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Genetic and developmental study of the notch locus of Drosophila melanogaster Foster, Geoffrey George 1971

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A GENETIC AND DEVELOPMENTAL STUDY OP THE NOTCH LOCUS OP DROSOPHILA MELANOGASTER by GEOFFREY GEORGE FOSTER B.Sc.j University of British Columbia, 1965 M.S., University of Washington, 1968 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 October, 1971 I n p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I ag ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and s tudy. I f u r t h e r agree t h a t p e r m i s s i o n fo r e x t e n s i v e copy ing o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r an ted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s under s tood that copy ing o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l owed w i thout my w r i t t e n p e r m i s s i o n . Department of Zoology The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date October 1971 ABSTRACT ii The sex-linked Notch locus plays an important role in embryogenesis and determination of many adult structures of the fruit fly, Drosophila melanogaster• Mutation at this locus can cause lethality in embryonic or later stages, as well as morpho-logical abnormalities of the adult eyes, wings, bristles and legs. Alleles of the Notch locus can be broadly grouped into three classes: 1) recessive lethal Notch (N) alleles, which may be deficiencies or point mutations, 2) Abruptex (Ax) alleles, which are probably point mutations and may be either lethal or viable, and 3) viable recessive alleles with visible phenotypes, which affect a variety of traits and are point mutations. The present investigation was initiated with a view to understanding the relationships between N and Ax alleles and the nature of their role in development, and has consisted mainly of the following approaches: 1) an examination of the phenotypes of certain unusual N alleles and the phenotypic responses to alteration of a. the dosage of these alleles in relation to wild-type (NT), 2) an examination of the interaction of Ax alleles with N alleles and with one another, and 3) developmental studies of the conditional (temperature-sensitive) phenotypes associated with certain Notch-locus genotypes. The results of the N-allele dosage study indicate that a single mutation in the Notch locus can affect different functions associated with this locus in fundamentally different ways. De-pending on the genotype and phenotype examined, the responses of iii various N alleles to dosage changes suggest that mutation at the Notch locus may result in reduced, increased or novel activity at the locus. Pour ethyl methanesulfonate-induced Ax alleles have been examined, none of which is cytologically abnormal in salivary gland chromosome preparations, and at least three of which map within the Notch locus. Depending on culture conditions and the alleles involved, Ax/N heteroallelic combinations may be viable or lethal. All Ax/N combinations studied exhibited less severe Abruptex phenotypes (bristle loss and wing vein gapping) than the respective Ax/Ax homozygotes. However, the Ax alleles differed from one another in their effects on the wing nicking of the N alleles, in that the viable allele Ax^B2 and the semi-F1 lethal allele Ax both suppressed wing nicking, whereas the two viable alleles AxE2 and AX^172 both enhanced wing nicking. Furthermore, heteroallelic combinations of Ax alleles which affected nicking in different direction, were lethal (AxE^/AxE2, AxE1/Ax16172, Ax9B2/Ax16172), whereas combinations of Ax alleles with similar effects on nicking were viable (AxEVAx^B2, AxE2/ Ax16172). The temperature-shift experiments have revealed an interest-ing pattern of temperature-sensitive periods (TSPs) for lethality or adult morphological abnormalities associated with various Notch-locus genotypes. TSPs for lethality may be monophasic occurring in the embryo (N6Qsll/N6QSi:L;Dp51b7), or the second larval instar (Ax16172/n264-40 ), or they may be polyphasic, occur-ring in embryo, larval and pupal stages (N2^~1Q3/fan0) . on the iv other hand3 the TSPs for all the adult morphological abnormali-ties examined occur during the third larval instar, including rough eyes and wing nicking (N60gll/+> N264-103/spl), leg segment fusion (^264-103/.^ N264~1Q3/spl), wing vein gapping (Axl6l72/+) and disturbance of bristle numbers (N264-103/splj Axl6l72/+)t Several molecular models are discussed in relation to the observations on N-allele dosage and interactions of the Ax and N alleles. The results are consistent with the hypothesis that the Notch locus is a regulator gene influencing many developmental processes, that mutations can affect the activity either of the entire gene or of various parts of the gene individually, and that N and Ax mutations usually affect this regulatory system in opposite ways from one another. V ACKNOWLEDGMENT It is a pleasure to acknowldege the support and encourage-ment of Professor Dave Suzuki, who let me do pretty much as I pleased during this investigation, but always was able to come up with the critical question. I also want to thank Professor Bill Welshons, who generously provided me with information and mutant strains, without which this project could not have been started. During this investigation I was the recipient of a Killam Predoctoral Fellowship, and I gratefully acknowledge this support. vi TABLE OP CONTENTS ABSTRACT ii ACKNOWLEDGMENT V LIST OP TABLES vii LIST OP FIGURES xii LIST OF PLATES xiv INTRODUCTION 1 MATERIALS AND METHODS 13 I. Description of strains and mutant stocks. 13 II. Use of the symbol D£. 22 III. Incubation temperatures. 23 IV. Procedure for temperature-shift experiments. 24 V. Preparation of specimens for scanning electron microscopy. 27 VI. Procedure for wing nick counts. 28 VII. Procedure for bristle and wing vein gap counts. 28 VIII. Statistical procedures used on nicking, bristle, and vein gap data. 28 IX. Calculation of index of phenotypic expression of bristle and wing vein gap phenotypes. 31 RESULTS 33 A. PHENOTYPES OF SELECTED N AND N^ COMBINATIONS 33 I. Effects of gene dosage on the phenotypes of n£ and N^ O.. 33 II. Effects of gene dosage on the phenotypes of N 1 Q3. 36 III. Effects of gene dosage on the phenotypes of NgU. 44 IV. Effects of gene dosage on the phenotypes of NCO. 58 V. The phenotypes of Nx/Ny;Dp combinations. 62 B. ORIGIN AND MAPPING OF THE ABRUPTEX MUTATIONS 66 C. PHENOTYPES OF ABRUPTEX MUTATIONS 74 I. AXe1. 74 II. AXE2. 78 III. Axl6l72. 80 IV. Ax?52 83 vii D. INTERACTIONS OF NOTCH AND ABRUPTEX MUTATIONS 89 I. Viability of N/Ax heterozygotes. 89 II. The effects of N mutants on the bristle and wing vein phenotypes of Ax mutations. 91 III. The effects of Ax mutants on the wing nicking phenotypes of N mutations. 98 E. INTERACTIONS BETWEEN DIFFERENT ABRUPTEX MUTATIONS 102 F. DEVELOPMENTAL STUDIES OF SELECTED GENOTYPES 109 I. NgH/+ - TSPs for wing nicking and eye facet disruption. 109 II. N1Q3/spl - TSPs for wing nicking, eye facet disruption, tarsal fusion, and bristle disruptions. 125 III. OR - radiation-induced rough eye phenocopy. 130 x v . Axl6l72/N40 _ TSP for lethality; Axl6l72/+ -TSPs for wing vein gapping and loss of ocellar bristles. 132 V. Nl03/fano - TSPs for lethality. 146 VI. Ngll/Ngi:L;Dp - TSP for lethality. 158 VII. Summary of temperature shift results. 161 DISCUSSION 169 LITERATURE CITED 214 APPENDIX 1 224 APPENDIX 2 227 APPENDIX 3 231 APPENDIX 4 236 APPENDIX 5 24l APPENDIX 6 249 APPENDIX 7 255 APPENDIX 8 259 APPENDIX 9 264 APPENDIX 10 269 APPENDIX 11 274 APPENDIX 12 279 APPENDIX 13 284 APPENDIX 14 289 LIST OF TABLES viii TABLE PAGE 1. Symbols and phenotyplc descriptions of non-Notch-locus mutations used. 16 2. Symbols and descriptions of Notch-locus alleles used. 18 3. Symbols and descriptions of chromosome rearrange-ments used. 20 Viability of N^°/N4o;Dp and N^Q/N2*0;Dp/Dp females in relation to siblings at different temperatures. 35 5. Summary of the effects of varying gene dosage on the wing phenotypes of and N^O. 37 6. Number of wings of N1Q3/+ females exhibiting nick-ing when raised at different temperatures. 38 7. Viability of N103/N1Q3;Dp females in relations to siblings at different temperatures. 43 8. Number of wings of N1Q3/N1Q3;Dp females exhibiting nicking when raised at different temperatures. 45 9. Number of wings of NSH/+ females exhibiting nicking when raised at different temperatures. 49 10. The dosage effect of N^ on wing and eye phenotypes of NgU-bearing females. 50 11. Viability of Ng11/Ng11;Dp and NS1:L/Ngll; Dp/Dp females in relation to their siblings, when raised at differ-ent temperatures. 53 12. Number of wings of Nx/N^Q;Dp females exhibiting wing nicking when raised at different temperatures. 63 13. Results of cross for the genetic localization of AxEl. 68 14. Results of crosses for the genetic localization of Axe^. 71 15. Results of crosses for the genetic localization of Ax9B2. 73 16. Counts of ocellar and postvertical bristles and wing vein gaps in various combinations of AxE1 at 20.5°C. 75 ix TABLE PAGE 17. Counts of bristles in eclosed AxE1/Y males raised at 20.5°C. 77 18. Summary of the bristle and wing vein gap pheno-types of A x E 2 . 79 19. Summary of the bristle and wing vein gap pheno-types of Axl6l72. 82 20. Summary of the bristle and wing vein gap pheno-types of Ax9B2. 85 21. Comparison of the bristle and wing vein phenotypes of the viable Ax alleles at 22°C. 87 22. Summary of statistically significant sex differences in bristle and wing vein gap frequencies observed in the viable Ax strains. 88 23. Summary of the viability of heterozygous combina-tions of different Ax and N alleles. 90 24. Summary of the bristle and wing vein gap pheno-types of AxE2/N heterozygotes. 92 25. Summary of the bristle and wing vein gap pheno-types of Ax16172/n heterozygotes. 93 26. Summary of the bristle and wing vein gap pheno-types of Ax9B2/N heterozygotes. 94 27. Counts of bristles in AxE1/Nl°3 females raised at 22°C. 97 28. Nicking frequencies in wings of Ax/N heterozygotes. 99 29. Summary of viability of various heterozygous combinations of Ax alleles at 22°C. 103 30. Relative viability of A x 9 B 2 / A x 9 B 2 > A x 9 B 2 / A x E 2 a a n d Ax9B2/Axl6l72 at 22°C. 104 31. Summary of the bristle and wing vein phenotypes of heterozygous combinations of different Ax alleles at 20-22°C. 107 32. Eye and wing phenotypes of adult females shifted from 20.5°C to 29°C at different successive intervals. 110 X TABLE PAGE 33. Eye and wing phenotypes of adult females shifted from 29°C to 20..5°C at different successive intervals. Ill 34. Eye and wing phenotypes of NS11/* adult females shifted from 20.5°C to 29°C at different successive intervals (Experiment 2). 113 35. Eye and wing phenotypes of NSJ-1/+ adult females shifted from 29°C to 20.5°C at different successive intervals (Experiment 2). 114 36. Positions of anterior boundaries of mutant tissue extending from the posterior rim of the eyes of Ngll/+ females, shift-up experiment 2. 118 37. Positions of anterior and posterior boundaries of mutant tissue in the centre of the eyes of N8H/+ females, shift-down experiment 2. 119 38. Correlation of the occurrence of wingtip nicking with position of the boundary of mutant eye tissue. 120 39. Number of rows of disrupted ommatidia in adult OR flies irradiated before or after puparium formation, at 20.5°C and 29°C. 133 40. Data indicating viability of Axl6l72/N40 females relative to their sibs when shifted from 22°C to 29°C at different successive intervals. 137 41. Data indicating viability of Axl6l72/N**° females relative to their sibs when shifted from 29°C to 22°C at different successive intervals. 138 42. Data indicating viability of AX16172/N40 females relative to their sibs when pulsed from 22°C to 29°C and back after 18 hours, at different successive intervals. 139 43. Data indicating viability of AX16172/N40 females relative to their sibs when pulsed from 29°C to 22°C and back after 24 hours, at different succes-sive intervals. 140 44. Data indicating viability of N10 3/fano females in relation to their sibs when shifted from 22°C to 29°C at different successive intervals. 148 45. Data indicating viability of Nlc>3/fano females in relation to their sibs when shifted from 29°C to 22°C at different successive intervals. 152 xi TABLE PAGE 46. Data indicating viability of N1Q3/fano females in relation to their sibs when pulsed from 22°C to 29°C and back after 18 hours, at different successive intervals. 155 47. Data indicating viability of N103/fano females in relation to their sibs when pulsed from 29°C to 22°C and back after 24 hours, at different successive intervals. 156 48. Data indicating viability of NSH/NSll;Dp females in relation to their sibs when shifted from 20.5°C to 29°C at different successive intervals. 159 49. Data indicating viability of NSi:L/NSi:L;Dp females in relation to their sibs when shifted from 29°C to 20.5°C at different successive intervals. 160 50. Data indicating viability of NS1:L/NSll;Dp females in relation to their sibs when shifted from 20°C to 28°C at different successive intervals during the embryo stage. 162 51. Data indicating viability of NSi:L/NS1:L;Dp females in relation to their sibs when shifted from 28°C to 20°C at different successive intervals during the embryo stage. 163 52. Expected product proportions of Ax/Ax, Ax/+, and Ax/N, according to activator:repressor model ^Figure 17). 190 LIST OP FIGURES xii FIGURE PAGE 1. Genetic map of the Notch locus. 4 2. Mating scheme used to synthesize isogenic wild-type strain (OR). 15 3. The anatomical positions of a) bristles and b) wing veins discussed in the text (LINDSLEY & GRELL 1968). 30 4. Summary of the effects of relative dosage and of temperature on the eye and wing phenotypes of Ngll-bearing flies. 56 5. Diagram used for scoring position of mutant eye tissue boundaries. 117 6. The eye and wing phenotypes of females shifted from a) 20.5°C to 29°C, and b) 29.°C to 20.5°C at different times before puparium formation. 123 7. The eye, wing, leg, and bristle phenotypes of N1Q3/spl females shifted from a) 20°C to 28°C, and b) 28°C to 20°C at different times prior to or after puparium formation. 128 8. The eye phenotypes of OR flies irradiated at different times before and after puparium forma-tion, at 20.5°C and 29°C. 135 9. Viability of AXi6i72/n40 shifted at different stages of development. 143 10. The number of ocellar bristles and wing vein gaps of &xlbl72/+ females when a) shifted from 22°C to 29°C, or pulsed from 22°C to 29°C for 18 hr., and then back to 22°C, and b) shifted from 29°C to 22°C, or pulsed from 29°C to 22°C for 24 hr., and then back to 29°C. 145 11. Relative proportions of viable N1Q3/fano adult females, late pupal NlQ3/fano female deaths, and early pupal (unscorable) deaths, in cultures shifted from 22°C to 29°C at different successive intervals. 150 12. Time of death of N1Q3/fano females in relation to time of shift from 22°C to 29°C. 154 xiii F I G U R E P A G E 13. Viability of NS 1 1/^ 1 1 ;Dp females shifted at different times during the embryo stage. 165 14. Temperature sensitive periods for lethality and adult morphological phenotypes of selected Notch-locus mutant genotypes. 167 15* Hypothetical molecular models to explain tarsal fusion and enhanced wing nicking in N1(->3/+ females at 29°C. 174 16. Hypothetical molecular model to explain opposite response to temperature of eye and wing phenotypes of NS11. 179 17. Model of Notch locus comprising antagonistic elements. 189 18. Correlation of the genetic map positions and complementation pattern of certain Notch-locus mutations. 195 19. "Range of function" model to explain lethality of A x 9 B 2 / a x E 2 and Ax9B2/ A xl6l72. 