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UBC Theses and Dissertations

Etudes du developpement de la drosophile: variations on a theme Grigliatti, Thomas A. 1971

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I 1220 ETUDES DU DEVELOPPEMENT DE LA DROSOPHILE: VARIATIONS ON A THEME by THOMAS A. GRIGLIATTI B.S., Santa Clara University M.A., San Francisco State College A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in Genetics in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1971 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis fo r scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Zoology (Genetics) The University of B r i t i s h Columbia Vancouver 8, Canada Date December 15, 1971 ABSTRACT As a holometabolous organism which has been highly exploited genetically, the f r u i t f l y , Drosophila, is an excellent model for the study of development in higher organisms. Conditional mutations that are sensitive to temperature differences were used to investigate problems of gene action in different tissues, regulation of specific tissue determination and differentiation, and the genetic regulation of development and functional integration of the nervous system. The f i r s t problem, a classical question in developmental biology, attempts to determine whether control of the activity of a structural gene is directly imparted by the tissue in which the gene product is active or whether, in fact, a freely flowing evocator of gene action exists. A temperature-sensitive ras al l e l e has an effective lethal phase at 29°C about 12 hours after pupation and a temperature-sensitive period (TSP) for lethality beginning midway through the third larval instar and ending around pupation. The mutation also alters the quantity of pteridines present in the eyes, testes and malpighian tubules at 29°C. The TSP for pigment production in the malpighian tubules occurs in the egg and f i r s t instar larvae, and in eyes and testes after pupation. The demonstrated autonomy of the mutant in the eyes implies the tissue-specific functioning of the gene. It is suggested that the different TSPs for a single mutation indicate tissue-specific activation of a gene at different times during develop-ment, although the possibility of activation of preformed polypeptides has not been eliminated. There exists in Drosophila a class of mutations called "homeotic" which cause changes in determination of the imaginal discs. The second problem investigated concerns the possibility of isolating a temperature-sensive homeotic mutant for the purpose of studying genes which regulate specific pathways of differentiation. The a40a homeotic mutant, ss , was found to have a temperature-sensitive transformation of the arista segment of the antennal complex to a tarsus of the leg. In a selected stock, penetrance was complete so that at 29°C, normal aristae were produced, whereas at 17°C, complete tarsi developed in a l l f l i e s . "Shift" studies revealed a temperature-sensitive period in the third larval instar. The temperature-initiated a40a action of ss does not appear to act on a ts receptor site within the disc c e l l s . In combination with another homeotic mutant, Antenna-pedia, the entire antennal complex is transformed to a complete leg at 22°C. Mutants affecting the nervous system were sought for the purpose of investigating the genetic regulation of the development and function of the nervous system. A temperature-sensitive mutation, ts para of Drosophila melanogaster causes an immediate but reversible paralysis only of adults when shifted from 22°C to 29°C. The mutation is a sex-linked recessive mapping 2.8 units to the l e f t of .f. Wild-type f l i e s observed for two hour periods exhibited normal mobility at a l l temperatures between 22° and 35°C. From 22° to 25°C, para t s f l i e s were wild-type in walking, climbing and flying a b i l i t y . At one ts degree intervals above 25°C, para f l i e s became increasingly deb i l i -tated and at 29°C, complete paralysis occurred. After prolonged maintenance at 29°C, recovery of some activity could occur at that temperature. Extensive studies of behavior of mosaics at 29°C revealed a requirement of the + al l e l e in the head for mobility and a thoracic component for proper leg movement. Normal electroretino-grams were obtained at both 22° and 30°C. The results suggest a temperature-sensitive defect in the central nervous system. ACKNOWLEDGEMENTS This becomes the most d i f f i c u l t part of the thesis. How does one thank Dave Suzuki? As my major professor, he has been more than an excellent scientist and teacher. He has been patient, considerate, enthusiastic, j o v i a l , and more than helpful in every way. In short, he has been and is a friend. I can say no more.* The work presented in Chapters 5 and 6 was done in collab-oration with Rodney Williamson. I wish to thank him for this and also for his helpful discussions on the rest of the work presented in this thesis. To the rest of the members of the lab, past and present, too numerous to mention, for their help and discussions, but more important for their camaraderie and for providing the unique atmosphere in which this work was done, my special thanks. It has been fun. Chapters 3, 4 and 5 of this thesis have been published in the Proceedings of the National Academy of Sciences, U.S.A. Chapter 6 is in press in Developmental Biology. except maybe to add that he's a good quarterback. vi TABLE OF CONTENTS ABSTRACT i i ACKNOWLEDGEMENT v LIST OF TABLES v i i i LIST OF FIGURES ix CHAPTER 1 REVIEW 1 A. Determination of Imaginal Discs 9 B. Transdetermination 15 C. Homeotic Mutants 22 D. In Vitro Cultures of Discs 29 E. Biochemistry Review 31 CHAPTER 2 GENERAL INTRODUCTION 33 CHAPTER 3 A TEMPERATURE-SENSITIVE MUTATION AFFECTING LEVELS OF PTERIDINES IN DROSOPHILA MELANOGASTER I. Introduction 39 II. Materials and Methods A. Genetic Analysis 40 B. Developmental Analysis 40 C. Spectrofluorometric Analysis of Pigment Production 41 III. Results A. Temperature Effects on Eye and Testis Pigmentation 46 B. Temperature Effects on Malpighian Tubule Pigmentation 50 C. Autonomy of Pigment Formation in Different Organs and Pigment Turnover 51 IV. Discussion 56 CHAPTER 4 THE TEMPERATURE-SENSITIVE HOMEOTIC MUTANT, ss I. Introduction 59 II. Materials and Methods 61 III. Results and Discussion 69 v i i CHAPTER 5 A TEMPERATURE-SENSITIVE MUTATION (para ) CAUSING ADULT PARALYSIS IN DROSOPHILA MELANOGASTER I. Introduction 77 II. Materials and Methods 78 III. Results A. Genetic Properties 79 B. General Biological Properties 79 C. Tissue Specificity 86 D. Visual and Flight Response 92 IV. Discussion 93 CHAPTER 6 DEVELOPMENTAL ANALYSIS OF THE PARALYTIC MUTATION, parats I. Introduction 95 II. Materials and Methods 96 III. Results 102 IV. Discussion 148 CHAPTER 7 SUMMARY AND CONCLUSIONS 154 LITERATURE CITED 157 LIST OF TABLES TABLE 1. Known Homeotic Mutants 2. Phenotypes of malpighian tubules and eyes after each kind of shift. 3. Expected and actual results from shifts during the TSP. 4. Results of screening adult offspring of mutagenized f l i e s for paralysis at 29°C. 5. A correlation of the position of mutant patches and the pattern of paralysis of mosaic females at 29°C. 6. Behavior at 29°C of mosaics in which the phenotype of abdominal tissue differed from the head and thorax. 7. Behavior of head mosaics at 29°C. 8. Behavior at 29°C of thoracic mosaics. 9. Categorization of 29°C activity of bilateral head-thorax and thorax mosaics. 10. Leg activity at 29°C in bilateral head-thorax and thorax mosaics. 11. Frequency of mutant or mosaic tissue in individual legs of leg-only mutants. 12. The behavior at 29°C of individual mutant legs in legs-only mosaics. ix LIST OF FIGURES FIGURE 1. General l i f e cycle of Drosophila melanogaster. 3 2. Imaginal organ rudiments: their position in mature third instar larva and the corresponding adult structures which they form. 5 3. General anatomy of adult f l y : lateral view. 8 4. A diagram indicating the directions in which trans-determination occurs with the corresponding transform-ations caused by homeotic mutants. 19 5. General position of homeotic mutants on the Drosophila genome. 28 6. Temperature-sensitive period for the E6 mutant. 43 7. Relative amounts of fluorescent pigments in E6 and Oregon-R f l i e s . 49 8. The temperature-sensitive periods in E6 for lethality and pigment levels in the eyes, testes and malpighian tubules. 53 a40a 9. A scanning electron micrograph of ss and Oregon-R f l i e s raised at different temperatures. 63 10. Diagram of the entire antennal complex. Shows the possible phenotypes of f l i e s hatched from various shift experiments. 68 a40a 11. Results of shift studies on ss f l i e s . 71 I O A • i . - • u .e A _ B a40a . a40a 11. A scanning electron micrograph of Antp , ss /+ ss raised at 22°C. 76 ts 13. Recovery of behavioral activity of para f l i e s at different temperatures during a 2 hour period. 85 14. Classification of mosaic females generated by somatic loss of In(l)w vC according to the ventral location of mutant tissue. 89 15. The method for generation of somatic mosaicism by loss of In(l)w v C. 98 16. Diagrams used for delineating during the scoring procedure. the area of mosaicism 101 X FIGURE 17. Dorsal view of a lateral section and sagittal section of Drosophila adult. 104 18. Activity of each leg in bilateral thoracic mosaics at 29°C. 116 19. Behavior of bilateral thoracic and head-thoracic mosaics at 29°C. 118 20. Location of different mutant patches on the dorsal surface of the thorax. 121 21. Location of mutant tissue in head-thorax catercorner mosaics. 129 22. Location of mutant tissue in three head-thorax mosaics which behaved unexpectedly at 29°C. 133 23. Location of mutant tissue in four head-thorax mosaics which behaved unexpectedly at 29°C. 135 24. Activity recovered with time at 29°C by different mosaics. 139 25. A diagram of the device used to test the optomotor response. 141 ts 26. Electroretinogram of para at 22°C and 30°C showing the response to a short (20 /Us) flash of light. 144 1 CHAPTER 1 REVIEW In the past 2 0 years, bacteria and bacteriophages have been intensively analyzed by molecular biologists. Their spectacular success in elucidating the molecular nature of gene structure and function has overshadowed phenomena in multicellular organisms. How-ever, with an understanding of the basics of replication, coding, trans-cription and translation, interest has been renewed in problems inherent to eukaryotic multicellular organisms. As one of the earliest and most extensively exploited organisms genetically, the frui t f l y , Drosophila melanogaster, is unique for the study of development. As a holometabolite, i t experiences a complete metamorphosis from a larval crawling form to an adult which walks, f l i e s and sees. A brief review of the l i f e cycle is shown in Figure 1. The anatomical structures of the adult and larvae are com-pletely different, and the differences between the two stages with respect to obtaining food, locomotion, and behavior in response to external stimuli are obvious. Nevertheless, the larval stage antici-pates the requirements for formation of the adult organism. During early embryonic development, packets of cells (imaginal cells or discs), which w i l l give rise to most of the external structure of the f l y during pupation, are set aside. The discs are formed from ectodermal cells during blastoderm formation (about 3 hours postfertilization). Cell proliferation in the discs continues until the late third instar when the final size and shape are attained (Figure 2 ) . Each disc consists of a large FIGURE 1 General l i f e cycle of Drosophila melanogaster. INSTAR 2nd INSTAR PUPA 3rd A INSTAR hz\ FIGURE 2 Imaginal organ rudiments: their position in mature third instar larva and the corresponding adult structures which they form. Adopted from Fristrom e_t a l . (1969). 6 number of small, histologically similar c e l l s . Three pairs of discs give rise to the adult head: the labial discs which give rise to the proboscis (Wildermuth and Hadorn, 1965), the clypeo-labrum (Gehring and Seippel, 1967) and the eye antennal discs. The antennal portion gives rise to the antennae, prefrons, rostrum and palpi (Gehring, 1966); the eye disc generates the rest of the head capsule, dorsal and posterior to the frontal suture including the eyes and vibrisse (Ouweneel, 1970a) (see Figure 3 for the anatomy of the adult f l y ) . The thorax arises from six pairs of discs. Three pairs of discs produce the six legs, the ventral thorax and the sternopleura (Stern e_t a_l., 1963). The humeral discs become the humeral regions of the prothorax (Lamprecht and Remensberger, 1966). Wing (Hadorn and Buck, 1962) and haltere discs (Loosli, 1959) produce the wings and halteres respectively, as well as the dorsal parts of the thorax. The unpaired male^or-female^genital disc forms both the genital apparatus (excluding the gonad), and the last abdomenal seg-ment (Hadorn et <al., 1949). The remaining adult abdomenal epiderm arises through proliferation of paired dorsal and ventral histoblast cells located within each abdomenal segment (Bodenstein, 1950). The imaginal discs can be readily identified by their posi-tion, size and shape. The final differentiation of the discs into adult structures is triggered during pupation by the release of the hormone, ecdysone (Mandaron, 1970). Discs provide a magnified i l l u s -tration of the phenomena of a "determined" stage which commits undiff-erentiated cells to a specific fate and "differentiation" which is the actual realization of that fate. The discs can be readily manip-ulated in a number of ways. Thus, whole or fragmented discs may be FIGURE 3 General anatomy of adult f l y : lateral view. Ant - antenna; Ast - arista; C^ _3 - coxa^_3; E - eye; HA - haltere; HP - hypopleura; Lp - labial palpus; MP - maxillary palpus; MS - mesopleura; MT - metanotum; 0 - ocellus; PT - ptero-pleura; SC - scutellum; ST - sternopleura; T - trochanter; V - vibrissae. implanted into adult hosts where proliferation without differentiation occurs or into larval hosts where both processes ensue. Recently, methods for culturing of discs ii\ vitro have been established. In addition, i t is possible to isolate large amounts of disc tissue at a level that permits quantitative biochemical studies (Fristrom and Mitchell, 1965). In most organisms, the processes of determination and differ-entiation follow one another in rapid succession, whereas in holo-metabolous organisms, these events may be widely separated temporally. Hadorn (1965) has defined determination as the "process which initiates a specific pathway of development by singling i t out from among the various possibilities for which a cellular system is com-petent". It i s , therefore, the regulative, rather than the structural aspect of differentiation. A. Determination of Imaginal Discs The processes of determination and transdetermination have been reviewed extensively (Wildermuth, 1970; Gehring, 1968; Ouweneel, 1970b). Hence, only a succinct review w i l l be presented in the next few pages. Among the basic problems of development is the question of whether determination occurs under the influence of an external stimulus provided by separate cells or group of c e l l s , or whether i t occurs within a c e l l without the influence of inducing factors. It would appear that the latter is the more likely possibility since embryonic or young 1st instar larval imaginal discs, cultured either in vitro (Gottschewski, 1960) or in an adult abdomen (Garcia-Bellido, 10 1965; Hadorn e_t a_l. , 1968; Schubiger et a l , , 1969), are determined in the same fashion as _in situ. That is to say, once the c e l l or small group of cells are recognizable as presumptive disc ce l l s , they undergo further determination autonomously. Several lines of evidence indicate that the actual time of determination is early in embryogenesis. Removal of imaginal cells of young embryos by surgical extirpation or irradiation leads to the absence of specific adult structures (Geigy, 1931; Howland and Child, 1935). In addition, various chemical treatments administered during embryogenesis can induce phenocopies of known disc mutants. Elegant studies have illustrated that 3 hour old embryos have been subjected to the determinative process. In these studies, cells from the anterior half of a three hour old embryo genetically marked with yellow (a mutant causing yellow body color, see Lindsley and Grell, 1968) were mixed with cells from whole embryos marked with the dark body color, ebony. These mixtures were then implanted into larval hosts, and i t was shown that cells marked with yellow gave rise only to structures found in the anterior half of the f l y , whereas ebony cells gave rise to both anterior and posterior structure (Chan and Gehring, 1971). It would appear, then, that determination had occurred by 3 hours postfertilization. Somatic recombination can be used to obtain spots of homo-zygous mutant tissue in an otherwise heterozygous (wild-type) back-ground (see Stern, 1968, for a review). Cells exhibiting a mutant phenotype in one area presumably result from a single somatic crossover event which was subsequently amplified mitotically. Bryant and Schneiderman (1969) found that the induction of somatic crossing over in three hour old embryos yielded marked clones of cells which were confined to a single appendage. Although clones were found which extended over several segments of the leg, for example, they were never found to extend uninterrupted from the leg to adjacent parts of the body. It seems, therefore, that certain cells of the blastula are already determined to form a specific part of an adult structure, such as a leg. However, since the patches often extended through several segments of the leg, i t is believed that the cells within a given disc Anlage have, at these early stages, the potential of producing many ce l l types within that appendage. Several lines of evidence indicate that the developmental potential of each disc c e l l becomes increasingly restricted as larval l i f e ensues. For instance, i f somatic crossing over is induced at various times during larva growth, the extent of mosaic patches which arise are restricted to increasingly smaller areas of a given structure as the larva matures (Bryant and Schneiderman, 1969). In earlier studies, Becker (1957) found that certain cells of the eye disc from late second instar larva s t i l l have the option to differentiate into either ommatidia or into vibrissae. By the third instar stage, these same cells had lost this potential, and were committed to form either ommatidia or vibrissae. By the late third larval instar stage, regional specificity is superimposed upon the prior determined state of a given disc. Thus, cells are committed to form specific regions and structures of the adult appendage. A disc may be removed from a third instar larva and cut into fragments which can then be implanted into other host third instar larvae. Such fragments are found to differentiate into specific segments of the adult structure (Schubiger, 1968). Similarly, when a small area of cells is removed from explanted discs by microbeam treatment, and the discs implanted into third instar larvae, adult appendages are formed which lack a specific structure (Ursprung, 1957, 1959). Using these techniques, i t has been possible to construct anlagen plans or fate maps of cells in the imaginal discs for: eye antenna (Vogt, 1946a; Gehring, 1966), wing (Hadorn and Buck, 1962), haltere (Loosli, 1959), leg (Bodenstein, 1941), male foreleg (Nbthiger and Schubiger, 1966; Schubiger, 1968), female genital (Hadorn and Chen, 1956; Ursprung, 1957), male genital (Hadorn, et a l . , 1949; Ursprung, 1959; Luond, 1961). Thus, a disc from a late third instar larva, though made up of histologically similar ce l l s , i s , in fact, comprised of cells already rigidly programmed to form different parts of a specific adult appendage and the cells committed to form one specific area are no longer able to generate parts of the organ supplied by other areas. Thus, by the end of the third instar, cells from different areas within a given disc bear the same relationship to one another as cells in whole discs did to each other in the late embryo. Nothing is known about the nature of determination within an individual c e l l because single cells haven't been amenable to experi-mentation. However, some information on state of determination of individual cells has been deduced by mixing cells from different discs. In such experiments, cells from two or more discs are mechanically or chemically dissociated, then mixed and reaggregated. The mosaic pellet is then transplanted into an adult host for several days to allow time for possible c e l l sorting, and then injected into a larval host where i t is allowed to differentiate during metamorphosis. Alternatively, 13 the pellet can be injected directly into the larval host. The cells from different discs are marked genetically with mutations affecting color mutants and often bristle and hair morphology. If cells from two genetically different discs of the same type (i.e., leg) are mixed, allowed to aggregate and then tested, they form patterns similar to the in situ patterns except that they are phenotypic mosaics (Nothiger, 1964; Tobler, 1966; Garcia-Bellido, 1966a). This indicates that the cells from the