s l Percentages of add A eggs which y i e l d adults a f t e r successive shift-ups administered at d i f f e r e n t times during development. t s l Percentages of add A white pre-pupae which y i e l d adults a f t e r successive shift-ups and shift-downs administered p r i o r to and a f t e r white pre-pupae formation. t s l Percentages of add A white pre-pupae which y i e l d adults a f t e r 12-hour heat pulses and 12-hour cold pulses administered p r i o r to and a f t e r white pre-pupae formation. Percentages of add A t S ^ mosaic survivors at 29°C. t s l Fate map of add A t s l Photomicrographs of add A and Ore-R embryonic development at 29 C taken at 2-hour i n t e r v a l s . old and 10-day old survivors at 29°C. and Ore-R c e l l cultures Sur v i v a l of 1-day o l d , 5-day ses E adults at 29°C. Percentages of ses E^ mosaic t s l Photomicrographs of add A at 29°C. ACKNOWLEGEMENT I am very g r a t e f u l to Dr. David T. Suzuki for providing me with a most i n t e r e s t i n g research problem, and for h i s f a i t h and help. I am e s p e c i a l l y indebted to Dr. Theodore Homyk, J r . , who gave so graciously and u n s e l f i s h l y of h i s time i n the completion of t h i s research. His e n t h u s i a s t i c encouragement, patient guidance, and a b i l i t y to s i m p l i f y a problem on more than one occasion, provided the necessary impetus to continue. 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 his h e l p f u l suggestions on maternal-effect studies and his c r i t i c a l reading of t h i s t h e s i s . I would l i k e to thank Miss C h r i s t i n e Beard for her tissue culture photographs. My parents and brother, and a number of f r i e n d s , have provided tremendous support throughout my years at UBC and I would l i k e p a r t i c u l a r to thank them. Also, they have been most tolerant of my "preoccupation". I wish to thank a l l members of Dr. Suzuki's laboratory, too numerous to mention, whose help and encouragement have made t h i s work both, possible and enjoyable. CHAPTER 1 GENERAL. INTRODUCTION The control of gene expression i n b a c t e r i a and t h e i r viruses i s now understood i n d e t a i l . In addition to providing us with information on regulation of gene a c t i v i t y , these studies emphasize the v i t a l r ole of each gene product i n sustaining the l i f e of an organism. A s p e c i f i c enzyme i n a metabolic pathway can be removed by a mutation i n the s t r u c t u r a l gene. This allows the determination of the c e l l u l a r e f f e c t s of the missing enzyme and often, the metabolic pathway. Such point mutations have also proven useful i n the study of complex developmental pathways such, as bacteriophage assembly (Edgar and Wood, 1966; Meezan and Wood, 1971), and the function of other macromolecular complexes including b a c t e r i a l membranes ( S i l b e r t , 19J5), ribosomes (Nomura and Morgan, 1977), f l a g e l l a (Silverman and Simon, 1977) and chemotactic receptors (Alder, 1975). However, phenomena that only occur i n m u l t i c e l l u l a r organisms such. as. c e l l to c e l l i n t e r a c t i o n and communication, tissue d i f f e r e n t i a t i o n , dosage compensation and co-ordinated behaviour are less w e l l understood, and hence must be studied i n them. One approach to study co-ordinated behaviour i n m u l t i c e l l u l a r organisms i s to use mutations which systematically disrupt neural development and function. The r a t i o n a l e behind this approach l i e s i n the fa c t that both assembly and function of the nervous system i s under the d i r e c t i o n of genes. Proper performance of a given behaviour 2 requ i re s the complet ion of s e v e r a l steps i n a pathway. Each step i n the behav i ou ra l pathway i s a p h y s i o l o g i c a l process dependent on p o l y -pept ides coded by genes. There fo re , an a l t e r a t i o n i n the s t r u c t u r e of a po l ypep t i de would a l l ow the s tudy ing of a l t e r e d behav iour w i t h respect to the ma l f unc t i on i n g of a component i n the behav i ou r a l pathway. The use of s i n g l e gene mutat ions . thus a l lows sequence dete rminat i on of the pathway by l ook i ng at these mutat ions one at a t ime. The gene t i c approach, to study behaviour v i a the d i s r u p t i o n of neu ra l f u n c t i o n has s e v e r a l advantages over the otherwise s u r g i c a l , chemical and developmental man ipu lat ions because (1). mutat ions are i n h e r i t a b l e , (.2) t h e i r e f f e c t s are ve ry s p e c i f i c , (3) genet i c mani -p u l a t i o n s are min imal when compared to other approaches and (4) s t a t i s t i c a l study of behav iour is. p o s s i b l e through the use of a c lone of mutants. To study the behaviour of m u l t i c e l l u l a r organisms, i t i s necessary to f i n d an exper imenta l animal possess ing a complex 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 pat te rned behav iour . In sects axe I d e a l candidates f o r t h i s purpose of s tudy ing the f u n c t i o n of the nervous system. Among them, D ro soph i l a i s chosen f o r ex tens i ve behav i ou ra l study because of i t s complex nervous system (approximately 10^ neurons v s . 10^-10 ' ' ' ^ neurons i n v e r t eb r a t e s ) and i t s w e l l s t ud i ed behav i ou ra l p a t t e r n s . For example, mating behaviour i s o f t en 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 female, tapping the f ema le ' s abdomen, extending and v i b r a t i n g a wing i n a s p e c i f i c grequency to produce a s p e c i e s - s p e c i f i c cou r t sh i p song (Hotta and Benzer, 1976). The stereotyped behaviour also includes a p o s i t i v e optomotor response demonstrated by f l i e s quickly adjusting t h e i r movement to changes i n d i r e c t i o n of a turning cylinder l i n e d with alternate black and white s t r i p e s (Heisenberg and Gotz, 1975). Moreover, f l i g h t behaviour i n Drosophila i s a complicated process Involving resonating the thoracic box by a l t e r n a t e l y stretching and r e l a x i n g d i f f e r e n t muscles (Levine and Tracey, 1973). In addition, Drosophila exhibit negative geotactic and p o s i t i v e phototactic behaviour (McEwen, 1918; Benzer, 1967). , The w e l l known genetics i n Drosophila which f a c i l i t a t e the induction, i s o l a t i o n and genetic c h a r a c t e r i z a t i o n of mutations i s another reason why i t i s chosen f o r behavioural studies. Potent chemical mutagens (for example, et h y l methanesulfonate (EMS)) which induce point mutations have allowed the detection and recovery of a wide array of mutations which- ahye provided an enormous amount of Information on processes such, as biosynthetic steps (Talk and Nash-, 1974), macromolecular assembly (Homyk, unpublished) and, more recently, behaviour. Behavioural mutants unable to perform one or a l l of the courtship steps have been i s o l a t e d and characterized s u c c e s s f u l l y (Hotta and Benzer, 1976). Mutants that have abnormal geotactic and phototactic responses have also been studied (McEwen, 1918; Benzer, 1967). In the l a t t e r studies, many non-phototactic mutants recovered were characterizable by abnormal v a r i a t i o n s i n t h e i r electroretinograms (ERGs). An electroretinogram i s an e x t r a c e l l u l a r recording of a l i g h t induced e l e c t r i c a l response from the eye. In f a c t , mutations have been i d e n t i f i e d that s p e c i f i c a l l y cause abnormalities i n laminal c e l l s and r e t i n u l a c e l l s that are indicated by an abnormal optomotor response and ERG respectively (Pak and Grabozski, 1978). Recent work has also focused on the f l i g h t behaviour of Drosophila v i a the use of several f l i g h t l e s s mutants (Homyk, unpublished). Thus, the use of these behavioural mutations, i t s genetic amendability, together with the new use of neurophysiology i n Drosophila, such as i n t r a c e l l u l a r recording techniques (Ikeda and Kaplan, 1970a), outweigh the disadvantages of i t s small s i z e . In f a c t , such techniques have helped to reveal the complex neural-motor c i r c u i t (Kaplan and Trout, 1969; Ikeda and Kaplan, 1970a; Ikeda and Kaplan, 1974; Jan and Jan, 1978) i n Drosophila. Behavioural abnormalities could be caused by mutations a f f e c t i n g the assembly of the nervous system. Hence, the development of the nervous system must be studied. Drosophila has been a favourite organism for • studying the genetic control of developmental processes i n a m u l t i c e l l u l a r eukaryote. Its developmental process (Zalokar, Audit 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 determination (Lewis, 1978) have been studied i n d e t a i l . I t i s chosen for developmental studies because of i t s complex l i f e cycle involving metamorphosis from egg to l a r v a to pupa to adult i n a r e l a t i v e l y short time (12 days at 22°C). Its development 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 and g e n e t i c i s t s owing to the vast differences i n the anatomical structures between the adult and the l a r v a as w e l l as the transformation process from the l a r v a to an adult w i t h wings, legs and compound eyes which i n i t i a l l y were never presented. Among the many tools used i n the studying of Drosophila development, temperature-sensitive (ts) mutations have proven to be a valuable asset. They allow expression of a mutant phenotype only at the r e s t r i c t i v e temperature while being normal at the permissive temperature (Suzuki, 1970). Temperature-sensitivity i n micro-organisms i s a consequence of a si n g l e amino acid s u b s t i t u t i o n i n a polypeptide 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 of a protein at d i f f e r e n t temperatures (Jockush, 1966) rather than an e f f e c t on the actual process of trans-c r i p t i o n or t r a n s l a t i o n . In Drosophila melanogaster, ts l e t h a l mutants s i m i l a r to those of micro-organisms- have been recovered (Suzuki et^ a].., 1967; B a i l l i e et ad., 1968). These mutants have temperature-sensitive periods (TSPs) which- are often r e a d i l y defined during development by appropriate s h i f t s of cultures from permissive to r e s t r i c t i v e temperatures. The u l t i l i t y of ts mutations Is manifold: they obviate the need to engineer s p e c i a l balanced stocks of l e t h a l s , they allow recovery of mutants that could not be detected otherwise (such as dominant l e t h a l s , Suzuki and Procunier, 1969) and most important of a l l , they permit the delineat i o n of TSPs to the time of gene action during development. One question often asked concerning behavioural mutations i s where i n the animal the mutation i s exerting i t s e f f e c t s . Answers to t h i s question are often complicated by problems evident i n the following example. The defective ti s s u e i n a non-phototactic mutant may be i n parts of the nervous system that are not d i r e c t l y relevant to the v i s u a l 6 physiology, but to a mere motor defect. Hence, i t i s necessary to investigate 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 individuals composed of genotypically d i s t i n c t types of tissue. The generation of mosaics often required manipulations of the f l y ' s genotype through the use of chromosome mechanics. The more sophisticated genetic tools available to Drosophila has allowed i t to become one of the most extensively developed mosaic systems of a l l organisms (for mosaic systems i n other organisms, see review by Pak and Pinto, 1976). Other uses of mosaics i n behavioural analysis have involved questions on s p e c i f i c interaction between normal and mutant tissues. Thus, the problem of whether the phenotype of a genotypically normal tissue 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 tissue 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 tissue within which behavioural mutants act has been described i n d e t a i l by Hotta and Benzer (1972). It i s based on the assumption that In a f e r t i l i z e d Drosophila egg, the polar orientation of the spindle i n the f i r s t mitotic d i v i s i o n i s randomly positioned within a aphere (Fig. IA). (Parks, 1936). Following t h i s , nine synchronous nuclear divisions occur without c e l l d i v i s i o n . The nuclear derivatives of the two n u c l e i formed a f t e r the f i r s t d i v i s i o n remain clustered with l i t t l e mixing (Fig. IB). Following these nine d i v i s i o n s , the n u c l e i migrate to the surface"of the b l a s t u l a where they divide three more times a f t e r which c e l l membranes form around them (Fig. IC). Once the blastoderm i s formed, the c o r t i c a l p o s i t i o n occupied by a c e l l determines i t s developmental f a t e ; the anterior region of the blastoderm gives r i s e to anterior structures of the adult f l y (Hathaway and Selman, 1961; Chan and Gehring, 1971). It i s obvious that for any two s i t e s on the blastoderm, the