201 20.. Mating scheme used to replace autosomes of recessive viable Ax stocks with OR autosomes. 205 LIST OP PLATES PLATE 1 Wings of a) b) c) +/+;Dg_, d) AxE2/AxE23 and e) OR females, raised at 22°C. xiv PAGE PLATE 2 Wings of N1Q3/+ females raised at a) 22°C b) 25°C, cT~29°C. 41 PLATE 3 Scanning electron micrographs of NS11/* females raised at a) 20.5°C, and b) 29°C, 47 PLATE 4 Wings of a) NCo/+, b) NCo/+;Dp, and c) NCO/NC o;Dp females raised at 22°C. 60 1 INTRODUCTION The sex-linked Notch locus (standard map position 3.0) plays an important role in embryogenesis and in the determination of many adult structures of Drosophila melanogaster. Mutation at this locus can result in a wide array of defects, such as lethality in embryonic or later stages, and morphological abnormalities of the adult eyes, wings, bristles, and legs. In spite of a great deal of information collected over a period of many years concerning various alleles of this, complex locus, no unified theory has yet been advanced which satisfactorily explains the nature of its role in development. With a view to understanding this role and the nature of recently recovered atypical mutant alleles, the present investigation was initiated. As outlined in more detail below, the main approaches to this problem have been: 1) examination of the phenotypes of certain mutant alleles of the Notch locus, their phenotypic responses to alterations in relative mutant:wild-type allele dosage, and their interactions with one another; and 2) developmental studies of various conditional (temperature-sensitive) phenotypes associated with certain Notch-locus genotypes. Owing to the incredible complexity of the Notch locus, it will be useful at this point to include a brief review of the findings of other investigators. Mutations in the Notch locus can be broadly grouped into three classes - the Notches, the Abruptexes, and the recessive visible mutations. Since mutations within either of the first two classes generally 2 exhibit qualitatively similar phenotypes, these groupings probably reflect, for the most part, similar functional defects. However, the recessive visible mutations are much more hetero-geneous with respect to phenotype, and thus cannot be regarded as a functionally similar group. The relative genetic positions of a number of alleles within the locus are summarized in Figure 1. Typical Notch (N) mutants, which define the locus, are recessive lethals with a dominant phenotype consisting of serra-tions at the tips and along the edges of the wings (Plate la) and/or delta-like thickenings at the ends of the longitudinal wing veins (Plate la,b), a generally increased number of thoracic microchaetae, and other bristle disturbances (BRIDGES AND BREHME 19^4). The lethality of flies homo- or hemizygous for N muta-tions is associated with a gross hypertrophy of the embryonic nervous system at the expense of ectodermally derived structures and a failure of mesodermal tissues to differentiate (POULSON 1939a, b, 1940). N mutations may result from cytologically visible deficiencies, position-effect inactivations of intact (wild-type) Notch loci associated with chromosome rearrangements, or cytologically invisible, genetically separable changes within the Notch locus (LINDSLEY AND GRELL 1968). Cytological analysis of deficiency Notches has provided convincing proof that the Notch locus is located in the polytene chromosome band 3C7, and that a deficiency for this band is sufficient to cause the Notch phenotype (MOHR 1932; SLYZINSKA 1938; DEMEREC 1939). Duplica-tions of genetic material which contain a wild-type copy of the FIGURE 1 Genetic map of the Notch locus. Lethal alleles are below the line, non-lethal alleles above. Solid lines represent information obtained from the following refer-ences: WELSHONS 1958a, b, 1965, 1971; WELSHONS & VON HALLE 1962; WELSHONS, VON HALLE & SCANDLYN 1963- Broken lines represent information obtained from data contained in the present report. The shorter lines above and below the main map indicate the approximate locations of mutations which have not been positioned critically with respect to all the sites on the main map. Note, however, that from the published data and information: 1) AxE2, Ax59b, Ax59d, NJ'1^ , Nfl°, and N h 2 1 are to the right of spl; 2) AxE1 is to the right of fano; 3) A x 9 B 2 is to the right of N ^ ; 4) A x 9 B 2 and Ax59d are to the left of N ^ ; 5) fa£ is between N e l 1 and fano. N I u I * ! X < Ijj o c J ci m M co" N V-c Tr-et PT W o c a CH bfi — Z o 0 Z Z h- 2 o • H Z X < X < Z Z Y-S r? (M o < - z a> PLATE 1 Wings of a) b) n S 1 1 / * , c) +/+;D£, d) AxE2/AxE2, and e) OR females, raised at 22°C. Magnification I8x. 7 Notch locus (If^ ) completely abolish the wing nicking phenotype in deficiency heterozygotes, allow N homo- and hemizygotes to live, and cause a new phenotype - Confluens, consisting of extra wing veinlets (Plate lc) - when their presence results in more than the usual number of loci, in either males or females (MORGAN, SCHULTZ AND CURRY 1941; LEPEVRE 1952; WELSHONS 1965). These observations have led to the hypothesis that the dosage of the 'N^  gene product determines the expression of the mutant phenotypes associated with the Notch locus (WELSHONS 1965). Generally, the point N mutants behave as though they were deficiencies and allow pseudodominant expression of the normally recessive Notch-locus eye mutants facet (fa), facet-glossy (fa£), split (spl), and the wing mutant notchoid (nd), when heterozygous with these mutants. In addition, in combination with the wing mutant facet-notchoid (fano), they cause lethality. Mutants of the N type can be classed as amorphs (WELSHONS 1965). However, there have been several reports in the literature of N mutants which do not behave entirely like deficiencies, in that expression of the wing nicking phenotype is variable and interactions with the intralocus recessive visible mutations are in some cases reduced or absent (LINDSLEY AND GRELL 1968; WELSHONS, personal communication). In all cases however, the N mutations are lethal when homo- or hemizygous (WELSHONS 1965). Additional atypical N mutants exist, which, besides having mild expression of one or more of the typical Notch phenotypes, are associated with abnormalities not seen in other N mutants (WELSHONS 1956a; WELSHONS AND VON HALLE 1962). The mild 8 expression of the typical Notch phenotypes of these classes of mutations, suggests that they may not be completely amorphic, j. but instead have intermediate activity between N_ and N deficiencies (i.e. are hypomorphic to N^) . However, the fact that certain of the N mutants have phenotypes not associated with deficiencies for band 3C7S 'suggest that they may be defec-tive in some way other than by producing less gene product or a product having decreased function. In addition to the N mutations having mild or atypical phenotypes, there exists with-in the Notch locus a number of recessive lethals which lack dominant adult phenotypes, but since they allow pseudodominant expression of the recessive visibles (WELSHONS 1965), and have embryological defects similar to those of N homozygotes (POULSON 1968), these lethals can also be included in the N class of mutations. Clearly the N class of mutations is not entirely homogeneous, since it includes dominant N amorphs, non-visible recessive lethals, and atypical Notches. The possibility that some non-amorphic N mutations may be hypomorphic or neomorphic, might be studied by investigation of the effects of gene dosage on the phenotypes of these mutants. Theoretically, these two mutant classes can be distinguished by their response to alteration of the relative dosage of mutant product with respect to wild-type product (cf MULLER 1932). A major difficulty with this approach is that so far the product of the Notch locus is unknown, so that the relative proportions of mutant and wild-type products can only be inferred by the genetic constitution and phenotypic expression of the genotype. 9 The insertion of duplications of the N^ locus into autosomes permits manipulation of the numbers of mutant and wild-type alleles. In the present investigation, the effects of altering N:N+ gene dosage have been observed for four different N alleles. The results indicate that this approach is valid, although there remain some reservations which have yet to be settled experi-mentally . The Abruptex mutations characteristically cause a reduction in the numbers of certain bristles, and interruptions in wing venation (Plate Id). The original Abruptex allele, Ax28a, is viable in the hemizygous and homozygous condition, and when heterozygous with deficiencies for the Notch locus, both the wing nicking phenotype of Notch and the bristle and wing vein ? fto phenotypes of Ax a are suppressed (MOHR 1932). Wing nicking does occur with a low incidence in Ax28a/N individuals (LEFEVRE, RATTY AND HANKS 1953). The suppression of the Notch phenotype by Ax28a, and the presence of an extra band adjacent to band 3C7 p So in salivary gland preparations of Ax a chromosomes led to the hypothesis that Ax28a was a duplication of the Notch locus with the Abruptex phenotype resulting from a position effect associat-ed with the proximity of the two loci (MORGAN, SCHULTZ AND CURRY 19^1). However, on the basis of mutational data and the fact that other known duplications of the locus produce a Confluens phenotype, LEFEVRE and co-workers suggested that the extra band 2 8 a in the Ax _ chromosome was not 3C7 (LEFEVRE et al_. 1953). Recently, extrapolating from studies on two recessive lethal Ax mutations, WELSHONS (1971) has suggested that Ax28a may indeed 10 consist of two 3C7 bands, one of which contains a dominant Ax mutation that would be lethal without the adjacent wild-type locus. The present report describes the results of investigations on five Ax mutations, their interactions with several N mutants and their interactions with one another, with a view to resolving the nature of Ax alleles and their relationship to Notch. While no decision can be made from the results of the present study as to whether Ax2^a really is duplicated for salivary band 3C7, the data to be presented show that in Ax/N heterozygotes: 1) N mutants suppress the phenotypes of Ax mutations, 2) not all Ax mutants suppress the wing nicking of N mutants, and 3) the sup-pression of wing nicking by certain Ax mutants does not appear to be associated with duplication of the Notch locus. Finally, and perhaps most significantly, these studies have revealed an unexpected system of lethal interactions among the Abruptex mutants. As indicated earlier, the third group of Notch-locus mutations, the recessive visibles, are phenotypically and there-fore probably also functionally very heterogeneous. Note also (Figure 1) that the two eye mutant sites fa (including fa£) and spl, are separated genetically by the site of the wing mutant fano, and that the wing mutant sites fano and nd, are separated by the position of the eye mutant spl. The eye mutations complement one another (i.e. the fa/spl and fag/spl heterozygotes are wild-type in appearance) as well as the two wing mutants. On the other hand, faS/fa, nd/nd2 and fano/nd have intermediate 11 phenotypes compared to the respective homozygotes (WELSHONS 1965). As mentioned in the discussion of N mutants, the recessive visible mutations are expressed pseudodominantly or result in lethality when heterozygous with N mutations. In addition to this, it has recently been reported that heterozygotes of fa^ with or Ax^^, also express the fas phenotype, although less so than in fas/faS flies (WELSHONS 1971). The present investigation has only concerned certain combinations of reces-sive visibles with N and Ax mutations, and has not dealt systematically with recessive visible mutations as a group. Nevertheless, as will be discussed later, the data allow certain conclusions about the nature of fano and spl. In the course of investigating the interactions between various Notch-locus mutations, outlined above, several instances of temperature-sensitive (ts) expressions of lethal or morpho-logical phenotypes, have been discovered. Mutations with pheno-types that are expressed conditionally as a function of temperature have been useful tools in the analysis of development in such diverse organisms as Drosophila (DRIVER 1931; TARASOFP AND SUZUKI 1970; SUZUKI 1970), bacteriophages (EPSTEIN, BOLLE, STEINBERG, KELLENBERGER, BOY DE LA TOUR, CHEVALLEY, EDGAR, SUSMAN, DENHARDT, AND LIELAUSIS 1963), and slime molds (LOOMIS 1969). The utility of ts mutants in developmental studies results from the ability to manipulate the temperature impinging on the organism, at specific time intervals during development. The present report describes experiments involving temperature shifts at different times during the development of selected 12 Notch-locus genotypes, which have been used to define tempera-ture-sensitive periods (TSPs) for several of the conditional lethal and morphological phenotypes. The results show that TSPs for lethality may be found at several stages of development, ranging from the egg to the pupa stage, whereas the TSPs for adult morphological phenotypes (including eye facet pattern, wing nicking, wing vein gaps, bristle loss, and fusion of leg segments) all occur in the third larval instar. The results of the present investigation are consistent with the hypothesis that the Notch locus is a regulator gene which controls many developmental processes. According to this hypothesis, the phenotypes associated with most N mutations reflect reduced repressor activity, whereas the phenotypes of Ax mutations appear to reflect increased repressor activity. Molecular models illustrating this concept are discussed in relation to the data. 13 MATERIALS AND METHODS I. Description of strains and mutant stocks. The wild-type strain (hereafter designated OR) used in all experiments was derived from the highly inbred strain Oregon by the series of crosses outlined in Figure 2. The OR stock was established early in 1969, from a single brother-sister mating of the type shown in the last line in Figure 2, and has been kept in mass culture since that time. This stock is vigor-ous and fertile at both 20°C and 29°C. OR individuals frequently exhibit slight branching of the posterior crossvein, which is typical of Oregon R strains (LINDSLEY AND GRELL 1968), and also occasionally have gaps in the posterior crossveins. No gapping of the longitudinal veins has been seen in this stock, and variation in bristle numbers is slight. Sample bristle counts of OR males and females are contained in Appendix 8 (lines 10-12). Brief descriptions of the mutations and chromosome re-arrangements used are presented in Tables 1-3. Except where noted with an asterisk (*) more information can be obtained in LINDSLEY AND GRELL 1968. FIGURE 2 Mating scheme used to synthesize isogenic wild-type strain (OR). FM6 pol SM1 TM2 spa T ( 2 ; 3 ) e pol spa FM6 pol SMI TM2 spa + > > + + + OREGON R369 FM6 SMI TM2 T ( 2 5 3 )e spa spa pol pol cr* pol + • SMI TM2 spa cr* Y ' + ' ' + ' > > + o* 16 TABLE 1 Symbols and phenotypic descriptions of non-Notch-locus mutations used. Name Bar Bar of Stone bobbed-lethal b rown-Vari e gat e d carnation crossveinless Curly deep orange deep orange-lethal ebony ebony-sooty forked Hairy wing lethal(l)J1 miniature-2 ruby sparkling-poliert Symbol Location B X-57.0 BS bb-V bw car dor dorJ Hw X-57.0 X-66.0 2-104.5 X-62.5 cv X-13.7 Cjr 2-6.1 X-0.3 X-0.3 3-70.7 3-70.7 X-56.7 X-0.0 1(1)J1 X-0.0 m£ X-36.1 rb X-7.5 Phenotype Narrow eye. Extremely narrow eye. Recessive lethal in XX females or XO males. Dominant mottled brown eye colour. Eye colour dark ruby; orange in combination with v. Wing crossveins missing. Wings curved upwards; recessive lethal. Orange eye colour. Recessive lethal dor allele. Black body colour. Black body colour; allele of e. Bristles shortened and bent. Dominant, extra bristles along wing veins and on head and thorax. Recessive lethal; cover-ed by duplications. Wing size reduced. Eye colour ruby; white in combination with wa. s^a p Q l 4-3.0 Eyes small, glazed. 