same region of a given disc type are able to co-operate to form a specific adult structure. On the other hand, when genetically marked cells from two different disc types, for example, wing and genital, are mixed and implanted, upon metamorphosis no integrated mosaics are formed (Nothiger, 1964). Thus, individual cells must be determined to the extent that like cells w i l l aggregate, whereas unlike cells w i l l sort out, perhaps by c e l l migration. Moreover, i f proximal and distal cells from the leg disc (Tobler, 1966) or the wing disc (Garcia-Bellido, 1966a) are mixed, segregation is also observed. Therefore, even cells from different areas of a given disc are able to segregate from one another. Finally, during these mixing experiments, in a few cases (Nothiger, 1964) a single c e l l or small number of cells from one disc type (genital) became trapped in a large area of cells of another disc type (wing). These trapped cells differentiated autonomously. Is the abi l i t y of unlike cells to segregate from each other a property inherent to larval disc c e l l s , or is i t acquired only in the metamorphosing cells? The abi l i t y of larval disc cells to segregate was rather convincingly demonstrated by Garcia-Bellido (1967). He mixed dissociated wing and leg disc cells from third instar larvae, 14 and placed the pellets in adult hosts which were starved so that no proliferation occurred. After two to ten days, they were examined histologically and the wing discs were found to have reaggregated into folded sheets like the pattern found in in situ third instar discs. Furthermore, Kuroda (1969) showed that in re-associated eye-antennal disc cells cultured in vitro, the presumptive omatidial cells were able to sort out and cluster separate from the presumptive antennal ce l l s . Although the cells of a specific disc from late third instar larva are determined to form only a small region of the adult structure, i t has not been proved that the developmental potential of these cells cannot be further restricted. That i s , they may not have reached their f i n a l stage of determination. In mixing experiments involving genetic-al l y marked leg discs, Garcia-Bellido (1966a) found small mosaics between bristle and bract cells (specialized hair c e l l ) . Since isolated brachts have not been observed in mixing experiments, i t was argued that they are induced by the bristle c e l l s . Furthermore, Lees and Waddington (1942) have shown that a bristle which is composed of two cells (the shaft and the socket cells) is formed during the early pupal stages by unequal division of the bristle mother c e l l . It seems, therefore, that in some cases further restrictions of developmental potential occurs during early pupa. In summary, the adult body structures are derived from a series of discrete packets of cel l s . These discs are ectodermal in origin and are i n i t i a l l y determined to produce a given adult structure at blastoderm formation. Their i n i t i a l determined state is further restricted during larval growth, such that specific cells within a 15 disc become programmed to produce particular sections of the adult appendage. Once the i n i t i a l determination is established, further development is autonomous. B. Transdetermination The experiments described so far have shown that once a state of determination is imposed, that state is stable and hereditary to a l l daughter cel l s . The length of time that this state of deter-mination would be retained in disc cells which were dividing but not differentiating soon became of interest. Hadorn and members of his group have maintained some lines of disc blastemas by culturing in vivo in adult fly abdomens for up to seven years. When these disc blastemas are tested by implanting fragments into larvae, the metamorphosed structures formed are the same as that of the original disc. Sometimes, when these in vivo-cultured blastemas are tested, structures arise which are different from the program dictated by the original deter-mination event. For example, cultures of presumptive genital disc cells occasionally produce leg or antennal structures (Hadorn, 1963). The new structures were called allotypic in contrast to the normally produced autotypic structures (Hadorn, 1965). The process whereby one state of determination is exchanged for another state of determin-ation has been called transdeterminat ion. If and when transdetermination occurs, not a l l the cells of the test implant undergo transdetermination. Moreover, there is a sharply defined boundary between the transdetermined (allotypic) and normal (autotypic) cel l s , although they may l i e adjacent to one another. An intermediate or mosaic structure has never been described on the border between the allotypic and autotypic structures. Like autotypic cell s , allotypic cells pass on their program for altered differentia-tion to their daughter cells over several generations (Gehring, Mindek and Hadorn, 1968). From the size and shape of allotypic regions with-in a disc, i t is generally believed that transdetermination occurs in several cells concomitently. However, i t has been impossible to suc-cessfully transplant single ce l l s . In addition, i t is d i f f i c u l t to identify a single allotypic c e l l in a wild-type background. If trans-determination occurred in a single c e l l , c e l l multiplication during late third instar or early pupa would lead to at least a small cluster of clonally-related ce l l s . By genetically marking the implanted c e l l s , it was possible to show that the allotypic structures always arose from the implanted disc cells and not from host cells (Gehring, 1968). The question then arose, do allotypic structures arise from already determined autotypic cells or from undetermined cells? From the state of determination fixed in late third instar discs, one would assume that the allotypic structures arise from determined autotypic c e l l s . Gehring (1967) induced somatic crossing over in larvae hetero-zygous for several genetic markers. He then implanted the eye-antennal disc into adult hosts for several days and then transplanted the cells back into larval hosts. After metamorphosis, several clones of marked cells contained both autotypic and allotypic c e l l s . In several cases, the palpus which is an autotypic structure formed by the antennal disc had both wild-type and marked cells and the clone of marked cells was continuous with a large area of allotypic wing ce l l s . From this i t was concluded that somatic crossing over occurred in an autotypic c e l l which proliferated and then underwent a transdetermination event to 17 give rise to the allotypic cells of the clone. Transdetermination has been observed in a l l disc types which have been cultured in vivo (Dubendorfer, 1969). Disc cells from any larval stage and even embryonic tissue (Hadorn et a_l., 1968) w i l l under-go transdetermination once they have completed their imaginal develop-ment in an adult host. It seems that the rate of transdetermination varies from one type of disc to another. For instance, the genital disc undergoes transdetermination events only after i t has been through several generations of adult hosts (Hadorn, 1966), whereas a labial disc w i l l undergo transdetermination after only one generation in an adult host (Wildermuth, 1968). Wildermuth (1968) also offered evidence for differ-ences in the rate of transdetermination induced in discs from various species of Drosophila. The most interesting fact associated with transdetermination is that i t is a directed process. That i s , once programmed by deter-minative events, a group of cells can undergo transdetermination to form only one or two specific allotypic structures. The change from^a given autotypic structure to an allotypic structure is predictable for both direction and frequency in a l l cases. For example, genital disc cells undergo transdetermination to form either leg or antennal struc-tures. Allotypic cells may themselves undergo transdetermination again to give rise to a second order of allotypic structures. For example, allotypic leg cells may form wing structures. The direction of these further transdeterminative events is also predictable. It is possible to construct a diagram showing the directions of transdetermination starting with any given autotypic or allotypic structure (Figure 4). FIGURE 4 A diagram indicating the directions in which transdetermination occurs with the corresponding transformations caused by homeotic mutants. transformations observed in transdetermination; transformations caused by homeotic mutants. For the f u l l name of the homeotic mutants, see Table 1. GENITAL bxd 20 It is interesting to note that many, but not a l l , of the possible steps in transdetermination are reversible. Transdetermination is known to occur within a disc, that i s , cells from a specific region of a disc which are determined to give rise to a specific part of the adult appendage, sometimes produce structures that generally arise from other cells within that disc (Gehring, 1966). One of the possible explanations of transdetermination, namely, somatic mutation, is generally ruled out as a possibility because the rate at which cells undergo transdetermination is much higher than the somatic mutation rate. From analyses of clones (Gehring, 1967), i t has been shown that allotypic structures can be made up of cells of two different phenotypes indicating that the allo-typic structures arise from two or more ce l l s . The possibility that mosaic allotypic structures arise from two independent transdetermina-tive events followed by aggregation is discredited because no salt and pepper mosaics are found. Instead, cells seem to group in mosaic clones within an allotypic structure. Thus, transdetermination is not due to somatic mutation. The possibility that the culture medium affects transdeter-mination cannot be ruled out, although i t seems unlikely. Disc cells that are cultured in larvae undergo transdetermination with the same frequency and the same direction as cells cultured in adult f l i e s , even though the composition of the hemolymph varies (Hadorn, 1963). In addition, i t has been demonstrated that the frequency of transdetermina-tion is directly related to the extent of a l l proliferation and not to the length of time spent in the hemolymph (Tobler, 1966). Apparently 21 the a b i l i t y of the programmed disc cells to undergo transdetermination is a property inherent to the discs themselves. However, Wildermuth (1968) has shown that the culture medium does have an effect on trans-determination in certain cases where discs from one species of Drosophila were cultured in another species. As mentioned, transdetermination seems to be associated with c e l l proliferation. By two different but unrefined techniques, it has been demonstrated that there is a linear correlation between growth rate and the frequency of transdetermination (Tobler, 1966; Wildermuth, 1968; Mindek, 1968). Wildermuth (1968) gave good evidence that trans-determination is restricted to dividing c e l l s . He observed that a labial disc which was cultured in an adult host for several days then implanted into a metamorphosing larva gave rise to a complete proboscis (generally this structure is formed from a pair of labial discs). The new half of the proboscis (a mirror image of the old half) arose by c e l l proliferation from the original labial disc. The new half of the proboscis could be distinguished from the old half by observing the orientation of the implant early in differentiation. Allotypic struc-tures frequently arose in these transplants and they always arose on the newly-formed half of the proboscis. Because of this correlation of transdetermination with ce l l proliferation, Hadorn has hypothesized that in cells undergoing transdetermination, specific carriers of determination which are normally replicated and passed on to their daughter cells to form autotypic structures, become diluted out when proliferation is rapid, thereby allowing for a shift in gene expression. One can speculate whether transdetermination occurs as a change from one determined state directly to another determined state 22 or i f the cells go from a state of determination to an undetermined stage (de-determination) and then choose a new state of determination. If the latter possibility is true, the mechanism of transdetermination might be more closely related to the mechanism of regeneration, except that in transdetermination one course of differentiation is exchanged for another. True regeneration (as distinguished from organ duplica-tion) has been known to exist in insects (see review, Bulliere, 1971) and has recently been discovered in Drosophila (Schubiger, in press). It is most interesting that some cases of transdetermination have been reported in vertebrate tissues which normally undergo regeneration (see Hay, 1968). C. Homeotic Mutants There exists a class of mutations whose phenotypic effects resemble the changes resulting from transdetermination. These mutations have been termed "homeotic" (Goldschmidt, 1945). Homeosis is defined as the replacement of one organ structure by another structure from an homologous organ (Bateson, 1894). One example of this is the replace-ment of part of the antenna by a leg tarsal segment in the mutant aristopedia (Balkaschina, 1929). Several different homeotic mutants have been described. These mutants, along with the structural modifications they effect, are listed in Table 1. Some of the homeotic mutants cited may not truly f i t the definition of homeosis owing to the d i f f i c u l t y in demonstrating homology between eye and wing tissue for example. In these homeotic mutants, as in transdetermination, the transformations of one structure into another is always complete. That TABLE 1 Known Homeotic Mutants Mutant Locus Reference Tissue Affected New Phenotype proboscopedia (pb) 3-47 Bridges & Dobzhansky (1932) probosc is antenna or tars' Antennapedia (Antp) 3-48 Lewis, 1956 antenna leg Nasobemia (Ns) 3-48 Gehring, 1966 antenna leg Polycomb (Pc) 3-48 Hannah-Alana, 1958 legs extra sex combs Multiple sex comb (Msc) 3-48 Tokunaga, 1966 2nd & 3rd legs 1st leg Extra sex comb (sex) 3-48 Hannah & Stromnaes, 1955 2nd & 3rd legs extra sex comb tetraltera (tet) 3-48.5 Goldschmidt, 1940 wing haltere tetraptera (ttr) 3-51.3 Astauroff, 1929 haltere wing aristapedia (ss a) 3-58.5 Balkaschina, 1929 arista tarsus bithorax (bx) 3-58.8 Bridges & Morgan, 1923 metathorax . mesothorax Contrabithorax (Cbx) 3-58.8 Lewis, 1964 mesothorax metathorax TABLE 1 (continued) Mutant Locus Reference Tissue Affected New Phenotype Contrabithoraxoid (Cbxd) 3-58.8 Lewis, 1968 metathorax abdomen (1st) Ultrabithorax (Ubx) 3-58.8 Lewis, 1964 metathorax mesothorax bithoraxoid (bxd) 3-58.8 Lewis, 1964 abdomen (1st) metathorax postbithorax (pbx) 3-58.8 Lewis, 1964 metathorax mesothorax loboid-ophthalmoptera ( l d O J * ) 3-102 Kobel, 1969 eye wing Haltere mimic (Hm) T (2,3) Lindsley & G r e l l , 1968 wing haltere leth a l (4)29 (1(4)29) 4 Gehring 1969 antenna 2nd & 3rd legs coxa, trochanter 1st legs, late pupal leth a l Hexaptera (Hx) 2 Herskowitz, 1949 prothorax wing (or leg) extra sex comb (esc) 2-54.9 S l i f e r , 1942 2nd & 3rd legs cf 1st legs Ophthalmoptera (Oph ) 2-68 Goldschmidt & Lederman-Klein, 1958 eye wing reduplicated sex combs (rsc) 1 Lindsley & G r e l l , 1968 2nd & 3rd legs d" extra sex comb ro 25 i s , no structures are formed which are intermediate between the auto-typic and a l l o t y p i c structures. This would seem to indicate that in both transdetermination and in homeotic mutants, s e l e c t i o n of one path-way of determination and d i f f e r e n t i a t i o n excludes the expression of other possible pathways. As was the case for a l l o t y p i c structures that resulted from transdetermination, the a l l o t y p i c structures produced by homeotic mutants are i d e n t i c a l to t h e i r autotypic counterparts in a l l respects. S i m i l a r l y , the c e l l u l a r a f f i n i t y of the a l l o t y p i c c e l l s is the same as' t h e i r autotypic counterparts. Thus, i f g e n e t i c a l l y marked c e l l s from t h i r d i n s t a r antenna discs of the homeotic mutant aristapedia are mixed with c e l l s from a normal leg disc and implanted into a larva, the marked a l l o t y p i c t a r s a l c e l l s aggregate and d i f f e r e n t i a t e with the wild-type leg c e l l to form a s a l t and pepper mosaic leg tissue (Garcia-Bellido, 1968). F i n a l l y , ordinary morphological mutations which a f f e c t normal legs or wings have the same e f f e c t on the a l l o t y p i c structures produce by the homeotic mutants a r i s t a p e d i a (Braun, 1940), bithorax and probos-copedia ( V i l l e e , 1946) and loboid ophthalmoptera (Cuweneel, 1970). It is concluded, therefore, that the new structure generated by a homeotic mutation i s b i o l o g i c a l l y complete and indistinguishable from i t s auto-typic counterpart. A l l homeotic mutants that have been tested, either by gener-ation of mosaics or by transplantation, have been shown to be autono-mous (Braun, 1940; Vogt, 1946b; Lewis, 1964; Roberts, 1964; Garcia-B e l l i d o , 1968). It is most in t e r e s t i n g to note that most of the known homeotic mutants map g e n e t i c a l l y within a rather r e s t r i c t e d segment of 26 the Drosophila chromosomes. Of the 22 mutants known, 17 reside on the 3rd chromosome between 47 and 58 map units, a genetic segment which comprises less than 5% of the total Drosophila genome.(Figure 5). Moreover, within this short region, there are two clusters of mutations; one between 47 and 48.5 contains 7 mutants and the other between 58.5 and 58.8 contains 7 mutants. Of the eight remaining mutants, one maps on the third chromosome at 102, another at 51.3 and a third, haltere mimic, is associated with the translocation between the second and third chromosome and thus may also reside on the third chromosome. This leaves five homeotic mutants known which do not reside on the third chromosome. Three of these are located on the second chromosome, one is on the X-chromosome and one maps on the fourth chromosome. These observations suggest that this small region on the right arm of the third chromosome is an important area involved with regulation of disc determination. The transformations caused by the homeotic mutations parallel the directed changes encountered in transdetermination (see Figure 3). However, some cellular transformations found in transdetermination experiments have no counterpart among the homeotic mutants. This could be due to the fact that some homeotic mutants are lethal and therefore not readily recovered. For example, the mutant, 1(4)29, which converts part of the antenna to the coxa of a leg and the second and third leg to the f i r s t leg structure, is generally a pupal lethal (Gehring, 1970). Similarly, spineless aristopedia (which convert the arista to a tarsus) is lethal when raised at 29°C ( G r i g l i a t t i , unpublished data). The converse is also true. Not a l l of the structural trans-formations caused by homeotic mutations have been observed in trans-FIGURE 5 General position of homeotic mutants on the Drosophila genome. X(l) - sex chromosome; 2L, 2R - left and right arms of the second chromosome; 3L, 3R - lef t and right arms of the third chromosome; 4 - the fourth chromosome. For the f u l l name of the homeotic mutants, see Table 1. X X 29 determination experiments. For example, the transformation of wing tissue to halteres (contrabithorax, tetraltera, and haltere mimic) has never been observed in transdetermination experiments. Nevertheless, the striking resemblance in characteristics at the phenotypic level produced by transdetermination or homeotic mutations, strongly suggests that the same molecular processes are being affected in the two processes. The best phenotypic expression of the homeotic mutation, oph-thalmoptera, is generated in the presence of other mutations which reduce the number of eye facets. The degenerated eye facets are often replaced by duplicated head structures which must arise by c e l l proliferation. Kobel (1968) suggested that these additional c e l l divisions might lead to the formation of allotypic structures by the same mechanism as trans-determination. Various theories as to the cause of homeotic mutations have been advanced. However, the causes of homeotic mutations and their relationship to transdetermination are s t i l l unresolved. D. In Vitro Cultures of Discs In addition to in vivo culture of imaginal discs,.various attempts have been made at maintaining the discs j j l vitro. However, i t is d i f f i c u l t and has been relatively unsuccessful until recently. Schneider (1964) was able to culture eye antennal discs in a medium which was defined except for the addition of serum albumin. The discs were cultured along with both cerebral hemospheres of the cephalic ganglion, the ventral ganglia, ring gland, aorta and lymph gland. She was able to obtain some differentiation of the antennal structure and a few cultures (about 10%) developed ommatidia and cornea and showed 30 some pigment deposition. A similar result was obtained by Hanly et a l . (1957). Hanly and Hemmert (1967) were able to show that the extent of bristle development in cultured eye discs was temporally related to the attachment of the eye disc to the