further apart they are, the more l i k e l y a mosaic boundary would l i e between them (Fig. ID). Hence, i n a population of mosaics, the frequency with which, any two adult landmarks are of d i f f e r e n t phenotypes i s r e l a t e d to t h e i r proximity In the blasoderm. A map of the blastoderm p o s i t i o n of structures on the external surface of adults can be constructed (Fig. 2) from the d i s t r i b u t i o n of mutant and wild type tissues i n a population of mosaics. The frequency with which any two surface landmarks are phenotypically d i f f e r e n t , i s said to be the "map distance" on an embryonic fate map. This i s analogous to mapping two linked genes based on the frequencies of crossing over between them. A fate map d i f f e r s i n that i t allows a two dimensional construction based on the t r i a n g u l a t i o n of three d i f f e r e n t structures (Fig. 31. The embryonic f o c i of behavioural mutants can also be determined by t r i a n g u l a t i n g a behavioural phenotype r e l a t i v e to several d i f f e r e n t external c u t i c u l a r markers. This i s done by measuring the frequency 8 Figure 1. Formation of a mosaic f l y by loss of one X chromosome. I n i t a l egg contains a ring-X chromosome plus a rod-X chromosome carrying recessive genes for marking the body surface as w e l l as the behaviour to be studied. (A) Loss of ring-X at 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 cortex (C) where c e l l membranes form to produce a one c e l l thick blastoder. (D) Surface view of the blastoderm with the mosaic boundary seperating the two c e l l types. (from Hotta and Benzer, 1972) 9 A B C D 10 Figure 2. Right h a l f of the fate map of Drosophila melanogaster for adult external body parts, constructed by mosaic mapping. A mirror image of the map corresponds to the l e f t h a l f . Dotted l i n e s i n d i c a t e distances to the nearest midline, as obtained by halving the distance measured between two homologous s i t e s on each side. Distances are indicated i n s t u r t s . One s t u r t i s defined as a distance such that the two external body parts are of d i f f e r e n t genotypes i n 1% of the mosaics. The s i z e of the c i r c l e i s proportional to the frequency with which the structure i s i t s e l f s p l i t by the mosaic boundary, (from Hotta and Benzer, 1972) DORSAL MIDLINE 12 Figure 3. P r i n c i p l e of mosaic fate 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 blastoderm. The farther apart 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 structures derived from these s i t e s w i l l be of d i f f e r e n t genotype. The distance between two s i t e s on the fate map i s indicated by the frequency with which two c u t i c l e landmarks are of d i f f e r e n t phenotypes. Right: Location 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 . Sites A and B are 10 sturts apart. If C i s 4 sturts from A and 8 s t u r t s from B, then C can be .. located. A choice between the two p o s s i b i l i t i e s requires mapping with, respect 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 external marker i s w i l d type or vice 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 for the mutant phenotype i s therefore possible. Emhryological studies have delineated the neural and muscular tissues i n the embryo (Poulson, 1965) and fate mapping of mutations with known neurological defects finds their mapped embryonic f o c i congruent with the cytological picture (Pig. 4). Thus, the genetic and developmental tools developed i n Drosophila provide a means of dissecting the components of behaviour. This report involves the use of new mutations and focus mapping i n an attempt to find out where, when and how s p e c i f i c genes control neurobiological phenomena. This includes how genes affect the assembly and function 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 action and determine whether i t i s affecting the muscles or the nervous system. Moreover, the time and duration of i t s action w i l l be investigated. 15 Figure 4. Right h a l f of the fate map of Drosophila melanogaster for adult external body landmarks. Dotted l i n e s , according to embryological studies (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 of mutations conferring s e n s i t i v i t y to mechanical shock have been reported (Judd, Shen and Kaufman, 1972; G r i g l i a t t i et a l . , 1973; F e i t e l s o n and H a l l , 1975). Such f l i e s are extremely s e n s i t i v e to s t r e s s . A l i g h t tap of the v i a l containing these f l i e s i s usually s u f f i c i e n t to induce immediate p a r a l y s i s . Early studies on stress-induced p a r a l y s i s i n the American cockroach, Periplaneta americana (Cook, 1967) revealed that shock induces production or release of a neuroactive substance, Factor 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 followed by a blockade i n the nervous system. The behavioural response of.the.insect to stress p a r a l l e l s the above p h y s i o l o g i c a l observations; an i n i t i a l h y p e r a c t i v i t y followed by p a r a l y s i s (Cook and Holt, 1974). The i n i t i a l r i s e i n nervous a c t i v i t y 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 of 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 post-synaptic response caused by repeated pre-synaptic a c t i v i t y . Since f a c i l i t a t i o n i s an insect's normal response to s t r e s s , an abnormal f a c i l i t a t i o n pattern might be expected among some s t r e s s - s e n s i t i v e mutants. Indeed, a bang-sensitive mutant, bas, exhibits an abnormal f a c i l i t a t i o n pattern at the neuromuscular j u n c t i o n (Jan and Jan, 1978). This f a c i l i t a t i o n pattern develops much f a s t e r and decays much more slowly than normal. Fate mapping analysis of bas indicated that i t s primary focus i s l o c a l i z e d to the prothoracic ganglion (Feitelson and H a l l , 1975). Thus, taken together, data for bas i n d i c a t e that the mutant defect i s most l i k e l y pre-synaptic i n o r i g i n . The s t r e s s - s e n s i t i v i t y phenotype may characterized a class of defects that w i l l be useful for studying neuromuscular junctions. In the present study, the properties of recessive s t r e s s -s e n s i t i v e mutations i n three sex-linked genes (ses B, ses D, ses E) are investigated. The mutants studied were a l l recovered by Homyk and Sheppard (1977) and Homyk, Szidonya and Suzuki (1980). They were analyzed f o r t h e i r embryonic f o c i by fate mapping, t h e i r response to d i f f e r e n t neurotropic drugs and, i n the case of temperature-sensitive a l l e l e s , t h e i r temperature-sensitive periods were delineated. I I . Materials and Methods Except where s p e c i f i e d , a l l cultures were grown on standard cornmeal-yeast-dextrose Drosophila medium at 22°C ± 1°C. The i s o l a t i o n 1 2 1 procedures, mapping and behavioural properties of ses B , ses B-, ses D , 2 1 ses D and ses E have been reported (Homyk and Sheppard, 1977; Homyk, Szidonya and Suzuki, 1980). The map positions of the genes are: ses B, 1-33.0; ses D, 1-20.0; ses E, 1-27.7. The behavioural 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 this study are summarized i n Table 1. Developmental Studies The mutations ses B^ " and ses E^ " were found to be heat-sensitive l e t h a l s , that i s , while surviving at 22°C, these f l i e s die at 29°C. Temperature-sensitive periods (TSPs) f o r heat-sensitive l e t h a l i t y are delineated by the e a r l i e s t s h i f t down which reduces v i a b i l i t y and the e a r l i e s t s h i f t up y i e l d i n g s i g n i f i c a n t l e v e l s of s u r v i v a l (Suzuki, 1970). A l l s h i f t experiments were performed between 22°C (permissive) and 29°C ( r e s t r i c t i v e ) . The developmental stage present i n each culture at the time of s h i f t was determined by inspecting developing f l i e s at that time. La r v a l i n s t a r s were determined by the morphology of t h e i r mout parts and anterior s p i r a c l e s (Bodenstrin, 1950). F l i e s from which eggs were to be c o l l e c t e d were placed i n empty b o t t l e s whose mouths were covered with a watch glass. The watch glass c a r r i e d a s t r i p of agar (1% agar i n b o i l i n g water, 2% ethanol Table 1 Behavioural characteristics of wild type Oregon-R and mutant flies used in this study. Mutants Total number of flies observed Behaviour before • tapping of vial Behaviour after 1 tap to make flies f a l l to the bottom of the vial Behaviour after 5 - 10 sec. of tapping ; ses 20 active Climb up the walls of the vial quickly Flies climb up slowly and occasionally f a l l back. Require 90 sees, for ful l recovery. 4 flies paralyzed, require 1-2 min. to recover. 2 ses D n I I I I I I ses E I I tt Climb, up the walls of the vial quickly All flies begin to climb up the walls of the vials after they were paralyzed for 5 to 10 sees.. ses B^  it inactive 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. 2 ses B n moderately active Climb up the walls of the vial 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. Ore-R I I active Climb up the walls of the vial immediately and very quickly None paralyzed. Flies climb up the walls of the vial immediately and very quickly. ro o and 1% a c e t i c acid) covered with yeast paste (yeast and tap water mixed). Since females can r e t a i n f e r t i l i z e d eggs before deposition, the f i r s t two-hour egg c o l l e c t i o n was discarded. The second two-hour c o l l e c t i o n was saved and the eggs removed from the agar s t r i p by washing. These developmentlaly synchronized eggs were c o l l e c t e d on a f i n e wire*mesh and subsequently transferred to a 20% sucrose s o l u t i o n which allows the debris (yeast paste and agar) to sink to the bottom of the beaker while the eggs f l o a t on top of the s o l u t i o n . The eggs were then re-c o l l e c t e d on the wire mesh and washed thoroughly with d i s t i l l e d water. The f i n a l washing of the eggs i s e s s e n t i a l to ensure that a l l traces of sucrose are completely removed to prevent mould growth. A f t e r the washing, the eggs were counted and l a i d on s t r i p s of 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 placed i n fresh v i a l s containing Drosophila medium. Forty-eight hours l a t e r , the paper s t r i p s were remoyed and the number of unhatched eggs noted. Three sets of cultures were established f or s h i f t experiments. Following egg c o l l e c t i o n , one set was l e f t at 22°C and another was immediately transferred to 29°C. At successive i n t e r v a l s of 12 hours, cultures were s h i f t e d up or down accordingly. 24-hour heat pulses were also administered to the t h i r d set of cultures at d i f f e r e n t times of development. A l l cultures were scored for the number of pupae formed and the number of adults eclosed. Genetic Mosaic Studies Since a l l of the mutations were located away from the t i p 22 of the X chromosome, the d i s t a l l y located external c u t i c u l a r markers, yellow and white were r e a d i l y combined with them by crossing over. Genetic mosaics were generated through the use of the somatically unstable ring-X chromosome, In(l)w V (" (Hinton, 1955). Females carrying the I n ( l ) w V ^ chromosome were mated to males carrying the s p e c i f i c behavioural mutations (ses) linked to the c u t i c u l a r markers yellow and white. These males are ;y_ w ses/Y. By crossing I n ( l ) w v C / y w s p l females to y_ w ses/Y males, 'In(l)w V^/y w ses female zygotes can be recovered. Loss of the I n ( l ) w V ^ ring-X chromosome i n the early cleavage stage (Garcia-Bellido and Merriam, 1969) of In(l)w V^/y w ses female zygote r e s u l t s i n patches of y w ses/O male tiss u e i n the adult f l y . Since t h i s tissue i s hemizygous, the mutant phenotype can be expressed provided the gene i s active i n those c e l l s . The l o c a t i o n and extent of mutant tissue are determined externally by expression of the yellow and white phenotype on a background of w i l d type t i s s u e . The areas of mutant tissue were recorded for each mosaic and then i t s behaviour at 29°C was noted before and a f t e r each shock treatment. If the mosaic f l y became completely paralyzed a f t e r a shock, the time for i t s recovery was recorded. P a r t i a l p a r a l y s i s was also recorded i n those cases where the f l y dragged i t s e l f along using one or more pai r s of legs. In t h i s manner, the mosaics were examined on a d a i l y basis for at least two weeks. Neurophysiological Studies In order to test whether synaptic functions are involved i n the ses mutant defects, the mutants were exposed to varying con— centrations of a v a r i e t y of drugs known to be psychotropic and neuro-tr o p i c i n mammals. The number of drugs used, t h e i r l a r v a l l e t h a l dose 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 are l i s t e d i n Table 2. Each mutant s t r a i n was subjected to at l e a s t 5 d i f f e r e n t concentrations of each drug. The concentrations were —, 1, 2 and 4 times the l a r v a l l e t h a l dose as reported by Howard, Merriam and Meshul (1975) or determined i n the present study. Where extra s e n s i t i v i t y or extra resistance was noted, the experiments were repeated with lower (— times the l a r v a l l e t h a l dose) or higher (8 times the l a r v a l o l e t h a l dose) concentrations, r e s p e c t i v e l y . 