17 Name tinylike Ultrabithorax-130 vermilion white apricot eosin yellow yellow-2 Symbol Location Phenotype tyl X-36 Small bristles. Ubx130 3-58.8 Large haltere size; recessive lethal. v w w1-w 2 2L. X-33.0 Eye colour bright scarlet. X-1.5 White eye colour. X-1.5 Apricot eye colour; allele of w. X-1.5 Yellowish-pink eye colour; allele of w. X-0.0 Yellow body and bristle colour. X-0.0 Yellow body, darker bristles; allele of 18 TABLE 2 Symbols and descriptions of Notch-locus alleles used. Allele *Abruptex-9B2 * Abruptex-59d5 *Abruptex-l6l72 *Abruptex-El *Abruptex-E2 facet-glossy facet-notchoid *facet-notchoid-E Notch-8 Symbol Used Description Notch-264-40 Notch-264-103 Notch-60gll Ax 9B2 Ax 59d Ax16172 Ax-El Ax E2 fa® fa no fa noE N N 40 N 103 N g H Bristle loss; wing vein gap-ping - see Results. Recessive lethal; bristle loss and wing vein gapping. Extensive bristle loss and wing vein gapping - see Results. Recessive lethal; bristle loss and wing vein gapping -see Results. Bristle loss and wing vein gapping - see Results. Irregular eye facet array and glazed eye surface. Nicked wings, thick wing veins; lethal when hetero-zygous with most N mutations. Like fano, but milder wing phenotype. Cytologically deficient for several salivary chromosome bands including 3C7. Reces-sive lethal; nicked wings and thick wing veins. Phenotypically like N£, but not cytologically deficient. Not cytologically deficient. Recessive lethal; temperature sensitive wing nicking, fusion of leg segments, and interaction with other alleles - see Results. Not cytologically deficient. Recessive lethal; temperature sensitive wing nicking and eye facet disarry - see Results. 19 Allele *Notch-70k27 Symbol Used N70k27 Notch-Confluens N Co split spl Description Recessive lethal; weak wing nicking, eyes normal; from origin, should also contain Ngll mutant site - see Appendices 3» Not cytologically deficient. Recessive lethal; weak wing nicking; strong extra wing vein (Confluens) phenotype in presence of extra N^ loci - see Results. Eyes reduced in size and with irregular facet array; miss-ing or doubled bristles frequent. •Alleles not described in LINDSLEY AND GRELL (1968). 20 TABLE 3. Symbols and descriptions of chromosome rearrangements used. Name Symbol Used Markers Carried Description Bar of Stone, white-plus Y Bar of Stone, yellow-plus Y delta-49 delta-49 Duplication (l;2)51b7 ^Duplication (l;Y)59k9(4) ^Duplication (l;Y)60dl9(l) •Duplication (l;Y)67g24(l) First Multiple-6 •lethal First Multiple-6 Muller-5 Second Multiple-1 BS w+•Y BS y+•Y dl49,y Hw n£ dl49»tyl bb1 D£ 59k9(4) 60dl9(1) DP67g24(l) FM6 1(FM6) M5 SMI w+-N+;BS y+-l(l)Jl+;BS y, Hw, m£ bb1, tyl w+, N+ y£, 1(1)J1+ xi, ki)JI+ y2-dor+ (inclusive) B Z.> B, 1 W a , B 9Z Insertion of X chromosome markers into Y chromosome. Insertion of X chromosome markers into Y chromosome. Inversion in central region of X. Inversion in central region of X. Insertion of X chromosome markers into right arm of chromosome 2. Insertion of X chromosome markers into Y chromosome #. Insertion of X chromosome markers into Y chromosome §. Insertion of X chromosome markers into Y chromosome #. Multiply inverted X; female sterile. FM6 chromosome carrying EMS-induced lethal. Multiply inverted X. Multiply inverted second chromosome. 21 Name Symbol Used Markers Carried Description ^Translocation T(1;Y)2E (1;Y)2E 1L> KDJi*; dor+ Insertion of X chromosome markers into Y chromosome (RAYLE & HOAR 1969) ^Translocation T(2;3)e (2;3)e Third TM2 Multiple-2 *Compound X Ubx13°, es *yellow-white- XX, w f/Y w, f forked Compound X Reciprocal trans-location between Second and Third chromosomes. Reces-sive lethal. Multiply inverted Third chromosome. Compounded X chromosomes. Compounded X chromosomes. * Rearrangements not described in LINDSLEY AND GRELL (1968). # GREEN, personal communication. II . Use of the symbol Dp . 22 As noted in Table 3» the symbol D£ is used to denote the presence of a second chromosome containing an insertion of the w+-N+ region. To avoid confusion over the number of N^ alleles present, the symbols Dp+ or + are not used in the text to denote a normal (non-duplication-bearing) second chromosome. For example, the notation N/+;Djo describes a female containing one mutant N allele and one N^ allele on the X chromosomes, and one N+ allele on the second chromosome. 23 III, Incubation temperatures. Several different temperatures were used for incubation of developing cultures in various parts of this investigation. The cultures kept at 20°C, 25°C, 28°C, and 29°C were all grown in incubators, each of which held the respective temperatures to ± 0.5°C. The other temperatures of 20.5°C, 21.5°C, and 22°C, were found in different areas within temperature-regulated rooms in which the average temperature controls were set at 22°C. In these rooms, the actual temperature depended largely on position within the room and, to some extent, on seasonal weather changes. The temperatures stated are those observed in checks made at the time of the experiment in question. In a previous report, the actual 20.5°C temperature was rounded off to 21°C (POSTER AND SUZUKI 1970). IV. Procedure for temperature-shift experiments. 24 The critical time during development of ts mutants when temperature induces mutant phenotypes (or temperature-sensitive period, abbreviated TSP), can be determined by shifting cultures from one temperature to another at different successive intervals. A sufficient number of eggs for experiments involving temperature shifts were obtained by mating approximately 100 aged females (3 to 8 days after eclosion) with 30 to 40 males, two to three days prior to egg collection. For collection of eggs, the parents were kept at room temperature in empty half-pint bottles lying on their sides. The bottles were capped with fresh yeasted petri plates containing Drosophila medium which were changed daily. These conditions ensured good egglays. For most shift experiments, eggs were collected within a two hour interval from approximately 100 fertilized females. In early experiments, the eggs were collected directly on medium in culture bottles, but in later experiments, they were collected on medium contained in petri dishes, then transferred to bottles containing more food. The use of plates facilitated immediate assessment of the success of an egglay, thereby conserving food in unsuccessful cases. It was found that the best egglays were obtained on plates containing fresh moist medium, whose surface had been scratched with a needle and sprinkled with live dried baker's yeast. Furthermore, the best results were obtained when egglays were performed in darkness with the surface of the medium held vertically. 25 The larval instars present in the cultures at the time of shifting were identified according to the morphology of their mouthparts. Samples of larvae were placed in a drop of water on a microscope slide, crushed under a coverslip, and examined microscopically. The larval instars could readily be determined by the number of teeth present on the mandibular hooks (BODENSTEIN 1950). No special techniques were needed to recog-nize non-larval stages. In experiments involving genotypes with known or suspected embryonic TSPs, eggs were collected on petri plates within a one hour period. The food in these plates was not transferred to bottles containing more food until after the temperature shifts. It was reasoned that the smaller volume of food and air and the thinness of the walls of the plates would allow more rapid temperature equilibration of the medium, thereby allowing a more accurate determination of the TSP. In one experiment, synchronization of developmental time in cultures was attempted through the isolation of first instar larvae immediately after they had hatched from the egg. However, by the third instar, larvae in such cultures were not particular-ly well-synchronized, so this method was not repeated. Accurate staging of third instar larvae relative to pupa-tion time was achieved by performing shift experiments in a manner different from that outlined above. In these experiments, several batches of eggs laid by a relatively small number of parents over several days, were collected on regularly changed petri plates, and shifted from one temperature to another at the 26 same time. White prepupae (a very transitory condition at the beginning of the prepupal stage (BODENSTEIN 1950)) were then isolated at defined times after the shift. Thus, if it can be assumed that development of third instar larvae takes about the same length of time for different larvae, the interval from a shift to pupation was determined very precisely. Details of the scoring of the various shift experiments are presented along with the results of the particular experi-ment concerned. 27 V. • Preparation of specimens for scanning electron microscopy. The ability of the scanning electron microscope to maintain objects at different heights within focus makes it a useful tool in the illustration of fine morphological detail. In the present study, the scanning electron microscope has been used to prepare illustrations of mutant eye phenotypes and, in one temperature shift experiment, to provide a record of the eye facet data. Eyes of adult flies were prepared for viewing with the scanning electron microscope in the following manner. The flies were decapitated and the heads placed in chloroform for three or more days. The heads were then removed from the chloroform and allowed to air-dry for at least three days. Omission of the chloroform treatment resulted in the collapse of many of the eyes upon dessication. The dried heads were mounted on aluminum discs with household cement, coated with gold-palladium alloy in a vacuum evaporator, and photographed with a Cambridge Steroscan scanning electron microscope. 28 VI. Procedure for wing nick counts. In order to obtain quantitative data on dosage and N/Ax interactions, counts were made of the number of individuals with 0, 1, or 2 nicked wingtips. Samples of flies for these counts were obtained by rearing the progeny of 3-10 pairs of parents (3-5 day egglays) in 1/4 pint bottles containing Drosophila medium. Except where noted otherwise, scoring was limited to the tips of the wings. VII. Procedure for bristle and wing vein gap counts. To obtain quantitative data on Ax phenotypes, and their interactions with N mutations and one another, counts were made of the number of individuals with 0, 1, 2, (etc.) of a given bristle, and of the number of individuals with gaps in the longi tudinal wing veins. See Figure 3 for the names and positions of the bristles and wing veins scored. Culture conditions were the same as described for wing nicking counts. VIII. Statistical procedures used on nicking, bristle and vein gap data. The mean bristle (or wing nick or vein gap) frequency per fly (x) was calculated by the formula On + n, + 2n„ + 3n + ... x - ° 1 2 3 n FIGURE 3 The anatomical positions of a) bristles and b) wing veins discussed in the text (LINDSLEY & GRELL 1968). O R B I T A L S O c E L L A RS V E R T I C A L S P O S T V E R T I C A L S THORACIC M I C R O C H A E T A E D O R S O C E N T R A L S ANTERIOR P O S T A L A R S S C U T E L L A R S L O N G I T U D I N A L VEINS POSTER IOR ANTER IOR CROSS VEINS 31 where nQ, n^, n^ are the number of flies with 0, 1, or 2 bristles (gaps, nicks), and n is the total number of flies. Standard deviation (S.D.) was calculated by the formula 1 k n-1 X ri^ (x. - x) i=0 1 where x^ = 0, 1, 2 (etc.) bristles (or nicks or gaps). One sided 95% confidence intervals for x were calculated according to the formula S.D. t confidence interval =, v .95 J n-1 where ^.95 is the percentile value for Student's distribution with n-1 degrees of freedom (SPIEGEL 1961). Two values of x were said to be significantly different at the 95$ level of confidence, if their confidence intervals did not overlap. IX. Calculation of index of phenotypic expression of bristle and wing vein gap phenotypes. The index of phenotypic expression, used to summarize bristle and wing vein gap phenotypes in Tables 18-20, 24-26, and 31, relates mutant bristle or wing vein gap frequencies to the respective wild-type frequencies. The wild-type index is de-fined as 1.00, and progressively lower indices indicate increas-ingly severe expression of the mutant phenotypes. Index of expression of bristle phenotypes is given by the formula mutant bristle frequency index = . wild-type bristle frequency 32 Index of expression of wing vein gap phenotypes is given by the formula index = 8-(mutant wing vein gap frequency) . 8 33 RESULTS A. PHENOTYPES OP SELECTED N AND N^ COMBINATIONS I. Effects of gene dosage on the phenotypes of and . One hundred six progeny from the cross N^/dl4_9, £ Hw £ x OR cr*, and 68 N^ <~>/+ progeny from the cross wf}_ N1*0 rb/dl49^y Hw m£ $ x OR cf*, were raised at 20.5°C and examined for wing nicking. Both wings of all individuals were nicked at the tips and along the proximal part of the trailing edges. Examina-tion of smaller samples of the same genotypes raised at 29°C revealed no marked effect of temperature either on the degree of o expression or number affected. Since N_ is deleted for more loci than are carried by Dg_, the genotypes N^/N^;Dp and N^/N^;Dp/Dp were lethal. However, since N ^ closely resembles phenotypi-cally, in both wing nicking and in its interactions with the recessive visible Notch-locus alleles, it was assumed that the dosage results obtained with N ^ would resemble those of a deficiency for the locus. This assumption is supported by the observation that females had a wing phenotype indis-tinguishable from those of either or N£/+, and by observa-tions (reported later) on similar combinations of and with other N mutants. In order to study the phenotypic effects of altering the :N+ ratio, zygotes containing varying numbers of and loci were generated by the crosses outlined in the footnote to 34 Table 4. Nlt0/Y;Dp males and N2|Q/N^°;Dp females are quite viable at both 20.5°C and 29°C (Table 4). The wings of the N ^ / N ^ D p females had serrations both at the tips and along the trailing edges of the wings (although occasionally a female was nicked in only one wingtip), a phenotype very similar to that of N^V+ heterozygotes. On the other hand, the N^/N^Dp/Dp and 1(FM6)/ N ^ D p females (both of which carried two N+ loci) had wild-type wings. Thus, N^°-bearing females which have only one dose of express the typical Notch mutant phenotype, whereas females with j . two doses of N_ are wild-type, m accord with the rules described by WELSHONS (1965). Furthermore, the N^°/Y;Dp/Dp males and 1(FM6)/N4Q ;Dp/Dp females (cross 2, Table 4), each of which had an extra dose of had a Confluens wing phenotype, again pre-dicted by WELSHONS (1965). Both N ^ / N ^ D p and N^/N40;Dp/Dp females appeared to be fully fertile. Four of the latter were mated to w/Y males, and no wa/w-eyed individuals were recovered among 135 female progeny, confirming that the parents were homozygous for Djd (note that N^O was linked to wf^  in these experiments, and that D£ carries w+). If only one dose of Djd were present, half of the females should have had the wa/w eye phenotype. While N^/YjDp males were fertile, their N^°/Y;Dp/Dp brothers were almost completely sterile. However, 2 out of 16 males tested did yield 3 and 7 progeny respectively, when mated to 3 wa/wa females each. The 40 infertility of N /Y;Dp/Dp males is not surprising in view of the triplication of all the loci (except N^) carried by the duplica-tion. TABLE 4 Viability of N^°/N1*0;Dp and N^/N210;Dp/Dp females in relation to siblings at different temperatures. PROGENY FEMALES MALES CROSS* TEMPERATURE 1(FM6)/N l(FM6)/N;Dp** N/N;DR N/N;D£;D£ N/Y;D£ N/Y;D£/D£ 1 20.5°C 26 26 20 - Hj -29°C 74 98 68 69 2 20.5°C 53 l6l 72 44 103 4l 29°C 65 157 94 62 168 42 * 1. l(FM6)/wa N40 rb ? x wf, N ^ rb;D£ <?