cephalic ganglia. Kuroda (1969b), using a synthetic medium (K-6) (Kuroda and Tamura, 1956), was able to demonstrate the differentiation of ommatidia from eye discs in cultures to which ecdysone analogs were added. Fristrom et a_l. (1970) grew leg and wing discs in Schneider's medium supplemented with ecdysone and reported that evagination of the leg discs took about 10 hours, approx-imately 4 hours longer than ^ri vivo. Growing eye-antennal, wing and leg discs plus the cerebral complex of a defined medium (S), Mandaron (1970) reported no differ-entiation or evagination of the discs in the absence of ecdysone. How-ever, in the presence of ecdysone, most of the discs evaginated and differentiated the normal bristles, hairs or claws characteristic of the epidermis and, in addition, there was some deposition of chitin by the leg structures. Interestingly, no musculature was found in the leg structures that were formed and these legs did not grow in length. Leg and wing discs cultured without the brain or ring glands in a different medium (M) again showed no evagination (Mandaron, 1971). However, with ecdysone, the discs evaginated and thus he suggested that evagination is "an active process related to an intrinsic property of the disc". This is substantiated by several studies in vivo which showed that evagination coincides with the change of disc c e l l shape from columnar through cuboidal to flattened cells (Fristrom e_t a l . , 1970; Poodry and Schneiderman, 1970). Fristrom et a l , (1970) also reported that actinomycin D and puromycin inhibit evagination initiated 31 by ecdysone and therefore suggest that molecular synthesis is needed for normal evagination. Instead of using the c r i t e r i a of differentiation, Robb (1969) developed a defined media based on the efficiency and continuity of labelled uridine uptake. Discs cultured in his medium show linear 3 (though decreasing) incorporation of H-uridine into RNA for 48 hours. Recently, Schneider has developed a media in which cells derived from embryonic tissue w i l l proliferate. To date, disc cells have not been able to grow in this media (W. Gehring and C. Laird, personal communication). These recent advances promise to make extensive studies of discs _in vitro quite possible in the near future. In fact, these recent advances in culturing of discs has already stimulated some i n i t i a l biochemical investigations in RNA and protein synthesis in discs. E. Biochemistry Review Study of the biochemistry of disc development became feasible with the development of culture mediums for _in vi tro studies and through the development of a system for mass isolation of discs (Fristrom and Mitchell, 1965). The general patterns and dynamics of protein and RNA synthesis in third instar leg discs have been investi-gated and found to resemble those in other eukaryotic cells (Fristrom and Knowles, 1967; Fristrom, Brothers, Mancebo and Stewart, 1968). The effects of ecdysone on RNA synthesis in vitro have been investigated (Fristrom e_t al_., 1970; Raikow and Fristrom, 1971) and have shown that ecdysone stimulates an increase in both the rate of RNA precursor uptake and the rate of RNA synthesis. It causes an in-crease in heterogenous RNA synthesis, a preferential increase in ribo-somal RNA synthesis and accelerates the processing of 38S ribosomal RNA precursors to 28S and 18S subunits. It is suggested that there may be a causal connection between increased RNA synthesis and evagination of the discs and also that the increase in ribosomal RNA synthesis may be in connection with the transport of messenger RNA to the cytoplasm. No difference in the species of protein or RNA synthesized in different discs or discs in various stages of development have been reported. It is clear that a great deal of work is needed before we begin to understand the molecular basis of determination and di f f e r -entiation. 33 CHAPTER 2 GENERAL INTRODUCTION Determination and differentiat ion of imaginal discs poses questions of fundamental importance to an understanding of development (see Chapter 1). We may pose several questions worthy of investigation: (1) what is the role of ce l l communication in the regulation of the determinative process? (2) how does the development of neural tissue proceed? (3) what are the mechanisms involved in neural integration? and (4) does the development of the nervous system affect the dif ferent iat ion of other, non-neural tissues? In a l iv ing organism, determination appears to be a step-wise process by which the possible pathways of di f ferent iat ion open to a given ce l l become increasingly l imited. However, cultured discs from late third instar or prepupae seem capable of switching from an i n i t i a l program to an alternate pathway of d i f ferent iat ion. Whether the deter-mination of an imaginal disc as a complete entity is firmly barred from alternative fates only at the onset of d i f ferent iat ion, or whether there is a lock put on the i n i t i a l decision, remains to be seen. If the latter is correct, then transdetermination would involve an i n i t i a l de-determination with subsequent redif ferent iat ion. In this respect, transdetermination would be a process akin to regeneration. The method whereby cel ls recognize each other has been studied by mixing experiments. Upon mixing cel ls of different third instar discs, only cel ls of the same type of disc aggregated. Indeed, the speci f ic i ty was so great that cel ls from different regions of the same disc apparently segregate from one another. The question then 34 arises as to how c e l l s are able to define and recognize " s e l f " versus "non-self". It may be possible that c e l l s of different disc types share some of the early events associated with anlagen determination so that, for example, particular c e l l s from the young leg disc recog-nize as " s e l f " some c e l l s of the immature genital disc. If some common c e l l types exist among developing discs early in la r v a l l i f e , perhaps the ontogenic relatedness of discs could be correlated with the s p e c i f i c stepwise pattern observed in transdetermination. There is some evidence that c e l l migration may act as a mechanism of metamorphosis (Tokunaga, 1962). Thus, the sorting out and aggregation phenomena observed in implantation studies may be of significance to disc development _in vivo. Once a c e l l has been i n i t i a l l y determined, subsequent deter-mination and d i f f e r e n t i a t i o n can occur autonomously. How do external factors, such as hormones, affect a c e l l or group of ce l l s ? Do hormones act as temporal control of the onset of subsequent steps of determination, or do they control the direction of the determinative process? I n t r i n s i c factors, such as the rate of c e l l d i v i s i o n , have been shown to be correlated with the frequency of transdetermination. whether the increase in the rate of c e l l u l a r p r o l i f e r a t i o n results from the transdetermination process or whether i t i s the pr i n c i p a l cause or i n i t i a t o r of the transdetermination process, has not been established. If c e l l p r o l i f e r a t i o n does trigger transdetermination, then one might ask i f the rate of c e l l d i v i s i o n can affect the choice of the new pathway of d i f f e r e n t i a t i o n . What role does i n t e r c e l l u l a r communication within a disc play in the determination and d i f f e r e n t i a t i o n of that disc? An 35 efficient biological system demands that each c e l l must recognize the position i t w i l l occupy in the adult structure in order to complete the standardized pattern of the adult framework. It seems reasonable, therefore, that the cells must not only know that they are determined for a particular state, i.e., "legness", they must also know what their positional relationship is to their neighboring cells and to the disc as a whole in order to produce a specific structure of the adult appendage. The problems of pattern formation and prepatterns have been considered in an excellent review (Stern, 1968) and theoret-ical models on how positional information and pattern formation might occur have been discussed (Wolpert, 1969). It would seem that communi-cation between cells within a disc may be instrumental in the deter-mination of the final phenotype. Indeed, there is anatomical evidence for gap junctions that allow cytoplasmic connection between disc cells (Poodry and Schneiderman, 1970). Transdetermination appears to occur in a number of cells simultaneously and i t therefore seems reasonable to assume the prevalence of a system of intercellular communication within discs. Additional support for such a scheme has been presented (Grig l i a t t i and Suzuki, 1971). The origin of disc cells is ectodermal. The adult appendages which they produce are primarily epithelial yet they have well developed musculature and neural connections. The question arises as to whether the disc cells are entirely epithelial, or whether they contain pre-sumptive muscle and/or presumptive nerve c e l l s . Early workers observed "mesenchymal c e l l s " juxtaposed to the disc epithelium and thought they may have "invaded" the disc tissue (Newby, 1942; Auerbach, 1936). In transplantation experiments, only the genital disc differentiates muscular tissue, i.e., the contracting ejaculatory duct (Hadorn et a l . , 1949). Muscles have never been observed in metamorphosed transplants of leg or wing cell s , although both are supplied with muscles in situ (Hadorn and Buck, 1962; Schubiger, 1968). Poodry and Schneiderman (1970), using the electron microscope, detected disoriented aggregates of perhaps partially histolized muscle cells in metamorphosed trans-plants of leg discs. They further identified adepithelial cells in third instar discs _in situ which they suggest may give rise to muscle tissue. However, careful examination of these cells throughout devel-opment must be made before any definitive conclusions may be drawn. Even i f these adepithelial cells represent presumptive muscle c e l l s , how they come to be found in developing discs is s t i l l unknown. Mutant affecting muscle development and function might be extremely valuable in analyzing the relationships of the muscle and ectodermal c e l l s . Some third instar discs posses a neural connection to the brain, for example, the f i r s t and second leg discs, while others, for example, the third leg disc, lack this association (Auerbach, 1936; Bodenstein, 1950). Recent experiments in the housefly, Musca domestica have shown that the attachment of nerves to muscles is associated with muscle development in the legs (Bhaskaran and Sivasubramanian, 1969). The significance of neural connection for muscle differentiation and tissue regeneration in insects has been reviewed by Niiesch (1968). It would be of interest to establish what role, i f any, innervation plays in the determination and/or differentiation of the discs. On the other hand, i t may be that neural connection to the discs is important in the determination or differentiation of the adult nervous system. Our information on the nervous system of Drosophila, in general, its 37 development and its functional integration, is lacking. The study of mutants affecting the development of the nervous system, the innerva-tion of disc and muscle t issue, interneural and neuro-myal transmission and neural degeneration and regeneration may provide a valuable founda-tion for the understanding of the nervous system of higher organisms in general. Of the several problems discussed above, three have been chosen as the material for this thesis. The f i r s t , a c lass ical problem in developmental biology, attempts to determine whether control of the act iv i ty of a structural gene is direct ly imparted by the tissue in which the gene product is active, or whether, in fact , a freely flowing evocator of gene action exists. That i s , given a gene whose act iv i ty is required in several different organs during development, is this gene activated concomitantly in a l l organs in response to a general humoral factor within the body, or is the gene activated at different times during development in different tissues in response to t issue-speci f ic triggers? The second problem investigated concerns the poss ib i l i ty of isolating a temperature-sensitive homeotic mutant that expresses a wild-type phenotype at the permissive temperature and a fu l ly penetrant mutant phenotype under restr ict ive conditions. Such a mutant might provide the firm foundation on which various biochemical techniques can be investigated or refined for the purpose of approach-ing a molecular understanding of determination and the regulation of di f ferent iat ion in higher organisms. Studies related to the temperature-sensitive aspects of such a mutant may provide information as to the time of action of regulatory genes and may also provide information 38 on c e l l communication and pattern formation. The t h i r d problem is the p o s s i b i l i t y of i s o l a t i n g mutants a f f e c t i n g the nervous and/or muscle systems of Drosophila. Such mutants may be used to begin to investigate some of the problems ju s t mentioned as to the development of the nervous system, i t s functional integration, and the e f f e c t of innervation on the develop-ment of various t i s s u e s . A more complete introduction to each of these problems is presented at the beginning of i t s respective chapter. The u t i l i t y of conditional mutations whose expression i s temperature-dependent i s amply i l l u s t r a t e d in these problems. They allow one to overcome the problem that c e r t a i n homeotic, neural and muscle mutations might be l e t h a l . In addition, the conditional aspect provides a means for determining the i n t e r v a l during which the gene product is used. This, coupled with experiments using somatic cross-ing over, might allow one to determine the duration or interim between the t r a n s c r i p t i o n of a given gene and the time at which the product is u t i l i z e d or converted to a functional form. Not only would such assays permit the inv e s t i g a t i o n into possible roles of masked messenger RNA, et cetera, in development, but they also allow the investigator to control the onset of the change and the s e l e c t i o n of the develop-mental pathway. Thus, the i s o l a t i o n of temperature-sensitive mutations in Drosophila melanogaster adds new depth to the investigations of c l a s s i c a l topics and promises to open up new areas of i n t e r e s t in Drosophila. 39 CHAPTER 3 A TEMPERATURE-SENSITIVE MUTATION AFFECTING LEVELS OF PTERIDINES IN DROSOPHILA MELANOGASTER I. Introduction A major question in developmental genetics is whether a single gene whose function is known to be required in different tissues, is activated concomitantly in a l l of these tissues in response to a single trigger or responds to tissue-specific stimuli at different development-al stages. Since many of the fluorescent pigments found in adult eyes and testes and in larval and adult malpighian tubules of Drosophila  melanogaster are regulated by the same set of genetic loci (Ziegler, 1961), a single mutation affecting pigmentation in a l l three tissues might shed light on the problem. Flies carrying a recessive sex-linked mutation, l ( l ) E 6 t s (Suzuki, 1970), cause lethality at 29°C but result in a mutant eye color of viable adults at 22°C. Subsequent tests have indicated that, in addition to temperature-sensitive (ts) lethality, the presence and quantity of pteridines in the eyes, testes and mal-pighian tubules are also affected by temperature. The level of pigment in these organs was measured in f l i e s grown at 29°C during different developmental intervals in order to delineate the temperature-sensitive period for the presence of pigment. 40 II. Materials and Methods A. Genetic Analysis ts The mutation 1(1)E6 (abbreviated as E|6) was induced by ethyl methanesulfonate and found to be viable at 17°C and 22°C, but was lethal at 29°C. At 22°, the eye color of E6 males was obviously mutant, whereas homozygous females were visually indistinguishable from wild-type. The lethal was i n i t i a l l y localized genetically (Suzuki, 1970) close to v (33.0) (the symbol of a mutation is followed by its genetic position in parenthesis; for a complete description of the mutants used, consult Lindsley and Grell, 1968) between cv (13.7) and v and subsequent-ly positioned to the left of v within 0.7 map units by the markers \z_ (27.7) and v. A test of allelism of E6 with ras (32.8) revealed that E6/ras females were viable at 29° but had a mutant eye color. In order to determine whether ts lethality and the ts pigment phenotypes are caused by a single mutation, females heterozygous for E6 and m (36.1) were mated at 29° and the male offspring scored for the presence of surviving crossover progeny with the mutant eye phenotype. This experi-ment showed that E6 was 3.67 units to the l e f t of m and that no separa-tion of lethality from the pigment variant was found among 3,421 males scored. If, in fact, the lethal and eye phenotypes result from separate mutations, an upper limit of 0.09 map units (Stevens, 1942) separates the two sites; this makes i t probable that the two phenotypes result from a single mutation at the ras locus. B. Developmental Analysis The "effective lethal phase" (Hadorn, 1961), that i s , the time at which E6 f l i e s die when raised at 29°, was established by maintaining 41 eggs from the E6 stocks collected within 2 hours of deposition at 29°. The developmental stages reached were determined by inspecting the culture at 12 hour intervals. It was found that obvious autolysis and death occurred prior to the formation of any distinguishable adult structures, approximately 12 hours after puparium formation. The actual developmental interval of sensitivity to the high temperature (TSP) during which growth at 29° irrevocably commits the f l i e s to death was established by reciprocal "Shifts" at successive 12 hour intervals in which 29° cultures were shifted to 22° (Shift-down) and vice versa (Shift-up). The f i r s t Shift-down which gave a reduction in survival was taken to indicate the beginning of the TSP and the f i r s t culture in a Shift-up which yielded viable adults delimited the end of the TSP. By these c r i t e r i a , the TSP was shown to begin midway through the third larval instar and terminate just prior to pupation (Figure 6). It was immediately noted that the eyes of f l i e s hatching from a culture shifted up just after the TSP for lethality were dark brown in color. This suggested that, 1) defective pigment synthesis in testes and malpighian tubules might also be affected by high temperatures, and that, 2) TSPs for mutant pigment syntheses might be established. The Shift studies carried out to determine the TSPs for pigment production wi l l be described with the Results. C. Spectrofluorometric Analysis of Pigment Production The fluorescent pigments (FP) of the various tissues were separated by thin layer chromatography, using MN 300 cellulose with CaSO^ binder (Machery and Nagel) spread to a thickness of approximately 250u on glass plates. The plates were developed by ascending chromato-FIGURE 6 Percent of E6pupae which eclose after Shift-ups and Shift-downs at different times during development. The developmental states present at each culture age at 22°C and 29°C are shown at the bottom. 100 -i_ 0 0 0 0 0 0 .30 60 90 120 150 ISO 210 240 AGE OF CULTURE (HOURS) AT SHIFT I EGG I 1st INSTAR I 2nd INSTARi 3rd INSTAR I PUPA I ADULT DEVELOPMENTAL STAGE 2nd INSTAR , |EG6|lsf INSTAR | * | 3rd IN STAR | PUPA 44 graphy in the dark at 22° for three to four hours in solvents of either propanol: 1% NH^  water (2:1), or on occasion, butanol: acetate: f^O (20:3:7). The latter solvent had the advantage of resolving FP-3 to FP-6 better than the propanol: NH^  water solvent; however, i t did not resolve (or only poorly resolved) FP-7 to FP-9. Consequently, the propanol: NH^  water solvent was used for the bulk of the experiments. The plates were allowed to dry for one hour in a fume hood and were then examined with a U.V. light source and scored visually. The quantity of each fluorescent pigment was determined spectrofluorometrically using an Aminco Bowman scanning fluoromicrophotometer with a Varicord recorder. Eye pigments were obtained from intact heads of adult f l i e s decapitated by a microscalpel. The heads were placed directly onto the thin layer cellulose plates and crushed with the end of a glass rod in order to release pigment directly onto the cellulose thin layer plates (Hadorn and Mitchell, 1961). Pigments from the testes and malpighian tubules were more d i f f i c u l t to obtain. Testes were removed from adult f l i e s by micro-dissection in Drosophila Ringer's solution (Ephrussi and Beadle, 1936) and transferred to the thin layer plates with a minimum of accompanying D. Ringer's solution. One pair of testes from a single fly was used per run. Malpighian tubules of either 3rd instar larvae or adult f l i e s were dissected and chromatographed in the same manner. While the fluor-escent pigment of malpighian tubules from single individuals (4 strands) was sufficient for quantitation, generally malpighian tubules from two identically-treated individuals were pooled to increase the amount of pigment per run and the accuracy of our measurements. Crushing of the testes and malpighian tubules was not necessary for good recovery of fluorescent pigment. 