5 ml. of each drug of appropriate concentrations (see above) were added to 1.4 gram of instant Drosophila medium (Boreal) to make 6 ml. of medium. Where possible, d i s t i l l e d water at room temperature was used to dissolve the drug. In order to prevent any heat i n a c t i v a t i o n when the s o l u b i l i t y of the drug at room temperature presented a problem, the water temperature was r a i s e d j u s t enough to dissolve the drug. To prevent b a c t e r i a l growth, two a n t i b i o t i c s , streptomycin and t e t r a c y c l i n e (10 mg./litre) were added to the d i s t i l l e d water while mould growth was i n h i b i t e d with tegosept (100 gm./litre ethanol). Newly deposited eggs were c o l l e c t e d and counted on s t r i p s of moistened b l o t t e r paper as outlined above and then placed into v i a l s containing drugs of d i f f e r e n t concentrations. These were subsequently scored f o r the number of pupae formed and the number of adults eclosed both, at 22°C and 29°C. Table 2 L i s t of drugs, t h e i r putative action and the Drosophila l a r v a l LD Taken i n part from Howard, Merriam and Meshul, 1975. Larva l l e t h a l nDiT/- T> _ ~- dose, mg/ml of DRUG Putative a c t i o n . ' 6 ' instant Dros. medium (Boreal) Carbamylcholine chloride Cholinonimetic 3 P i l o c a r p i n e n i t r a t e " 0.8 Succinylcholine chloride " 2.5 Nicotine " o06 Atropine s u l f a t e Cholinergic blocker 6 Hexamethonium bromide " " 2 d-tubocurare M M 4 Eserine s u l f a t e Inhibits acetylcholinesterase .025 Reserpine Reduces transmitter storage .2 Nialamide Inhibits monoamine oxidase 1 T • • J II II II 1 Iproniazid J-Caffeine Inhibits c y c l i c nucleotide phosphodiesterase 0.5 Theophylline " " " " 0.5 Strychnine s u l f a t e Glycine antagonist 2 P i c r o t o x i n GABA antagonist .01 A l l y l g l y c i n e Convulsant 5 Amino oxyacetic acid Inhibits GABA degradation .8 Methionine sulfoximine Convulsant .5 Sodium b a r b i t a l Nerve, muscle depressant .5 Urethane Muscle depressant 1 Dopamine Neurotransmitter 5 dl-Norepinephrine " 7 Tyramine False transmitter 3 dl-Octopamine " " ' 10 Histamine Neurotransmitter 7.5 GABA " 25 Glutamic acid " 25 Catechol Adrenergic blocker 4 Yohimbine " " 2 . I I I . Results ses B Characterization of TSPs: The temperature-sensitive phenotype of the ses B mutation enabled analysis of gene a c t i v i t y by temperature-2 s h i f t experiments. The ses B mutant exhibit a ts maternal-effect. 2 2 A ses B stock maintained 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 of males to females i n 2 each, generation whereas the stock i n which, homozygous ses B females have been kept at 29°C y i e l d s no adult progeny at a l l . This ts maternal-2 e f f e c t was further investigated by mating ses B homozygous females to Oregon-R males at 29°C Only 4 males were recovered with 291 females 2 survived (Table 3). In addition, males from the cross ses B /Y X f_: = /Y 2 2 2 attached-X females and ses B /Y X ses B /ses B females survive well at 22°C (90% and 73% v i a b l e progeny r e s p e c t i v e l y ) . This ts maternal-e f f e c t was also investigated ny s h i f t studies. Adult females from the homozygous stock 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 and counted while t h e i r subsequent development was scored. Females, kept at 29°C for more than 3 days, l a i d eggs unable to give r i s e to adults. Before the 3rd day, about 10% of the eggs produced adults. However, eggs c o l l e c t e d from females which had been held at 29°C for more than 3 days and s h i f t e d down immediately to 22°C yielded 50% adult eclosion. These females, which had been kept at 29°C for some time, were then transferred back to 22°C and i n subsequent c o l l e c t i o n s , eggs were monitored Table 3 Maternal-effects of ses B . The progeny were reared at the same temperature the cross was made. Cross 2 ses B females X Ore-R males at 29°C 2 ses B females X Ore-R males at 22°C eggs col l e c t e d 930 750 pupae formed 327 726 Progeny recovered males females males females 4 291 296 403 Cross 2 ses B /Y males X y f:=/Y females at 29°C 2 ses B /Y males X y f:=/Y females at 22°C males females males females Progeny recovered 135 236 186 205 Cross 2 2 2 ses B /Y males X ses B /ses B females at 29°C 2 2 2 ses B /Y males X ses B /ses B females at 22°C males females males females Progeny recovered 0 0 152 175 for further development. More than 4 days at 22° C were required before s u r v i v a l reached the normal value (more than 80%) of adult eclosion (Table 4). Eggs of control Oregon-R females held at 29°C for any period of time always had more than 75% adult eclosion. 2 S h i f t studies also indicated that ses B has a 3rd i n s t a r -pupal TSP. A shift-up a f t e r 240 hours at 22°C gives a s i g n i f i c a n t l e v e l of survivors, thus marking the end of the TSP. However, the beginning of the TSP could not be we l l defined since the duration of the 3rd ins t a r -l a r v a l period could not be determined. When raised at 29°C, a large f r a c t i o n of the 3rd i n s t a r larvae can survive without pupating for as long as three weeks before they begin to die. Nevertheless, s h i f t -down experiments Indicate that a f t e r 19-2 hours at 29°C (the end of 2nd i n s t a r - l a r v a l period i s 144 hours at 29°C) the l e v e l of s u r v i v a l to adulthood Is reduced (Fig. 5, Table 5). This may r e f l e c t e i t h e r the beginning of the TSP or 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 possible. Therefore, the TSP i s estimated to be between the 3rd i n s t a r l a r v a and beginning of pupation. It should be noted that In successive shift-up experiments, most of the larvae were able to pupate but f a i l e d to eclose as adults. In contrast, i n the shift-down experiments, a large number of the larvae s h i f t e d during l a t e r stages of development were unable to pupate (Table 2 5). When ses B embryos or 1st i n s t a r larvae were subjected to 29°C heat shocks, t h e i r a b i l i t y to pupate was greatly reduced. Therefore, 2 i t seems that ses B could have another TSP during the embryonic and Table 4 Temperature-sensitive maternal-effect of ses B . ses B /ses B females was crossed to ses B /Y males. females l i v e d at 22°C f o r days females l i v e d at 29°C for days eggs l a i d at °C f o r days eggs developed at °C Number of eggs co l l e c t e d number of pupae pupae a/ eggs number of adults adults c/ eggs 2 22. 1 22 279 258 92 250 90 3 22 1 29 205 135 66 21 10 1 29 1 29 275 209 76 37 13 2 29 1 29 100 68 68 10 10 3 29 1 29 124 59 48 9 7 5 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 N3 00 29-Figure 5. Percentage of ses B*" adults eclosed from hatched eggs a f t e r , • , shift-ups and T, shift-downs administered at d i f f e r e n t times during development. The developmental stage 22°C at the time of s h i f t i s shown at the bottom. The duration.of 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 of t h i s mutant at 29°C, hence, the curve for the shift-down cannot be drawn. 100 "D CU CO _o o cu CO *-> ~3 T3 CO 50 j embryo | 1st 2nd hours at 29° 24 48 72 84 114 144 I I i i — r i — ±_. i i i i i i i i i i 24 48 72 96 120 144 168 192 216 240 264 288 312 hours at 22° 336 360 embryo | 1st 2nd 3rd pupa adult L O O -31 Table 5 2 Temperature-sKift studies of ses B . Eggs were c o l l e c t e d at 0 hour. For s h i f t -up experiments, eggs were c o l l e c t e d at 22°C over a 2-hour period and l e f t at 22°C u n t i l subsequent s h i f t s . For shift-down experiments, eggs' were c o l l e c t e d at 22°C over a 2-hour period, Immediately transferred to 29°C and l e f t at that temperature u n t i l subsequent shift-downs. number number number hours of hatched q { q £ pupae_ % adults % adults % eggs e g g S pupae adults l a r v a e P u p a e l a r v a e SHIFT-UP'EXPERIMENTS 0 egg c o l l e c t i o n 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 c o l l e c t i o n 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 to stress and usually requires more than 3 minutes to recover a f t e r p a r a l y s i s . 271 mosaics were c o l l e c t e d and the behaviour of each were examined i n several t r i a l s . Mutant or normal behaviour could be un-equivocally assigned to only 109 mosaics. Of the 109 mosaics, 50 were c l a s s i f i e d as mutant and 59- were c l a s s i f i e d as normal. The remaining 162 mosaics were l e f t u n c l a s s i f i e d due to the inconsistency of t h e i r behaviour i n seperate t r i a l s . Thus on one day they might be very bang-s e n s i t i v e while on another day, they would be completely normal. The i n a b i l i t y to assign a behavioural phenotype to these mosaics prevents the a p p l i c a t i o n of a conventional fate mapping an a l y s i s . However, the r e s u l t s suggest that the behavioural phenotype i s most l i k e l y c o r r e l a t e 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 extent, the phenotype of the abdomen (Table 6). Drug Studies: Only those s t r a i n s i n which drug treatment resulted i n a s i g n i f i c a n t a l t e r a t i o n of the percent pupariation or adult eclosion as compared to the wild type s t r a i n , Oregon-R, w i l l be discussed. The r e s u l t s f o r the drugs are shown i n Table 7. A l l responses to d i f f e r e n t concentration of various drugs which are not shown i n the table did not d i f f e r from the Oregon-R wild type (Appendix). The re s u l t s can be summarized as follows:-A mutant i s more s e n s i t i v e or less 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 lower v i a b i l i t y than Oregon-R at the same Table 6 Correlation of the three body segments 1 of ses B mosaics with t h e i r behaviour. CUTICLE GENOTYPE head thorax abdomen mutant normal mutant normal mutant normal BEHAVIOUR mutant 23 5 24 2 13 5 inconsistent 39 28 34 16 22 24 normal 6 33 6 20 3 16 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 various 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 are also shown. Drugs were dissolved i n water to make d i f f e r e n t concentrations before they were added Ore-R pupae „, adults larvae " pupae 2 ses B pupae „, adults „, larvae pupae ses B''" pupae „ adults „ larvae " pupae ses D^ " pupae „ adults „ larvae " pupae „1 ses E pupae ^ adults larvae pupae Reserpine .05 .1 .2 .4 .8 33.3 92.3 54.1 80.0 17.1 . 71.4 2.9 100.0 0.0 20.0 75w0 4.8 100.0 0.0 0.0 0.0 19.0 62.5 2.5 100.0 0.0 0.0 0.0 Nicotine .0156 .03125 .0625 .125 .25 83.7 97.2 94.3 97.0 71.4 88.6 2.9 100.0 0.0 81.0 73.0 64.0 80.0 0.0 0.0 0.0 88.0 97.0 97.0 94.0 74.0 100.0 34.0 100.0 6.0 100.0 P i l b - .2 c&rpine .4 n i t r a t e .8 1.6 3.2 72.2 66.7 62.5 100.0 60.0 100.0 0.0 0.0 75.0 70.0 52.5 76.2 35.0 85.7 29.3 91.7 44.7 29.4 Carbamyl- . 75 choline 1.5 chloride 3.0 6.0 12.0 73.8 96.8 67.4 100.0 33.3 100.0 0.0 0.0 82.1 91.3 69.0 100.0 89.3 100.0 60.7 70.6 70.0 100.0 H20 control no drugs 87.2 91.2 72.1 90.3 82.5 100.0 50.0 84.0 94.9 94.6 78.4 93.1 51.3 65.0 75.0 100.0 80.0 96.9 85.0 94.1 94.9 94.6 97.4 100.0 57.1 91.7 96.4 92.6 100.0 82.5 J O concentration of drug. Si m i l a r l y , a mutant i s less sensitive or more resistant to a part i c u l a r drug i f i t s larvae have a greater v i a b i l i t y than Oregon-R at the same concentration of drug. Only mortality i s examinied here. The behavioural effects of these drugs at concentrations lower than l e t h a l dose were not studied. The ses B"^" mutation i s more sensitive to reserpine (reduce catecholamine transmitter storage) and i s more resistant to nicotine (an agonist of acetylcholine). 2 The ses B mutation i s more sensitive to reserpine (same as the ses B"*" mutation) but unlike the ses B^ mutation, i t i s more sensitive to nicotine as wel l . ses D 2 Genetic Mosaic Studies: ses D i s not temperature-sensitive and i s moderately sensitive to stress compared to the other mutants 2 1 studied. Paralyzed ses D f l i e s normally recover within l-^ minutes. 