> 2. l(FM6)/wa N1*0 rb;D£ ? x N ^ rb;D£, cr* ** includes 1(FM6)/N;Dp/Dp females (cross 2 only). u> VJ1 36 The wing nicking phenotypes of and N ^ combinations are summarized in Table 5. The genotypes n5/+, N2*0/*, N^/N^Pp, and n^ Q/n^ Q-Dp/pp females were wild type in terms of wing nicking, as has been reported for N/Y;D]d males and N/+;D]d females (LEPEVRE 1952; WELSHONS 1965). Thus, in terms of the characteristic Notch wing nicking phenotype, N ^ responded to changes in gene dosage in the manner expected of a complete amorph. In passing, it should be noted that minor eye facet dis-ruptions frequently occurred in the N^°/Y;Dp, N^°/Y;Dp/Dp, N^O/N^Dp and N^/N1*0;Dp/Dp progeny of cross 2 (Table 4), but seldom in the progeny of cross 1. The irregularities were more extensive and more frequent in N/Y;Dp/Dp than in N/Y;Dg_ males, but no such difference was detectable between N/N;Dp/Dp and N/N;Dp females. These observations suggest that homozygosis of some factor on the duplication-bearing chromosome may be re-sponsible for the eye roughness, although it cannot be ruled out that some kind of maternal effect on Djd resulted in the facet disarray, since Djd came from the female parent in cross 2 but not in cross 1. Similar mild eye facet irregularities have also been seen in +/Y;Djd males and +/+;Djd females, although no careful studies were made of these genotypes. This point will be refer-red to when the N ^ ^ gene-dosage results are described. II. Effects of gene dosage on the phenotypes of N103 103 The frequency of wingtip nicking in N _/+ females is much lower at 20°C and 22°C than at 25°C and 29°C (Table 6). The 37 TABLE 5 Summary of the effects of varying gene dosage on the wing phenotypes of n£ and NUMBER OP GENOTYPE N + LOCI WING PHENOTYPE* FEMALES 1 1.0 N Nlt0/+ 1 1.0 N N8/N4°;Dp 1 1.0 N N40/N40.Dp i 1.0 N 40 N /+;Dp 2 .+ N4°/N4°;Dp/Dp 2 + N2*0/*; Dp/Dp 3 Co MALES N^°/Y;Dp 1 + N4Q/Y;Dp/Dp 2 Co +/Y;D£ 2 Co * 1.0 N = all individuals have nicked wings; + = wild-type wings; Co = Confluens wings 38 TABLE 6 Number of wings of N103/+ females exhibiting nicking when raised at different temperatures. TEMPERATURE % NICKED CROSS* INDIVIDUALS MEAN NUMBER OP NICKED WINGTIPS PER FLY (+ 95% CONFIDENCE INTERVAL) NUMBER OF FLIES EXAMINED 20°C 1 2 74 80 0.97 ± .12 1.25 ± .09 103 213 22°C 1 2 71 82 1.03 ± .14 1.34 ± .09 91 218 25°C 1 2 100 100 1.97 ± .05 1.99 ± .02 143 337 29°C 1 2 100 100 2.00 2.00 90 228 * 1. OR ? x £ wf N103;Dp. cf 2» M5/2. wf; N 1 0 3 $ x OR err 39 difference in nicking observed between crosses 1 and 2 (Table 6) at the two lower temperatures probably reflects a difference in genetic background between the two crosses, since the stock was outcrossed to M5/M5 females to generate the female parents for cross 2. Although the frequencies of wing nicking appear to be similar at 25°C and 29°C, the wings of N1Q3/+ females were more extensively nicked at 29°C than at 25°C, with serrations at the tips and along both the leading and trailing edges (Plate 2). The degree of nicking of these females at 29°C was definitely more extreme than that of or N4(V+ flies, whereas the nick-ing phenotype in N1Q3/+ females raised at 25°C was less pro-nounced than in N°/+ and females, the wings frequently being only slightly incised at the tips. The enhanced nicking seen in females at 29°C has a counterpart in the pheno-type of N1Q3/Y;Dp males raised at this temperature. At the three lower temperatures the wings of such males are wild;type, as is the case with most other N alleles (including the deficiency Df(wRJ3)/Y;Dp, which was recently examined). However, N1Q3/Y;Dp males raised at 29°C frequently had thickened wing vein tips (usually L5), reminiscent of the thickenings seen in typical N/+ heterozygotes. From these observations, and the fact that N1Q3/fano is viable at 20°C-25°C (Appendix 1), it is clear that N103 does not behave like a deficiency at any of the temperatures studied. At the lower temperatures the relatively mild expres-sion of the wing nicking phenotype suggests that N103 is hypomorphic rather than amorphic at these temperatures. On the other hand, at 29°C the mutant wing expression of females PLATE 2 Wings of N1Q3/+ females raised at a) 22°C, b) 25°C, c) 29°C . Magnification 20x. \ 42 is even more pronounced than of heterozygotes for N deficiencies, the Notch wing phenotype even being expressed in males. This may indicate that at 29°C a defective product of N103 competes with or partially inactivates the N^ allele product. At 29°C only the N 1 0 V + females (Table 6) exhibited fusion of tarsal segments, whereas the legs of their ;Dp sisters and n1Q3/Y;Dp brothers had the normal number of distal segments (1 metatarsus and 4 tarsi). At 20°C-25°C- all three genotypes had normal legs. Thus the mutant leg phenotype behaves like the wing phenotype, in that it is suppressed both in males and heterozygous females in the presence of a duplication for N+. It is pertinent here to note that all N103/fano females raised at 25°C, and some when raised at 20°C or 22°C, also had fused tarsi (Appendix 1). Furthermore, occasional heterozygotes of another Notch allele (N70k3°) with fano survive and at least some of these also exhibit tarsal fusion (Appendix 4). These results lead one to suspect that the leg phenotype may be associated with greatly reduced function at the Notch locus (i.e. intermediate between N/+ and N/N), although it must be pointed out that surviving Ngll/fano heterozygotes had normal legs (Appendix 1). N103/N103 ;D£ females have good viability at 20°C-25°C, but at 29°C considerable mortality (Table 7) results from the sticking to the medium of newly eclosed females. Six n103/n103-Dp females recovered at 29°C exhibited much more severe wing nicking and tarsal fusion than their 29°C N^Q3/+ sibs. Wing nicking data for the N103/N103 ;Dp females raised at the three TABLE 7 Viability of N103/N103 ;Dp females* in relations to siblings, at different temperatures. FEMALES . MALES TEMPERATURE M5/N;D£ M5/N N/N;D£ M5/Y;D£ M5/Y N/YjD£ 20° C 74 59 70 42 63 72 22°C 89 85 75 62 77 81 25°C 110 106 128 91 130 110 29°C 68 56 6 26 88 98 * Progeny of the cross M5/y wa N 1 0 3 $ x ^  wf; N1Q3;Dp o* -t UJ lower temperatures are presented in Table 8. At all three temper-atures the incidence of wing nicking was significantly lower than in N 1 0 3 A females raised at the same temperatures (Table 6). The reduction of nicking at 20°C and 22°C was much greater than that at 25°C. Thus at temperatures at which the wing nicking of N103/+ is milder than that of n£/+ or N1*0/*, doubling the ratio of N ^ 3 significantly reduces the expression of the wing nicking phenotype. On the other hand, at 29°C N103/+ flies have more extensive wing nicking than N®/+ or females, and the extent of wing nicking is further increased in N^3/NlQ3;Dp females. III. Effects of gene dosage on the phenotypes of N g l 1. When raised at 25°C or 29°C, Ngll/+ females- resemble typi-cal N heterozygotes but have weaker wing nicking phenotypes than or heterozygotes. At these temperatures the eyes possess a wild-type facet pattern with only occasional minor ir-regularities (Plate 3a). When raised at 20°C-22°C on the other hand, all females have a lower incidence of wing nicking and exhibit a "rough" eye phenotype (consisting of disarrayed ommatidia and duplicated interommatidial bristles) which extends over the posterior three-quarters to four-fifths of the eye (Plate 3b). In an earlier investigation of females obtained from the cross OR $ x wf^  NS11 rb/Bs w"1" • Y crf, 78.4$ (530/676) of the individuals raised at 29°C, and 3.5$ (24/681) of the individuals raised at 20.5°C, had one or both wingtips 45 TABLE 8 Number of wings of N103/N103 ;Dp females exhibiting nicking when raised at different temperatures. MEAN NUMBER OP NICKED NUMBER OP NICKED WINGTIPS PER FLY (± 95$ FLIES TEMPERATURE INDIVIDUALS CONFIDENCE INTERVAL) EXAMINED 10 20°C 10 0.10 ± .07 63 22°C 11 0.15 ± .10 72 25°C 90 1.64 ± .10 128 PLATE 3 Scanning electron micrographs of females raised at a) 20.5°C, and b) 29°C. Magnifications a) 270x, b) 290x. ft^vv: - V c • ' • / / ' • ' l ^ m W m 48 nicked (POSTER AND SUZUKI 1970). More recent data obtained from flies raised at several temperatures confirm these findings (Table 9). No significant difference in wingtip nicking was apparent between 20°C and 22°C, but the frequency of nicking increased progressively at 25°C and 29°C. The differences between the progeny of crosses 1 and 2 at 25°C and 29°C, may re-flect genetic background differences (see footnote to Table 9). Thus, the mutant eye phenotype of females is only expressed at lower temperatures, while expression of the wing nicking phenotype is increased at higher temperatures. The effect of an extra N^ locus on the expression of N^11 phenotypes was examined in the female progeny of the cross w/w ? x wf NS11 rb/Y;Dp o^ (Table 10). Since the duplication contains the allele, duplication-bearing progeny could be distinguished unambiguously from their non-duplication-bearing sisters on the basis of their eye colours. All female progeny raised at 20.5°C were mutant in both eyes and 6% of individuals exhibited wingtip nicking, while at 29°C all eyes were wild type and 95% of individuals had nicked wings (Table 10). In n£^L/+;D£ females no wing nicking was observed at either temperature, and at 29°C the eye facet pattern was wild type. On the other hand, considerable facet disarray was observed at 20.5°C in these females. The mutant phenotype in the Ngll/+;Dp flies classed as "R" (Table 10) was not as marked as in their Ngll/+ sisters, and 78 of the 108 "R" females were mutant in only one eye. Nevertheless, the eye roughness of N g lV+;Dp females is much more extensive than that seen in the occasional +/+;Dp 49 TABLE 9 Number of wings of Ngll/+ females exhibiting nicking when raised at different temperatures. TEMPERATURE % NICKED CROSS* INDIVIDUALS MEAN NUMBER OP NICKED WINGTIPS PER FLY (± 95$ CONFIDENCE INTERVAL) NUMBER OF FLIES EXAMINED 20° C 1 2 0 1 0.00 0.01 ± .02 152 241 22°C 1 2 2 1 0.02 ± .02 0.01 ± .01 l6l 273 25°C 1 2 10 24 0.11 ± .05 0.28 ± .05 184 287 29° C 1 2 67 76 0.85 ± .12 1.19 ± .06 103 257 * 1. OR $ x wf NS11 rb/B^ w\y cf 2. M5/wf; N g l 1 rb ? x OR cf Note that the M5/N parents for cross 2 were obtained by the cross M5/M5 ? x wf NS11 rb;'D£ c?> 50 TABLE 10 The dosage effect of on wing and eye phenotypes of earing females. PHENOTYPE GENOTYPE NgH/+ Ngll/+;Dp TEMPERATURE 20.5°C 29°C 20.5°C 29°C apricot apricot wild-type wild-type NUMBER IN EACH CLASS EYE COLOUR N+R* NR N+R+ NR+ 179 0 11 0 0 0 7 155 108 0 115 0 0 0 177 0 *N = one or both wings nicked; N = wings not nicked; R = one or both eyes with extensive facet disarray; R+ = no extensive eye facet disarray. 51 female (or in N ^ / N ^ P p and N^/N^O;Dp/Dp females, Table 4), so ell it can be assumed that the eye phenotype of N& is not complete-ly suppressed in the presence of an extra IT^  locus, in contrast to the complete suppression of the Notch wing phenotype in such cases. It is instructive to describe the appearance of Ns~^/Y;Dp males. The wings of such males are wild type, with no wing nicking or thickened veins. However, males of this genotype have essentially the same eye phenotype and temperature-sensi-ell / tivity as /+ females. Thus, in the presence of the duplica-tion, N^^^-bearing males exhibit no wing nicking or thickened wing veins but express the mutant eye phenotype. This is similar to the addition of an extra dose of N^ to N^H-bearing females (Table 10), except that in the females the mutant eye phenotype is considerably diminished. Frequently, Ngll/Y;Dp/Dp males are found in stock cultures (XX,£ w f; D£ ? x wa NS11 rb/Y;D£ cf*, incubated at 20°C-22°C). Such males are relatively infertile, have Confluens wings, and exhibit a much more regular eye facet pattern than their NSll/Y; Dp brothers. The eyes of such males are not completely wild-type, but since this is also true of N^°/Y;Dp/Dp males, it cannot be ascertained whether the eye facet disruption is due to the presence of or to homozygosis of Dg_. However, it is signifi-cant that in spite of any irregularities caused by homozygosis for Djd, increasing the dose of decreases the mutant eye pheno-type of N g l 1 in males. This is similar to the effect seen in ;Dp females. 52 Further studies on the effects of gene dosage on the pheno-types of N^11 have revealed that the addition of an extra dose of N^H affects the eye and wing phenotypes in opposite direc-tions and, unexpectedly, that flies with the genotype Ngll/Ngll. Dp have a temperature-sensitive lethal phenotype (Table 11). The lethality will be discussed in a later paragraph. Expression of the mutant eye phenotype in the few NSH/NSH;Dp females which survived at 20.5°C and 22°C was much more extreme than in NS11/* females, in contrast to the reduction of the mutant eye phenotype caused by the extra N^ in NgH/+;Dp females (Table 10) . The eyes of surviving N^H/NSH^Dp females were reduced in size by about two-thirds and showed an irregular facet pattern over the whole eye with greatly multiplied numbers of interommatidial setae, giving a brush-like appearance to the eye surface. Furthermore, the enhancement of the mutant eye phenotype was apparent in Ngll/Ngll.Dp females raised at 25°C and 29°C, unlike Ngll/+ females, which are wild type at these temperatures. In contrast to the eye phenotypes, expression of the Notch wing phenotype was reduced in Ngll/NgH;Dp compared to at all tempera-tures. No thickening could be detected in the wing veins of the Ngll/Ngll ;Dp females recovered at 20°C-22°C, whereas NgH/+ females raised at the same temperature have detectable, though often small thickenings at the tips of the veins (Plate lb). At 25°C no wing nicking was seen in 43 scorable females, compared to 10% and 24$ of the Ngll/+ females (Table 9). At 29°C 22% (10/42 from experiment 1, and 6/32 from experiment 2, Table 11) of the N^VNgl^Dp females showed wingtip nicking, compared to TABLE 11 Viability of Nsll/Nsll;Dp and nS11/^11;Dp/Dp females in relation to their siblings, when raised at different temperatures. PROGENY FEMALE MALE CROSS* TEMPERATURE M5/N;D£ M5/N N/N;D£*« M5/Y;D£ M5/Y . N/Y.;:Dj 1 20.5°C 102 91 1 90 87 92 29°C 53 53 42 29 85 65 1 20° C 164 158 - 0 128 157 118 22° C 168 159 2 173 191 195 25°C 97 102 44 74 104 101 29°C 43 35 32 10 40 48 2 20.5°C 131 42 24 83 35 113 29°C 49 13 37 17 9 57 * 1. M5/wf NS11 rb ? x wf; NS11 rb/Y;D£ x?1 2. M5/wa nS11 rb;D£ ? x wf; NS11 rb/Y;D£ o» ** Includes N/N;Dp/Dp females (cross 2 only). VJl UJ 54 67$ and 76$ in the samples (Table 9). Thus it can be seen that increasing the dose of N^ -*-1 relative to N^ causes reduction of the Notch wing phenotypes (as observed with N ^ 3 at 20°C-25°C), whereas expression of the mutant eye phenotype is enhanced. Not surprisinglyj the addition of a second dose of N^ to flies already carrying 2 doses of N&-1-1 and 1 of N^, causes con-siderable reduction in the severity of the mutant eye phenotype seen in Nsll/NSi:L;Dp females. The eyes of all N^/NS 1 1;Dp/Dp females (cross 3, Table 11) were much less mutant in appearance than those of N g l l/Ng u ;Dp females raised at the same tempera-ture, being larger in size and having less severe facet and bristle effects. However, the eyes of NS11/NS1:1-;Dp/Dp females were not