46 III. Results A. Temperature effects on eye and testis pigmentation. The TSP for eye and testis pigmentation was determined in the following manner: 15-20 prepupae from a 22° culture were collected within a 15 minute interval and shifted up to 29°. Starting twelve hours after this Shift-up, different vials of pupae were shifted down again at successive one hour intervals until 70 hours after prepupal formation. Adult f l i e s from each v i a l were counted and their eyes and testes analyzed for pigment production as described above. The percent eclosion (number of adults/number of pupae) for each v i a l was calculated in order to show that the TSPs for lethality and eye-testes pigmentation are definitely separable. The heads of a total of 362 E6 and 173 Oregon-R f l i e s raised at different temperatures (17°C, 22°C, 29°C) were chromatographed and compared. It was found that anywhere from 7 to l l fluorescent pigments could be separated from both the mutant and + strain, depending on the preparation and the quality of the thin layer plate. Generally, 8 pig-ments were resolved easily with the propanol: ammonia solvent. These pigments were designated as FP-1 for that fluorescent pigment running closest to the origin (smallest Rf) to FP-8 for the fluorescent pigment running nearest the front. No attempt was made to characterize the pig-ments by their absorption or fluorescent spectra. However, by comparing our plates with the data and descriptions of other workers (Hadorn, 1958, 1962; Narayanan and Weir, 1964), some pteridine pigments could be tenta-tively identified. The two eye pigments consistently affected in E6 by growth at high temperature were FP-1 and FP-8; FP-1 corresponds to drosopterin. The drosopterin level in the mutant raised at 29° was 47 decreased by about 30% as compared to Oregon-R (Figure 7A). At 22°, E6 female eyes were visually wild-type whereas males were distinguishably mutant; at 17°, the eyes of both females and males were indistinguishable from wild-type. Chromatographically, drosopterin levels in f l i e s raised at 17° were 80% in E6 males and 90% in E6 females of those in comparable Oregon-R f l i e s . It is interesting to note that the drosopterin level in females raised at 29° was approximately equal to that in males raised at 22° and the level in females raised at 22° approximated that of males raised at 17°. From Shift studies, the TSP for eye pigment formation was determined to begin 52-56 hours after pupation at 29°. The end of the TSP was not measured but i t was assumed to continue until eclosion (Counce, 1957). The pigment content of the testis differed somewhat from that of the eye and malpigian tubules. Only five fluorescent pigments were consistently separable from the testes of both the E6 mutant and Oregon-R males. The drosopterin pigment was either missing, or masked by the bright blue FP-1 (isoxanthopterin) of the testes in both the E6 mutant and Oregon-R males. While FP-2 was present in E6 males grown at 22° or 17°, i t was absent in the E6 males shifted to 29° at the onset of pupa-tion (Figure 7B). The level of FP-1'found in males raised at 22° or 17° never reached the level of the wild-type controls. The Shift studies showed that the onset of the TSP for pigment synthesis in testes at 29° occurred 56-60 hours after pupation which was very close to the TSP for eye pigment. The end of the TSP for pigment formation in testes was not determined. The percent eclosion in the various vials varied from 70% to 90% of the rate of survival determined for E6 at 22°. Therefore, the TSP for lethality was definitely prior to FIGURE 7 The drosopterin (FP-1) levels (with confidence limits) in the eyes of _E6 females relative to the level of Oregon-R females raised at 29°C. The distribution of fluorescent pigments in thin layer chromatograms of testes of E6 and Oregon-R males raised at different temperatures. The arrow indicates the pigment (FP-2) measured to determine the TSP. The drosopterin (FP-1) levies (with confidence limits) in the malpighian tubules of E6 f l i e s relative to the level in Oregon-R f l i e s raised at 29°C. DROSOPTERIN L E V E L (% OF CONTROLS) Ol O o o o o — cn o o 0 0 o 10 o 50 the TSPs for eye and testis pigmentation. B. Temperature effects on malpighian tubule pigmentation The effects of temperature on pigment production in the mal-pighian tubules was determined by growing cultures of E6 at the restric-tive temperature until 3rd instar and then shifting them down to 17°. Pigments in malpighian tubules from either 3rd instar larvae or adult E6 f l i e s were chromatographed following the Shift-down and compared with those of E6 larvae or adults raised entirely at 17° and Oregon-R f l i e s from high or low temperatures. The beginning of the TSP for pig-ment synthesis in the malpighian tubules was determined by collecting eggs laid within one hour at 29° and shifting individual cultures down to 17° at successive four hour intervals until the 36th hour. The mal-pighian tubules of 3rd instar larvae were scored for pigment by quanti-tative TLC. The end of the TSP for malpighian tubule pigment synthesis was delineated by sequential Shift-ups at successive 12 hour intervals of cultures established from two hour egg lays. Shift-ups were carried out from the time of egg deposition to late third larval instar stages and malpighian tubules removed from late third instar larvae were assayed for pigmentation. The fluorescent pigments found in the malpighian tubules were the same as those in the eyes. FP-1 (drosopterin) was nearly absent in malpighian tubules in third instar E6 larvae raised at 29°, while i t was detectable in E6 larvae raised at 22° and increased to approximately 50% of that found in Oregon-R larvae at 17° (Figure 7C). FP-8 was not present in sufficient quantity in either Oregon-R or E6 malpighian tubules to permit quantitative comparisons. 51 Shift studies indicated that the TSP for pigment synthesis was from approximately 12 hours after egg deposition at 29° to about 120 hours at 17°. The end of the TSP for pigment synthesis (delineated at 17°) corresponded to the end of the 2nd larval instar. To summarize, for f l i e s growing at 22°, the TSP for malpighian tubule pigmentation was 12 hours to 90 hours, for lethality 153 hours to 187 hours, for eye pigmentation 224 hours to eclosion, and for testis pigmentation 228 hours to eclosion (Figure 8). C. Autonomy of pigment formation in different organs and pigment turnover Since the TSPs for pigment synthesis varied from organ to organ, i t is tempting to conclude that the time of gene activity varies from organ to organ. Indeed, Stern and Tokunaga (1968) demonstrated the autonomous pleiotropy of the spl mutation in eyes and mesonota. However, in our experiments, the presence of pigment in a tissue does not preclude transport of the molecules from other tissues and therefore, the presence of pigment in a tissue cannot be assumed to reflect its synthesis in that tissue. A demonstration of tissue autonomy for pigment production would strengthen the suggestion that pigments are synthesized at the site of deposition. The f i r s t experiment consisted of a series of Shift studies. Cultures collected within a two hour period at 29° were shifted down to 22° at 80 hours (mid-3rd instar) just prior to the TSP for lethality. Half of these f l i e s were left at 22° until eclosion (Group A) while the other half was shifted back up 12 hours after pupation (Group B). Thus, f l i e s in Group B were kept at 29° except during the TSP for lethality. FIGURE 8 The temperature-sensitive periods in E6 for lethality and pigment levels in the eyes, testes and malpighian tubules (on a 22°C time scale). M T i 1 1 1 —i h - — i 1 1 0 30 60 90 120 150 180 210 240 |<-EGG->|«-lst lNSTAR-^2nd INSTAR>|« L 3 r d INSTAR >|« PUPA ADULT AGE (HOURS AT RT) AND DEVELOPMENTAL STAGE FOR EACH TSP 54 Concomitantly, eggs were collected at 22° and half were shifted up 12 hours after pupation (Group C) and the other half left at 22° until eclosion (Group D). The adult f l i e s that emerged from these Shift studies were scored for eye, testis and malpighian tubule pigmentation. The results of the Shift studies are shown in Table 2. It can be seen that the mutant malpighian tubule phenotype was determined irreversibly by early exposure to 29° whereas growth at low tempera-tures up to pupation did not affect eye or testis pigmentation after a Shift-up. These experiments ruled out significant transport of pigment from the malpighian tubules to the eyes and testes and vice versa, a result supporting the autonomy of each tissue. Tissue autonomy of pigment synthesis was also tested in V c mosaic females using the unstable ring X chromosome, In(l)w (Hinton, V c t s 1955). In(l)w /1(1)E6 females were raised continuously at 29° and the adults scored for the presence of mutant eye tissue. Since the VC somatic loss of In(l)w produced cells hemizygous for E6, the observa-tion that mutant patches of eye tissue were readily detected in a wild-type background constituted the unequivocal genetic proof of autonomy of E6. Several bilateral mosaics for external genitalia were also observed. Both approaches suggest that the E6 gene is acting autonom-ously in a l l three organs. 55 TABLE 2 Phenotypes of malpighian tubules and eyes after each kind of Shift (see Figure 3). Temperature Shifts Type of Shift Malpighian Tubules* Phenotype Eyes** Testes-C D Shift-down before TSP Shift-down before and after TSP Shift-up after TSP 22°C control mutant mutant wild-type-like wild-type-1 ike wild-type wild-type mutant mutant mutant mutant wild-type wild-type based on chromatographic determined visually profile 56 IV. Discussion It has been shown that a mutation which results in a mutant phenotype in different tissues acts autonomously in each tissue (Stern and Tokunaga, 1968). Our experiments have corroborated this and have demonstrated that the amounts of those fluorescent pigments affected by E6 are sensitive to temperature in different organs at different times during development (Figure 8). Since the genetic mapping studies indicated that a single mutation was responsible for both lethality and pteridine synthesis or deposition, at least three separable TSPs have been delineated during development. We assume that the lethal effect of E6 occurred in tissues other than the organs scored chromatograph-ic a l l y since mutant patches of eye and external gonadal tissue were observed in mosaic f l i e s raised at 29°C. The significance of these results is dependent upon the inter-pretation of the TSP in molecular terms. In micro-organisms, ts lethal-ity has been shown to be a consequence of missense mutation which results in the thermolability of the polypeptide gene product (Jockusch, 1966). The genetic properties of ts lethal mutations in Drosophila strongly suggest a basis for temperature-sensitivity similar to that in micro-organisms (Suzuki, 1970). The genetic proof that expression of the E6 phenotype in each organ was autonomous suggests that the E_ locus does indeed function in the organs in which pigment was measured. However, i t should be stressed that the concentrations of the pigments measured were quite likely an indirect result of the activity of the E6_ locus since many different pteridine and ommachrome eye mutants affect the amount of drosopterin produced (Hadorn and Mitchell, 1951). Furthermore, the complete via-57 b i l i t y of white eyed mutants which are totally lacking in pigments shows that the fluorescent compounds per se are not necessary for via-b i l i t y . Hence, i t could be suggested that a single period of genetic activation (transcription) occurs early in development in a l l tissues; the resultant gene product could be a long-lived masked messenger RNA (Tyler, 1967) or an inactive polypeptide (Neurath, 1964; Steiner and Oyer, 1967; Steiner e_t al_., 1967) with a temperature-sensitive trans-lational or activation mechanism, respectively. Although these possi-b i l i t i e s cannot be eliminated, their contrivance and special assump-tions required, render them unattractive. Waddington's experiments (Waddington and Robertson, 1969) showed that pigment synthesis in optic discs was suppressible by actinomycin D treatment in early pupation, a result strongly supporting the hypothesis that the synthesis of RNA and proteins involved in pigment formation occur close to the time of pig-ment production in the eye i t s e l f . It is therefore suggested that the E6 locus is activated in different organs in response to tissue specific stimuli at different developmental stages. The occurrence of the TSP for a mutant phenotype following pupation suggests a number of experiments. The effects of ecdysone on macromolecular synthesis in imaginal discs may be tested on the induc-tion of pigmentation in explanted and transplanted imaginal discs. The discs in which E6 manifests its lethality may also be indirectly detected using somatic elimination of the unstable ring X chromosome. For example, the recovery at 29° of viable mosaics in which one eye was wild-type while the other was mutant in color or which carried male genitalia indicates that lethality due to E6 does not occur in tissues derived from eye and genital imaginal discs. Nolte (1959, 1961) has shown by light and electron microscopy that ras , a non-ts al l e l e of E6_ which also manifests depressed levels of drosopterin, has a disorientation of the secondary pigment cells of the eye and some irregularly shaped pigment granules. Of immediate interest is whether temperature affects the normal formation of secondary pigment cells and pigment granules or transport and deposition of pigment in the eyes of the E6 mutant. 59 CHAPTER 4 a h d a THE TEMPERATURE-SENSITIVE HOMEOTIC MUTANT, ss I. Introduction Hadorn's (1965, 1967) demonstration of "transdetermination" in Drosophila, that i s , a change in determination of larval imaginal discs which results after repeated transplants through adult hosts, has renewed interest in the problem of disc determination. His experi-ments demonstrated that the rigid program in the discs could, neverthe-less, be altered at the phenotypic level. Furthermore, the sequence in changes was highly specific, that i s , genital discs could not be transdetermined directly to wings, rather they f i r s t had to be altered to legs and then to wings. Biochemical studies of transdetermination are d i f f i c u l t owing to the mechanical limitations of transplantation techniques. However, a class of mutations called "homeotic" mimics the kinds of phenotypic changes in determination resulting from serial trans-plantation (Goldschmidt, 1945). By correlating the time of X-ray-induced somatic crossing over during antennal disc development, the switch in disc determination from antennal to leg triggered by the homeotic mutant, Antennapedia, was inferred to occur after the beginning of the 3rd larval instar stage (Postlethwait and Schneiderman, 1969). The expression of the homeotic mutant, proboscopedia (p_b), was found to be dependent upon temperature (Villee, 1944). By shifting cultures from one temperature to another, the temperature-sensitive period (TSP) for the expression of this mutation was also found to occur in the third instar stage 60 (Vogt, 1946a). Extensive genetic, developmental and biochemical studies of the mechanism of homeotic effects have been hampered by low pene-trance and variable expressivity of such mutants. This chapter concerns the developmental effects of a selected strain of the temperature-a40a sensitive (ts) homeotic mutation, ss , which was completely penetrant. 61 II. Materials and Methods In a search for a fu l l y penetrant ts homeotic mutant, a l l homeotic mutations currently available from stock centers were tested for their penetrance and the degree of expression of the mutant pheno-type at 29°, 22° and 17°C. Flies homozygous for an a l l e l e of spineless-a40a aristapedia, ss (see Lindsley and Grell, 1968), a mutant which transforms the arista of the antennal complex to a tarsus-like struc-ture characteristic of the distal part of legs, were totally wild-type when raised at 29°C and showed a high penetrance of the mutant pheno-type at 17°C. Individuals expressing a total transformation of aristae to tarsi at 17°C were selected and inbred for 8 generations to produce the stock reported here. The fourth and f i f t h antennal segments and the arista (the sixth antennal segment) were tarsus-like at 17°C. The normal legs generally did not show swelling and fusion of the four tarsal joints usually characteristic of s s 3 f l i e s and in males, the sex combs were neither enlarged nor present on the second pair of legs. The bristles were essentially wild-type. Virtually a l l of the f l i e s had normal aristae when raised at 28°C (Figure 9a). At 22°C, about 85% of the f l i e s had a protrusion of a leg-like structure from the third antennal segment with a normal though somewhat shorter arista extending from the tarsal elements (Figure 9b). Expressivity of the mutant phenotype at 22°C varied from a slight swelling at the proximal end of the arista to a leg-like struc-ture which was approximately half the total length of the arista. About 15% of the f l i e s grown at 22°C had normal aristae with no leg-like structures. At 17°C, the aristae of over 98% of the f l i e s were complete-ly transformed to tarsal segments of the leg (Figure 9c). Although FIGURE 9 scanning electron micrograph of ss raised at: 28°C 22°C 17°C a wild-type Oregon-R raised at 22°C, magnification approx-imately 280X. 65 transformation of aristae to leg-like structures was not complete in a few f l i e s , the mutation was 100% penetrant at 17°C. a40a The temperature-sensitivity of ss permitted a delineation of the actual developmental interval during which the expression of mutation could be altered. This was accomplished by collecting eggs on petri plates for a one hour period, transferring groups of 50 to 75 eggs to individual vials and shifting such cultures established at 17° and 28°C to 28° and 17°C, respectively, at successive times (Suzuki, 1970). This provided a crude delineation of the TSP, which was then determined more accurately by a second series of experiments which were carried out in the following manner. Asynchronously developing cultures were established at 28° and 17°C. As soon as the f i r s t pupa was detected, the cultures were shifted to 17° and 28°C, respectively. Newly formed prepupae were collected at 1 hour intervals, placed into vials and lef t to develop with no further shifting. In this manner, the interval between the shift and pupation could be accurately defined The phenotypes of f l i e s hatching after the shift were then determined. The developmental interval during which temperature affected a40a the expression of ss was determined from the shift experiments by the following rationale. Flies hatching from cultures shifted down prior to the TSP wi l l pass through that interval at the low temperature and therefore will express the mutant phenotype, whereas cultures shift down after the TSP w i l l yield wild-type adults. Reciprocal results are expected from the shift-up experiment. The TSP could be readily timed in this way. The phenotypes of f l i e s shifted within the TSP were of special interest. The discs producing jointed appendages represent con centric rings of cells which evaginate to form each segment. We may 66 ask what cells within the disc are affected by temperature. The obser-vation of a change at the aristal base at 22°C rather than a salt and pepper mosaic of leg and ari s t a l structures showed a polarity of effect on the adult organ. If the events leading to the transformation of aristae to leg structures resulted from a wave or gradient of deter-minative substance passing through the antennal disc, then one might expect that aristae of f l i e s shifted up during the TSP would show a leg-like structure in the place of the proximal portion of the arista with a normal ari s t a l segment protruding distally from a leg-like base (Pattern I, Figure 10). Similarly, f l i e s hatching from cultures shifted down during the middle of the TSP might be expected to show a normal arista from which a leg-like structure protruded distally (Pattern II, Figure 10). Therefore, f l i e s hatching from shift cultures were scored for the relative amount and location of leg and arista tissue and each arista was placed into one of ten categories shown in Figure 10. Finally, in order to enhance the change of the antennal com-a40a plex to leg structures, ss was combined with the homeotic mutant, Antp (Lewis, 1956) which converts the antenna into leg structures. T, _ a40a ^ . a40a B J . ^  a40a ^ , The structure of ss +/ss Antp was compared with ss +/ , * . B a40a . a40a t „„„_, + Antp and ss +/ss + at 22°C. FIGURE 10 Diagram of the entire antennal complex. Shows the possible pheno-types of f l i e s hatched from various shift experiments. Pattern I (a) normal aristaj (b) proximal 1/4 of arista trans-formed to tarsus, distal 3/4 normal arista; (c) prox-imal 1/2 transformed to tarsus, distal 1/2 arista; (d) 3/4 tarsus, 1/4 arista. Pattern II (a) proximal 3/4 of arista is normal while distal 1/4 is transformed to tarsus; (b) proximal 1/2 of arista normal, distal 1/2 transformed to tarsus; (c) proxi-mal 1/4 arista, distal 3/4 tarsus; (d) complete transformation to tarsus; (e) salt and pepper pattern of mixed arista and tarsus tissues; (f) stripes of arista and tarsus tissues. 