2 When homozygous ses D f l i e s are under stress, a l l s i x legs are often 2 paralyzed. However, i t was noticed 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 wild type legs were never paralyzed. The other mosaics under stress, i n which some of the 6 legs are of different phenotypes, often have their walking w i l d type legs dragging their paralyzed mutant ones. Thus the behaviour of each leg i s independent of the others with behaviour and cuticular genotype having a high degree of concordance, so the behaviour of each leg of the mosaic f l y was scored independently. This allowed the focus mapping of the behaviour f o r each leg. Analysis of 1608 legs of mosaics revealed that the behavioural focus of each leg has i t s own s i t e within the presumptive nervous system of the blastoderm fate map. The 6 f o c i occupy 6 seperate, non-interacting s i t e s i n the blastoderm aligned with t h e i r respective legs (Fig. 6). Drug Studies; ses D"*" i s more r e s i s t a n t (less s e n s i t i v e ) to p i l o c a r p i n e n i t r a t e (an agonist of the c h o l i n e r g i c system). (Table 7) 2 ses D response did not d i f f e r from the Oregon-R w i l d type. ses E Characterization of TSPs: ses E"^  i s the least s t r e s s - s e n s i t i v e as paralyzed ses E"^  f l i e s u s ually recover i n a matter of 5 to 10 seconds. The temperature-sensitive l e t h a l phenotype of ses E mutation enabled analysis of gene a c t i v i t y by temperature-shift experiments. Reciprocal s i n g l e s h i f t studies at 24-hour Intervals from permissive to r e s t r i c t i v e temperatures and v i c e versa revealed that ses E"^  has a TSP during the t r a n s i t i o n of 1st to 2nd Instar larvae (Fig. 7). The l e t h a l phase (LP) f o r this TSP Is pupariation. At the r e s t r i c t i v e temperature, few pupae form; most 3rd i n s t a r larvae wander around the v i a l or b o t t l e and die on the sides without forming a pupal case. This TSP s t a r t s at approxiamtely 45 hours at 22°C a f t e r o y i p o s i t i o n and ends at 117 hours, a duration of approximately-70 hours. ses E"^  also 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, anterior notopleural b r i s t l e ; hu, humerus; oc, oc 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, leg 1 focus; II, leg 2 focus; III, leg 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 shift 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, four teen c u l t u r e s were synchronized at egg c o l l e c t i o n , mainta ined at 22°C f o r 6 days i n order to pass through the l a r v a l TSP, then s h i f t e d up to 29°C and mainta ined there . At 24-hour i n t e r v a l s , 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 at 29°C had been t r a n s f e r r e d . Th is e s t a b l i s h e d heat pu l ses of va r y i ng d u r a t i o n . " These heat pu l ses r e vea l ed that the second TSP s t a r t s at approx imately 198 hours at 22°C. Standard s h i f t - u p experiments revea led t h i s TSP ends at 288 hours at 22°C, a time corresponding to the e a r l y pupa l stage ( F i g . 8 ) . Thus, ses E"^  has a 3 rd i n s t a r l a r v a l - p u p a l TSP approx imately 90 hours l ong . To conf i rm the 3rd i n s t a r l a r v a l - p u p a l TSP and to examine the s e n s i t i v i t y of the 2 TSPs to heat - induced l e t h a l i t y , 24-hour heat pu l ses were admin i s tered at va r i ou s times dur ing development. No 24-hour heat pu l se caused death, of a l l developing f l i e s . The most s e n s i t i v e time I n t e r v a l dur ing the second TSP was at the 3rd i n s t a r -pupa l t r a n s i t i o n stage dur ing which. 24-hour heat pulses s i g n i f i c a n t l y reduced the number of adults : from 85% i n c o n t r o l s to 15% ( F i g . 9 ) . There were no phenotypic abno rma l i t i e s a s soc i a ted w i t h these 15% s u r v i v o r s . The 24-hour pu l se 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 exper iments, thereby i n d i c a t i n g that a longer p o r t i o n of t h i s TSP mustJbe spent at r e s t r i c t i v e temperatures to induce death . Genet ic Mosaic S tud ies : Apart from the s t r e s s - s e n s i t i v e phenotype, ses E i s a l s o a ts drop-dead. The 230 mosaics recovered 42 Figure 8. Percentage of ses E adults eclosed a f t e r , A , shift-ups and v, shift-downs administered at d i f f e r e n t 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 r e s p e c t i v e l y . 44 Figure 9. Percentage of, , ses E adults and , Oregon-R adults eclosed a f t e r 12 hour heat pulses (29 C) administered at d i f f e r e n t times during development. The developmental stages at the time of the pulse are shown at the bottom. The lengths of the l i n e s represent the duration of the pulses at 29°C. 100 T3 CD CO _o O CD to "D CO 50 JL 24 48 72 96 120 144 168 192 216 240 264 288 312 336 time in hours emb. 1st 2nd 3rd pupa were observed for behaviour and v i a b i l i t y at 29°C. During the course of observation, i t was noted that i n d i v i d u a l legs of mosaic f l i e s became paralyzed. Hence, the mosaic data of ses E"*" can be examined i n two ways. Assuming the d i f f e r e n t phenotypes of ses E"*" are a consequence of a same point mutation, then both adult l e t h a l i t y and s t r e s s - s e n s i t i v i t y may be due to e f f e c t s on the same tissues or on two d i f f e r e n t t i s s u e s . By focus mapping both drop-dead and s t r e s s - s e n s i t i v e phenotypes, these a l t e r n a t i v e s might be distinguished. Unfortunately, exposing the mosaics to high, temperature to test the p a r a l y t i c behaviour caused death, i n many f l i e s before p a r a l y s i s could be observed. Thus, the occurence of l e t h a l phenotype before leg p a r a l y s i s obscurred quantitative assessment of leg phenotype. As a r e s u l t of t h i s , only 27% of the legs were judged to have become paralyzed before the time of death and conventional focus mapping using the leg phenotype was: not possible. Examination of the s u r v i v a l curve (Fig. 22, Chapter 3) shows that 73% of the mosaics died during the f i r s t 13 days of incubation at 29°C. S p e c i f i c a l l y , most of these died between days 5 and 13, a f t e r which time the s u r v i v a l curve l e v e l s o f f . Conventional mosaic analysis f o r the l e t h a l phenotype was undertaken, assuming that this 73% f r a c t i o n represents mosaics with a l e t h a l focus (or f o c i ) . Inspection of the data 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 three body segments, died prematurely. On the basis of these data, the ses E^ mutant f o c i are judged to be domineering. Using the mathematical analyses derived for 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. The p o s i t i o n shown i n Figure 10 was determined to best f i t the data. This p o s i t i o n places the f o c i near the posterior regions of the presumptive nervous system. It can be seen, however, that discrepancies for several of the distances appear i n t h i s fate map. Thus, i t seems inconsistent-that the distance between the humeral and ses E f o c i (17 s t u r t s ) should be less than the distance between the humeral and anterior notopleural b r i s t l e (24 s t u r t s ) . A possible explanation f o r t h i s inconsistancy i s that the focus i s d i f f u s e , that i s , i t occupies a r e l a t i v e l y large area of the blastoderm. It i s also complex i n that there are 2 f o c i , one on each side of the blastoderm. The f o c i are b i l a t e r a l l y domineering which means that only one of these two f o c i need be mutant for the mutant behavioural phenotype to be f u l l y expressed. This i s i n contrast to b i l a t e r a l l y submissive f o c i i n which. Both, f o c i must be mutant i n order to confer a mutant behavioural phenotype. Using the same c a l c u l a t i o n which had placed the f o c i i n the v i c i n i t y of coxa 2 and coxa 3, the distance between the l e f t and r i g h t f o c i i s found to be about 50 s t u r t s . This would place them i n the middle of the blastoderm fate map. However, i f the focus i s d i f f u s e , the chance that i t w i l l have a mixed genotype (that i s , -|- mutant, normal) w i l l increase. And i f a small amount of mutant tissue i s s u f f i c i e n t to produce a mutant phenotype, the chances of the focus appearing mutant w i l l increase. Since ses E"^" f o c i are b i l a t e r a l and domineering, the portion of mosaics ex h i b i t i n g mutant behaviour should be large. As Hotta and Benzer (.1972) show, th i s *ure 10. Fate map of ses E . a, antenna; anp, anterior notopleur 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 sternite ; J | S ^ , ses E focus. 49 would ultimately increase the distance between 2 b i l a t e r a l l y domineering f o c i . As noted before, conventional focus mapping of the leg p a r a l y s i s phenotype of ses E"*- could bot be performed due to the early onset of l e t h a l phenotype. However, using the 27% of the legs that showed the p a r a l y t i c phenotype consistently before death, i t was possible to c o r r e l a t e the phenotype of the leg c u t i c l e with t h e i r p a r a l y s i s . This showed that leg 'paralysis did not always occur i n a mutant leg and implies that the f o c i f o r leg p a r a l y s i s and the thoracic c u t i c l e are separable. The c o r r e l a t i o n analysis did show, however, that a paralyzed prothoracic leg was most commonly associated with mutant tis s u e i n the anterior notopleural b r i s t l e (ANP), coxae of the f i r s t , second and t h i r d legs i n that order (Table 8). Likewise, p a r a l y s i s of legs 2 and 3 were more often associated with, mutant c u t i c l e i n the coxae of each respective leg, than with mutant c u t i c l e i n the respective t a r s i or other thoracic parts. These data suggest that the f o c i for leg p a r a l y s i s l i e somewhere below the thoracic hypoderm on the b l a s t u l a fate map, i n the general area where the adult l e t h a l f o c i were l o c a l i z e d . Therefore, using both modified and conventional mosaic analyses, i t i s possible to map out the same area i n the ventral nervous system of the f l y which i s believed to be the primary focus for the s t r e s s -s e n s i t i v e and drop-dead phenotypes. Drug Studies: Results (Table 7) indicated that ses E^ i s TABLE 8 The distance of ses E focus with respect to various surface landmarks. The distances are ranked from 1 to 10 with 1 being closest to and 10 being furthest away from the focus. Distance from leg 1 leg 2 leg 3 Tot a l combined-focus to ranking ranking ranking ranking Antenna 8 10 9 27 9 Humerus 9 9 10 28 10 ANP B r i s t l e 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 Sternite 3 10 8 8 26 8 tn I—1 more r e s i s t a n t to an agonist of c h o l i n e r g i c system, carbamylcholine chl o r i d e . IV. Discussion Single gene mutations which a f f e c t the motor a c t i v i t y of f l i e s have proven to be useful i n pinpointing the genetic re g u l a t i o n of behaviour and i t s neuromuscular o r i g i n . While physiologists*study the d i r e c t e f f e c t of stress on normal ins e c t s , the present study has made use of mutations which exaggerate s e n s i t i v i t y to stress to the point of pa r a l y s i s to allow us to pinpoint the c e l l s affected f o r further p h y s i o l o g i c a l studies. Temperature-sensitivity i s a useful property of the ses B mutation. Developmental studies in d i c a t e that the ses B mutant gene product i s required during the l a r v a l stage and possibly i n the ovary during oogenesis. This conclusion i s reached a f t e r temperature-shift 2 studies revealed that a ts maternal-effect exists f o r ses B . Eggs maintained at 29°C from female parents which have been kept at 29°C for more than 3 days do not develop into adults (less than 10%). Since approximately 3 days are required f o r complete oogenesis from stage 1 to stage 14 oocyte (see King, 1970 for d e f i n i t i o n s of the stages) i n Drosophila, the re s u l t s could suggest that a thermolabile product which i s e s s e n t i a l throughout the l a t e r stages of development i s synthesized by and Incorporated i n t o the early oocyte. However, i f these eggs were s h i f t e d down immediately a f t e r the egg i s l a i d , about 50% of them develop into adults. This indicates that e i t h e r the thermo-l a b i l e product can be reactivated by a change of temperature or the embryonic c e l l s make enough product at the permissive temperature to "rescue" the embryos. The l a t t e r p o s s i b i l i t y i s supported by s u r v i v a l of heterozygous females from homozygous mothers at 29°C. It has also been shown that when ses B embryos or 1st i n s t a r larvae are exposed to 29°C heat pulses, l a r v a l development i s prolonged and i n some instances, development i s arrested i n the 3rd i n s t a r . In contrast, exposure of 2nd or 3rd i n s t a r larvae to such temperatures r e s u l t s i n l i t t l e prolonged 3rd i n s t a r l a r v a l period. Temperature-shift studies also indicated a 3rd instar-pupal TSP and that s h i f t downs as l a t e as 2nd Instar are capable of forming adults. These r e s u l t s i n d i c a t e that 2nd i n s t a r larvae which had been ra i s e d at 29°C are rescuable by allowing them to develop throughout the 3rd Instar-pupal TSP at 22°C. These observations, together with the r e s u l t from maternal-effect studies 2 2 2 (which i n d i c a t e that +/ses B heterozygotes. derived from ses B /ses B 2 mothers are capable of surviving) suggest that