identical to those of NSH/+, in that they generally had a more mutant facet array and were smaller at all tempera-tures . The effects of D£ on eye facet pattern, noted earlier, may partly account for the former observation, and inspection of certain other stocks (wrb^, spl, and vr^  spl rb) has sug-gested that the reduction in eye size may be at least partly related to some factor carried in the rb-containing stocks. Nevertheless, the fact that the Ngll/Nsll;Dp/Dp females have less severely affected eyes than N^VnS11 ;Dp females, is consistent with the other observations on gene dosage. A summary of the main effects of gene dosage and tempera-ture on the eye and wing phenotypes of N 5 ^ , is presented in Figure 4. It can be seen that the mutant eye phenotype is en-hanced both by lower temperatures and by increased N6^1:N+ ratio, whereas the wing nicking phenotype is decreased by these \\ FIGURE 4 Summary of the effects of relative N:N+ dosage and of temperature on the eye and wing phenotypes of Ngll-bearing flies. The relative size and pattern of the eyes indicates the relative degree of expression of the mutant eye phenotypes wild-type eye facet arrangement; exten-sive facet disarray occasional facet disarray. Wing phenotypes: + = wild-type (no nicks, no thickened wing veins); .2N, etc., indicates approximate frequency of Notch-winged in-dividuals having a nick in one or both wing tips; Co = Confluens wings. G E N O T Y P E 20 - 22 C E Y E S 25°C 29 °C 20-22 C W I N G S 25°C 29°C N ^ y N S " ;DP 9 + O N .2 N N sn/N S";DP/DP 9 + N g y + N g n / y ; D p 9' .02 N .1 -.2 N ' ,7-.8 N N g " / + ; D p + N ^ " / Y ; Dp / D p Co Co Co 57 conditions. Thus, in terms of wing nicking the N^^ allele behaves more like N^ (i.e., becomes less hypomorphic) at progres-sively lower temperatures. On the other hand, in terms of the eye phenotype becomes progressively more mutant at lower temperatures and increased Ng-*--*-:N* ratio, a result which is opposite to the behaviour expected of a hypomorph. Turning to the lethal phenotype, the data in Table 11 (crosses 1, 2) show that Ngll/Ngll;Dp females usually die at 20°C-22°C but survive at higher temperatures. No significant pupal mortality was observed in these crosses, indicating that death of Ng-^/N^^Dp females occurs at some stage prior to puparium formation. The possibility that the lethality might be due to a temperature-sensitive bobbed-lethal (bb^) allele on the Ngll-bearing chromosome (which would be covered by bb* on the Y chromosome in Ngll/Y ;Dp males, and therefore would not cause lethality), was excluded by the absence of lethality in the cross l(FM6)/we bb1 $ x wf. NS11 rb/Y;D£ 0^(165 w^ bb^/Y a", 150 l(FM6)/wa NS11 rb $ , 178 wf_ bbVwf^ NS1* rb % , disregarding Pp). Moreover, the results of experiment 3 (Table 11) suggest that N^H/NgH;Dp/Pp females survive at 20.5°C, which in turn suggests that the relative dosage of Ng11 and determines the viability in this case. Test matings of 11 fertile females raised at 20.5°C (cross 3, Table 11) to w/Y males, yielded no wa/w female or wa/Y male progeny (out of 248 female and 98 male total progeny), confirming that these females were homozygous for Pp_. The lethality of Nsll/Nsll;Pp females at low tempera-tures contrasts strikingly with the observed viability of 58 N^O/N^Dp (Table 4) and N103/N103;Dp (Table 7) flies. Thus, lethality in the presence of N^11 is not due to some defect in the N^ allele carried by Dg_, but to the properties of N^11 itself. The results of developmental studies on this lethality will be reported in a later section. In addition to the phenotypes already described, it was noted that Nsll/Nsll;Dp females raised at 20°C-22°C had certain abnormalities, such as sparse thoracic microchaetae and frequent-ly missing ocellar bristles, generally characteristic of Abrup-tex mutations. Expression of this phenotype was reduced at 25°C and was absent at 29°C, which is the same response to tempera-ture observed for the eye phenotype. This aspect of N^11 will be referred to again. IV. Effects of gene dosage on the phenotypes of . The stock used for the present study N C o) was derived by recombination from a w^ N C o rb chromosome, after it was discovered that the latter contained both a bb1 allele and an enhancer of wing nicking on the X chromosome (Appendix 2). The derived w^ N C o stock lacks both the lethal and the modifier. In a sample of 100 NCo/+ females obtained from the cross OR $ x w^ NCo/Y;Dp cr*at 22°C, 42$ had a nick in one or both wingtips. The non-nicked NCo/+ females could be reliably dis-tinguished from their non-nicked N("!o/+;Dp sisters (97 were examined), which exhibited a characteristic strong Confluens phenotype (compare NCo/+ and NCo/+;Dp wings in Plate 4), like PLATE ij Wings of a) NCo/+, b) NCo/+;Dg, and c) NCo/NCo;Dp females raised at 22°C. Magnification l8x. 61 that of NCo/Y;Dp males. The enhancement of the Confluens pheno-type in the presence of an extra locus is similar to that seen in NCo/Dp(l;l)Co flies (WELSHONS 1956a, b), and contrasts with the reduction of the eye phenotype in the presence of an extra N^ locus. The effect of increasing the NCo:N+ ratio was determined from the progeny of the cross: M5/wa NCo $ x w^ NCo/Y;Dp cf , at 22°C. The number of NCo/NCo;Dp females (36 out of 240 off-spring, with 40 expected) indicates that NCo/NCo;Dp is not a lethal genotype at 22°C. However, these females were weak, moving about very slowly and frequently becoming mired in the food medium. Of 28 flies whose wings could be scored (not mired in food), 5 (18%) exhibited wingtip nicking, compared to 42% in the NCo/+ females. The Confluens phenotype of these females was even more extreme than that of NCo/+;Dp females (Plate 4), and the wings themselves were curved downwards and held at right angles to the body. Thus, the Confluens phenotype is enhanced by increasing or decreasing the :N+ ratio, whereas expression of the wing nicking phenotype is reduced or abolished. In addition, the NCo/NCo;Dp females had a disrupted eye facet phenotype not seen in the other NCo-bearing combinations. To summarize briefly, the observations on the response of N^0 to changes in the ratio suggest that this allele is hypomorphic in terms of the defect responsible for wing nicking, but not so far as the Confluens phenotype is concerned. More-over, the fact that Confluens is known to result from increased N* dosage suggests that may be hypermorphic in this regard, 62 especially when it is remembered that NCo/Y;Dp males have a more extreme phenotype than N+/Y;Dp males. Thus, the properties of NCo indicate a clear functional distinc-tion within the Notch locus for the wing nicking and Confluens phenotypes. V. The phenotypes of Nx/Ny;Dp combinations. An examination of the phenotypes of combinations of NCo, and N g l 1 with and D]d, has yielded results consistent with the findings of the gene dosage investigations and with the assumption that N2^ can be considered an amorphic allele. Com-parison of the wingtip nicking data with those for the respective N/+ heterozygotes (compare Table 6 and cross 1, Table 12, and compare Table 9 and crosses 2 and 3, Table 12) shows that the frequencies and patterns of temperature sensitivity of wing nicking in ;Dp females are remarkably similar to those for Nx/+. The few statistically significant differences are likely due to differences in genetic background. The frequency of wingtip nicking of N^^/N^O ;Dp and NCo/N40 ;Dp females from crosses 4 and 5 (Table 12) is also quite similar to the frequency among the respective Nx/+ females (note that 8l% of 74 NCo/+ progeny of the cross OR $ x wf^  NCo rb/Y;Dp C*, had nicked wing-tips) . Furthermore, the disordered eye facet phenotype seen in females at low temperature was also expressed in the Ngll/N40.Dp females at 20°C-22°C, and the strong wing nicking and tarsal fusion seen in N103/+ females at 29°C was expressed 63 TABLE 12 Number of wings of Nx/Nlt0;Dp females exhibiting wing nicking when raised at different temperatures. GENOTYPE CROSS* N1Q3/N40;Dp 1 Ngll/N40.Dp Ng^/N^SDP NCo/N4o;Dp % NICKED TEMPER INDIVID--ATURE UALS MEAN NUMBER OP NICKED WINGTIPS PER FLY (± CONFIDENCE INTERVAL) NUMBER OF FLIES EXAMINED 20°C 82 1.38 + .14 104 22°C 73 1.22 + .18 67 25°C 99 1.84 + .07 88 29°C 100 2.00 72 2 20°C 7 0.10 + .09 59 22°C 7 0.08 + .08 59 25°C 38 0.55 + .18 55 29°C 82 1.4 + .3 22 3 20° C 4 0.04 2; .05 52 22°C 5 0.05 + .05 65 25°C 33 0.40 + .19 55 29°C 61 0.8 + .3 18 4 20.5°C 9 mm 81 29°C 83 - 86 5 20.5° C 73 45 * 1. 2 . 3. 4 . 5 . M5/wf N ^ rb $ x wf; N1Q3/Y;Dp .<?» M5/wf; rb $ x wf; N g l 1 rb/Y;D£ o* M5/wf; rb ;D£ ? x wf; N^ 1 1 rb/Bf_ w^. Y c^ 1 (FM6)/wa NS 1 1 rb ? x wf; rb/Y;D£ <f l(FM6)/wa N C o rb E-N 7 0 k 2 7 bb1 £ x wf; N ^ rb/Y;D£..<^ 64 in the N 1 0 3 / N^ Q;Dp females at this temperature. Also, the wings of the NCo/N40 ;Dp females were similar to those of N /+ in appearance. The recessive behaviour of to the wing nicking, eye, and leg phenotypes of the other N mutants, and the absence of a strong Confluens phenotype in NCo/N40 ;Dp females, strongly supports the assumption that N^^ can be regarded as an amorphic allele. Observations made on limited numbers of and NCo/N8;Dp females raised at 20.5°C (24 and 5 flies from the crosses N8/dl49,y Hw m£ $ x wf^  NS11 rb/Y;D£ a*, and N8/dl49,y Hw m£ £ x w^ rb/Y;Dp cr^respectively), confirmed the reces-siveness of N^ to the N s l 1 eye phenotype, to the wing nicking phenotypes of both N^H and NCo, and the abolition of the Confluens phenotype. Observations on the phenotypes of trans combinations of the three N mutants with mild wing nicking, confirm and extend the results obtained in the gene dosage investigations. As would be predicted if the mild wing nicking of the N/+ hetero-zygotes reflects hypomorphic activity, no wing nicking was observed among 89 N1°3/NS11;Dp (22°C), 33 N103/NCo;Dp (22°C), and 213 NS1l/NCo;Dp (20.5°C)* females, a striking reduction compared to the respective N/+ heterozygotes. Moreover, the eye ^Heterologous combinations obtained from the respective crosses: m 5 / j r N 1 0 3 $ x w ^ N s 1 1 rb/Y;D£ <? M5/y N 1 0 3 x wf: NCo/Y;Dp <? 1 (FM6)/wa NS11 rb % x wf N ^ rb/Y;D£ cf9 65 phenotype of N1°3/NS11;Dp and the Confluens phenotype of N1Q3/ ]vjCo .pp females were intermediate between those of the respective N / + and N / + ; D j d combinations, indicating that N 1 0 3 is intermediate between N^ and n£ or N*^ in these respects also. The eye and wing vein phenotypes of Ngll/NCo ;Dp, on the other hand, were essentially identical to those of the respective N/+ heterozy-gotes, suggesting that N^11 is amorphic or at least very hypo-morphic in terms of the Confluens function, and likewise in terms of the mutant eye function, even though both alleles have considerable activity in the function whose absence causes extensive wing nicking. Observations such as these, combined with the results of the gene dosage investigation, support the notion that mutations at the Notch locus may only affect certain of the functions of this locus as determined phenotypically. In fact, it appears that a single mutation can affect different Notch-locus functions in fundamentally different ways, as exem-plified by the wing nicking and Confluens phenotypes of . 66 B. ORIGIN AND MAPPING OP THE ABRUPTEX MUTATIONS Pour of the five Ax alleles studied (Ax51, AxE2, Axl6l72 and Ax9B2) have been examined in detail with respect to the phenotypes of the wing veins and of certain bristles. The first two of these mutants were recovered among the progeny of ethyl methanesulfonate (EMS)-treated OR males in this laboratory (Appendix 3), and Ax1^1?2 and Ax9B2 were also EMS-induced (LEFEVREj WELSHONS, personal communications). AxE1 is semilethal as a hemi- or homozygote and in trans heterozygous combinations with most N mutants. The few individuals which do manage to eclose are very weak and usually become mired in the food medium shortly after hatching. AxE2 is not as extreme as AxE1 and is both viable and fertile as a hemi- and homozygote. The Ax alleles, Ax1^1?2, Ax962 and Ax^9d, were kindly supplied by Dr. W. J. WELSHONS. Ax l 6 1 7 2 and Ax9B2 are viable in the hemi-zygous and homozygous condition, although homozygous Ax9B2 females are poorly fertile and this stock is kept by crossing hemizygous Ax/Y males to compound-X females. The fifth mutant, A x£^, which was not examined in great detail, was maintained in cis combination with the recessive visible mutant fa£. Ax59d is almost a complete recessive lethal, has cytologically normal salivary gland chromosomes, and has been mapped genetical-ly within the Notch locus between spl and N C o (WELSHONS 1971). The salivary gland chromosomes of AxE1, AxE2, and Ax!6l72 have also been examined cytologically, and all appear to be normal in the Notch region of the X chromosome (KAUFMAN, person-67 al communication). In addition, as will be described below, AxE1, AxE2, and Ax9B2 have been mapped genetically at sites with-in the Notch locus. In the mapping of AxE1, advantage was taken of the facts that Axe-*- is viable and fertile when heterozygous with the non-Abruptex recessive visible mutations in the Notch locus, and that the A x E Vn and fano/N genotypes are lethal. Thus, in the cross wi fano spl rb/w^. AxE1 rb+ g x wf N ^ rb/B^ w^.Y , the only surviving female progeny should be non-disjunctants, "break-through" AxE1/N and fano/N females, or fano+ Ax+ crossovers between fano and AxE-*-. The results of two such crosses are pre-sented in Table 13. It can be seen that there were appreciable p 1 numbers of surviving Ax /N and fano spl/N females, which were very weak and sterile and could easily be recognized phenotypic-ally. The B^ class of females results from non-disjunction of the maternal X chromosomes. The w^ rb+ class of exceptions was unexpected, since these would at first appear to be recombinants within the Notch locus unaccompanied by recombination for the closely linked flanking eye-colour markers. However, some of these females differed from wild type in that they were Abruptex-like (wing vein gaps) or had nicked wings. Seven of the excep-tional females survived long enough to be test-crossed to wa spl rb males, and five of these crosses yielded progeny. The results of these and subsequent crosses, which are recorded in Appendix 5, indicated that the w^ rb+ exceptions were not the products of recombination, but were triploid females. The pertinent observations supporting this conclusion are that: 68 TABLE 13 Results of cross for the genetic localization of AxE1. NUMBER OF MALE GENOTYPE OF SURVIVING FEMALE PROGENY SERIES* PROGENY w^ S£l rb w^ rb^ Bf. N/AxE1 N/fano spl 46,986 31 160 17 11,542 25 184 10 TOTALS 58,528 14 56 344 27 Series 1: 24 cultures in half-pint bottles, 25-30 females per culture; mostly 3 day broods but some were longer (5-6 days); total eglaying period 24-28 days. Series 2: 40 cultures in quarter-pint bottles, with 5 sets of 8 bottles each having 1, 2, 3, 4, or 5 parent females, respectively. Neither the total number of progeny nor the ratio of rb:rb+ male progeny showed any effects of increasing numbers of females, so the data were pooled. Eggs were collected in 3 day broods for a total of 6 broods 69 1) these females were poorly, fertile compared to the spl rb females, and they yielded male, female and sterile intersex progeny; 2) some of the F^ female progeny from the test crosses were also semisterile and yielded intersexes, while others were fully fertile and yielded only male and female progeny; 3) from the phenotypes of the exceptional females and those of their progeny and one subsequent generation, at least four of the original fertile w^ rb+ females must have carried the chromosomes wa fano spl rb, w a N2*0 rb, and w^ AxE1 rb+. All the w^ spl rb female exceptions tested (see Appendix 5) behaved like normal diploids and passed the spl rb chromosome to their progeny. These represent true crossovers between fano and AxE1, and posi-tion AxE1 to the right of fa110. From these data no decision can be made as to the position of AxE1 with respect to spl, although it would seem that if AxE1 is to the right of spl, the two mutants must be extremely closely linked. This is based on the fact that 14 crossovers between fano and AxE-L were recovered, but none between AxE-L and spl. The frequency of recombination between fano and AxE1 (Table 13) is 0.05$, which is greater than the map distance between fano and spl (0.03$) recorded by WEL-SHONS (1958). Unfortunately, the present results and the data of WELSHONS cannot be compared in order to position AxE1 with respect to spl, since genetic background, temperature and other culture conditions were likely different in the two investiga-tions. Nevertheless, it is probably safe to conclude that AxE1 maps within the Notch locus, close to or at spl. F P In order to map Ax with respect to other mutants at the 70 Notch locus, all of the male progeny of wf: fano spl rb/w* AxE2 rb+ females were scored for their visible phenotypes. The results of two experiments are presented in Table 14. For positioning AxE2, the relevant recombinants recovered were the wa fano AxE2, spl rb, wa fano spl AxE2, and rb_ classes, which place AxE2 to the right of spl (see Appendix 6 for progeny tests of these recombinants). When the double crossover classes wa fano rb and fano AxE2 are included, the genetic map distance between fano and spl is calculated to be 0.05 unit and between spl and AX e 2 0.01 unit. This observation, combined with the fact that spl lies approximately equidistant between fano and nd in WELSHONS' map (1965) suggests strongly that AxE2 lies within the presently defined limits of the Notch locus. It should be noted at this point that when linked in the cis position, fano completely suppresses the wing vein but not the bristle pheno-types of AxE2. This and other interactions of AxE2 with fano and spl are described briefly in Appendices 6, 7. Although Ax16172 was not mapped extensively, the available data suggest that this mutant probably also maps within the Notch locus. Use was made of the discovery that both the AxE2/ Ax9B2 and Ax1^172/Ax9B2 combinations are lethal, while AxE2/ Axl6l72 females are both viable and fertile. Thus, in the cross wa AX E 2 rb/w^ Ax 1 6 1 7 2 rb+ $ x w^ Ax9B2 rb/Y CF*, the only surviving female progeny should be Ax* recombinants or non-disjunctants, whereas all males should survive. In one experi-ment of this kind 12,873 males were recovered and no recombinant females were observed. Crossing over in the wa-Ax and Ax-rb 71 TABLE 14 Results of crosses for the genetic localization of AxE2. MALE PROGENY GENOTYPES CROSS* PERCENT CROSSOVERS wa fano S£l rb 8 ,185 8,295 + A x e 2 + 8 ,052 8,045 wa AxE2 + 179 130 + fano spl rb 164 150 wa fano A x e 2 + 3 3 + spl rb 2 7 wa fano spl A x e 2 + 0 2 + + rb 1 0 wa fano s£l + 493 464 + A x e 2 rb 609 463 wa fano rb 0 1 + fano A x e 2 + 1 0 TOTALS 17,689 17,560 1.77 0.05s* 0.01*** 5.76 * 1. w fano spl rb/AxE2 ? x £ wf/Y. <? 2. w^ fano spl rb/AxE2 $ x wf fano spl rb/Y cfl Cross 1 consisted of 12 cultures, cross 2 of 18, in half-pint bottles, 5 pairs of parents per bottle. Eggs were collected in 3-5 day broods over a total period of 27 days, ** Includes double crossovers wa fano rb and + fano AxE2 + *** Includes double crossover wa fano rb 72 regions as observed in the males occurred with normal frequencies. This indicates that AxE2 and Ax16172 must be situated close to one another, probably within the Notch locus. The lethal inter-actions mentioned above will be described more fully in a later section. The results of two crosses used to map Ax^52 are reported in Table 15. The five Ax-N recombinants recovered place Ax9B2 between and . The observed frequencies of crossing over within the Notch locus (0.09% between both N1*0 and Ax$B2 3 and Ax9B2 and N ) were rather high when compared with the 0.03-0.04% between N ^ and observed by WELSHONS (1958b), although recombination with the flanking markers wf^  and rb occurred with near-normal frequencies (Table 15). This could reflect reduced viability of Ax^62 males compared to Ax+, especially when one considers the possible interaction of A x 9 B 2 with genetic modi-fiers, which undoubtedly exist in both N stocks and in the w a fano spl A x e 2 rb stock. Nevertheless, the available crossover data and the origin of Ax^ , indicate that Ax9B2 i s a point mutant which maps within the Notch locus. Thus the genetic data strongly suggest the inclusion of all four Ax alleles tested within the limits of the Notch locus. 73 TABLE 15 Results of crosses for the genetic localization of Ax9B2. PROGENY CROSSOVER CLASS CROSS* GENOTYPE NUMBER % GENOTYPE ' NUMBER % + Ax + 3017 + Ax rb 22 59 wa-Ax w Ax + 55 1.72 wa Ax rb 28 1.24 Ax-rb Ax-N + Ax rb 125 3.91 + + rb 3 0.09 + Ax + wa + rb 91 4.02 2 0.09 * Male progeny of crosses: 1. wf; N ^ rb/+Ax9B2 + ? x wf fano spl AxE2 rb/Y *f 2. wf; N £ f _ +/+Ax9b2 rb ? x wf; fano spl AxE2 rb/Y cH Cultures were incubated at 22°C in quarter-pint bottles (11 bottles, cross 1; 20 bottles, cross 2), with 5-8 female parents per bottle. Since fertility was variable, especially in cross 2, brood length was adjusted (3-7 days) to increase the number of progeny in some cultures. Total egg collection period was 10-15 days. 74 C. PHENOTYPES OF ABRUPTEX MUTATIONS I. A x e 1 . FT The most conspicuous phenotypic features of Ax /+ females raised at 20.5°C, are the presence of terminal gaps in at least one of the L5 wing veins in nearly all individuals and the high incidence of missing ocellar and postvertical bristles, the post-verticals generally being more strongly affected than the ocellars (consult Figure 3 for the positions of wing veins and bristles scored). Other bristles are not affected so strongly, although the postorbital setae and thoracic microchaetae are noticeably sparser and less regularly arrayed than in OR flies. Sample wing vein gap and ocellar and postvertical bristle counts of AxE1-bearing flies are presented in Table 16 (see Appendix 8 for sample bristle counts of OR flies). The females in lines 1 FT and 2 are genotypically similar with respect to Ax but derived from different crosses. Thus, the slight differences in values probably reflect background genotypic variation. It can be seen El that in the Ax /+ heterozygotes, the numbers of postvertical and ocellar bristles are reduced and the incidence of wing vein gaps is high. Addition of an locus (compare sibs in lines 2 and 3) strikingly increases ocellar bristle number and reduces the number showing postvertical bristle or vein gap phenotypes. By comparing lines 2 and 4 (remembering that the back-ground genotypes are different), the effect of autosomal inser-ts tion of the Notch locus can be measured since N° is a deletion 75 TABLE 16 Counts of ocellar and postvertical bristles and wing vein gaps in various combinations of AxE-*-at 20.5°C. NUMBER OP PROGENY IN EACH PHENOTYPIC CLASS WING VEIN NUMBER OF NUMBER OF GAPS** GENOTYPE CROSS* OCELLARS POSTVERTICALS - 1 OR MORE L4 & 0 1 2 0 1 2 0 L5 L5 AxE1/+ ? 1 55 34 8 94 3 0 4 89 4 AXE1/+ $ 2 72 16 2 72 9 9 0 75 15 AxE1/+;D£ ? 2 1 5 88 32 29 33 46 48 0 AxE1/N8;D£ $ 3 15 2 2 19 0 0 0 18 1 AXE1/N4o;D£ £ 4 62 10 1 62 8 3 0 73 -AXE1/Y;D£0^ 4 60 7 1 68 0 0 11 57 -* 1. OR ? x AxE1/Bs w^.Y o^ 2. wf; rb/w^ rb ? x wa AxE1/Y;Dpcr;? 3. N8/dl-49,y Hw m£ $ x w^ AxE1/Y;Dpcr? 4. l(FM6)/AxE1 $ x wf; N ^ rb/Yj^cr' * Scoring of wing vein gaps: 1) progeny of crosses 1-3 were scored either for no gaps, or for gaps in L5 only (1 or both wings), or for gaps L4 and L5 (1 or both wings); 2) progeny of cross 4 were only scored for the absence or presence of wing vein gaps. \ \ 76 of the entire locus. In general, the two sets of results are similar in that vein gapping is increased and the bristle numbers decreased. The point mutant N2^ (line 5) interacts similarly to N8, thereby behaving as an amorph with respect to its phenotypic interaction with AxE-L. In terms of both allelic dosage and phenotype the AxE-*-/Y;Dp males (line 6) are similar to the females in lines 1 and 2. In contrast to the results with N^ and N2*0, 92 AxE1/NCo;Dp females from the cross l(FM6)/AxE1 ? x wf^  NCo rb/Y;D£ & exhibit-ed no wing vein gaps. Thus, behaves like a dose of N^ in •pi terms of its morphological interaction with Ax . Death of flies hemizygous or homozygous for AxE1 occurs in the late pupa or partially-eclosed adult stages with the few "escapers" rapidly becoming mired in the food. By placing the culture bottles on their sides, emerging adults could be prevent-ed from falling into the medium, and specimens thus be obtained for examination. Such flies have deformed wings and crippled legs and usually die within three days. Although the genital systems of A x E 1 / Y males are normal and motile sperm are produced (KAUFMAN, personal communication), they made no attempt at copulation when placed with aged virgin wild-type females. The bristle data for A x e : l / Y hemizygotes presented in Table 17 show that none of the males examined possessed ocellars, postverticaLs, or anterior scutellars, and that the dorsocentrals were usually entirely missing. Orbitals and posterior scutellars were re-duced in frequency by one-third to one-half, and about two-thirds, respectively. In contrast to the drastic reduction in 77 TABLE 17 Counts of bristles in eclosed AxE1/Y males* raised at 20.5°C. NUMBER OP PLIES IN EACH MEAN NUMBER OF PHENOTYPIC CLASS BRISTLES PER TYPE OF BRISTLE 0 1 2 3 4 5 6 FLY (± 95% CONFIDENCE INTERVAL) ORBITALS 0 0 9 32 36 10 1 3.57 ± .16 OCELLARS 88 0 0 - - - - 0.00 POSTVERTICALS 88 0 0 - - - - 0.00 VERTICALS 0 0 0 1 84 - - 3-99 ± .02 DORSOCENTRALS 78 6 1 0 0 - - 0.09 ± .06 ANTERIOR SCUTELLARS 85 0 0 — — — — 0.00 POSTERIOR SCUTELLARS 37 35 13 0.72 ± .14 Data are the pooled observations on AxE1/Y male progeny which survived in 5 crosses performed for other purposes. The general format of these crosses was l(FM6)/AxEl x "x"> where "x" was s£l A x E 2 , fano A x E 2 j fano spl Ax^,"~two fafto~~AxE2 recombinants derived independently, and A x 9 B 2 t 78 numbers of the other bristles examined, the verticals were pre-sent in wild-type frequency. Homozygous AxEl females (which occasionally eclosed in l(FM6)/AxE1 £ x AxE1/Bs w+.Y cf^ stock cultures) were not examined in detail, but their phenotype was generally like that of the males. Wing vein gap frequencies could not be obtained in these flies owing to the extreme deform-ity of the wings, but little wing venation and no wing nicking was observed in a few individuals whose wings were sufficiently extended to permit examination. II. A x e 2 . F2 The most characteristic features of Ax hemi- and homozy-gotes are the nearly complete absence of anterior orbital bristles and the presence of terminal gaps in the L5 (and often L4) wing veins (see Figure 3). Other bristles are also affected, but to a lesser extent. The results of sample bristle and wing vein gap counts of hemizygous and homozygous AxE2 individuals raised at 22°C and 29°C are summarized in Table 18. It can be seen that at 22°C the number of orbital bristles was reduced by one-third, while the verticals, postverticals, dorsocentrals, and scutellars were present in nearly wild-type frequencies. The ocellar bristles were slightly reduced in numbers at the low temperatures, and there was a small but significant sex-differ-ence in ocellar frequency, females having a lower frequency than F ? males. At 22°C every k-xr^  hemi- or homozygote had gaps in from two to four wing veins, with no significant differences between E2 TABLE 18 Summary of the bristle and wing vein gap phenotypes of Ax * # ORBITALS OCELLARS POSTVERTICALS VERTICALS GENOTYPE 22°C 29°C 22°C 29°C 22°C 29°C 22°C 29°C AxE2/Y cf* 0.66 0.64t 1.00 0.46T 1.00 0.38T 1.00 0.99T AXE2/AXE2 $ 0.67 0.62t 0.97 0.15TS 1.00 0.l4TS 1.00 0.99 AXE2/+ % 0.99H 0.95th 1.00 0.95™ 1.00 1.00H 1.00 1.00 SCUTELLARS DORSOCENTRALS ANTERIOR POSTERIOR WING VEINS GENOTYPE 22° C 29°C 22°C 29°C 22° C • 29°C 22°C 29°C AxE2/Y a* 1.00 0.89T 1.00 0. 7^T 1.00 0.97T 0.72 0.39T AxE2/AXE2 £ 1.00 0.91T 1.00 0.86TS 1.00 0.97T 0.71 0.54TS AXE2/+ ? 1.00 0.97™ 1.00 1.00H 1.00 1.00H 0.88H 0.81™ * The numbers presented represent the "index of phenotypic expression" of the bristle or wing vein phenotypes, with 1.00 equal to wild-type, and smaller numbers indicating progressively more severe expression of the mutant phenotypes. See Methods and Materials for the formu-lae which define "index of phenotypic expression" for both bristles and wing vein gaps. # See Appendix 8 for actual counts of bristles and wing vein gaps. T Statistically significant differences between the 29°C and the 22°C frequency. S Statistically significant difference between homozygous females and hemizygous males at this temperature. H Statistically significant difference between heterozygous and homozygous females at this temperature. 80 the sexes. At 29°C the frequencies of all the bristles except the verticals were reduced, the ocellar bristle sex-difference was much more pronounced than at lower temperatures, and two other bristles, (postverticals and anterior scutellars) showed significant sex-differences which were not apparent at lower temperatures. It is also noteworthy that while the ocellar and postvertical frequencies were lower in females than in males, the anterior scutellar frequency was higher in females. The frequency of wing vein gaps increased significantly at 29°C, and here also there was a sex-difference, the males having more gaps than the females. Thus, females were more mutant than males with respect to the ocellar and postvertical phenotypes, but less mutant than males with respect to the anterior scutellar and wing vein gap phenotypes. The last line of Table 18 summarizes the wing vein and bristle data obtained from A x e 2 / h - heterozygotes at 22°C and 29°C. All mutant phenotypes were greatly reduced compared to those of the homozygotes at each temperature, and temperature sensitivity was only detectable for the orbitals, ocellars, dorsocentrals, and wing vein gaps. III. Ax16172. The characteristic appearance of Ax^ 1^ 2 hemi- and homo-zygotes is one of markedly reduced head and thoracic bristle frequencies, and extensive gaps in wing venation. These pheno-types are much more extreme than those of AxE2. In addition, 81 the wings of Ax-1-^1^ often are curved or drooped downwards, deformed at the bases, and frequently contain prominent bubbles, and the eyes frequently contain regions of ommatidial disruption or are irregular in outline. The results of bristle and wing vein gap counts of Ax1^172 males and females raised at 22°C and 29°C are summarized in Table 19. In the samples examined no postvertical and no or very few ocellar bristles appeared in either sex at either temperature. There appeared to be a slight increase in ocellar frequency at 29°C, the difference between the 22°C males and 29°C males being significant at the 95$ level. Other than this, the ocellar, postvertical, and vertical bristle frequencies were not affected by either sex or temperature. The frequencies of the other bristles examined (orbitals, dorsocentrals, and scutellars), and of wing vein gaps, were affected significantly by both tempera-ture and sex in the presence of Ax-*-^72. However, it can be seen that there did not appear to be a consistent pattern, either of temperature sensitivity or of sexual dimorphism, except that temperature sensitivity, if it occurred in both sexes, was always in the same direction in males and females for a given phene. The orbital frequency was increased in both sexes at 29°C