69 I I I . Results and Discussion These studies demonstrate that highly penetrant ts homeotic mutations can be recovered. This result is of considerable importance since bulk preparations of discs in a homogeneous state are required for biochemical studies of the mechanisms of determination. The mutant a40a expression of ss occurs at 17°C; therefore, the f i r s t culture in shift-up experiments which produces adults with a leg-like transforma-tion of the arista indicates the beginning of the TSP. Similarly, the f i r s t shift-down culture which yields wild-type f l i e s signals the end of the TSP. By these c r i t e r i a , the TSP was approximately 6 hours in duration at 28°C and 18 hours in duration at 17°C, and occurred during the second quarter of the third larval instar stage (Figure 11). This is a very short TSP and may be suggestive of a c r i t i c a l event during disc development when the final "lock" on the fate of those cells occurs. The TSPs of the other two alleles of spineless-aristapedia and of proboscopedia also have been shown to occur sometime during the third larval instar stage (Vogt, 1946a; Vill e e , 1943). Mosaic spots generated by somatic crossovers have shown that Antp also affects the antennal discs during the third larval instar stage (Postlethwait and Schneiderman, 1969). Clearly, the change in disc fates caused by homeotic mutants occurs during the third larval instar long after the i n i t i a l determinative events have occurred (Gehring, 1969). These mutants suggest that c r i t i c a l determinative events occur early in the third larval instar at a time when extensive c e l l division and prolifer-ation take place in the imaginal discs. We are currently attempting to determine whether ts homeotic mutations which affect earlier determina-FIGURE 11 Results of shift studies - closed circles are from shift-up experiment; open circles from shift-down experiment. growth curve(28°) 72 tive events can be recovered. What is s t i l l uncertain is whether the homeotic mutations represent a class of genes which are active from the post embryonic stages (after the time of i n i t i a l determination of the discs) through the 3rd instar stage; or whether, in fact, these genes are active only during the third larval stage of development. Experi-ments are under way which may distinguish between these two alternatives, It is interesting to note that of the homeotic mutants which are affected by temperatures, a l l are sensitive to cold temperature and only one, proboscopedia, is also affected by exposure to high tempera-ture. In micro-organisms, cold sensitivity of mutations appears to be a property more common among those concerned with regulation or mole-cules which can self-assemble (Guthrie, _ t al., 1969). It is of interest whether this suggests the regulatory mechanism of homeotic l o c i . Selection for cold-sensitive homeotic mutations may increase the yield of such mutants. Transplantation experiments have shown that the mutant s s a a40a (an allele of ss; ) acts autonomously (Braun, 1940). This indicates a40a that the temperature-modifiable changes in determination of the ss does not result from a freely circulating "humoral" factor. Shift studies during the TSP of N____, a temperature-sensitive mutation which disrupts eye facet arrangement, revealed a wave of facet orientation that proceeded from the posterior rim of the eye anteriorly (Foster and Suzuki, 1970). It was asked whether such a wave or gradient of determination also occurred in the aristae in res-a40a ponse to ss . Shifts up and down were carried out at different times during the TSP and the aristae of the adults examined. It was found that individuals shifted up during the early part of the TSP had leg-73 like structures only in the proximal part of the transformed arista, whereas after shifts up at successively later times within the TSP, the leg structures increased distally as the size of the arista retreated. However, unlike the facet pattern, shifts down during the TSP did not yield a reciprocal pattern. Shifts down early in the TSP yielded aristae that were transformed to legs proximally with aristal tissue di s t a l l y . Successively later shifts down gave more arista proceeding proximally. These results are summarized in Table 3 and indicate that the change in determination is not simply the reflection of a wave or gradient of determination passing through the disc or to a ts receptor site for such a gradient. The results may indicate that there is significant c e l l movement within late third instar discs. Alternatively, there could be a site within the antennal disc which, in response to cold temperature, produces a substance which alters the final deter-mination of the existing pre-determined aristal c e l l s . „ , . , _ - I I A , - b a40a , a40a , , Flies which were genotypically Antp ss /+ ss had the entire antennal complex transformed into a complete leg-like structure at 22°C (Figure 12). The mutant phenotype was completely penetrant. In a few cases, the transformation was so complete that the males had sex combs on their antennal legs. These two mutations supposedly affect different segments of the antenna and when studied separately in the original stocks, expression of each mutant at 22°C was quite low. The fact that the two mutants interacted for 100% expressivity and a total transformation of antenna to leg, leads us to ask whether c e l l communication within discs may be important in determining the final phenotype. This question is currently under investigation. 74 TABLE 3 Expected and actual r e s u l t s from s h i f t s during the TSP.* S h i f t down S h i f t up A Pattern I d > a Pattern I a > d B Same as above Pattern II a > d C Pattern II d >a Pattern I a >d A. These are the actual r e s u l t s from the s h i f t experiments. These do not agree with ei t h e r of the expected r e s u l t s i f the change in determination i s due s o l e l y to a wave or gradient of determination passing through the d i s c s . B. These are the r e s u l t s expected from s h i f t experiments i f a gradient of determination passes from anterior to posterior through the d i s c . C. These are the r e s u l t s expected from s h i f t experiments i f a gradient of determination passes from posterior to anterior through the d i s c . * The term "Pattern" re f e r s to diagrams in Figure 10. FIGURE 12 1 <- • u ^ . B a40a. a40a . . A scanning electron micrograph of Antp , ss /+ ss raised 22°C, magnification approximately 210X. 77 CHAPTER 5 A TEMPERATURE-SENSITIVE MUTATION (para t S) CAUSING ADULT PARALYSIS IN DROSOPHILA MELANOGASTER I. Introduction The genetic regulation of neural structure and function and its relationship to behavior are of fundamental biological interest. The ready induction and recovery of mutations affecting behavior and their genetic manipulation in Drosophila are now being exploited in a number of laboratories (Hotta and Benzer, 1969, 1970; Pak e_t a l . , 1969; Ikeda and Kaplan, 1970a). By selecting f l i e s manifesting the phenotype of paralysis, we fel t that mutations affecting nerves and/or muscles could be efficiently obtained. However, f l i e s exhibiting such a mutant phenotype would not be expected to be viable; therefore, we searched for mutations that showed conditional paralysis that was temperature-dependent (Williamson et a l . , 1970; G r i g l i a t t i et a l . , 1970). This chapter concerns the discovery and properties of such a mutation, ts paralytic-temperature-sensitive (para ), in Drosophila melanogaster. 78 II. Materials and Methods Newly eclosed adult Oregon-R males were fed the mutagen, ethyl methanesulfonate (0.025M) dissolved in a 1% sucrose solution (Lewis and Backer, 1968). Twenty-four hours later, they were mated at 22°C to attached-X-bearing females in quarter pint bottles. Adult progeny were then placed in a preheated plexiglass screening apparatus which allowed the ready separation of immobilized f l i e s from those re-taining normal movement (Williamson, 1971). One to eight thousand adults at a time were placed in the box and lef t for 1/4 to 2 hours before selection. A l l motionless f l i e s were then returned to 22°Cj any which regained mobility were mated (males to XX females, females to Oregon-R males) and a l l offspring of f e r t i l e individuals were tested for paralysis at 29°C. The specific properties and details of analysis of the ts paralytic mutant recovered w i l l be discussed in the next sect ion. 79 II I . Results Table 4 shows the results of the screening. The bulk of the immobilized f l i e s recovered were dead or sterile at 22°C. However, out of an estimated quarter of a million f l i e s screened, one was detected which carried a mutation causing a temperature-sensitive paralysis. A. Genetic Properties The ts paralyzed fly was a male and a l l of its male progeny were paralyzed upon shifting to 29°C whereas the females were not, thereby showing this mutation to be sex-linked. Females heterozygous for the mutation were not paralyzed whereas homozygous females were. Consequently, the mutation was named paralytic-temperature-sensitive, ts ts para The mutation was mapped genetically by crossing para males to females carrying the markers (see Lindsley and Grell, 1968) (followed by their genetic positions): y_ (0.0), cv (13.7), v (33.0), f_ (56.7) and ts car (62.5) and testcrossing the F^ females at 22°C. The para and + male progeny of the testcross were separated at 29°C and then scored for the visible markers. The mutation was readily located 2.8 units to the left of f_ (2,993 males scored). B. General Biological Properties ts The effects of temperature on para f l i e s were then studied in detail. At 22°C, the mutants exhibited normal walking and flying ts a b i l i t y . When adult para f l i e s were shifted from 22°C to 29°C, com-plete paralysis was induced in less than 5 seconds; upon shifting the paralyzed f l i e s back to 22°C, mobility was recovered in less than 2 seconds. Paralysis and recovery could be induced repeatedly in the 80 TABLE 4 Results of screening adult offspring of mutagenized f l i e s for paralysis at 29°C. Type of f l i e s Number Total f l i e s screened (estimated) 250,000 Immobilized at 29°C 293 Dead 200 Recovered mobility at 22°C 93 Fertile at 22°C 34 Temperature-sensitive paralytic mutations 1 81 same individuals with no apparent harm. Flies maintained at 29°C for several hours recovered normal mobility immediately upon shifting down to 22°C. While paralysis was i n i t i a l l y complete at 29°C, a recovery of some movement at this temperature could be seen. After 30 minutes at 29°C, the f l i e s were able to right themselves and regained limited walking a b i l i t y . After an hour or more the f l i e s were capable of climb-ing up the sides of the via l s . However, i t must be emphasized that these f l i e s were visibly weak and never regained the strength and co-ts ordination which para f l i e s showed at 22°C. Flies which had regained mobility after 2 hours at 29°C immediately recovered wild-type behavior upon shifts to 22°C and were paralyzed again upon shifting back to 29°C. ts Preliminary tests suggest that para f l i e s l e f t at 29°C for 12 hours, then shifted down to 22°C for 5 minutes and back up to 29°C are not paralyzed. Thus, i n i t i a l 29°C recovery remains temperature labile, whereas long-term recovery at 29°C suggests a different basis for move-ment . ts In order to determine whether para also affected larval mobility, two tests were carried out. A direct test was made by placing 3rd instar larvae reared at 22°C into Drosophila Ringer's (Ephrussi and Beadle, 1936) solution at 29°C. The larvae continued to move normally for several hours and were totally unaffected by the increased tempera-ts ture. A second method compared the relative fitness of para individuals with wild-type f l i e s at 29°C. Attached-X-bearing females (which were ts ts wild-type with respect to para ) were crossed to para /Y males for 24 hours at 22°C. The inseminated females were allowed to lay their eggs for 3 days in fresh bottles at 29°C. The F^ progeny were left to develop at 29°C until adults began to emerge, whereupon 219 late pupae 82 were collected, placed in fresh vials and half were shifted to 22°C. ts Twelve hours later, the f l i e s had eclosed and the ratio of para /Y males to wild-type females was scored. At 22°C, the sex ratio was ts 97c/:122$, showing that para males had competed successfully with their wild-type sisters up to pupation at 29°C (females tend to hatch sooner than males). In the sample left at 29°C, a ratio of 83c/:215? was obtained. However, when the number and sex of adults found dead in the food and unhatched in their pupal cases was scored and included in the sex ratio, the ratio returned to 230c/:240$. It was concluded ts that the para mutation does not interfere with the development of the larval or early pupal stages and therefore is expressed only in differentiated adults. The fact that some males did eclose at 29°C ts could be accounted for by the earlier observation that para adults recover some mobility after prolonged exposure to 29°C. The hatched males had obviously recovered sufficiently to crawl out of their pupal cases. ts The effect of prolonged exposure of para adults to different temperatures was then examined in order to determine the degree to which movement, co-ordination and recovery of activity were affected over a wide temperature range. A dissecting microscope was mounted over two identical water-tight chambers immersed in a temperature-controlled ts circulating water bath. Adult para and + f l i e s (at least 40 each) which had been raised at 22°C were placed into separate chambers and observed continuously for two hours at each temperature. Observations were made on different f l i e s at one degree intervals between 22°C and 35°C. A l l movements from flying to tarsal twitching of individual legs were recorded; however, for analytical purposes, the behavior during 83 recovery has been classified into six categories: complete paralysis, kicking, a b i l i t y of f l i e s to right themselves, walking, climbing and flying. The results are presented in Figure 13. The units separating the categories on the ordinate are arbitrary so that the shapes of the curves are not especially significant. Oregon-R wild-type f l i e s were not visibly affected over the entire range of temperatures from 22°C ts to 37°C On the other hand, para f l i e s behaved normally only up to 25°C. They were visibly debilitated between 26° and 28°C and completely paralyzed at higher temperatures. With increasing temperatures, the phenotypic effects of the mutation became more severe and the length of time required for recovery was prolonged. For example, at 29°C, f l i e s began to climb after 70 minutes, whereas those at 31°C took 105 minutes to regain the same a b i l i t y . At 33°C, the f l i e s showed only a weak capacity for walking toward the end of the observation period and at 34°C, only 5% of the f l i e s were able to even right themselves. It should be added that after two hours at any temperature up to 33°C, a l l ts para f l i e s shifted down to 22°C recovered. However, after 2 hours at 34°C, only 10% of the f l i e s remained alive after shifting down to 22°C. ts Since para f l i e s can be maintained at 33°C for up to 2 hours, i t was assumed that a l l v i t a l organs s t i l l function in the paralyzed f l y . Indeed, the heart, which can be easily seen through the dorsal wall of the abdomen, was observed to beat quite normally in paralyzed individuals. Similarly, slight movements of the abdomen which might result from gas exchange through the spiracles could be observed in ts para f l i e s at 29°C. FIGURE 13 Recovery of behavioral activity of para f l i e s at different temp-eratures during a 2 hour interval. Each point represents the time at which at least half of the f l i e s observed exhibited the behavioral t r a i t . 86 C. Tissue S p e c i f i c i t y ts It was then asked whether the para mutation functions autonomously and whether any tissue s p e c i f i c i t y of the mutation could be determined. These questions could be answered by generating somatic mosaics of p a r a t S and + t i s s u e . The r i n g chromosome, I n ( l ) w V ^ (see Lindsley and G r e l l , 1968), is somatically unstable and is l o s t with a high frequency in m i t o t i c a l l y d i v i d i n g nuclei (Hinton, 1955). By ts marking a normal rod X chromosome bearing para with y_ (a recessive mutation which produces yellow c u t i c l e and b r i s t l e s ) , the loss of the + + ts r i n g chromosome (which c a r r i e d the y_ and para a l l e l e s ) could be de-tected on the e x t e r i o r c h i t i n as yellow patches in a wild-type back-ground (Bryant and Schneiderman, 1969). In(l)w v C,+ +/In(l)dl-49,y w s p l females were crossed to y para t s/sc^•Y,y + males at 22°C. Mosaics of In(l)w V <",+ + /y_ p a r a t s zygotes were then recovered and the exact area of yellow tissue of each mosaic recorded. Each mosaic was then placed in a coded v i a l , and another person allowed to note the behavior of each f l y under a d i s s e c t i n g microscope for 20 minutes at 29°C. Non-mosaic wild-type females were placed at random in coded v i a l s as controls, After 150 mosaics had been scored, the data on the location of the mutant patches and the behavior at 29°C were compared. The r e l a t i o n between s p e c i f i c behavioral patterns and the location of the patches was quite clearcut. From the mosaic data one could accurately predict the behavior of each f l y at 29°C. A t o t a l of 300 mosaics was scored with the sizes of yellow tissue ranging from ts tiny patches involving one or two b r i s t l e s to almost complete y_ para /0 males. A d e t a i l e d analysis of the mosaic data w i l l be presented in the next chapter. The pertinent data has been summarized by placing the 87 mosaics into the following classes based on the location of yellow tissue: I - abdomen, II - head, I I I - thorax and IV - head-thorax. Each class could be further subdivided into s p e c i f i c subgroups which differed in behavioral patterns (Figure 14). A description of the behavior of each class of f l i e s indicated in Figure 14 can be seen in Table 5. A l l Class I mosaics were wild-type in behavior at 29°C. F l i e s of the reciprocal class in which only the abdomen was wild-type were completely paralyzed. Complete head mosaics (Class l i b ) assumed a normal stance at 29°C but could not move, + ts thus showing that para head tissue i s necessary for normal leg move-ment. F l i e s with mutant tissue in the legs only (Class I l i a ) could move, but the legs were s t i f f during movement. This class i s actually a composite of 38 different mosaics involving from 1 to 5 mutant legs. Of these, 19 had one mutant leg, 11 had mutant regions on 2 legs, 5 on 3 legs, 1 on 4 and 2 on 5 legs. B i l a t e r a l thoracic mosaics (Class 11 lb) moved their legs on the wild-type side, but the mutant legs were para-lyzed in an extended position. Mosaics having completely mutant thoraces (Class IIIc) were paralyzed, as were the complete head and b i l a t e r a l thoracic mosaics (Class IVc). In addition, 18 f l i e s having mutant tissue on the dorsal surface of the thorax only, were completely normal in mobility at 29°C. In another, the entire dorsal portion of the thorax and two legs were mutant yet the four + legs moved normally. These data c l e a r l y demonstrate dual components for normal leg movement -wild-type head for motion and wild-type thorax and legs for the posture and normal movement of each leg. The use of mosaics to locate the regions governing normal mobility depends upon the detection of mutant tissue on the external FIGURE 14 C l a s s i f i c a t i o n of mosaic females generated by somatic loss of In(l)w yC according to the ventral location of mutant tis s u e . The s t i p p l e d area represents y_ p a r a t s tissue in which the r i n g i s l o s t . I abdomen only 90 TABLE 5 A correlation of the position of mutant patches and the pattern of paralysis of mosaic females at 29°C. Number of Class Mosaics Location of Mutant Patch Behavioral Characteristics at 29°C I I la 6 Abdomen 9 Bilateral head wiId-type walks, climbs in helical pattern l i b Entire head stands normally, cannot move I l i a I l l b 38 30 Legs only Bilateral thorax walks in s t i l t e d manner, legs s t i f f mutant legs paralyzed, wild-type legs continue to move H i e 7 Entire thorax IVa 17 Bilateral head and thorax legs paralyzed mutant legs paralyzed, wild-type legs continue to move IVb Bilateral head, entire thorax completely paralyzed IVc 26 Entire head, bilateral thorax completely paralyzed IVd Entire head and thorax completely paralyzed 91 surface of the f l y . It has been assumed that the external mutant patch is an indication that the underlying internal tissue is also mutant. Indeed, a good correspondence has been found in certain non-neural and some neural tissues (Ikeda and Kaplan, 1970a). Supporting evidence for this contention derives from our screening of 216 pheno-typically non-mosaic In(l)w V C,+ +/_ para t s females and over 200 pheno-ts typic y_ para /0 males at 29°C. If, in