ses B gene product i s synthesized and required during oogenesis as w e l l as embryonic, 1st i n s t a r and 3rd instar-pupal stage. These TSPs are r e l a t e d i n such a way that the reduced l a r y a l v i a b i l i t y due to exposure to 29°C at e a r l i e r TSPs can .be a l l e v i a t e d by further developing them at 22°C during l a t e r TSPs. Many examples of sex-linked maternal-effect mutations e x i s t i n Drosophila including sortless (Colaianne and B e l l , 1972), deep orange (Garen and Gehring, 1972)., cinnamon (_Baker, 19.73), fused and rudimentary (Pausto-^Sterling, 1971).,' murkv_ and singed (Mohler, 1977), and l ( l ) E 2 5 t S 2 (Tarasoff and Suzuki, 1970). ses B i s p e c u l i a r i n that i t s ts gene product i s not only important p r i o r to and at the embryonic stage, but during l a t e r stages as w e l l . Also, i t d i f f e r s from other non-conditional mutants i n that i t s gene product i s p a r t i a l l y rescuable by avoiding subsequent development at the r e s t r i c t i v e temperature. Although mosaic analysis had suggested that mutant tissues i n the head and the thorax w i l l most l i k e l y confer a mutant behavioural phenotype, the vast amount of tissues i n these regions make i t almost impossible to pinpoint the abnormal behaviour to a s p e c i f i c tissue. On the other hand, the presence of a large number of mosaics with inconsistent behaviour (Table 6) could suggest that the inconsistency i s a c t u a l l y an i l l u s i o n of intermediate behaviour between the two extreme phenotypes. Thus they were c l a s s i f i e d as mutant on one day and normal on another. This, together with the fact that there i s only a minority of mosaics with, completely mutant thoraxes showing mutant behaviour (for example, of the 64 mutant thoraxes (Table 6), 24 had mutant behaviour, 34 had Inconsistent behaviour and 6 had normal behaviour, suggest that ses B gene could act non-autonomously, that i s , the mutant gene does not-act d i r e c t l y on the motor regions of the thoracic ganglia, but i n d i r e c t l y through a c i r c u l a t i n g substance produced elsewhere i n the f l y . In f a c t , i n the American cockroach, Periplaneta americana, p h y s i o l o g i c a l studies have shown that such a c i r c u l a t i n g substance c a l l e d Factor S does e x i s t i n the insect's haemolymph and i s released i n l a rger amounts during stress (Beament, 1958; Helsop and Ray, 1959; Sternburg, 1963; Davey, 1964; Samaranayaka, 1974). At low concentrations, Factor S i n i t i a t e s a state of h y p e r a c t i v i t y i n the nervous system of the organism while at higher concentrations (which i s the case when the organism i s exposed to prolonged periods of stress or higher l e v e l of s t r e s s ) , Factor S causes a neural blockade (Cook, 1967; Cook, de l a Cuesta and Pomonis, 1969). Therefore, i t i s l i k e l y that a c i r c u l a t i n g substance analogous to that i n the cockroach, exists i n Drosophila, bearing i n mind that there are s t i l l p o s s i b i l i t i e s of mutant receptor c e l l s being extra s e n s i t i v e to Factor S and/or mutant c e l l s having a lower threshold for release of Factor S. These r e s u l t s point to future pursuits to test the gene autonomy of ses B. An easy way would be to perform r e c i p r o c a l ovary transplants between w i l d type host and mutant ovaries, and v i c e versa. I f the ses B gene does act non-autonomously during oogenesis, then wild type o v a r i e s . i n ses B host w i l l be predicted to be temperature-s e n s i t i v e , and ses B ovaries i n w i l d type host w i l l be predicted to produce eggs at 29°C that w i l l give r i s e to adults. The see B mutation i s also more s e n s i t i v e to reserpine. The pharmacological action of reserpine i s to release (and thus reduce) catecholamine (a biogenic amine) storage i n insects (Bertler, Carlsson and Rosengren, 1956; Burn and Rand, 1957, 1958, 1959a,b. Burn, 1960). In s p i t e of several e f f o r t s by Cook (1967) and Sternburg and his co-workers (Sternburg, Chang and Kearns, 1959), the exact biochemical determination of Factor S was unsuccessful. However, both studies concluded that Factor S i s a biogenic amine. Biogenic amines i n insects are apparently stored and released to act i n at l e a s t two ways: (1) from conventional neurons where t h e i r e f f e c t s are l o c a l i z e d to the immediate synaptic region, (2) released from neurosecretory organs where t h e i r e f f e c t s may be more generalized. The extra s e n s i t i v i t y of ses B to reserpine therefore suggests that stress p a r a l y s i s i n Drosophila could be mediated by a biogenic amine. Based on this r e s u l t and the f a c t that the paralysis-causing agent i s a c i r c u l a t i n g biogenic amine, the non-autonomous nature of the ses B gene becomes more probable. Thus, i n addition 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 , the ses B mutation also a f f e c t s oogenesis, embryogenesis and pupariation. It represses pupariation by prolonging the 3rd Instar l a r v a l period for as long as 3 weeks before death commences. The present studies suggest t n a t s ^ s B could be an excellent candidate f o r studying hormones i n insects because hormones are known to play important roles i n f e r t i l i t y , development and metabolism. Before any further speculation, more experiments are needed. For example, i f the ses B gene express i t s e l f non-autonomously, then perhaps quantitative analysis on the stress factor by e x t r a c t i o n and subsequent assay for I t s a c t i v i t y by i n j e c t i o n into normal f l i e s , or a p p l i c a t i o n to i s o l a t e d insect nerve 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 larvae to a pupation hormone, ecdysone. One of the other two s t r e s s - s e n s i t i v e mutations studied 2 2 i s ses D . Mosaic analysis on ses D suggested a neural dysfunction. 2 Mosaic for ses D exhibit independent behaviour of each leg upon s t r e s s . This d i f f e r s from ses B"1" mosaics where the e f f e c t i s a l l or none, that i s , either a l l 6 legs are paralyzed or they are not. The 2 1 f a c i for ses D map to the same area of the fate map as those for Hk (Hotta and Benzer, 1972). Hk^ mutants over-react to s t i m u l i such as sudden movements and often shake t h e i r legs vigorously under ether anaethesia. The embryonic f o c i f o r Hk^ " l i e near t h e i r corresponding legs and each are located at positions v e n t r a l to t h e i r adult c u t i c u l a r s i t e s . This area c l o s e l y corresponds to the region of the blastoderm destined to form the v e n t r a l nervous system. This i n t e r p r e t a t i o n was further supported by e l e c t r o p h y s i o l o g i c a l studies (Ikeda and Kaplan, 1974) which showed that a l l action potentials 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 bursts of a c t i v i t y i s observed. The frequency of t h i s endogenous, a c t i v i t y i s also found to be s i m i l a r to that of the shaking legs. Hence, the congruence of the ses D f o c i with those of Hk""" suggests that ses D p a r a l y s i s could be due to hyper-a c t i v i t y i n the thoracic ganglia as w e l l . 2 The behavioural phenotype of the ses D mutant resembles that of another bang-sensitive mutant, bas, whose embryonic focus i s i n the nervous system (Feitelson and H a l l , 1975). Further e l e c t r o -p h y s i o l o g i c a l studies on this mutant revealed that there i s an abnormal f a c i l i t a t i o n pattern a f t e r mild stimulation. F a c i l i t a t i o n i s an enhanced post-synaptic response caused by one or more previous synaptic trans-missions. The neuronal f a c i l i t a t i o n observed i n bas develops much f a s t e r and decays much slower and i s associated with a prolonged release of transmitter at the pre-synaptic neuron caused by unknown reasons (Jan and Jan, 1978). The insect's h y p e r a c t i v i t y as a preliminary response to stress i s known to be a phenomenon of f a c i l i t a t i o n (Helsop and Ray, 1959; Sternburg, Chang and Kearns, 1959; Cook, 1967) where sustain l e v e l s of i t can produce p a r a l y s i s (Cook, de l a Cuesta and Pomonis, 1969). 2 1 Therefore, the s i m i l a r l o c a t i o n of f o c i of ses D , Hk and 2 bas and the resemblance i n phenotypes of bas and ses D suggest that there could be an enhanced or a f a s t e r development of f a c i l i t a t i o n 2 and/or a prolonged time course of f a c i l i t a t i o n i n ses D as w e l l . The 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 (an agonist of acetylcholine) r e s i s t a n c e to the abnormal response to stress of ses D, i f any, i s unknown. However, i f the ses D defect i s due to abnormal f a c i l i t a t i o n caused by prolonged release of transmitter at the neuromuscular juncti o n , then these r e s u l t s could suggest the existence of an i n h i b i t o r y i n t e r -neuron acting on the motor neuron v i a the c h o l i n e r g i c system. This conclusion i s reached a f t e r several independent e f f o r t s f a i l to provide evidences of c h o l i n e r g i c neuromuscular junctions i n insects (Hildebrand, H a l l and Osmond, 1979; Eldefrawi, 1976; Jan and Jan, 1976). The l a s t of the s t r e s s - s e n s i t i v e mutations studied, ses E \ had p l e i o t r o p i c e f f e c t s including s t r e s s - s e n s i t i v e and drop-dead pheno-types. Although conventional fate mapping with mosaics did not f i r m l y p o s i t i o n an embryonic focus for the adult l e t h a l phenotype, i t did suggest a general region of the blastoderm which i s more neural than mesodermal i n o r i g i n . The focus f o r ses E"*" i s quite d i f f u s e and i s located i n an area of the blastoderm destined to form the thoracic ganglia. The importance of the i n t e g r i t y of the thoracic ganglia i n providing a proper response to stress has already been emphasized. Therefore, i n addition to i t s e f f e c t on stress-induced p a r a l y s i s , the thoracic ganglia might be involved i n the drop-dead phenotype as w e l l . It should be noted that the temperature-sensitive l e t h a l i t y of ses E could be explained by not only being extremely s e n s i t i v e to mechanical stress but to other forms of stress such as heat as w e l l . Drug studies indicated that ses E^ i s more r e s i s t a n t to another agonist of ace t y l c h o l i n e , carbamylcholine chloride (carbachol). There-fore, l i k e ses D, a ch o l i n e r g i c system suppressing the s t r e s s - s e n s i t i v e phenotype and, i n this case, the drop-dead phenotype as well cannot be r u l out. A better understanding on the neural defect of these mutants i s to i s o l a t e the c h o l i n e r g i c receptors (Eldefrawi, 1976) and perform a study on the binding properties of d i f f e r e n t neurotransmitters to t h e i r receptors. If such studies i n d i c a t e the binding properties of ch o l i n e r g i c receptors are d i f f e r e n t , then defects other than abnormalities at the neuromuscular jucntions must operate i n these s t r e s s - s e n s i t i v e mutations. In summary, there are at le a s t two types of s t r e s s - s e n s i t i v e mutations: one i s generalized, a f f e c t i n g a large amount of neural tissue while the other Is more l o c a l i z e d , a f f e c t i n g a sin g l e neuron c o n t r o l l i n g leg movements. Hence, one has to be very s e l e c t i v e i n choosing the 61 mutant for further studies because s t r e s s - s e n s i t i v e mutations are believed to act v i a three ways (Feitelson and H a l l , 1975; Homyk, 1977): (1) mutant c e l l s are hypersensitive to the stress factor, (2) mutant c e l l s release an excessive amount of s t r e s s f a c t o r during s t r e s s , and (3) mutant c e l l s f a i l to produce an enzyme or enough enzyme which normally i n a c t i v a t e the f a c t o r . The present study has l o c a l i z e d the defects of several s t r e s s - s e n s i t i v e mutations to t h e i r blastoderm fate map and has; delineated TSPs for mutations with ts developmental phenotypes. More techniques i n anatomy, biochemistry and histochemistry are therefore necessary to determine the s p e c i f i c role of genes i n development and provide i n t e r e s t i n g r e l a t i o n s h i p s between genes, development and behaviour. 