compared to the lower temperatures, and in both cases the frequency was significantly lower in females than in males. It should be noted here than in an earlier sample of Ax 1^ 7 2 flies raised at 20.5°C, the frequency of wing vein gapping and loss of certain bristles differed from the present sample (Appendix 9). Since the two sets of data were obtained at different times, the TABLE 19 Summary of the bristle and wing vein gap phenotypes of Ax * § ORBITALS OCELLARS POSTVERTICALS VERTICALS GENOTYPE 22°C 29°C 22°C 29° C 22° C 29°C 22°C 29°C Axl6l72/y # 0.26 0.53T 0.00 0.03 0.00 0.00 0.97 0.98 Axl6l72/Axl6l72 % 0.16s 0.42TS 0.00 0.01 0.00 0.00 0.98 0.99 Ax16172/+ £ 0.68H 0.70H 0.80H 0.39™ 0.43H 0.31H 1.00H 1.00H SCUTELLARS DORSOCENTRALS ANTERIOR POSTERIOR WING VEINS GENOTYPE 22° C 29°C 22° C 29°C 22°C 29°C 22°C 29°C Ax16172/y j 0.76 0.38T 0.35 0.l4T 0.90 T 0.37 0.17 0.14 Axl6l72/Ax16172 0.68s 0.20TS 0.56S 0.24TS 0.96 0.49TS 0.44S 0.33TS Ax16172/+ ^ 1.00H 0.99H 1.00H 0.94™ 1.00H 1.00H 0.71H 0.58™ * See footnotes to Table 18. § See Appendix 9 for actual counts of bristles and wing vein gaps. co (V> 83 difference could result from modifying genes accumulated during the interval. The data for Axl6l72/+ heterozygotes (Table 19) show that the expression of the Abruptex phenotype was much milder in heterozygous than in homozygous females, and that where temperature differences existed in both homozygotes and hetero-zygotes, they were in the same direction. The data for Ax16172 can be summarized briefly as follows: 1) the higher temperature pushes the frequency of the orbital bristles closer to wild type, whereas the frequencies of dorsocentrals, scutellars, and wing vein gaps become more mutant at 29°C; 2) females are more mutant than males in terms of the orbital and dorsocentral frequencies, whereas males are more mutant than females with regard to the scutellar frequencies and wing vein gap frequencies; and 3) ex-pression of all the mutant phenotypes of Axl6l72 1 s reduced in Ax/+ heterozygotes compared to homozygotes. IV. Ax9B2. The characteristic features of Ax9B2 hemi- and homozygotes are marked reductions in the frequencies of ocellar, postverti-cal, and dorsocentral bristles. Other bristles are usually present in near-wild-type frequencies, although the anterior scutellars are often missing. The extent of wing vein inter-ruptions at 22°C range from mild to moderate, often overlapping wild-type. At higher temperatures occasional nicks appear in the wing tips. The results of bristle, wing-vein gap, and wing nick counts 84 of Ax^B2 hemi- and homozygotes are summarized in Table 20. It can be seen that at the lower temperature, homozygous females had higher frequencies of dorsocentrals and posterior scutellars than males. No significant sex differences were seen for the other bristles or for the wing vein gaps at low temperatures. In another sample of Ax9B2 males and females raised at 20.5°C, the frequencies of certain bristles differed significantly from the present 22°C sample and there was a sex-difference in the post-vertical but not the posterior scutellar frequency (Appendix 10). These differences likely result from genetic background differ-ences, since the two sets of data were obtained from different crosses (see Appendix 10). Comparing the 22°C and 29°C data, it can be seen that at the higher temperature, the frequencies of postvertical and dorsocentral bristles and of wing vein inter-ruptions were significantly raised, whereas the frequency of posterior scutellar bristles was lowered. Also note that at 29°C but not at 22°C, a few flies (which were included in the "6" class, Appendix 10) possessed an extra median orbital bristle. No sex-differences were observed at 29°C, but this may only be due to the small number of individuals examined. Ap-parently an autosomal temperature-sensitive modifier of Ax which resulted in lethality of Ax^B2 hemi- and homozygotes at 29°C, was present in the Ax9B2 stock. This is inferred from the re-sults of certain crosses performed to investigate lethal inter-actions among Abruptex mutants (see Table 30). The data for Ax9B2/+ females (Table 20) reveal an interest-ing contrast between the orbitals and the other bristles. The TABLE 20 Summary of the bristle and wing vein gap phenotypes of Ax9B2 * # ORBITALS OCELLARS POSTVERTICALS VERTICALS GENOTYPE 22°C 29°C 22°C 29°C 22°C 29°C 22°C 29°C Ax502/Y cf 0.99 0.99 0 .00 0.04 0.00 0.22T 0.99 0.99 AX9B2/Ax9B2 ? 0.99 0.99 0.01 0.00 0.00 0.20t 0.99 1.00 AX5B2/+ ? 0.91H - 0.06H - 0.01 - 1.00 -SCUTELLARS DORSOCENTRALS ANTERIOR POSTERIOR WING VEINS GENOTYPE 22°C 29°C 22°C 29° C 22°C 29°C 22° C 29°C Ax9B2/y ^ 0.27 0.48t 0.79 0.69 0.92 0.77T 0.78 0.51T Ax9B2/Ax9B2 ? 0.35S 0.50t 0.84 0.74 0.99S 0.85T 0.79 0.45T AX9B2/+ ? 0.80H - 1.00H - 1.00 - 0.99H -* See footnotes to Table 18. # See Appendix 10 for actual counts of bristles and wing vein gaps. co VJ1 86 orbital frequency in the Ax9B£/+ females was significantly lower (more mutant) than in homozygous females, whereas the other bristle frequencies were significantly higher or unchanged in the heterozygotes. It is possible that this sort of observation might result from heterozygosis of a recessive modifier of Ax caused by outcrossing the stock, but such a modifier would have to be specific for the orbital effect of Ax^B2. A summary of the phenotypic differences between the three viable Ax mutant strains is presented in Table 21. Comparison reveals that besides the obvious quantitative differences, there appear to be qualitative differences between Ax9B2 3 0n the one hand, and AxE2 and AX161725 on the other. This is particularly obvious with AxE2 and Ax9B2, since AxE2 has a fully penetrant orbital phenotype, with only minor loss of other bristles, whereas Ax9B2 has fully penetrant expression of ocellar, post-vertical, and dorsocentral phenotypes, with only minor orbital loss. Furthermore, in both AxE2 and Axl6l72 there were instances of females being either more or less mutant than males for a QB2 given phene, whereas in Ax^ females were less mutant than males in every case where a significant sex-difference appeared (Table 22). As will become apparent in the results to follow, these observations are not the only manifestation of what must be basic differences between Ax^B2 and the other two Ax mutants. 87 TABLE 21 Comparison of the bristle and wing vein phenotypes of the viable Ax alleles at 22°C. Ax E2 Ax 16172 : : A x 9 b 2 ORBITALS 0CELLARS POSTVERTICALS DORSOCENTRALS ANTERIOR SCUTELLARS WING VEIN GAPS XX 0 0 0 0 XX XX XX XX X X XX 0 XX XX XX X 0 = no or only mild expression of mutant phenotype. X = moderate mutant expression; penetrance not complete. XX = complete penetrance of mutant phenotype (i.e., no individuals possess the wild-type number of bristles). 88 TABLE 22 Summary of statistically significant sex differ-ences in bristle and wing vein gap frequencies observed in the viable Ax strains. ORBITALS 0CELLARS POSTVERTICALS DORSOCENTRALS ANTERIOR SCUTELLARS POSTERIOR SCUTELLARS WING VEIN GAPS Ax-E2 9 > 0» ¥ > ( f i 9 < o* Ax16172 Ax9B2 9 > d>* 9 < o* ? > a* 9 < a» 9 < <? ? < o* 9 < tf 9 < a* 9 < cr7 * 9 > o" = females more strongly mutant than males. ? < cf = females less mutant than males. 89 D. INTERACTIONS OP NOTCH AND ABRUPTEX MUTATIONS I. Viability of N/Ax heterozygotes. The viability data for the heterozygous N/Ax combinations examined are summarized in Table 23. As WELSHONS (1971) report-ed, all of the combinations of A x ^ d with N alleles were lethal. However, AxE1, which was originally detected on the basis of its lethality with , was lethal when heterozygous with N^^1, N C o, and but viable with N 1 0 3 at 20°C-22°C. Death of Ax59d/N F X and Ax *VN females occurs mainly in the pupal stages, as is the case with Ax-^^ and AxE^ hemizygotes and homozygotes. In con-trast to these two mutants, AxE2, Ax 1^ 7 2, and Ax^62, which survive as hemi- and homozygotes, were viable as Ax/N heterozy-gotes at 20.5°C-22°C, although there did appear to be some reduced survival of AxE2/Nlj0, AxE2/NCo, and Ax l 6 l 7 2/N 1 0 3 flies. The mortality of the latter combinations did not appear to be correlated with a noticeable incidence of pupal lethality. At 29°C, on the other hand, most of the Ax/N combinations, parti-cularly those involving Ax1^172, were poorly viable, the reduced survival being associated with a corresponding increase in pupal death. The relatively greater lethality of Ax 1^ 1 7 2^ at 29°C is not paralleled by lethality of Ax-^172 hemizygotes or homozygotes at this temperature. 90 TABLE 23 Summary of the viability of heterozygous combina-tions of different Ax and N alleles. A B R U P T E X A L L E L E T E M P E R A T U R E N O T C H A L L E L E a ! N ^ • • • N . 1 0 . 3 . N ® 1 1 - NC° Ax59d 20.-22° C L * L L _ 2 9 ° C - - L L -Ax E 1 2 0 - 2 2 ° C L L V L L 2 9 ° C - - L L -AxE 2 2 0 - 2 2 ° C V V V V V 2 9 ° C V V R V V V R A x 1 6 1 7 2 2 0 - 2 2 ° C V V V V V 2 9 ° C V R L L L L A x 9 6 2 2 0 - 2 2 ° C V V V V V 2 9 ° C V V R V R V V * L = lethal or semilethal (0-5$ survival in relation to siblings). •p V^ = reduced viability (5-30$ survival in relation to siblings). V = viable (greater than 30% survival). 91 II. The effects of N mutants on the bristle and wing vein phenotypes of Ax mutations. In order to study further the phenotypic interactions of N and Ax mutants, a detailed examination was made of the bristle and wing phenotypes of the viable Ax/N combinations. The results show that all five N mutants act similarly on the expression of the Abruptex phenotypes, although it will be noted that there are differences that appear to be N allele-specific. The bristle and wing vein data obtained from heterozygous combinations of AxE2, Ax1^1?2, and Ax^B2 with five different N mutants, are summarized in Tables 24, 25, and 26 respectively. Comparison with the data for the respective Ax homozygotes (Tables 18, 193 and 20) shows that where Ax alone caused marked bristle loss or wing vein gapping, most of the combinations with N had bristle and wing vein gap frequencies significantly closer 28a to wild type. This resembles the suppression of Ax pheno-types observed in Ax28a/N8 heterozygotes (MOHR 1932), and indi-cates that the suppression of Abruptex phenotypes by N mutants appears to be a general phenomenon. There do appear to be several exceptions, particularly with N 1 0 3 and (Tables 24, 26); however, analysis of the H ^ ^ and N^^" exceptions tends to confirm the rule that N mutants suppress Abruptex phenotypes, and most of the other exceptions appear to reflect bristle loss caused by the N mutants themselves. Dealing with the latter exceptions first, it can be seen that at 29°C, heterozygotes of Ax9B2 with N ^ , and N ^ E2 TABLE 24 Summary of the bristle and wing vein gap phenotypes of Ax /N heterozygotes.*# ORBITALS OCELLARS POSTVERTICALS VERTICALS GENOTYPE 22°C 29°C 22° C 29°C 22°C 29°C 22°C ' 29°C AxE2/N8 0.97Su O.78TSU l.OOSu O.99SU 1.00 1.00Su 1.00 0.99 AXE2/N4° 0.96Su 0.79TSU 1.00su O.95SU 0.99 1.00Su 1.00 0.99 AXE2/NCo 0.98SU 0.86TSU 1.00Su 0.99Su 1.00 1.00Su 1.00 1.00 AxE2/n103 0.68^ 0.76TSu l.OOSu O.99SU 1.00 1.00Su 1.00 1.00 Ax^/N®11 0.95Su 0.98Su 0.99 1.00Su 1.00 1.00Su 1.00 1.00 SCUTELLARS DORSOCENTRALS ANTERIOR POSTERIOR WING VEINS GENOTYPE 22°C 29°C 22°C 29°C 22° C 29°C 22°C 29 °C AxE2/n8 1.00 1.00Su 1.00 O.99SU 1.00 1.00 0.95Su 0.93Su AX E 2/N^ 1.00 1 . 0 0 S u 1.00 1.00Su 0.99 1.00 0.98Su 0.98Su AxE2/nCO 1.00 1 . 0 0 S u 1.00 0.97Su 1.00 1.00 0.99Su 0.98Su AXE2/N103 1.00 O.99SU 1.00 O.99SU 1.00 1.00 0.73SuL 0 ,88TSu AxE2/Ngll 1.00 1.00Su 1.00 0.99SU 1.00 0. 99 0,82SuL 0.93TSu * See footnote to Table 18. # See Appendix 11 for actual counts of bristles and wing vein gaps. T Statistically significant difference between the 29°C and the 22°C frequency. Su Statistically significant suppression of Ax phenotypes compared to Ax/Ax females. L Suppression of Ax phenotypes significantly less than with N°, N^O. V£> TABLE 25 Summary of the bristle and wing vein gap phenotypes of heterozygotes. * # ORBITALS OCELLARS ' POSTVERTICALS VERTICALS GENOTYPE 22° C 29°C 22° C 29° C '22° C 29°C 22° C 290 c AX16172/N8 0.70Su 0.68Su 1.00Su 1.00Su 1.00Su O.97SU 0.99 0.98 Ax16172/n40 O.73SU 1.00Su - 1.00Su - 1.00Su -Ax16172/nCO 0.82Su 1.00Su - 0. 99Su - 1.00Su -Ax16172/n103 0.57SuL 0.12sUL - 0.60SuL - 1.00Su -Axl6l72/Ngll 0.63SuL - O.93SUL - 0.38SuL - 1.00Su -SCUTELLARS DORSOCENTRALS ANTERIOR POSTERIOR • ' WING VEINS GENOTYPE 22°C 29°C 22°C 29° C 22° C 29° C 22°C 290 c Axl6l72/N8 1.00Su 0.99Su l.OOSu 0.98Su O.99SU 1.00Su 0.75Su 0.75Su Ax16172/n40 1.00Su O.99SU - O.99SU - 0.75Su -Ax16172/nCO 1.00Su 1.00Su - 1.00Su - 0.76Su -Axl6l72/N103 1.00Su 0.97Su - 0.99 - 0.72SuL -Axl6l72/Ngll 1.00Su 0.80SuL - 1.00Su - 0.62SuL -* See footnotes to Tables 18, 24 § See Appendix 12 for actual counts of bristles and wing vein gaps. UJ TABLE 26 Summary of the bristle and wing vein gap phenotypes of Ax9B2/N heterozygotes. * # ORBITALS OCELLARS POSTVERTICALS ' VERTICALS GENOTYPE 22°C 29°C 22°C 29°C 22°'C 290 c • 22° C 29 0 c Ax9B2/n8 0.99 0.91te 0.11su 0.17Su 0.82SU 0.3^ 1.00 0.90TE Ax9B2/n40 0.99 0.87te 0.08Su 0.11Su 0.8lSu 0.51TSU 1.00 1.00 Ax9B2/nCO 0.99 0.94te 0.63Su o . o 6 S u 0.84s^ 0.64TSu 1.00 1.00 Ax9B2/n103 0.74e 0.95t 0.63Su 0.00T 0.92Su 0.80Su 1.00 1.00 A x9B2 / NgH 0.85e 0.97t 0.00 0.02 0.01 0.92TSu 1.00 1.00 SCUTELLARS DORSOCENTRALS ANTERIOR POSTERIOR WING VEINS GENOTYPE 22°C 29°C 22° C 29°C 22°C 29°C 22° C 29° C AX9B2/N8 0.8lSu 0.58TSu 1.00Su O.99SU 1.00 1.00Su 0.86Su 0 .63TSu AX9B2/N40 0.82Su 0.62tSu 1.00Su 1.00Su 0.99 1.00Su 0.93Su 0 .90Su Ax9B2/nCO 0.75Su 0.51TL 1.00Su O.99SU 0.99 1.00Su 0.89Su 0 .8lTSu Ax9B2/n103 0.82Su 1.00TSu 1.00Su 1.00Su 0.98 1.00Su 0.60E A x 9 1 3 2 ^ 1 1 0.51SuL 0.54l 0.32E 1.00TSu 0.99 0.96 0. 