fact, considerable internal mosaicism existed with was not indicated externally, we would have expected many of the externally non-mosaic females to exhibit aberrant behavior and some of the presumed X/0 males to move at 29°C. This was not observed. A l l of the X/0 males were completely paralyzed at 29°C. Twelve females i n i t i a l l y screened as non-mosaic were observed to walk abnormally at 29°C; upon re-examination, 10 were found to be missing a leg, 1 had a mutant patch which had been overlooked and only 1 appeared to be completely wild-type. When different classes of mosaics (Classes l i b , IIIc, IVc and ts IVd) were compared with para f l i e s for 2 hours at 29°C, there were no detectable differences in the rates of recovery of co-ordinated move-ment in the mutant areas. This finding, in conjunction with the observ-ation that Class l i b , IIIc and IVb mosaics (Figure 14) cannot move at 29°G, shows that paralysis is not induced by the loss of a freely c i r -culating factor necessary for movement. Moreover, the a b i l i t y of Class I and Ila mosaics as well as the wild-type side of Classes IIB and IVa mosaics to move, rules out the existence of a freely circulating ts inhibitor of movement produced by the para mutation. D. Visual and Flight Response Flies having mutant tissue around one eye (Class Ila, Figure 3) could walk at 29°C, but upon climbing vertically, invariably followed a helical path, always keeping the mutant eye up. We asked whether this behavior indicated blindness in the mutant eye. This could be tested easily by examining its optomotor response at 29°C. Wild-type f l i e s invariably turn in the direction of moving stripes (Kalmus, 1948). When both eyes and o c e l l i of the wild-type f l i e s were painted, these f l i e s no longer showed a positive optomotor response. The wild-type eye and o c e l l i of bilateral head mosaics (Class Ila, Figure 14) were then painted. At 22°C, the f l i e s showed a positive optomotor response, whereas the results at 29°C were ambiguous. Cn occasion, the f l i e s did respond positively but much less strongly and more slowly. We therefore measured the electrical response of the eyes of ts para f l i e s to light by recording the electroretinogram (ERG) (Hotta and Benzer, 1969, 1970). The ERG was obtained by Drs. Yoshiki Hotta and Seymour Benzer. A positive ERG was obtained at both 22° and 30°C. ts This result indicated that the eyes of para f l i e s do transduce light into electrical responses at 30°C. 93 IV. Discussion ts The genetic and biological properties of the mutation, para , point to a specific defect in the nervous system of adults which regulates both flight and walking. The rapidity with which paralysis and recovery can be induced by temperature shifts argues against a direct involvement of de novo macromolecular synthesis. Co-ordinated activity at 29°C gained after a 2 hour exposure to 29°C is eliminated by a 5 minute exposure to 22°C (shift-down-arid-up). This suggests that early recovery from restrictive temperatures does not involve processes different from those affected by an i n i t i a l shift-up. ts The lack of effect of para abdomens on paralysis of f l i e s having a wild-type thorax and head (Class I) shows the absence of some freely circulating inhibitor of mobility which might be made in any part of the f l y . Furthermore, the complete autonomy of each half of bilateral thoracic mosaics (Class Illb) in both paralysis and recovery time rules out a temperature-induced humoral inhibitor or temperature-resistant promoter of movement. On the assumption that the external phenotype demarcates the boundaries of internal nervous tissue of the same genotype, the mosaics demonstrate two components that govern normal movement; the head and the ventral thorax including wings. These regions correspond to the location of the cephalic and thoracic ganglia. The normal ERG obtained at 30°C indicates that light can be transduced by the eye into electrical responses and rules out any generalized physiological effect of temperatures on CO^  levels. The 1 mutant, Hyperkinetic-1, Hk , has a defect which causes rapid rhythmic impulse generation in the motor areas of the thoracic ganglion (Ikeda and Kaplan, 1970a). These impulses induce a characteristic leg twitching 94 in lightly etherized f l i e s (Ikeda and Kaplan, 1970a). The double 1 ts mutant Hk para , exhibits leg shaking at 22°C which immediately ts stops upon shifting to 29°C. This suggests that para causes a dis-order which affects neural tissue. Recent studies on the time of appearance of activity of acetyl choline esterase and choline acetyl transferase during development (Dewhurst et a l . , 1970), prompted a ts study of these enzymes in para f l i e s . Activity of both enzymes was not reduced at 30°C (Williamson et a_l.). Of crucial interest is a determination of whether transmission within a neuron, between neurons or across a neuromyal junction is affected by high temperatures. Such studies are currently under investigation by Dr. K. Ikeda, The phenotype of temperature-dependent paralysis permitted the recovery of a hereditary behavioral defect. Selection schemes for paralytic larvae can also be readily constructed. Methods for large-scale mutagenesis and screening should make the search for such mutations feasible and promise to yield a variety of mutations of considerable biological interest. CHAPTER 6 DEVELOPMENTAL ANALYSIS OF THE PARALYTIC MUTATION, para t S I. Introduction Numerous physiological and cytological investigations into the structure and function of nerves and muscles have been carried out, yet the genetic control of these tissues has not been well characterized. It occurred to us that such information might be obtained by the recovery of mutations in Drosophila which exhibit a phenotype of paralysis, which could result from a lesion in either the nerves or muscles. However, since paralysis would be a lethal phenotype, selec-tion for a reversible temperature-dependent paralysis was carried out. A recessive, sex-linked mutation, paralytic-temperature-sensitive, ts para , was recovered from a quarter of a million zygotes (Williamson et a l . , 1970; G r i g l i a t t i ej: a l . , 1970). ts The genetic properties of para was described in the previous chapter. This chapter presents a detailed analysis of the relationship between the position of mutant tissue and the pattern of aberrant behavior at 29°C, with a view to an anatomical localization of the i . , ts lesion caused by para 96 II. Materials and Methods The X chromosome aberration, In(l)w V^, is a ring which is somatically unstable and lost with a high frequency in mitotically dividing nuclei (Hinton, 1955). This permits the generation of individual mosaics of X/0 (male) and X/Jn_____ (female) cells in a zygote which has an i n i t i a l genotype of X/In(l)wV(". X/0 cells can be recognized externally in such mosaics when the normal rod X chromosome ts carries para and the mutation, y_ (a recessive which produces yellow C + + cuticle), since In(l)w carries the dominant alleles, y_ and para . It can be seen that the absence of the ring chromosome can be detected by the yellow phenotype in a wild-type background (Figure 15). Loss of the ring chromosome during the f i r s t division of the zygote generates a bilateral gynandromorph in which the half of the f l y which is hemi-ts zygous (X/0), male, and yellow, and can express the para phenotype (Figure 15a). The other half of the f l y is female and wild-type ts since i t is s t i l l heterozygous for the yellow and para genes. Loss of the ring chromosome later in development results in a smaller patch of yellow, X/0 tissue (Figure 15b). The assumption that tissue under-lying the cuticular surface is genotypically similar to the exterior does have some experimental support (Ikeda and Kaplan, 1970b;Hotta and Benzer, 1970j Merriam, personal communication). C + t + In the actual cross, In(l)w ,y ,para /In(l)dl-49,y w spl, females were mated to v_ para t s/sc^•Y,y +males (for a complete description of the mutations used, consult Lindsley and Grell, 1968). Mosaics of In(l)w V^, + +/y_ para t S zygotes were recovered at 22°C. Females in which there was no loss of the ring in cuticular tissues were pheno-typically wild-type and were also tested for possible paralysis or FIGURE 15 The method for generation of somatic mosaicism by loss of In(l)w vC; (a) early loss (b) late loss during cleavage divisions. The stippled area is mutant X/0 tissue. r 99 debilitation at 29°C which could be an indication of undetected internal mosaicism. In addition, completely yellow, patroclinous, X/0 males which could result from loss of the ring X chromosome during maternal meiosis or loss in a l l cells which generate external cuticle were also recovered. A l l patroclinous males were also tested for activity at 29°C to determine possible internal mosaicism. The area of yellow tissue in each mosaic was recorded on diagrams as shown in Figure 16. Each mosaic was then placed into a coded v i a l . The behavior at 29°C was observed for up to 20 minutes. Non-mosaic, Oregon-R wild-type females were also placed at random in coded vials as controls. Methods for the optomotor response, electroretinogram, and flig h t response wi l l be detailed as needed in the Results. FIGURE 16 Diagrams used for delineating the area of mosaicism during the scoring procedure. DORSAL VENTRAL 102 III. Results A total of 371 mosaics was detected. The amount of yellow tissue ranged from tiny patches involving one or two bristles to almost ts complete y_ para /0 males. Of these, 310 were selected for observation at 29°C. Many of these mosaics could be placed into one of the follow-ing classes based on the location of the mutant tissue: I - abdomen-only mosaics, II - head-only mosaics, III - thorax-only mosaics, IV - head-thorax mosaics. These four classes were further subdivided into ten specific subgroups (Figure 14). Since the data point to a neural lesion, i t is instructive at this point to describe the basic elements of the nervous system with respect to their position in the f l y . An anatomical description of the nervous system in Drosophila has been reviewed by Poulson (1950), Bodenstein (1950), and Miller (1950) and is shown in Figure 17. The central nervous system (CNS) consists of the brain and subesophageal ganglion which is fused to the posterior end of the thoracic ganglia. The brain, with its optic lobes, lies dorsal to the esophagus but is connected to the subesophageal ganglion which lies ventral to the esophagus. The subesophageal ganglion is connected to the thoracic ganglia or ventral nerve cord, via a pair of fused nerves running through the cervical connection ventral to the esophagus. Each of the three fused thoracic ganglia gives off, among others, a pair of ventral nerves which innervate each pair of legs. The abdominal ganglion is fused to the caudal portion of the metathoracic ganglion and innervates the abdomen, genitalia and internal reproductive organs. The stomodeal ganglion of the sympathetic or visceral nervous system, rests between FIGURE 17 (a) Dorsal view of a lateral section, and (b) sagittal section of Drosophila adult. Darkened areas are nervous tissue. Symbols: Ab ns - abdominal nerves; C^ _3 - coxal cavity; Cn - crop nerve; Es - esophagus; Gn - ganglion; Lni-3 - leg nerves, f i r s t , second and third; meso - mesothoracic ganglion area; meta - metathoracic ganglion area; OL - optic lobe; pro - prothoracic ganglion area; S Gn - stomodeal ganglion; Se Gn - subesophageal ganglion; Sgn - salivary gland nerve; V - ventriculus; Vn^_2 - ventral nerves one and two innervating the f i r s t and second leg respectively. Adapted from Miller (1950). 105 the esophagus and aorta dorsal to the thoracic ganglia. It sends two nerves anterior to innervate the protocerebrum of the brain and the esophagus, as well as posterior nerves to innervate the cardia, the stalk of the crop, and the salivary gland. The fly can be divided into the three body parts: head, thorax, and abdomen. Since no ganglion of the central nervous system is located in the abdomen, i t was of interest i n i t i a l l y to determine whether mutant tissue in the abdomen affected the behavior of otherwise wild-type f l i e s . Conversely, would the presence of wild-type tissue ts in the abdomen in any way rescue an otherwise para fly? The results are shown in Table 6. Seventeen f l i e s had totally wild-type head and thoracic tissue and abdomens ranging from completely mutant to mosaic for small mutant patches (Class I, Figure 14). Of these, one fly had totally mutant abdominal tissue. Its behavior at 29°C was indistinguish-able from wild-type, that i s , i t could walk and climb the vertical sides of the v i a l normally, and was even observed to f l y . Sixteen f l i e s had wild-type head and thoracic tissue but mosaic abdominal tissue that ranged from yellow male genitalia through patches or bands of yellow tergites and sternites to almost totally yellow abdomens. A l l 16 of these mosaics displayed normal behavior at 29°C. Therefore, mutant tissue in the abdominal region does not visib l y alter the behavior of otherwise + f l i e s . Eighteen f l i e s had totally mutant tissue in the head and thorax, and abdominal mosaicism ranging again from almost totally wild-type to almost totally mutant abdomens. Upon exposure to high tempera-ture, a l l of these mosaics were paralyzed with a characteristic con-traction of a l l legs. 106 TABLE 6 Behavior at 29°C of mosaics in which the phenotype of abdominal tissue differed from the head and thorax. No. Flies Phenotype of Body Regions Head Thorax Abdomen Behavior at 29°C 1 16 18 mutant mutant mutant mosaic mosaic + (flew) a l l + a l l paralyzed -legs generally contracted 16 mosaic mutant + or mosaic a l l paralyzed -legs generally contracted 107 Sixteen individuals in which the thorax was totally mutant while the head tissue was mosaic, had abdominal tissue which ranged from either totally + to mosaic with only small patches of + tissue. At 29°C, these mosaics were a l l paralyzed, and again the legs were contracted. A l l of these data suggest that wild-type tissue in the ts abdomen in no way rescues otherwise para f l i e s from paralysis. ts Since the presence or absence of para tissue in the abdomen does not counteract the genotype of the head and thorax with respect to walking or flight behavior in any way, we can dispense with any further con-sideration of mosaicism in the abdomen from this point on. Attention was then focussed on the behavior of 17 f l i e s with mosaic heads and wild-type thoraces (Class l i b , Figure 14) at 29°C. Of these, two f l i e s had completely mutant heads and wild-type thoraces and abdomens (Table 7). Both were paralyzed and did not walk at 29°C although they maintained a normal stance. If the v i a l was tapped with sufficient force to topple them, they would often right themselves immediately but could not walk or move; furthermore, even gentle prodding with a blunt probe failed to induce walking. Thus, these two mosaics had a wild-type posture but simply did not move. A third fly having a small patch of + tissue above the lef t eye, was very similar to the preceding in both its stance and behavior phenotype. Five f l i e s were bilateral head mosaics; that i s , either the left or the right side was completely mutant both dorsally and ventrally (Class Ila, Figure 14). They a l l walked normally at 29°C; however, when they climbed the vertical surface of the v i a l , they invariably followed a helical pathway always keeping the mutant side of the head up. The possibility that these mosaics were blind in the eye on the 108 TABLE 7 Behavior of head mosaics at 29°C. No. Flies Location of Mosaicism Head Thorax Abdomen Behavior at 29°C completely mutant paralyzed -legs normal posture small + spot above left eye paralyzed lef t or right bi lateral a l l walk -climb - helix single quadrant mosaic a l l + mutant antennae a l l + 109 mutant side of the head was tested using both an optomotor response and an electroretinogram which w i l l be discussed later. Seven mosaics had mutant tissue in one of four quadrants of the head: dorsal right, dorsal l e f t , ventral right or ventral l e f t . A l l of these mosaics exhibited normal behavior at 29°C, including climbing in a relatively straight line or diverging randomly from a straight line. Finally, two mosaics were found that had mutant antennal tissue only. Both of these mosaics exhibited normal walking and climb-ing behavior. The final gross segmental mosaic class involved f l i e s with completely mutant thoraces (Class IIIc, Figure 14). Nine f l i e s with completely or almost totally mutant thoraces and wild-type head tissue were recovered (Table 8). Seven f l i e s had completely mutant thoraces. Of these seven, five were totally paralyzed at 29°C, another, though paralyzed, occasionally pawed with the f i r s t l e f t leg, and the seventh was paralyzed on the lef t side, but was able to drag i t s e l f forward slowly in a right diagonal direction. In contrast to the head-thorax mosaics of Table 6, the legs of these mosaics were not always contracted but varied from normal posture to contraction of the coxa, femur, and t i b i a . The movement of two of the mosaics may suggest the presence of internal + tissue not represented in the chitinous surface. This wi l l be discussed later. Finally, two other thoracic mosaics which each had only a small patch of + tissue in the dorsolateral area of the ts thorax near the humerals, were paralyzed at 29°C. Thus, para tissue located only in the thorax was sufficient to paralyze the f l i e s at 29°C. In summary of the results from mosaics considered so far, f l i e s with completely mutant head tissue are unable or unwilling to 110 TABLE 8 Behavior at 29°C of thoracic mosaics. Location of Mosaicism No. Flies Head Thorax Behavior at 29°C 5 paralyzed -legs extended + completely mutant 1 paralyzed -legs extended, IL paws 1 lef t side paralyzed -walks in r t . diagonal 2 + mutant with small dorsolateral + patches 2 paralyzed I l l walk but the f l y retains a normal stance. Incomplete head mosaics show varying degrees of activity. Mosaics with mutant thoraces only are paralyzed, generally unable to stand, and vary in the amount of contraction in the legs, from normal posture to f u l l coxa-femur-tibia contraction. Complete head-thorax mutants are paralyzed, and their legs are generally contracted. Finally, mutant abdominal tissue does not affect the walking, climbing or flying a b i l i t y of otherwise wild-type f l i e s . From the preceding analysis, we would expect that f l i e s with completely mutant thoraces and varying degrees of mosaicism in the head would be paralyzed at 29°C. Indeed, sixteen individuals with mosaic heads, completely yellow thoraces, and either + or mosaic abdomens were a l l paralyzed at 29°C. Two of these 16 individuals did move their f i r s t legs sporadically, but neither of them was able to right i t s e l f . Wild-type thoracic abdominal tissue cannot rescue a f l y with a mutant head (Class l i b , Figure 14) from paralysis at 29°C. Similarly, wild-type head abdominal tissue cannot rescue a thoracic mutant (Class IIIc, Figure 14) from paralysis at 29°C. These facts suggest that paralysis at 29°C cannot be due to the absence of a freely c i r -culating factor necessary for walking. The behavior of bilateral mosaics within a body segment can perhaps give us additional informa-ts tion on the tissue autonomy of para Autonomy in Drosophila is defined by the phenotypic reflection of a tissue's genotype in spite of its juxtaposition to cells of a different genotype. If the mutation is indeed autonomous, then thoracic mosaics can provide information ts about the regional specificity of para and perhaps indicate affected areas within these regions. Twenty-eight complete bilateral thoracic mosaics (Class I l l b , Figure 14) were recovered. Fourteen were totally mutant in the le f t half and 14 in the right half of the thorax (Table 9). Legs on the wild-type side invariably were active whereas the mutant legs were para-lyzed. In order to f a c i l i t a t e the analysis of their behavior at 29°C, and to compare their behavior with other mosaic classes, each mosaic was placed into one of the following six categories based on the amount of activity at 29°C; paralyzed, very weak, weak, moderate, strong, or very strong (Table 9). These are somewhat subjective divisions and are defined as follows: paralyzed - f l i e s did not move; very weak -attempted to move using the wild-type legs, but could not right them-selves or walk; weak - slowly pulled themselves with their wild-type legs; moderately-active - able to walk continuously using their + legs, generally walked in a diagonal direction pulling themselves forward and dragging the mutant side, or in a circular fashion with the + legs on the outside arc of the c i r c l e ; strong - walked faster and were generally more active then the preceding class, and sometimes, unsuccessfully attempted to pull themselves up the vertical surface of the v i a l ; very strong - pulled themselves a short distance up the vertical surface of the v i a l . In most cases, individuals could be readily placed into one of these categories. None of the bilateral thoracic mosaics was either completely paralyzed or wild-type in behavior. Clearly then, the wild-type half cannot restore total activity to the mutant side, nor does the mutant side suppress movement on the wild-type side. However, the variable expression of movement in what appeared to be similar mosaics could 113 TABLE 9 Categorization mosaics. of 29°C activity of bilateral head -thorax and thorax Number of Mosaics Class Characteristics bi lateral thorax only bilateral head-thorax a paralyzed no movement 0 2 b very weak attempted to move using + legs 1 9 c weak slowly pulled themselves with + legs 6 7 d moderate moved continuously using primarily + legs; moved diagonally or in ci r c l e 13 5 e strong more active than preceding, some use of mutant legs, unsuccessful climbing attempts 2 4 f very strong very active, pulled themselves short distances up a vertical surface 6 2 Total 28 29 114 reflect either some non-autonomy or variable internal mosaicism. In order to quantify the extent of behavioral activity, each category was assigned a di g i t a l value from 0 (paralyzed) to 5 (very strong). By multiplying the number of f l i e s in each category by its value and dividing the sum of these products by the number of mosaics, an average of 3.2 was obtained. This value indicates that, on the average, bilat-eral thorax mosaics exhibit a moderate amount of activity at 29°C. It may then be asked whether a l l three legs on each side of bilateral thoracic mosaics behaved similarly. The front, middle and hind legs on each side of bilateral thoracic mosaics were each placed into one of the five categories of behavioral activity seen in Rows A and C (Table 10). The legs on the wild-type side of the mosaic gener-ally exhibited normal walking a b i l i t y or, in a few cases, a slight debilitation. On the other hand, the legs of the mutant side were strikingly enfeebled. It is of considerable interest to note that the f i r s t leg on the mutant side, though debilitated, invariably showed much more activity than the second and third legs, which were both inactive (Figure 18). This may suggest a difference in innervation between the f i r s t leg and the second and third. In comparing activity of bilateral head-thorax mosaics (Class IVa, Figure 14) with bilateral thoracic mosaics (Class I l l b , Figure 14), a further comparison of the behavior of the f i r s t with the second and third legs could be made. Twenty-nine bilateral head-thorax mosaics were found and they were classified by the same c r i t e r i a applied to bilateral thorax-only mosaics (Table 9). Addition of head mosaicism ipsilaterally clearly reduced activity of the thoracic mosaics. Thus, activity of a quarter of the thoracic mosaics was weak or less whereas FIGURE 18 Activity of each leg in bilateral thoracic mosaics at 2 9 ° C . + legs, mutant leg 1, mutant leg 2 , mutant leg 3. 