62 CHAPTER 3 DROP-DEAD MUTATIONS I. Introduction The components of behaviour, from the sensory receptors i n the peripheral nervous system to the ce 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 of genes. Therefore, 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 behaviour. Hence, by s e l e c t i o n of mutants on the basis of t h e i r abnormal behaviour, s p e c i f i c components of the nervous system can be analyzed. One group of mutations with a s t r e s s - s e n s i t i v e (ses) phenotype has already been described. The second class studied f o r t h i s thesis i s characterized by death of adults (drop-dead) at r e s t r i c t i v e temperatures. Other temperature-sensitive adult drop-dead mutations have been reported (Tarasoff and Suzuki, 1968). The f i r s t adult drop-dead mutant recovered i n the present study was i s o l a t e d as a recessive ts l e t h a l on the X chromosome and only l a t e r , the ts adult drop-dead t s l (add A ) phenotype was found. A second ts drop-dead mutant, ses E, was found among f l i e s 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 . Non-condit i o n a l mutations with an adult l e t h a l phenotype, including drd (Hotta and Benzer, 1972), dmd (Flanagan, 1977) and 1(4)18 (Hochman, 1973) have been described. They a l l develop and eclose normally but die precociously soon a f t e r e closion. Analysis of genetic mosaics showed that the primary l e t h a l focus of drd l i e s i n the b r a i n tissue and of dmd i s i n the thoracic ganglion. Hotta and Benzer (1972) were able to confirm t h e i r conclusions based on mosaic analysis and by h i s t o l o g i c a l work which showed that a l l drd f l i e s have degenerated b r a i n t i s s u e . Thus, the drop-dead phenotype may prove to be a useful way to i d e n t i f y neural defects. t s l The temperature-sensitive l e t h a l phenotypes of add A and ses E~^  provide advantages over s i m i l a r mutations with non-conditional l e t h a l phenotypes (Suzuki, 1970). F i r s t , the ts nature of the l e t h a l phenotype allows one to perform s h i f t and pulse experiments between the permissive and non-permissive ( r e s t r i c t i v e ) temperatures to determine the temperature-sensitive periods (TSPs). A TSP i s a period during which development at the non-permissive temperature r e s u l t s i n the mutant phenotype, i n this case l e t h a l i t y . TSPs are generally regarded as the time of synthesis and/or function of a mutant gene product (Suzuki, 1970). In the present case, temperature-shift studies revealed that the add A mutation has TSPs i n the embryonic and l a t e l a r v a l to early pupal stages as well as i n the adult stage. S i m i l a r l y , the ses E mutation was found to have TSPs i n the m i d - f i r s t to mid-second l a r v a l i n s t a r s , mid-third l a r v a l i n s t a r to early pupal stage and of course, the adult stage. Furthermore, quantitative recovery and examination of adult mosaics for l e t h a l i t y and behaviour at the non-permissive temperature, allowed the l o c a l i z a t i o n of the add A adult l e t h a l phenotype to f o c i i n the mesoderm and suggested that the f o c i for the ses E phenotype are most l i k e l y i n the v e n t r a l nervous system. I I . Materials and Methods A complete de s c r i p t i o n of the i s o l a t i o n and properties of t s l 1 add A and ses E mutations used i n this study has been reported (Homyk and Sheppard, 1977; Homyk, Szidonya and Suzuki, 1980). The map positions of the genes are: add A, 1-20.9:; ses E, 1-27.7. Except t s l for the adult ts l e t h a l c h a r a c t e r i s t i c , add A mutants have develop-mental rates, behavioural a c t i v i t y and longevity i d e n t i c a l to Oregon-R at 22°C. In contrast, ses E"^  f l i e s are detectably less active and develop much more slowly than Oregon-R. As w e l l , f l i e s carrying e i t h e r t s l 1 add A or ses E have ts developmental l e t h a l phenotypes. Unless otherwise stated, a l l c u l t u r i n g conditions, heat pulses, temperature s h i f t s , drug studies and genetic mosaic analyses are i d e n t i c a l to those described i n Chapter 2. Examination of adult drop-dead behaviour t s l 1 add A , ses E and Oregon-R w i l d type f l i e s of d i f f e r e n t ages were s h i f t e d to 29°C and each day, t h e i r v i a b i l i t y at 29°C was examined by recording the number of survivors l e f t i n each v i a l . Each v i a l contained 10 f l i e s of one age group. Approximately 300 f l i e s divided equally into three age groups were used with the exception of Oregon-R where only 100 f l i e s of one age group was used. Examination of embryonic development In order to determine the nature of embryonic l e t h a l i t y of t s l add A , embryos were photographed at 2-hour i n t e r v a l s beginning from 66 o v i p o s i t i o n and continued u n t i l hatching. Eggs were c o l l e c t e d within one-hour periods and dechorionated i n 3% NaOCl (50% bleach). The embryos were then l a i d on the s l i d e s which were fixed on dry i c e and photographed using bright f i e l d i l l u m i n a t i o n . Stains were not used. However, a p o l a r i z i n g l i g h t f i l t e r was used to aid i n observation of the muscles which w i l l be biregringent under such conditions. A l l pictures were made with Kodak Panatomic-X f i l m i n a C a r l Zeiss photomicroscope with a b u i l t - i n camera set at the automatic exposure s e t t i n g . The exposed f i l m was developed and printed according to the manufacturer's s p e c i f i c a t i o n s . I I I . Results ,, A t s l add A t s l C haracterization of adult drop-dead behaviour: When add A f l i e s were s h i f t e d to 29°C, almost 95% died within 24 hours (Fig. 11) whereas only 7% mortality rate was observed for Oregon-R f l i e s a f t e r 20 days at 29 C (Fig. 12). Characterization of TSPs: TSPs of t h i s mutant were i n i t i a l l y determined by studying the consequences of 6-hour heat (29°C) pulses administered at d i f f e r e n t developmental stages. Anytime during the f i r s t 24 hours (embryo stage), such, pulses s i g n i f i c a n t l y reduce egg t s l h a t c h a b i l i t y (Fig. 13). More than 75% of add A eggs kept at 22°C hatch (data from s h i f t studies, F i g . 15) whereas as few as 9% of heat pulsed embryos survive. In contrast, h a t c h a b i l i t y of Oregon-R eggs i s unaffected by temperature; more than 75% of the eggs usually hatch. The e f f e c t of heat pulses on post-embryonic development was monitored by c a l c u l a t i n g the f r a c t i o n of hatched eggs which develop into adults. The data ( f i g , 14) show that a 6-hour heat pulse administered near the end of embryonic development or during the early 1st i n s t a r l a r v a l stage reduces the frequency of adult survivors. The same data also hint that a d d i t i o n a l TSPs ex i s t at the mid-third i n s t a r stage (120 hours at 22°C) and early pupal stage (216 hours at 22°C). S h i f t studies were performed to i d e n t i f y and describe the Figure 11. Survival of A , 1 day old; o, 5 day old; 10 day old add A t s l adults at 29°C and , 1-5 day old add A t s l adults at 22°C. ON VO 70 i Figure 12. S u r v i v a l of 1-5 day old Oregon-R adults at 29°C. 72 Figure 13. Percentage o f , — — , add A t s l 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. embryo 100 1st instar to a> o o ~o <D JZ o JC 50 12 24 36 time in hours 74 Figure 14. Percentage of add A eggs which y i e l d adults a f t e r 6 hour heat pulses (29°C) administered at d i f f e r e n t times during development. The developmental stages at 22 C are shown at the bottom. Approximately 100 eggs were used. % adult survivors 76 Figure 15. Percentage of,0—O, add A t s l eggs which hatch a f t e r successive shift-ups administered at d i f f e r e n t times during development and,»—•, control add A t s ^ eggs which hatch a f t e r developing at 22°C throughout. Approximately 100 eggs were used for each experiment. Developmental rates did not d i f f e r i n both experiments. the TSPs more p r e c i s e l y . Cultures synchronized to within 2 hours of deposition were established at 22°C and the v i a l s were s h i f t e d to 29°C at successive 6-hour i n t e r v a l s ( s h i f t - u p ) . Any shift-up at or before the 24 hour embryonic period s i g n i f i c a n t l y reduces the egg h a t c h a b i l i t y (Fig. 1.5). The r e c i p r o c a l shift-down experiments were also performed by c o l l e c t i n g eggs at 22°C, immediately t r a n s f e r r i n g the cultures 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 only culture i n which, eggs hatched (9%) was s h i f t e d down at 6 hours. In a l l other cultures, no eggs hatched. These experiments corroborate the pulse experiments i n defining an embryonic TSP for l e t h a l i t y . The shift-ups also revealed a s i g n i f i c a n t increase i n s u r v i v a l to adulthood i n cultures s h i f t e d up a f t e r 150 hours and an even more pronounced increase i n s h i f t ups a f t e r the white pre-pupae (wpp) stage at 216 hours (Fig. 16). Thus, the data Indicate the existence of TSPs during the embryonic, mid-3rd i n s t a r and early pupal stages. In order to characterize the pupal TSP more f u l l y , s h i f t s were made at 6-hour i n t e r v a l s on cultures synchronized by c o l l e c t i o n of wpp at 22°C and 29°C. Shift-up and shift-down experiments covering the 60-hour i n t e r v a l preceeding wpp formation were performed using 2 sets of cultures. One set was incubated at 22°C u n t i l wpp began to appear, then s h i f t e d up to 29-°C. White pre-pupae were c o l l e c t e d at 6 hour i n t e r v a l s and t h e i r incubation continued at 29°C for the shift-up experiment. The second set was s h i f t e d up to 29°C during the 3rd l a r v a l i n s t a r and incubated there u n t i l wpp began to appear. At this time, the cultures 79 t s l Figure 16. Percentages of add A eggs which y i e l d adults a f t e r successive shift-ups administered at d i f f e r e n t times during development. The developmental stages at 22°C at the time of shift-ups are shown at the bottom. were s h i f t e d down to 22°C and wpp c o l l e c t e d at 6-hour i n t e r v a l s f or the shift-down experiments. For shift-ups and shift-downs covering the 60-hour i n t e r v a l a f t e r wpp formation, wpp were c o l l e c t e d from cultures at 29°C and 22°C and s h i f t e d down and up.respectively at 6-hour i n t e r v a l s . This technique of synchronizing the cultures at the wpp stage y i e l d s r e s u l t s which indi c a t e a s i g n i f i c a n t increase of adult survivors i n those cultures s h i f t e d up at 6 hour or more a f t e r wpp formation (Fig.. 1.7). This signals the end of a TSP. S i m i l a r l y , decreases i n adult eclosion begin to appear when shift-downs are made 6 hours or less before wpp formation (Fig. 17) and t h i s indicates the beginning of a TSP. These experiments delineate a TSP extending from 6 hours before to 6 hours a f t e r wpp formation. Twelve-hour heat and cold pulses were made str a d d l i n g the wpp stage. The data c l e a r l y indicated that the frequencies of adult survivor increase when the cultures develop at permissive temperatures during the period 12 hours before and a f t e r wpp formation (Fig. 18). Data for cold pulses administered at 42 hours or more a f t e r wpp formation are not shown because of too few observations to be s i g n i f i c a n t , for example, a 50% s u r v i v a l i n a cold pulse 54 to 66 hours a f t e r wpp formation was based on 2 survivors out of 4 wpp. Twelve-hour heat pulses beginning as early as 54 hours before wpp formation were found to reduce the f r a c t i o n of eclosing adults, while the same heat pulses 12 hours a f t e r wpp formation had no e f f e c t on adult ec l o s i o n (Fig. 18). The most severe reduction of adults was observed when the heat pulses were 82 Figure 17. Percentage of add A t s l white pre-pupae (wpp) which eclosed as a d u l t s , A - — A , a f t e r successive shift-ups and,v—v, a f t e r successive shift-downs administered p r i o r to and a f t e r wpp formation. JZ 2 50 JZ o CL Q. 84 72 60 48 36 24 12 hours before wpp format ion •ntr--M^Nt / *Y J L 0 12 24 36 48 60 72 84 96 W D D hours a f te r K K wpp formation 84 Figure 18. Percentage of add A white pre-pupae (wpp) which eclosed as adults, O O, a f t e r 12 hour heat pulses (29°C) and, > «, a f t e r 12 hour cold pulses (22°C), administered p r i o r to and a f t e r wpp formation. 