1 Number of legs fn each Category FIGURE 19 Behavior of bilateral thoracic and head-thoracic mosaics at 29°C. thoracic, head-thoracic; letters correspond to classes of activity shown in Table 17. Number of Mosaics in each Class co CD zr Q < o ' Q co o O <T> 04 I a i - i _j •^1 CD i _ L _ o I ro i 04 • 1 I 119 TABLE 10 Leg activity at 29°C in bilateral head-thorax and thorax mosaics*. Number of mosaics in each category of activity Tissue Type of Con- Ex-Type Row Mosaic Leg tracted tended twitch s t i f f walk total 1st 5 4 12 6 1 38 A thorax 2nd 11 14 3 0 0 28 mutant 3rd 13 13 2 0 0 28 1st 11 3 7 7 1 29 B head-thorax 2nd 13 12 1 3 0 29 3rd 13 14 1 1 0 29 1st 0 0 «- 5 —» 23 28 C thorax 2nd 0 0 «- 5 —> 23 28 wi ld- 3rd 0 0 <— 5 —> 23 28 type 1st 2 5 3 4 15 29 D head-thorax 2nd 2 5 3: 4 15 29 3rd 2 6 2 4 15 29 * Classes are delineated as follows: contracted - coxa, femur and ti b i a , or at least femur and tibia were contracted; extended - pre-ceding joints not contracted but the leg did not move; twitch -occasional sporadic flexure of the joints but the leg was not used in walking; s t i f f - the leg though used for walking did not show normal articulation of the joints; and walking - normal use of the leg. FIGURE 20 Location of different mutant patches on the dorsal surface of the thorax. The cross hatched patch is the only region not encompassed by mutant tissue. almost two-thirds (18/29) of the head-thorax mosaics were weak or less. Also, six out of 28 of the thoracic mosaics were very strong whereas only two out of 29 of the head-thorax mosaics were. This can be seen in Figure 19. The digital index of behavior of the head-thoracic mosaic was 2.2, a further indication of the decreased activity relative to the thorax-only mosaic (3.2). A comparison of movement of legs in head-thorax mosaic reveals two interesting points: (1) while there is somewhat more debilitation of the mutant f i r s t leg when the head is also mosaic, i t s t i l l retains considerably more activity than either the second or third legs (Rows B and D, Table 10); (2) addition of mutant tissue to the head of thoracic mosaics results in a reduction of movement of the legs on the wild-type side. This suggests that there is some contralateral connection from the subesophageal ganglion to the thoracic ganglia. Furthermore, i t seems that there is an ipsilateral connection from the head to the thorax which may indirectly affect the activity of the f i r s t pair of legs. The extent to which the thorax is involved in mobility might be delineated by the behavior of mosaics carrying mutant patches on the dorsal surface of the thorax. Fifteen of such mosaics, a l l of which behaved normally at 29°C, were observed. No mosaic which was mutant over the entire dorsal surface of the thorax only was found. However, when the mutant patches on the dorsal surface of the thorax were super-imposed (Figure 20), they overlapped to extend over an area laterally down to the wings and over the entire dorsal surface of the thorax with the exception of a small patch of tissue on the right anterior dorsal surface of the thorax above the humeral bristles. However, several mosaics were found that had mutant tissue in that same area on the le f t side of the thorax. From the behavior of these mosaics, i t can be inferred that mutant tissue on the dorsal surface of the thorax in no way debilitates the f l y . This offers further support for ts an effect of para on the CNS since the thoracic and abdominal ganglia l i e in the ventral portion of the thorax. The different pattern of behavior of the f i r s t pair of legs from that of the second and third legs, made the early development of each leg of interest. Thirty-seven mosaics with mutant tissue in the legs only (Class I l i a , Figure 14) were examined and the frequency with which each leg had mutant tissue was determined (Table 11). There was a curious absence of mosaics with mutant tissue in the second leg only. Also, there were no mosaics with mutant tissue simultaneously in the f i r s t and second legs or in the f i r s t and third legs even though eight f l i e s had mutant tissue in both the second and third legs. Since mosaics with mutant tissue in the third leg alone but none in the second leg alone were recovered, we conclude that whenever mutant tissue is generated in the second leg, the third leg also contains mutant tissue. Therefore, i t is possible that the third leg cells are deter-mined later in development than the second leg cel l s , and that, in fact, the presumptive second leg cells give rise to the presumptive third leg c e l l s . Since we recovered nine mosaics with a l l three legs on one side mutant, i t is quite probable that the f i r s t and second leg cells are derived from a small number of the same progenitor c e l l s . We did not delineate the area of mutant tissue in the leg mosaics to permit an estimate of the number of cells from which the three leg discs were derived. In addition to the developmental data, i t is 124 TABLE 11 Frequency of mutant or mosaic tissue in individual legs of leg-only mutants. total of mosaic in mosaic in mosaic in mosaics in one leg only two legs three legs each leg 1 2 3 1,2 2,3 1,3 1,2,3 1 2 3 Number of mutant or 12 0 8 0 8 0 9 21 17 25 mosaic 125 interesting to note that though a l l of the leg-only mosaics could walk at 29°C, some were slightly enfeebled (Table 12). Thirty mosaics of both the head and thorax with different degrees of mosaicism ranging from small patches of mutant tissue in both the head and thorax to large patches covering most of the head and thorax were studied. The behavior of each of these mosaics was completely predictable on the basis of the results from mosaics in single body segments already discussed. In addition, 21 mosaics were isolated with completely mutant heads and only small patches of wild-type thoracic tissue. A l l were paralyzed as expected. Eighteen mosaics were found that had completely mutant heads and bilaterally mosaic thoraces (Class IVc, Figure 14). A l l were paralyzed at 29°C. In 17, the legs on the mutant side were contracted whereas those on the wild-type side assumed normal posture. Thus, combined mutant tissue in the head and thorax ensures the contraction of the mutant legs. Four f l i e s had completely mutant heads, wild-type thoraces, and mosaic legs. In one of these f l i e s , only the f i r s t right leg was mutant. It did not walk at 29°C and the f i r s t right leg was contracted. The f i r s t l e f t leg showed some slight femur-tibia contraction and both the second and third pair of legs had normal posture. The second f l y had mosaicism in both the second and third pair of legs. It took a few very small and slow steps, l i s t i n g to the left and moving slightly counterclockwise. Fly 3, like the f i r s t , had a mutant f i r s t right leg. The f i r s t right leg was completely contracted, and the f l y took two very small and slow steps using the other legs then did not move. Fly 4 also had a mutant f i r s t right leg which was contracted. The f l y 126 TABLE 12 The behavior at 29°C of individual mutant legs in leg-only mosaics. Number of mosaics in each category of activity leg contracted extended twitch s t i f f walk total 1 0 0 0 4 2 6 2 0 2 0 6 2 10 3 0 7 0 5 5 17 127 did not move at a l l , but just stood in one spot and listed to the l e f t . These four f l i e s reconfirm the inability of head mutants to move at 29°C and demonstrate the specificity of the head-leg mutant combination for the leg contraction. Three individuals that were bilateral head mosaics and bi-lateral thorax mosaics were found in which the mutant patches in the body segments were in opposite halves of the body (Figure 21). The behavior of these f l i e s is particularly interesting. If the nerves joining the cephalic ganglion with the thoracic ganglia are crossed, then one would predict that the mutant legs would be paralyzed and con-tracted while the wild-type legs should pull the f l y as in a bilateral thorax mosaic. On the other hand, i f the innervation of the thorax from the head is ipsilateral - not forming a chiasma - then one would predict that the fly would not walk at 29°C and that both the wild-type and the normal legs would assume the normal posture. The f i r s t individual had the right half of the head and the left half of the thorax mutant (Figure 21a). The le f t legs were completely contracted, i.e., coxa, femur, and t i b i a ; the f i r s t right leg was missing, and the second and third right legs showed a more normal posture with some femur-tibia contraction. The f l y never moved at 29°C. The second individual had the le f t half of the head and the right half of the thorax mutant (Figure 21b). It was paralyzed on the right side with some use of the f i r s t right leg, less use of the second right leg, and least use of the third right leg. It moved by dragging i t s e l f with the l e f t legs and slightly using the f i r s t right leg. The last individual in the group, like the f i r s t , had mutant tissue in the right half of the head and le f t half of the thorax and in addition the f i r s t left FIGURE 21 Location of mutant tissue in head-thorax catercorner mosaics. The stippled area indicates mutant X/0 tissue. a Dorsal Lef t S ide Right S i d e . L e f t Side Right Side Dorsal Dorsal Ventral Ventral 130 was mosaic and the f i r s t right was very slightly mosaic (Figure 21c). It moved using the right legs, and i t had some use of the f i r s t l e f t leg and the second l e f t leg was s t i f f . The results from these three individuals do not s t r i c t l y conform to either prediction. It is likely that there is a cross innervation of head to thorax but perhaps linear innervation exists as well. Interpretation of data using mosaics depends on the assumption that external phenotypes delineate internal tissue of the genotype. Indeed, a good correspondence between external and internal mosaicism has been found in some neural and non-neural tissues (Hotta'and Benzer,. 1970; Ikeda and Kaplan, 1970b;Merriam, personal communication). We can test this contention further in two ways: (1) by determining how many individual mosaics exhibit behavior that digresses markedly from the patterns established by the majority of the mosaics, and (2) by testing the behavior of non-mosaic females and yellow (XO) patroclinous males that were generated along with the mosaics. The non-yellow females have not lost the ring X chromosome in any external chitinous cells whereas the yellow males have lost the ring X chromosome in a l l the external chitinous c e l l s . If internal mosaicism in neural tissue exists, then some females might be enfeebled at 29°C, while some males should not be completely paralyzed at 29°C. Over 200 yellow patroclinous males were observed at 29°C and a l l were paralyzed. Over 300 wild-type females were tested at 29°C and of these 12 walked abnormally. Upon re-examination, 10 were found to be missing a leg, one had a mutant patch that had been overlooked, and only one completely wild-type, and therefore true exception, was found. At f i r s t this f l y walked and then, after about 10 seconds, i t 131 slowed down and walked occasionally. The femur and t i b i a of the third pair of legs often contracted, the second pair of legs was s t i f f , and the f i r s t pair of legs would sometimes show femur-tibia contraction. We conclude that this fly must indeed have had some internal mosaicism. Of the more than 300 mosaics observed, only seven deviated to some extent from the behavior patterns predicted on the basis of the general observations. Fly 1 (Figure 22a) was expected to be immobile. However, i t did move very slowly using the f i r s t pair and second lef t legs. This very weak activity might be expected i f the ventral patch of + tissue in the head extended dorsally through the cephalic ganglia. In another exception (Figure 22b), the right side was predictably paralyzed. However, the f l y dragged i t s e l f using the le f t legs and, what is surprising, could even climb the vertical side of a v i a l . Again, this suggests more wild-type tissue internally than was indicated by the external chitinous structure. A third mosaic (Figure 22c) was expected to be virtually immobilized, and the left side was, in fact, paralyzed. However, it dragged i t s e l f with the right legs. We con-clude, therefore, that i t had some wild-type tissue in the head not delineated by the external mosaicism or perhaps that the patch of + tissue on the right surface of the head continued down through the brain and subesophageal ganglion but not to the external ventral surface. On the basis of the visible mosaicism, another f l y (Figure 23a) was expected to move very slowly with the second and third right legs extended; however, i t walked well, climbed, and even flew once. While i t is true that from the external mosaicism we would predict that the cephalic as well as a good part of the thoracic ganglia were FIGURE 22 Location of mutant tissue in three head-thorax mosaics which behaved unexpectedly at 29°C. The stippled area indicates mutant X/0 tissue. Ventral Ventrol Ventrol FIGURE 23 Location of mutant tissue in four head-thorax mosaics which behaved unexpectedly at 29°C. Lightly stippled area indicates mosaic tissue. b c d 136 probably wild-type, we did not expect that the f l y would be quite as active as i t was. Another f l y (Figure 23b) walked f a i r l y rapidly. What was unexpected was that i t used its f i r s t and second l e f t legs well and listed to the right when walking. A sixth f l y (Figure 23c) was expected to be f a i r l y active. However, i t crawled forward very slowly at f i r s t and was then motionless except for cleaning behavior with the third pair of legs. Finally, the seventh f l y (Figure 23d) was expected to exhibit normal behavior. It did walk fast. However, i t slowed down occasionally, the second pair of legs seemed slightly s t i f f and i t was reluctant to climb the vertical surface of the v i a l . In summary, seven of the more than 300 mosaics observed at 29°C exhibited behavior that diverged somewhat from the behavior we would have predicted, based on their markings and the behavior of the other mosaics. One of approximately 500 non-mosaic females and yellow patroclinous males exhibited non-predictable behavior. This evidence indicates that there is a very good correlation between the amount of external and internal mosaicism in this system. ts When para f l i e s are shifted from 22°C to 29°C, they are i n i t i a l l y paralyzed. However, they wi l l recover some co-ordinated activity over a two hour period at that temperature. A possible measure of non-autonomy could be the rate of recovery of mosaic f l i e s at 29°C. ts Mosaics with para tissue in the head-only did seem to recover co-ts ordinated activity at 29°C faster than complete para f l i e s (Figure 24). Mosaics with totally mutant head and thorax, but wild-type abdomen (Class IVd, Figure 14), wild-type head and mutant thorax (Class IIIc, Figure 14), and mutant head and bilateral thorax (Class IVc, Figure 14) ts did not recover co-ordinated activity any faster than totally para 137 f l i e s (Figure 24). This finding, in conjunction with the observation' that thorax-only (Class IIIc), bilateral head-mutant thorax (Class IVb) and mutant head-bilateral thorax (Class IVc) mosaics could not move at 29°C, shows that paralysis is not induced by the loss of some freely circulat-ing factor necessary for movement. Moreover, the fact that abdominal mosaics (Class I), bilateral head mosaics (Class Ila), as well as the wild-type sides of bilateral thorax and head-thorax mosaics (Classes I l l b and IVa) can move, rules out the existence of some freely circulat-es ing inhibitor of movement produced by the para mutation. Thus, the mutation seems to be autonomous. The observation that five bilateral head mosaics a l l walked normally at 29°C, but invariably followed a helical pathway always keeping the mutant eye up when climbing the vertical side of a v i a l , prompted us to ask whether this behavior was an indication of blindness in the mutant eye. If the f l i e s were indeed blind, their attempts to balance the amount of light striking the two eyes might result in a helical climbing pattern. This suggestion could be tested quite easily by examining the optomotor response (Kalmus, 1948) of these mosaics at 29°C. When presented with a moving pattern of stripes, wild-type f l i e s w i l l invariably turn and walk in the direction in which the stripes are moving. This was tested by placing a fly whose wings had been amputated into a cylinder whose walls were striped vertically (Figure 25). The cylinder was then rotated to the right or the l e f t . Wild-type f l i e s gave a strong positive response at both 22°C and 29°C. When the eyes and o c e l l i of wild-type f l i e s were covered with white enamel paint, they ceased to show a positive optomotor response at both 22°C and 29°C. FIGURE 24 Activity recovered with time at 29°C by different mosaics. para t s control, mosaics, (a) head only mosaics (b) thorax only (c) complete head and bilateral thorax, and (d) abdomen only Behavioral Function Exhibited c. 30 co o o < CD 3 CD 3 C CD 01 CD 1 (A i c •o o o FIGURE 25 A diagram of the device used to test the optomotor response. The hollow cylinder with alternating black and white striped is rotated either clockwise or counterclockwise. A wild-type f l y placed in center of the cylinder will walk in the same direction in which the cylinder is rotated. 