85 100 r CO T3 ro if) ro TJ 0 jS 50 _c u l c Q. Q. cN -- -o o. o o-o — -o - f c — • -o 0 © • - -o o -o o o o- -o © -o o o o-o •©• o — -o 72 60 48 36 24 hours before wpp formation 12 0 wpp J I I I I I 12 24 36 48 hours after wpp formation 60 72 performed i n the i n t e r v a l from 30 hours preceeding wpp formation to 6 hours following wpp formation. Although there are minor discrepancies between the s h i f t and pulse experiments defining the s t a r t of the TSP, these experiments in d i c a t e a TSP around the wpp stage which commences at least 6 hours p r i o r to and ends 6 hours following wpp formation. Thus, we have t s l shown that the add A mutant has 3 TSPs; one embryonic TSP which occupies the whole embryonic period of 24 hours, one 12 hour TSP which, begins at 6 hours before wpp formation and ends at 6 hours a f t e r wpp formation, and another i n the adult stage. Genetic Mosaic Studies: The 292 mosaics used i n th i s analysis were recovered from the cross y_ w add At"S"^/Y males X In(l)w V^/y w s p l females. The mosaics were developed and mosaic patches were i d e n t i f i e d at 22°C. A l l mosaics were s h i f t e d to 29°C within 2 days of eclosion and observed f o r 19 days f o r drop-dead behaviour. t s l It was found that s u r v i v a l (or l e t h a l i t y ) of the add A mosaics s h i f t e d to 29°C do not show any signs of l e v e l l i n g o f f during the 19 days of observation (Fig. 19). Therefore, i t i s not possible to assign a drop-dead phenotype to these mosaics due to the p o s s i b i l i t y that not a l l of them with a premature ts l e t h a l phenotype were scored. Fortunately, each mosaic was also observed for the onset of behavioural abnormalities. It was noted that some mosaics demonstrated i n d i v i d u a l leg p a r a l y s i s beginning the f i r s t day a f t e r the s h i f t up to 29°C (Fig. 19). In most cases, the leg p a r a l y s i s phenotype was observed at least 87 Figure 19. Percentage of, 292 add A S mosaic survivors at 29°C; •» ^5 y w add A"1" mosaic survivors at 29°C (control); and A , % p l o t of 292 X 6 = 1752 legs f o r non-paralysis. percent o 00 00 two days before l e t h a l i t y . The major exceptions were those mosaics dead on day two and a f r a c t i o n of those dying on day two. Unfortunately, no observations were made before day one. It was noted, however, that a l l s i x legs of many mosaics dying on days two and three had become severely paralyzed the previous day. Thus, i n p l o t t i n g the data f o r leg p a r a l y s i s (Fig. 19), i t was assumed that those mosaics which died during the f i r s t three days, but had not shown signs of motor p a r a l y s i s , were mutant f o r a l l leg f o c i . This assumption seems reasonable f o r the following reasons: (1) In those mosaics which did show leg p a r a l y s i s and early death, a l l s i x legs were paralyzed. (2) In general, there i s a good c o r r e l a t i o n between time of death and the number of paralyzed t s l legs. (3) Very few y w add A males which, survived incubation at 29°C f o r several days, died before showing any motor d e b i l i t a t i o n . (.4) Mosaics dying early were generally mutant f o r c u t i c l e i n the lower t s l thorax, tissue nearest the add A f o c i as determined below. (5) tsl+ Few control, y_ w add A , mosaics died during the f i r s t 14 days of incubation at 29°C. If the number of non-paralyzed legs were pl o t t e d against the days of observation (Fig. 19), i t was found that the curve l e v e l s o f f at the 50% mark at about day 16. This i s consistent with the in t e r p r e t a t i o n that the behaviour of each leg i s con t r o l l e d by a sing l e focus, 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 to c l a s s i f y those legs that were paralyzed on day 0 to day 16 i n c l u s i v e as mutant and 90 the remaining legs normal. Fate mapping analysis showed that the primary f o c i for leg p a r a l y s i s l i e s i n the mesodermal tissues of the blastoderm. Each leg has i t s own independent focus located at about 22 sturts from i t s respective coxa (Fig. 20). Drug Studies: In order to test whether synaptic functions are involved i n the drop-dead mutations, the mutants were exposed to varying concentration of d i f f e r e n t psychotropic and neurotropic drugs known to a f f e c t neural functions i n mammals. The r e s u l t s of the drug studies are summarized i n Table 9. Complete r e s u l t s of the drug studies are presented i n the Appendix, t s l add A i s more s e n s i t i v e to Yohimbine, an adrenergic blocker, 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 agonist of c h o l i n e r g i c system. t s l Photomicrographic Studies: Because add A mutants have an t s l embryonic TSP, the embryonic development of add A and Oregon-R wi l d type were compared and examined for abnormalities using photo-micrography . The r e s u l t s of photomicrography of embryos kept at 29°C for varying lengths of time can be seen i n F i g . 21. The pictures with a darker background were taken using a p o l a r i z e r while those with a brighter background were taken using standard bright f i e l d i l l u m i n a t i o n . The malpighian tubules and muscles w i l l be b i r e f r i n g e n t under p o l a r i z e d l i g h t to provide an easier way of i d e n t i f y i n g these 91 t s l Figure 20. Fate map of add A . a, antenna; anp, anterior notopleural 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 t s^. 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 of two drop-dead s t r a i n s . The effects of the same drug on Oregon-R are also shown. Drugs were dissolved i n water to make d i f f e r e n t concentrations before they were added to the instant Drosophila medium (3oreal). Cultures with no drugs dissolved i n the water serve as controls. Ore-R pupae „ adults „ larvae ° pupae add A pupae c / adults „ larvae ° pupae ses pupae „ adults „, larvae ° pupae DRUG C ° f ' mg/ml P i l o - .2 carpine .4 n i t r a t e .8 .1.6 3.2 72.2 66.7 62.5 100.0 60.0 100.0 0.0 0.0 79.5 54.8 71.1 88.9 56.4 81.8 63.4 65.4 Carbamyl- .75 choline 1.5 chloride 3.0 6.0 9.0 73.8 96.8 67.4 • 100.0 33.3 100.0 0.0 0.0 82.1 91.3 69.0 100.0 89.3 100.0 60.7 70.0 70.0 100.0 Yohimbine .5 1.0 2.0 4.0 8.0 88.5 100.0 54.1 100.0 51.4 68.4 0.0 0.0 16.2 33.3 11.4 100.0 H 20 control no drugs 87.2 91.2 72.1 90.3 82.5 100.0 50.0 -84.0 58.3 76.2 88.2 92.4 44.7 94.1 51.4 100.0 100.0 ' 82.5 57.1 91.7 96.4 92.6 91.6 90.9 94 Figure 21. Photomicrographs of add A and Oregon-R embryos, developed at 29°C, at 2 hours p o s t - f e r t i l i z a t i o n : add A s embryos under I, polarized l i g h t ; I I , bright f i e l d i l l u m i n a t i o n ; Oregon-R embryos under I I I , polarized l i g h t ; IV, bright f i e l d i l l u m i n a t i o n . 9.6 F i gu re 21 ( c o n t . ) . Photomicrographs of add A and Oregon-R embryos, developed at 29 C, at 4 hours p o s t - f e r t i l i z a t i o n : Oregon-R 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 i l l u m i n a t i o n ; 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 , b r i g h t f i e l d i l l u m i n a t i o n . 98 Figure 21 (cont.). Photomicrographs of add A and Oregon-R embryo^, developed at 29°C, at 6 hours p o s t - f e r t i l i z a t i o n : add A embryos under IX, polarized l i g h t ; X, bright f i e l d i l l u m i n a t i o n ; Oregon-R embryos under XI, polarized l i g h t ; XII, bright f i e l d i l l u m i n a t i o n . 100 Figure 21 (cont.)- Photomicrographs of add A and Oregon-R embryo developed at 29 C, at 8 hours p o s t - f e r t i l i z a t i o n : add A S embryos under XIII, polarized l i g h t ; XIV, bright f i e l d i l l u m i n a t i o n ; Oregon-R embryos under XV, polarized l i g h t ; XVI, bright f i e l d i l l u m i n a t i o n . 102 Figure 21 (cont.). Photomicrographs of add A and Oregon-R embryos developed at 29 C, at 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 olarized l i g h t ; x v i i i , bright f i e l d i l l u m i n a t i o n ; Oregon-R embryos under xix, polarized l i g h t ; xx, bright f i e l d i l l u m i n a t i o n . o 104 t s l Figure 21 (cont.)- Photomicrographs of add A and Oregon-R embryos. developed at 29°C, at 12 hours p o s t - f e r t i l i z a t i o n : add A S embryos under XXI, polarized l i g h t ; XXII, bright f i e l d i l l u m i n a t i o n ; Oregon-R embryos under XXIII, polarized l i g h t ; XXIV, bright f i e l d i l l u m i n a t i o n . XXJV T i p 6 Figure 21 (cont.). Photomicrographs of add A and Oregon-R embryo.Sj developed at 29 C, at 14 hours p o s t - f e r t i l i z a t i o n : "add A embryos under XXV, polarized l i g h t ; XXVI bright f i e l d i l l u m i n a t i o n Oregon-R embryos under XXVII, polarized l i g h t ; XXVIII, bright f i e l d i l l u m i n a t i o n , mu, muscles; ma, malpighian tubules. X X V I I X X V I I I ,108 Figure 21 (cont.)- Photomicrographs of add A and Oregon-R embryoj^ developed at 29 C, at 16 hours p o s t - f e r t i l i z a t i o n : add A embryos under XXIX, polarized l i g h t ; XXX, bright f i e l d i l l u m i n a t i o n ; Oregon-R embryos under xxxi, polarized l i g h t ; x x x i i , bright f i e l d i l l u m i n a t i o n , mu, muscles; ma, malpighian tubules. •,110 t s l Figure 21 (cont.)- Photomicrographs of add A and Oregon-R embryos developed at 29°C, at 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 ; xxxiv, bright f i e l d i l l u m i n a t i o n ; Oregon-R embryos under xxxv, polarized l i g h t ; xxxvi, bright f i e l d i l l u m i n a t i o n . Figure 2 1 (cont.)- Photomicrographs of add A embryos, developed at 29 C: at 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 , bright 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 xxxix, polarized l i g h t ; xu, bright f i e l d i l l u m i n a t i o n . XXXVII structures i n the developing 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 dorsal and v e n t r a l segmentation t s l (8 hour), there were no detectable differences between the add A and Oregon-R embryos. However, a f t e r 10 hours at 29°C, the malpighian t s l tubules and muscles of add A embryos appear to develop at a f a s t e r rate since Oregon-R have none at t h i s time while the 22°C controls of t s l both Oregon-R and add A embryos developed at exactly the same rate. t s l It should be noted that the add A embryos may have an o v e r a l l increase i n rate of development of other tissues as w e l l , but only the muscles and malpighian tubules were e a s i l y detected by the techniques used t s l here. By 12 hours, the add A embryos have more prominent but poorly defined muscles and malpighian tubules and the segmentations once v i s i b l e e a r l i e r have disappeared. In contrast, Oregon-R at t h i s stage have w e l l defined muscles and malpighian tubules. At 14 hours, Oregon-R embryos s t a r t to show advanced muscle development and segmentation t s l while the add A embryos completely lack segmentation. At 16 hours, t s l the muscles and malpighian tubules i n the add A embryos become less organized and more dispersed while i n the Oregon-R embryos w e l l defined 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 sides of the embryos. At t h i s stage, the Oregon-R embryos also have trachae t s l 1 while add A have none. By 17~2 hours, when most Oregon-R embryos t s l have hatched, the muscles and the malpighian tubules of the add A embryos, though disorganized, are s t i l l present but the embryos f a i l to hatch. At 22 hours, these structures s t i l l p e r s i s t i n add Al~ii± embryos but no larvae hatch. In summary, i t appears that the muscles and malpighian tubules t s l of add A embryos, although formed at an e a r l i e r stage than Oregon-R, are never w e l l organized and are also i l l defined when compared* to t s l Oregon-R embryos. Therefore, i t seems that the l e t h a l i t y of add A at 29°C i s not due to a lack of muscle or malpighian tubule development but to a f a i l u r e of the muscular system and the malpighian tubules to maintain t h e i r i n t e g r i t y and to organized themselves. For example, the muscular sysstem f a l l s to form d i s t i n c t bands. It should be noted that the malpighian tubules 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 l i g h t . Moreover, these are very gross morphological differences as observed through the l i g h t microscope and the more subtle differences, i f present, are beyond the c a p a b i l i t y of the methodology used here. ses E Characterization of adult drop-dead behaviour: When ses E^ " f l i e s are s h i f t e d to 29°C, almost 95% die within 6 days (Fig. 22) whereas 95% of Oregon-R f l i e s usrvlve for at l e a s t 17 days at 29°C (Fig. 12). Characterization of TSPs: The d e s c r i p t i o n of TSPs of ses E"*" has already been presented i n Chapter 2. To r e c a p i t u l a t e , ses E"*" f l i e s have 2 TSPs - a 70-hour TSP extending from mid-lst 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 during the t r a n s i t i o n 116 Figure 22. Survival of v, 1 day old; o, 5 day old; •, 10 day old ses E 1 adults at- 29°C and , 1-5 day old ses E 1 adults at 22°C. 11' of 3rd i n s t a r l a r v a l to pupal stage (Fig. 4, F i g . 5). Genetic Mosaic Studies: 229 mosaics were recovered among of f s p r i n g of the cross y_ w ses E~*"/Y males X In(l)w V^/y w s p l females. The d e t a i l s of the mosaic analysis have already been mentioned i n the previous chapter. The s u r v i v a l curve of the ses mosaics (Fig. 23) suggests that those mosaics which die on or before the 13th day are drop-dead i n phenotype. This allows a more simple and s t r a i g h t forward f a t e mapping an a l y s i s . Control XO males recovered from the same cross, are a r e s u l t of f e r t i l i z a t i o n of a nullo-X egg by a v_ w ses E"^" sperm. These XO males a l l die before day 10. It was found that the embryonic focus of ses E~^  i s located i n the area of neuroblasts about 9 sturts below coxa 2 and coxa 3 i n the blastoderm fate map (Fig. 10, Chapter 2). Drug Studies: ses i s more r e s i s t a n t than Oregon-R to carbamylcholine chloride, an agonist of c h o l i n e r g i c system (Table 9). 