142 We then painted only the eye on the wild-type side and the o c e l l i of bilateral head mosaics. At 22°C, these painted mosaics s t i l l exhibited a strong positive optomotor response, whereas the results at 29°C were ambiguous. Most of the time, the mosaics did not respond to changes in the direction of rotation of the stripes. On a few occasions the f l i e s did stop moving when the direction of the rotation of the stripes changed, and occasionally did orient themselves in the general direction of the new rotation of the stripes. Thus, on a few occasions these f l i e s did respond positively but much less strongly and more slowly. They were clearly debilitated but our evaluation of the extent of their response was too subjective to make a definitive assessment of their v i s i on. A more definitive test of sight is the response of the eyes ts of para f l i e s to short flashes of light as shown by an electroretino-gram (ERG) (Hotta and Benzer, 1969). The ERG was obtained by the generosity of Dr. Yoshiki Hotta through the co-operation of Dr. Seymour Benzer. The ERG was positive and remained unchanged for 20 minutes at both 22°C and 30°C (Figure 26). This indicated that the eyes of para t S f l i e s do transduce light into impulses at both 22°C and 30°C and that these impulses can at least travel down the axons of the retinula c e l l s . Since the optomotor response was n i l or at best very weak, these signals may not reach or be recognized by the brain with any regularity. It must now be asked whether the optic nerves are transmitting the im-pulses to the brain and/or whether the cephalic ganglia can interpret such messages and send the appropriate messages of response to the thoracic ganglia and legs. At this time attempts are being made to ts determine i f interneural and neuro-myal transmission occurs in para FIGURE 26 Electroretinogram of para at 22°C and 30°C showing the res-ponse to a short (20 ^ s) flash of light. o 145 f l i e s at 29°C. ts The effect of para on the activity of afferent neurons was studied by examining flying a b i l i t y . The tips of toothpicks were trimmed and glued perpendicular to the center of the dorsal surface of the thorax. When these tethered f l i e s were l i f t e d , they began to " f l y " ts as soon as their feet l e f t a solid surface. At 22°C, para f l i e s initiated flight immediately upon l i f t i n g , whereas at 29°C, flight was not initiated. However, once flight was initiated at 22°C, upon shift-ing to 29°C, flight would continue for about 5-10 seconds. Bilateral head and f u l l thorax mosaics, as well as bilateral head-thorax mosaics, terminated flight upon shifting from 22°C to 29°C and were unable to initiate f l i g h t at 29°C. However, bilateral head-only mosaics can f l y at 29°C. This implies that a wild-type thorax and at least partially wild-type head are needed to initiate flight at 29°C. A more interesting question is whether a fly which had initiated flight at 22°C and was shifted to 29°C, would terminate flight when only its mutant legs touched a solid object. Although bi-lateral head-thorax mosaics cannot initiate flight at 29°C, i f flight is initiated at 22°C, upon shifting to 29°C they w i l l continue to fly for periods up to 15 seconds. Three bilateral head-thorax mosaics, and two bilateral head-full thorax mosaics were tested. Flight was initiated at 22°C, then the f l i e s were shifted to 29°C and less than 7 seconds later, the mutant legs were allowed to touch a plate. In a l l cases, the f l i e s terminated flight immediately. This may indicate that the afferent neurons and the inhibitory neurons to the flight muscles s t i l l function at 29°C. An alternate explanation exists; since the f l i e s are able to see, perhaps the proximity of the approach-146 ing platform caused the cessation of f l ight in a learned response to distance and landing. A similar experiment was attempted with com-ts plete para f l i e s . On occasion, f l ight in i t iated at 22°C was not terminated at 29°C when the f l i e s ' feet touched the platform. In retrospect, since some f l ights , once ini t iated at 22°C, continue for only several seconds when the f l y is shifted to 29°C, and paralysis takes a few seconds (less than 5 seconds) to be complete, these experi-ments seem of dubious value in the information they may y ie ld . ts If the afferent neurons of para tissue do indeed function, b i lateral thorax and b i latera l head-thorax mosaics (which can move at 29°C) might be expected to attempt to crawl away i f touched on the mutant side. Using a small hair , the legs and body of the mutant side and alternatively the wild-type side, were prodded. When the f ly was touched on the mutant side there was generally no reaction. Occasion-al ly the f ly made a weak attempt to crawl away. However, the intrusion of the hair on the + side of the body always caused a directed movement away from the probe. Likewise, i f a hair was placed in the direct path of the mutant side of the body, the f ly continued in the direction in which i t was t ravel l ing, allowing the mutant legs to brush by the hair . If the same hair was placed in the path of the + side of the body, the f ly invariably veered in a direction away from the object. These tests seem to indicate that the afferent neuron on the mutant side of the body is not working, or i f i t is functioning, the thoracic ganglia cannot sort out the message. These poss ib i l i t ies cannot be def in i t ive ly resolved without the use of electrophysiology. F inal ly , one experiment was performed to test the possib i l i ty that inhibitory neurons were malfunctioning at 29°C and thus induced 147 the paralysis. Wild-type f l i e s can walk after decapitation. If the lack of movement in complete head mosaics is caused by the malfunction-ing of inhibitory neurons, then decapitation might remove the inhibi-tion and permit walking. Only two complete head mosaics were found and at the time were not used for this experiment. However, four complete head and bilateral thorax mosaics were available for the experiment. This class of mosaics is inactive at 29°C, while bilateral thorax mosaics are f a i r l y active. Upon decapitation of the bilateral head-thorax mosaics at 29°C, leg movement on the wild-type side was not initiated. However, this does not c r i t i c a l l y rule out the possi-ts b i l i t y that inhibitory neurons are affected by the para mutations. 148 IV. Discussion The i n a b i l i t y of abdominal genotype to a f f e c t behavior of a head and thorax of a d i f f e r e n t genotype (Table 6) and the complete autonomy of each half of b i l a t e r a l thoracic mosaics shows the absence of a f r e e l y c i r c u l a t i n g i n h i b i t o r of movement which might be made in a l l parts of the f l y . S i m i l a r l y , the fa c t that Classes IIIc, IVc and IVd mosaics (Figure 14) were paralyzed at 29°C and did not recover co-ts ordinated a c t i v i t y any faster than complete para f l i e s suggests that p a r a l y s i s i s not due to a temperature-sensitive humeral factor neces-sary for movement. The more rapid recovery of complete head-only ts mosaics than complete para f l i e s does not negate the above conclusions. Only two of such mosaics were found and though the recovery times were repeatedly obtained for each, there was considerable v a r i a t i o n from one in d i v i d u a l to another and we consider the sample s i z e to have been too small to be d e f i n i t i v e at this point. The recovery data taken in con-junction with the behavior of several classes of mosaics a l l point to ts the autonomy of the para mutation. ts The genetic and b i o l o g i c a l properties of the para mutation point to a s p e c i f i c defect in the nervous system of the adult which regulates both f l i g h t and walking. The autonomous a c t i v i t y of the mutation permitted an analysis c o r r e l a t i n g behavior with the p o s i t i o n of somatic mosaicism. This l o c a l i z e d the components involved in the ts p a r a l y t i c e f f e c t of para to cephalic and v e n t r a l thoracic areas which correspond to the s i t e s of the cephalic (brain and subesophageal ganglion) and thoracic ganglia (Figure 17). The fact that complete head mosaics as well as mosaics with completely mutant head tis s u e and mosaic legs could not walk at 29°C suggests that the mutation 149 certainly affects the CNS. The mobility of leg mosaics minimizes the possibility that the mutant has defective muscles. The positive ERG ts of para f l i e s at 29°C shows that mutant eyes can transduce light into impulses, yet the poor optomotor response of the mutant eye in bilateral head mosaics suggests that the brain does not receive or ts cannot interpret these messages. The epistasis of para over Hk-1, a mutation whose phenotype results from an abnormal pattern of nerve ts f i r i n g (Ikeda and Kaplan, 1970a) supports the contention that para causes a lesion in the central nervous system (Suzuki _ t al_., 1971). Electrophysiological tests are being attempted to determine whether there is transmission across neuro-myal and interneural junctions at 29°C. The abi l i t y of the eyes to transduce light into impulses led us to ask whether other sense receptors, or afferent neurons in general, functioned at 29°C. The observation that tethered bilateral mosaics ts as well as para f l i e s , with few exceptions, terminated flight at 29°C, would indicate that sense receptors and afferent neurons function at ts 29°C in para However, the observations from the prodding experiments on bilateral mosaics suggest that i f , in fact, the afferent neurons function at 29°C, then their messages are not received and/or inter-preted by the ganglia of the CNS. The question of whether or not afferent neurons function at 29°C remains unresolved. ts On the assumption that para does indeed affect nerves, inferences on CNS circuitry can be made. The existence^ of an i p s i -lateral and/or contralateral connection of the subesophageal ganglia with the prothoracic ganglion and its relationship to the behavior of the f i r s t pair of legs is of interest. While the fused nerves of the 1 5 0 subesophageal ganglia send off a pair of ce r v i c a l nerves which innerv-ate the muscle about the neck, i t is unlikely that they also innervate the f i r s t pair of legs. The addition of mutant head tissue i p s i l a t e r a l to the mutant tissue of b i l a t e r a l thorax mosaics reduced the a c t i v i t y of the mutant f i r s t leg but s t i l l did not render i t as deb i l i t a t e d as the second and t h i r d legs. This suggests that while the head tissue does affect the behavior of the f i r s t leg, i t s control is not direct. The results might be rationalized as a simple additive effect of mutant ts tissue. Perhaps the para mutation, while inducing paralysis, may allow occasional neuronal a c t i v i t y ( i . e . , i t may be leaky). In a b i -l a t e r a l thorax mosaic (Class I l l b ) , the incoming signals from the wild-type head may affect some f i r i n g of the motor neurons to the f i r s t leg. The amount of signal reaching the mesothoracic ganglia from the head could be reduced or garbled by the p a r t i a l l y mutant prothoracic ganglia and thus may not influence f i r i n g of the motor neurons associated with the mesothoracic and metathoracic ganglia. The addition of mutant tissue to the head might lessen the number and strength of the signals reaching the prothoracic ganglion and thus have a reduced effect on the f i r s t leg. ts The proposal that the para mutation allows some, though greatly reduced, neural a c t i v i t y may be supported by the results from the optomotor response. In the b i l a t e r a l head mosaics in which the + eye was painted, there was a positive (though much less strong) opto-motor response occasionally. The opticon (optic lobe of the brain) on one side of the head, in addition to being connected to the proto-cerebrum, i s connected to the opticon of the opposite side of the head by fibres in the optic t r a c t . The occasional weakly positive - or allow-151 ing for anthropomorphism, perhaps confused - response may be due to an occasional s i g n a l reaching the wild-type side of the brain from the eye on the mutant side of the head. The fact that the wild-type legs of b i l a t e r a l head-thorax mutants are more d e b i l i t a t e d than the + legs of b i l a t e r a l thorax mosaics suggest that there is c o n t r a l a t e r a l connection of the head and thorax. This c o n t r a l a t e r a l connection i s c l e a r l y not a d i r e c t innervation and control of the r i g h t side of the thorax by the l e f t h a l f of the head. It may be that there i s simply an e f f i c i e n t exchange of information c o n t r a l a t e r a l l y as well as i p s i l a t e r a l l y and the lowered a c t i v i t y of b i l a t e r a l head-thorax mosaics may simply be an a d d i t i v e e f f e c t r e s u l t -ing from an increasing loss of CNS a c t i v i t y . As stated, the mobility of leg-only mosaics minimizes the l i k e l i h o o d that the mutant has defective muscles. However, while the leg-only mosaics were not paralyzed, they were often v i s i b l y a ffected. If i t i s true that the v e n t r a l nerves o r i g i n a t i n g in the thoracic ganglia continue to the d i s t a l segments of the legs without synapsis with neurons extending from the leg d i s c s , then the observed abnormal behavior of mutant legs on a wild type thorax is puzzling. There is always the p o s s i b i l i t y that i n t e r n a l mosaicism was not detected, since the invagination of the leg buds during embryogenesis occurs in the proximity of the ventral nerve formation. A l t e r n a t i v e l y , the e f f e c t ts of para on interneural transmission may be postsynaptic. Inter-ts neural and neuro-myal transmission being s i m i l a r , para may also have some postsynaptic e f f e c t on neuro-myal transmission, though i t s e f f e c t may be reduced in muscles. F i n a l l y , the afferent neurons from the sensory receptors may be affected in leg-only mosaics with the r e s u l t that the f l y tends to favor the mutant leg. ts The restriction of the effect of para to the adult f l y is of considerable developmental significance. Power (1952) noted that during pupation there is no manifest regression of CNS material which can be correlated with histolysis of specifically larval tissue. After the onset of pupation, there is an increase in the amount of tissue associated with the neuropile of the CNS and a concomitant decrease in cortex material. Thus, there is a decrease in the size or amount of neuron bodies of the cortex with a concomitant increase in their aggregated fibrular processes which make up the neuropile. The adult ts specificity of para suggests the possibility of separate genetic regulation of movement in larvae and adults. ts Since para f l i e s survived prolonged exposure to high temp-erature, i t was assumed that the v i t a l organs s t i l l functioned in the paralyzed f l y . Indeed, the heart, which could be easily seen through the dorsal wall of the abdomen, was observed to beat quite normally in paralyzed individuals. According to Miller (1950), there appears to be a dorsal median nerve present in the f i r s t chamber of the heart. No nerve connection has been traced, but the heart may be innervated from the stomodeal ganglion of the sympathetic nervous system. This ts suggests that the para mutation has l i t t l e or no effect on the auton-omic nervous system. The use of somatic mosaicism to delineate anatomical sites affected by a mutation depends upon the validity of the assumption that external genotype reflects the genotype of cells underlying the chitin. The resolution of questions such as: where in the CNS does ts the para lesion occur; is the autonomic system affected; are 153 ts inhibitory neurons affected; and, is the effect of para indeed post-synaptic, w i l l require a method for distinguishing internal mosaicism. One promising method is the use of quinacrines to stain heterochromatic bodies (Lewis, personal communication). If such a heterochromatic body was inserted into the unstable ring X chromosome, its loss could be detected. Alternatively, a distally located heterochromatic element on a wild-type rod X chromosome could be eliminated by mitotic crossing ts over which would also homozygose para Other methods of ce l l markers using enzyme variants may also be possible (Hotta and Benzer, 1970). ts The mosaic analysis has led us to conclude that para affects the CNS. However, behavioral mutants exhibiting this pheno-type could quite easily affect muscles as well as sensory neurons. Several mutants affecting one organ of sensory input, the eye, have been isolated and described (Hotta and Benzer, 19691). Ikeda and Kaplan (1970a)have described a mutant, Hk-1, which results in a lesion in specific motor neurons of the thoracic ganglia associated with the legs. Thus, mutations affecting behavior may provide a useful probe into the properties of elements of the nervous system. The property of temperature-sensitivity made it possible.to detect and recover the behavioral defect of paralysis. The availability of methods for mutagenesis and screening on a large scale make the search for mutations affecting nerves and muscles based on a paralytic phenotype quite feasible. Selection schemes for paralytic larvae can also be readily constructed. Thus, the technique of isolating temperature-dependent behavioral.mutants promises to yield various mutations of considerable biological interest. 154 CHAPTER 7 SUMMARY AND CONCLUSIONS Three problems of gene action during development have been investigated: (1) the temporal activity of a single gene known to function in several different tissues, (2) the effect of a gene known to affect determination or regulation of differentiation of a specific imaginal disc and (3) the genetic regulation of the nervous system. The availability of temperature-sensitive mutations permitted the analysis of each of these problems. With regard to the f i r s t problem, i t was demonstrated that the product of a specific gene which is required in at least three different tissues during development is produced at different times during development in each tissue. These results, in conjunction with those of other workers, led to the conclusion that the gene is probably transcribed at different times during development in the different tissues in response to tissue specific stimuli. It may be worth noting that in the l i f e of any given c e l l , the gene is only turned on and off once, whereas in the animal as a whole, there appears to be a succession of activation and inactivation. Some evidence was presented that suggested that this gene may affect membrane and/or pigment granules in differentiating eye tissue. a40a The temperature-sensitive mutation, ss , was shown to affect the choice of the pathway of differentiation of the antenna. a40a When raised at 28°C, ss f l i e s grew normal aristae. However, when raised at 17°C during the third instar, there was a change in deter-mination of the cells which produce the arista so that a tarsus was 155 produced. Shift studies showed that this effect was not due simply to a gradient of a freely flowing determinative substance. This seems to offer evidence against the simple models for positional information and subsequent determination suggested by Wolpert (1969). However, one may invoke c e l l migration as an effector of final phenotype or a more complex model of positional information may be posed. The shift studies also showed that the gene product was active during third instar. This information, in conjunction with induced somatic crossing over experiments, may t e l l us when the gene was transcribed. a40a B The combination of ss and Antp produced a complete leg at 22°C. Since each of these two genes alone exhibit poor expression at 22°C, and are supposed to affect different segments of the antennal structure, i t was suggested that their effect, when combined, may be evidence for c e l l communication in imaginal discs. It is possible to test whether this extreme phenotype is due to c e l l communication or is an epigenetic effect. Antennal disc cells from differently marked s sa40a a n (^ Antp^ stocks, can be dissociated, mixed and implanted into a larval host. The metamorphosed structure is then compared to the a40a , a40a B , , structure formed from + ss /+ ss ; Antp +/+ +; + +/+ + and . _ B a40a , a40a .. _ • • • • , Antp ss /+ ss disc cells treated in a similar manner but not mixed. If c e l l communication occurs, the mixed implant should resemble Antp^ _s__^/+ s s a ^ a control. If the mixed cells look like a40a , a40a . B , , the + ss /+ ss and Antp +/+ + controls, then epigenesis may be the cause of the extreme phenotype observed in the combinant at 22°C. a40a The high penetrance and expressivity of _s at the two temperatures, make i t ideal for initiating biochemical studies with an 156 aim to understanding the molecular basis for determination and regula-tion of differentiation in higher organisms. The last study was an attempt to determine whether mutants affecting the nervous system and/or muscles could be obtained. Such a mutant was successfully obtained. From the studies so far, i t seems that this mutant affects the central nervous system and affects only adults. The fact that its effects are confined to the adult suggests either that in adults and larvae the mode of neural transmission or that the functional anatomy is quite different. The existence of a ts neural mutant (para ) which acts only in adults, suggests that other mutants of the nervous system might be found to affect only the larval stages. Likewise, there might be a class of neural mutants that affect both adults and larvae. Some of the nerve or muscle mutants that affect either adults or larvae may acquire the additional value of functioning as animal models for some human diseases. For instance, one could select for mutants which cause degeneration of only the adult nervous system as a model for Huntington's Chorea. ts The para mutations quickly disrupts neural activity, and thus i t probably does not depend on any d_e novo macromolecular synthesis for its effect. It may be argued that mutants which require a long exposure to high temperature to cause paralysis may be lesions in macromolecules whose continual synthesis is required for normal neural activity. 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