119 Figure 23. Percentage of^ o, ses E mosaic survivors at 29°C; •, y w ses E /0 male survivors at 29°C and •, y w ses E mosaic survivors at 29 C. IV. Discussion Temperature-shift studies i n d i c a t e that the add A mutant has successive TSPs during the embryonic, 3rd i n s t a r l a r v a l - p u p a l and adult stages. The ses E"^  mutant has TSPs between the 1st and 2nd l a r v a l i n s t a r and the 3rd i n s t a r larval-pupal stages and the adult stage. The embryonic and the 3rd larval-pupal stages represent periods of considerable a c t i v i t y of developmental genes (Hadorn, 1951) so these r e s u l t s are not s u r p r i s i n g . The microscopic observations made t s l on embryos of add A provide 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. It was observed that the embryos develop normally 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 differences t s l between the Oregon-R and add A embryos were f i r s t v i s i b l e with the l i g h t microscope. This coincides with the TSP i n f e r r e d from s h i f t studies. A f t e r 12 hours, an amorphous mass of tissue reminiscent of Notch embryonic l e t h a l s (Shellenberger and Mohler, 1978) appears. t s l E a r l i e r studies of add A embryonic tis s u e cultures revealed that there i s a poor as 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 other d i f f e r e n t e e l l types (G. Beard, personal communication, F i g . 24). In addition to poor neuromuscular junction formation, the number of neural and muscle c e l l s i s reduced r e l a t i v e t s l to the controls. These observations suggest that add A could a f f e c t both! muscle and nerve development. Reduced numbers of nerve and muscle c e l l s (Buzin _elt a_l., 1978) and poor development (Cross and Sang, 1978) 122 Figure 24. C e l l cultures of I, Oregon-R at 29°C; II, same as I but at a higher magnification; I I I , add A t s l at 29°C; IV, same as III but at a higher magnification. 12* have also been observed with c e l l cultures of another embryonic l e t h a l , t s l s h i . It i s tempting, therefore, to suggest that the developmental t s l abnormalities of add A stem from a defect i n c e l l membranes that f a c i l i t a t e c e l l to c e l l connections. Among other p o s s i b i l i t i e s , the lack of or reduced production of chemical signals i n c e l l s responsible for i n t e r c e l l u l a r communication could r e s u l t i n some of the observed phenotypes. This i n turn could be due to a lack of or a reduced l e v e l of enzymes which are responsible f o r synthesis, release or reception of such chemical s i g n a l s . For example, Levi-Montalcini et a l . (1968) demonstrated that a protein, c a l l e d the nerve growth factor (NGF), plays an important r o l e i n the architecture of the nervous system both i n vivo and i n v i t r o . Results from temperature-shift studies suggest the p o s s i b i l i t y of an a l t e r e d protein. It i s possible to test the existence of a d i f f u s i b l e ts gene product by mixing c e l l cultures from normal and mutant embryos and determining whether there i s any influence of the wil d type c e l l s on the development of mutant myocytes and neurocytes. I d e a l l y , a c e l l marker system would be incorporated into such an experiment so that the genotype of i n d i v i d u a l c e l l s can be unequivocally determined. Unfortunately, such a system i s not a v a i l a b l e at the present time. But, i t i s possible to compare r e l a t i v e c e l l counts before and a f t e r mixing with w i l d type c e l l s (Cross and Sang, 1978). Altogether, tissue culture and temperature-shift studies suggest a ts product which i s e s s e n t i a l for the development of nerve t s l and muscle c e l l s most l i k e l y operates i n add A . More concrete t s l conclusions on the add A defect came from fate mapping analysis which indic a t e that i t s abnormalities l i e i n the muscular system. Therefore, the lack of coordination between the nerve and muscle c e l l s i n developing c e l l cultures could be a t t r i b u t e d to a membrane defect because membranes are known to be important i n c e l l to c e l l communications such as neuro-muscular ju n c t i o n formations during development. t s l If 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 neuro-transmissions would be expected to have an e f f e c t t s l t s l on add A . The fact that add A does not respond to any of these drugs other than drugs that a f f e c t the CNS and smooth muscles argues t s l against the somatic muscle o r i g i n of this mutation. add A i s more s e n s i t i v e to Yohimbine, an adrenergic blocker, and i s 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 , an agonist of ch o l i n e r g i c system. Both adrenergic and c h o l i n e r g i c system have an excitatory e f f e c t on the insect's CNS and smooth muscles (Pichon, 1 9 7 4 ) . Therefore, the existence of a t s l r e l a t i o n s h i p between add A drug responses and i t s muscular defect i s doubtful. However, these r e s u l t s point to future pursuits i n genetic mapping studies of the a l t e r e d drug resistance phenotype. Unless mapping of t h i s locus coincides with the drop-dead locus, the altered t s l drug resistance could w e l l be due to another locus affected by add A Mosaic analysis c l e a r l y indicated that the behaviour of each t s l leg of add A mosaic was independent of the others. Also, once paralyzed, these legs often remained so during subsequent t r i a l s . These observations suggest that the behaviour of each leg must has i t s own focus i n the somatic muscular system responsible for c o n t r o l l i n g the movement of each leg. Lethal 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 Drosophila tis s u e cultures has been documented (Wright, 1960), but adult l e t h a l i t y due to muscular defect i s s t i l l unheard of. In the present case, the seemingly unresolved r e s u l t s from drug studies and genetic mosaic analysis t s l demand the need f o r confirmation of the muscular defect i n add A This can be done by using.enzyme markers that w i l l allow i d e n t i f i c a t i o n of mosaicism i n t e r n a l l y . Indeed, such techniques have been used to i d e n t i f y mosaicism i n the nervous system of Drosophila (Kankel and H a l l , 1976). These i n t e r n a l markers are usually n u l l enzyme markers on the rod-X chromosome. Somatic loss of an unstable ring-X chromosome from the ring/rod heterozygote would lead to XO tissues which would not s t a i n for the enzyme i f these mosaics are scored i n t e r n a l l y by sectioning and histochemistry. It would be predicted that the absence of a c e r t a i n type of mutant muscle c e l l s would be found among survivors at 29°C. Unfortunately, an enzyme marker that w i l l s t a i n for muscles has yet to be discovered. The other drop-dead mutation studied, ses E \ i s d i f f e r e n t t s l from add A i n that i t s defect i s neural rather than mesodermal i n nature. Fate mapping analysis indicates that the defect of ses mutation i s i n the nervous system. The demonstration of the neural basis for 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 fate mapping studies to be due to a l e s i o n i n the b r a i n tissues (Hotta and Benzer, 1972) while the dmd l e t h a l i t y was due to a defect i n the thoracic ganglion (Flanagan, 1977). Drug studies showed that the ses E"^  mutant i s more r e s i s t a n t to carbamylcholine chloride (an agonist of the c h o l i n e r g i c system) whose action i n the i n s e c t s ' CNS i s p r i m a r i l y excitatory on the post-synaptic c e l l (Pichon, 1974). This, together with temperature-shift studies, would suggest a c h o l i n e r g i c system playing a r o l e i n t r i g g e r i n g c e r t a i n signals that cause the release of, or synthesis of c e r t a i n proteins which are e s s e n t i a l both during and a f t e r development. The neural nature of this mutant d i r e c t s future studies to fate map i n t e r n a l structures using the aforementioned enzyme marker technique i n order to l o c a l i z e i t s defect more p r e c i s e l y . t s l Therefore, the add A gene product could be a component of the muscular system which f a i l s to function at a higher temperature. The ses E"^  product seems to be involved i n the synthesis of or the release of a thermolabile p r o t e i n important i n the functioning and i n t e g r i t y of the nervous system. 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Ore-R ses B"" 2 ses B ses D"" ses D ses E^ " add A Control 47* 75 51 85 68 100 58 no drugs 50 84 95 95 80 57 51 83 85 78 97 78 96 45 87 - - 56 — 92 88 72 — - - - 31 -Carbamyl- .75 74 86 77 92 92 82 . 65 choline 1.5 67 68 89 - 100 69 44 c h l o r i d e 3.0 33 64 50 68 87 89 36 6.0 0 16 - 47 29 61 0 12.0 0 0 0 0 0 70 0 P i l o - .2 72 80 51 75 97 80 carpine .4 62 82 - 53 _ — — n i t r a t e .8 60 - 3 35 — 71 1.6 0 21 0 29 - — 56 3.2 0 - 0 45 - - 63 Succinyl- .625 32 60 90 100 63 25 choline 1.25 70 80 94 - 68 83 70 chloride 2.5 49 92 85 100 3 65 67 5.0 78 93 86 90 50 85 46 10.0 22 80 17 67 36 . 25 . 24 Nicotine .0156 84 88 81 97 81 81 63 .03125 94 97 64 75 100 71 60 .0625 71 74 0 58 67 69 68 .125 3 34 0 13 . 0 9 8 .25 .0 6 0 0 0 0 3 Atropine 1.5 57 63 56 95 83 88 63 s u l f a t e 3.0 31 30 74 87 77 61 56 6.0 6 21 24 46 60 78 28 12.0 0 4 0 — 18 10 34 24.0 0 0 0 0 0 0 -Hexa- .5 84 — 57 _ 90 methonium 1.0 88 83 40 90 — 95 70 bromide 2.0 21* 45 13 83 — 93 69 4.0 "0 15 7* 80 — 43 11 8.0 0 0 0 0 - 0 0 d- 1.0 80 64 79 69 80 92 73 Tubocurare 2.0 34 79 75 81 84 88 74 4.0 86 100 50. 69 ?0 84 74 8.0 50 33 0 52 14 41 0 — — — -STRAINS DRUGS CONC. Ore-R ses B^ 2 ses B ses D^  . 2 ses D ses E"^" J J At S l add A Eserine .00625 0 83 85 76 72 82 100 s u l f a t e .0125 0 75 55 53 67 90 59 .025 0 82 76 - 90 45 63 .05 0 - 63 3 34 3 13 .1 0 3 82 0 0 0 5* Reserpine .05 33 19 20 _ 60 - 0 .1 54 3 5 62 — 100 78 .2 17 0 - 13 _ 0 .4 3 0 - - — 62 0-.8 0 0 0 26 - 0 0 Nialamide .25 91 87 79 100 _ 80 54 .5 21 92 92 88 90 97 61 1.0 67 66 58 95 100 77 58 2.0 - 79 - 51 - 28 71 4.0 0 75 21 ' 87 50 62 32 Iproniazid .25 60 83 82 68 85 80 55* .5 40 68 47 64 33 63 68 1.0 34* 21 40 38 29* 39 46 2.0 0 27 23 23 53 26 54 4.0 0 0 5 3 5* 11* 18 Caffeine .125 33* 53* 68 66 87 33 .25 60 13 0 20 — 88 48 .5 73 51* 90 80 95 28 1.0. 18* 0 0 67 — 83 13 2.0 79 0 0 - - 2 5 Theo- .125 73 88 78 70 87 43 p h y l l i n e .25 - - - 70* _ _ .5 • 50 75 46* 59* — _ 55* 1.0 13* 0 0 16 • — 0 0 2.0 0 0 0 0 _ 61* 0 Strychnine .5 62 83 71 88 68 100 s u l f a t e 1.0 29* 83 92 98 — 80 28 2.0 0 35 0 54 — 83 42 4.0 "0 27 0 - - 38 36 8.0 0 — 46 43 45 0 P i c r o - ' .0025 36 56 95 88 100 90 43 toxin .005 58 75 86 95 75 87 44 .01 50 78 82 82 69 55 64 .02 67 69 42 76 57 90 26 .04 41 11 24 15 21 50 23 STRAINS DRUGS CONC. Ore-R ses B''" 2 ses B ses D"*" 2 ses D ses E^ " JJ At S l add A A l l y 1 - - 1.25 58* 71* 80* 25* 38* 64* 5* glycine 2.5 72* 10* 49* 0 0 50* 0 .5.0 2* 0 16* 0 0 0 0 10.0 0* 0 0 0 0 0 0 Amino . 2 52 88 66 36 46 84 - 33 oxyacetic .4 0 35 37 86 73 90 0 a c i d .8 0 35 30 0 0 92 0 1.6 11* 0 0 0 0 0 0 3.2 - 0 0 0 0 0 0 Methionine .125 42 89 56 85 59 53 35 sulfoximine .25 54 58 36 30 37 58 5 .5 66 81 73 75 80 — 42 1.0 16* 58 81 28 47 46 5 2.0 28 68 55 53* 7 73 0 Sodium .125 76 87 65* 95 66 59 b a r b i t a l .25 66 ' 78 41* 93 77 48 .5 62 71* - 63* — - 36* 1.0 - - 0 81* — 72 0 2.0 11 0 0 0 .- . 0 0 Urethane .25 77 92 73 97 73 66 26 .5 64 95 50 83 76 84 40 1.0 61 92 53 57 95 73 38 2.0 13 8 0 0 0 5 0 4.0 .0 0 o. 0 0 0 0 Dopamine 1.25 57 72 85 92 90 82 43 2.5 - 9.0 55 85 85 55 55 5.0 77 66 76 90 79 — 44 10.0 58 76 63 58 — 54 15 20.0 8 7 82 0 31 ... .0 0 dl-Nor- 1.75 53 72 60 87 74 83 45 epinephrine 3.5 42 65 63 85 100 86 34 7.0 4 57 66 56 95 56 25 14.0 23- 21 46 30 51 42 8 Tyramine .75 — 90 90 63 1.5 100 82 58 — - 55* 54 3.0 26 36 45 22 • — 40 63 6.0 0 0 3* 0 — 0 3 12.0 0 0 0 0 < — 0 0 138 STRAINS DRUGS CONC. Ore-R ses B"^  2 ses B ses D^ " 2 ses D ses E^ " AA At S l add A d l - 2.5 64 77 92 97 73 87 54 Octopamine 5.0 61 44 78 95 100 89 0 10.0 31 56 70 18* 76 39 10 20.0 0 11 9 0 0 0 0 Histamine 1.8 54 89 62 0 65 3.7 - 84 - - 90 87 83 7.5 62 - - - 0 15 22 15.0 11 38 38 0 52 5 0 30.0 0 0 - 0 0 0 0 Gama 6.0 _ 92 91 _ amino- 12.0 - — 88 - 70 _ 79 b u t y r i c 25.0 13 61 56 80 59 42 10 a c i d 50.0 - — 0 — - - -Glutamic 6.25 55 81 66 100 87 97 33 acid 12.5 61 47 69 — 72 85 63 25.0 37 56 61 80 92 96 60 50.0 37 33 22 33 28 8 27 100.0 11 0 0 13 0 .0 .0 Catechol 1,0 90 78 78 49 73 80 38 2.0 82 92 49 72 95 81 18 4.0 - 31 0 0 21 20 30* o • 8.0 0 0 0 0 0 0 0 16.0 0 0 0 0 0 0 0 Yohimbine .5 89 66 39 61 93 88 16* 1.0 54 72 0 42 86 53 11 2.0 51 45 16 26 62 24 0 4.0 0 0 0 0 0 0 0 8.0 0 0 0 0 0 0. . . . .0 A l l experiments have over 80% adult/pupae with, the exception of *, which have less than 50% adult/pupae. —, data not ava i l a b l e due to either extensive mould growth In culture or experiments not performed. A l l experiments were performed at 22°C. A l l mutant s t r a i n s f a i l to develop at 29°C. 

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