68e 0 #92TSU * See footnotes to Tables 18, 24. # See Appendix 13 for actual counts of bristles and wing vein gaps. E Significantly greater bristle loss or wing vein gapping than in Ax9B2/Ax° 2 homozygotes. VO .t 95 (Table 26), had lower (more mutant) orbital bristle frequencies than A x ^ b 2 homozygotes (Table 20). Similar orbital loss is observed in the respective N/+ heterozygotes raised at 29°C. Note also that in the heterozygotes of AxE2 with these N mutants, orbital bristle frequencies xvere significantly reduced at 29°C compared to 22°C, although they were still less mutant than A x e 2 homozygotes grown at 29°C (Table 24). The loss of vertical bristles in heterozygotes of Ax^B2 with N^ at 29°C, but not with other N alleles, may reflect an interaction between Ax^B2 and the verticals (vt) locus, located adjacent to N in bands 3C5-6 (GERSH 1965). This possibility arises from the fact that vt is o hemizygous in N_ heterozygotes, and that a homozygous deficiency for vt causes reduction of the number of vertical bristles (GERSH 1 9 6 5 ) . The other exception (non-suppression of the dorso-centrals phenotype in Ax-^^/N^0, Table 26) cannot be explained in Co , terms of the phenotype of N /+, and probably is specific for this Ax/N combination. Inspection of Tables 24-26 shows that there are several instances where Ax combinations with N"^3 and N^*^, either do not suppress the Ax. phenotypes at 22°C, or cause significantly less suppression than n£ or . It is significant that at this temperature, both N 1 0 3 and NS-1-1 express relatively mild Notch phenotypes, both in extent of wing nicking (Tables 6, 9), and in their interactions with the recessive Notch-locus mutations (WELSHONS, personal communicationj and see Appendix 1). Further-more, in most (9 out of 12) of the cases for which sufficient data are available (Tables 24, 26), significantly greater sup-96 pression of the Ax_ phenotypes occurs at 2 9 ° C , the temperature at which both N 1 0 3 and nS-H exhibit stronger Notch phenotypes (Tables 6 , 9 ) . Furthermore, the few Axl6l72/Ngll flies which survived at 2 9 ° C , also showed reduction of the Abruptex pheno-types when compared to 2 2 ° C flies (Appendix 1 2 ) . The most glar-ing exception to this (the ocellars of A x 9 B 2 / n 1 0 3 s Table 2 6 ) , can be accounted for by the observation that N103/+ heterozygotes also show drastically reduced numbers of ocellar bristles at 2 9 ° C . Thus, it appears that the exceptional behaviour of N-1-^ 3 and ^gll occurs mainly at temperatures at which the Notch phenotypes of these mutants are only mildly expressed, with more typical Notch behaviour (in terms of suppression of Ax phenotypes) occurring at higher temperatures at which Nl03 and Nfill behave more like the amorphic N alleles. Bristle counts made on AxE1/N1Q3 females raised at 22°C, (which were viable, in contrast to the lethality of the other AxE^/N combinations), strengthen the rule that N mutants suppress Abruptex phenotypes. Comparison of the data on A x E 1 / N 1 0 3 females (Table 27) with those from AxE1/Y males (Table 17) reveals that each bristle frequency was closer to wild type in the Ax/N females than in the Ax/Y males (with the exception of the verti-cals, which were essentially wild type in each case). It should be remembered, however, that N103 does not behave as a deficiency for the Notch locus at 22°C, and that the suppression of AxE1 phenotypes by may reflect the presence of significant levels of activity in the N1Q3 gene product. Comparison of the Ax EV N 1 0 3 data (Table 27) with that for AxE1/+ (Table 16), shows that TABLE 27 Counts of bristles in AxE1/N103 females* raised at 22°C. NUMBER OF FLIES IN EACH PHENOTYPIC CLASS MEAN NUMBER OF BRISTLES PER FLY TYPE OF BRISTLE 0 1 2 3 4 5 6 (± 95$ CONFIDENCE INTERVALS) ORBITALS 0 0 0 1 58 22 13 4.50 ± .13 OCELLARS 84 8 2 - - - - 0.13 ± .07 POSTVERTICALS 25 26 43 - - - - 1.19 ± .15 VERTICALS 0 0 0 0 94 - - 4.00 DORSOCENTRALS 0 1 33 29 31 - - 2.96 + .15 ANTERIOR SCUTELLARS 0 1 93 - - - - 1.99 ± .02 POSTERIOR SCUTELLARS 0 0 94 4.00 •Progeny of the cross M5/£ wf. N 1 0 3 £ x AxE1/BS w^-Y cf \o —] 98 the ocellars are quite comparable in the two genotypes, but the postverticals are more wild type in Ax/N than in Ax/+, and more comparable to Ax/+;Dp (Table 16). It may be that both the par-tial inactivation and the partial function associated with possibly acting by different mechanisms, are responsible for the suppression of the Ax^ phenotypes. Prom the foregoing observations, notwithstanding the ex-ceptions noted, it can be stated in summary that: 1) when in trans heterozygous combination, N mutants generally reduce expression of the bristle and wing vein phenotypes of Ax muta-tions, and 2) high temperatures, which enhance the interactions of N 1 0 3 and N&H with the recessive visible mutants at the locus, also increase the suppression of the Abruptex mutant phenotypes by these two N alleles. III. The effects of Ax mutants on the wing nicking phenotypes of N mutations. The examination of the phenotypes of the different avail-able Ax/N heterozygotes has revealed that the Ax mutants studied fall into two classes with respect to their effects on the N mutants. Both AxE2 and Ax-^172 enhance wing nicking, whereas Ax^B2 and AxE1 suppress nicking. The enhancement of nicking by A x e 2 and Ax16172 is evident in the frequencies of nicking with N 1 0 3, NCo, and N^11 (compare the data in Table 28 with Table 6 for N1Q3/+, and Table 9 for NS11/*; and note that H2% of NCo/+ TABLE 28 Nicking frequencies in wings of Ax/N heterozygotes.* Ax E2 N ALLELE 22° C 29°C Ax 16172 22°C 29°C A x 9 B 2 22 °C 29°C N N 40 2.00 2.00 2.00 2.00 2.00 2.00 2.00 0.02 ± .01L 1.48 + .17L 0.22 ± .08L 2.00 N Co 1.99 ± .02H 2.00H 1.99 ± .02 H 0.00J 0.94 ± .24 N 1 0 3 1.60 + .11H 2.00 1.63 ± .19 H 0.07 ± .04L 2.0 N s l 1 0.19 ± .06H 1.97 ± .05H 0.07 ± .06 0.00 0.42 + .21 * The figures presented are the mean number of nicked wingtips per fly, ± 95$ confidence intervals. Absence of a confidence interval indicates no variability in the samples examined. See Appendices 11-13 for actual counts of nicked wings. H Significantly higher frequency of wing nicking than in N/+ heterozygotes. L Significantly lower frequency of wing nicking than in N/+ heterozygotes. V£> vo 100 individuals had nicked wings, c£ page 58 ) . In the heterozygotes of A x E 2 and A x - ^ 1 7 2 Wj_th and the frequency of nicking was the same as for the N/+ heterozygotes (100%), but the wing serrations were much deeper in the presence of Ax_. The enhance-F? ment of the wing nicking phenotype of N mutants by Ax c and Ax16172 is the opposite of what has been reported for Ax "d (MOHR 1932). On the other hand, Ax^B2 suppresses both the in-tensity and frequency of nicking due to N (Table 28). The enhancement of nicking in Ax9B2/N heterozygotes at 29°C compared to 22°C (Table 28) likely is related to the occasional nicking seen in Ax^B2 hemi- and homozygotes at this temperature (WELSHONS, personal communication). Accurate data concerning the wing phenotype of AxEl/N103 could not be collected, owing to the frequent deformity and crumpling of the wings observed in flies of this genotype. How-ever, in those flies whose wings were sufficiently extended to permit observation, wing nicking occurred less frequently than in n103/+ females at this temperature. The suppression of wing nicking has generally been observed when AxE1/N heterozygotes have been available, including the breakthroughs re-covered in the mapping of AxE1 (Table 13). The facts that AxE1 and A x 9 b 2 possess normal salivary chromosome banding and map within the Notch locus suggest that duplication of the Notch locus is not a necessary condition for suppression of wing nick-ing by an Abruptex mutant. In summary, the foregoing observations indicate that there are two classes of Abrupt x mutation: 1) those which enhance 101 the wing nicking of Notch mutants (AxE2 and A > and 2) those which suppress wing nicking and Ax9B21 as well as Ax2^a). It has already been noted that Ax^B2 differs qualita-tively from A x E 2 and Ax161?2 (Tables 21, 22), and it will be seen that the interactions among the different Ax mutants, analyzed in the following section, provide further evidence that these two classes of Abruptexes must differ in fundamental ways. 102 E. INTERACTIONS BETWEEN DIFFERENT ABRUPTEX MUTATIONS Viability data for all the heterozygous Axx/Axy combina-tions tested, and for the Ax homozygotes, are summarized in Table 29. The time of death of the lethal genotypes is mainly pupal, with some also dying as newly eclosed adults. It can be seen that combinations of the lethals AxE^ and Ax59d with the viable mutations AxE2 and A x 1 6 1 7 2 5 are lethal (as are heterozygotes). This type of lethal interaction is reminiscent of the lethality of most N mutants with fano, and is hence per-haps not unexpected. Surprisingly, however, the viable mutation Ax9B2 is lethal when heterozygous with the other two viable Abruptexes (AxE2 and Ax16172), while both AxE1/Ax9B2 a n d AxE2/ ^xl6l72 a r e viable, This type of lethality is most unusual, and can be called "negative complementation", as opposed to the usual type of complementation in which two lethal alleles (x,y) produce a viable heterozygote (x/y) . The lethality of Ax962/ A x E 2 and Ax9B2/Axl6172 has been confirmed at both 22°C and 29°C (Table 30). However, it can be seen in the control cross ( M5/A x9B2 $ x Ax9B2/Y cP ) that Ax962 homo- and hemizygotes ex-hibit significant lethality (also pupal) at 29°C. This cross was started by crossing M5/M5 females to stock Ax9B2/Y males, then backcrossing F ^ females to stock A x 9 B 2 / Y males. Since the A x 9 B 2 / Y male progeny are fully viable in the other two crosses, it can be concluded that there is at least one recessive auto-somal temperature-sensitive modifier of Ax in the Ax9B2 stock, which kills Ax9B2 individuals at 29°C. 103 TABLE 29 Summary of viability of various heterozygous combinations of Ax alleles at 22°C. AxEl Ax59d Ax962 A X E 2 A x ^ 2 A x E 1 L* L V L L A x ^ L - L L Ax952 V L L AxE2 V V Ax16172 v * L = lethal V = viable 104 TABLE 30 Relative viability of Ax9B2/Ax9B2, Ax9B2/AxE2, and Ax9B2/Axl6l72 at 22°C, PROGENY GENOTYPES'* FEMALES MALES Axx TEMPERATURE M5/Axx Ax9B2/Axx M5/.Y ; Ax9B2/Y AXE2 22°C 124 0 100 122 29°C 131 0 101 121 Ax 1 6 1 7 2 22°C 86 0 76 94 29°0 136 0 76 98 Ax9B2 22° 0 101 96 95 128 29° 0 151 23 85 38 * Progeny of the cross M5/AX962 £ x Axx/Y <f , where x = E2 16172, or 9B2. 105 Correlation of the observed pattern of viability and lethality with the effects of Ax mutants on the wing nicking of N mutants, reveals the striking fact that only those Ax mutants which affect wing nicking in the same way give viable phenotypes when heterozygous with each other, whereas those Ax alleles which affect wing nicking in opposite directions from one another, are lethal in heterozygous combination. The effects of Ax^9d o n wing nicking could not be determined, since none of the Ax^^/N combinations tested were viable. The viability of has not yet been checked. It is also noteworthy that the viable combinations of the Notch-suppressing Ax's investigated in the present study (Ax9 B 2/Ax9 B 2 a ancj A x e 1 / A x 9 b 2 ) are essentially female-sterile. In other experiments, furthermore, it has been observed that AxEVAxE1;Dp females are sterile, although they lay many eggs. This is similar to the observation that the combina-tion Ax59b/Ax59d;Dp js also relatively infertile (WELSHONS 1971). Notwithstanding the possible effects of the modifier present in the A x 9 b 2 stocks, the observed correlation of Ax_ properties with respect to lethality and sterility, with the effects on Notch wing nicking, must reflect some fundamental difference between the two classes of Abruptex mutations. Further information concerning the two types of Abruptexes was obtained from examination of the bristle and wing phenotypes of the viable combinations and of a few surviving "break-through" individuals from the lethal genotypes. The Ax9 B 2/A heterozygotes examined were recovered among progeny of the map-ping cross wf; A x E 2 rb/+ A x 1 ^ 1 ? 2 + £ x w^ A x 9 B 2 rb/Y cf 106 performed at 22°C (all of the observed breakthroughs from this 9 B 2 F ? cross were considered to be Ax /kx^"1, since no extreme Ax females with wild-type eye colour were recovered). The results (Table 31) show that the overall mutant appearance of surviving •p1 *i TT O Ax /Ax individuals is generally more extremely Abruptex than "p "1 IT O is the case for Ax males or Ax^^ males or females (compare Table 31 with Tables 17 and 18). Similarly, the AxE2/Ax9B2 females which managed to eclose were much more extremely mutant than either homozygote. Comparison of the AxE2/Ax9B2 bristle data (Table 31) with those for AxE2 and Ax9B2 homozygotes (Tables 18, 20), shows that this is particularly obvious in the fre-quencies of the orbitals, verticals, dorsocentrals, and scutel-lars. Only two AxEl/Ax-^172 females were recovered, and these possessed no bristles except the verticals (Appendix 14). Com-parison with the data in Tables 17 and 19 shows that this genotype also was more mutant than the respective hemi- and homo-zygotes. In addition to the above observations, it was noted that the meso- and meta-thoracic legs of AxE1/AxE2, AxE2/Ax9B2, and AxE1/Ax16172 females were often deformed, the thoracic micro-chaetae were much sparser than those of the homozygotes, and the wings of these females were so deformed that venation could not be examined. In contrast to the more extreme appearance of the above three genotypes, the phenotypes of the viable combinations AxE2/Axl6l72 and AxE1/Ax9B2 were intermediate between the res-pective homozygous (or hemizygous) genotypes (compare the data for the heterozygous genotypes in Table 31, with the respective hemi- or homozygous genotypes in Tables 17-20). Detailed vein TABLE 31 Summary of the bristle and wing vein phenotypes of heterozygous combinations of different Ax alleles at 20-22°C *#. GENOTYPE ORBITALS OCELLARS POSTVERTICALS VERTICALS AxE1/AxE2 0.01e 0.00 0.00 1.00 AxE1/Ax9B2 0.56 0.00 0.00 1.00 AxE2/Ax161?2 0.451 0.00 0.00 0.99 AXE2/AX9B2 0.00E 0.00 0.00 0.55E GENOTYPE AxE1/Ax E2 AXE1/AX9B2 AXE2/Ax!6172 AxE2/AX9B2 SCUTELLARS DORSOCENTRALS ANTERIOR POSTERIOR WING VEINS 0 .08 0.301 0.801 0 .00E 0.00 0.071 0.891 0.43E 0.98 0.871 1.00 0.79 E 0.43 * See footnote to Table 18. # See Appendix 14 for actual counts of bristles and wing vein gaps. E Frequency significantly more mutant than in respective hemi- or homozygotes. I Frequency intermediate between that of respective hemi- or homozygote. 108 gap counts were not made of the AxE1/Ax9B2 females, but it was noted that the vein gapping was also intermediate between AxE1/Y males and Ax9B2/Ax9B2 females. Prom the foregoing account, it can be seen that the extent of expression of mutant phenotypes of the available heterozygous Abruptex combinations is correlated with the viability of those combinations. The lethal genotypes (AxE1/AxE2, AxE1/Ax16l723 AxE2/Ax9B2) all exhibit more extreme mutant phenotypes than those of the individual homo- or hemizy-gotes, whereas the viable genotypes (AxE1/Ax9B2 and AxE2/Axl6l72) exhibit intermediate phenotypes. Presumably the lethality of the three former genotypes actually results from the enhanced mutant phenotypes of these combinations. 59d Disregarding Ax for the moment, the observation on Abruptex mutant interactions can be summarized as follows: 1) trans-heterozygotes for Ax mutations which have opposite effects on the wing nicking of N mutants, exhibit negative com-plementation, having more extreme mutant phenotypes than either Ax homozygote alone and resulting in lethality, and 2) heterozy-gous combinations of Ax mutations which have the same effect on wing nicking, are viable, and their phenotypes are intermediate between those of the respective Ax homozygotes. Ax^9d cannot as yet be placed in either of these classes of Abruptexes, since this Ax is completely lethal when heterozygous with AxE1, AxE2, Axl6l72s and all N mutants tested. 109 P. DEVELOPMENTAL STUDIES OP SELECTED GENOTYPES I. _ TSPs for wing nicking and eye facet disruption. As reported above, the mutant eye phenotype of females is only expressed at low temperatures, whereas the wing nicking phenotype is expressed weakly at low temperatures and is enhanced at higher temperatures (Figure 4). As will be discus-sed, the results of several shift experiments show that both the eye and wing phenotypes of females have third larval in-star TSPs. In addition, the data have revealed a temperature-dependent polarized pattern of eye facet arrangement. In the first experiment, cultures established from 2-hour egglays (see footnote to Table 32) were shifted from 20.5°C to 29°C, and vice versa, at successive intervals after egg collec-tion, and the developmental stage of the flies at the time of each shift was noted. Adult females emerging in these cultures were scored for wing nicking and the eye phenotype, eyes being scored as mutant if any part of the eye contained disrupted ommatidia. The results show that in shift-ups the number of mutant eyes rose sharply between 120 and 144 hours, when only third instar larvae were present, while at the same time the proportion of nicked wings dropped drastically (Table 32). Simi-larly, only third instar larvae were present when shift-downs (between 72 and 84 hours of development) decreased the number of mutant eyes and increased the frequency of wing nicking (Table 33). Thus the TSPs for both the eye and wing phenotypes of NS11 TABLE 32 Eye and wing phenotypes of Nsll/+ adult females shifted from 20.5°C to 29°C at different successive intervals. NUMBER OF FLIES IN TIME OF SHIFT EACH PHENOTYPIC CLASS CULTURE CULTURE DEVELOPMENTAL NUMBER AGE (HR) STAGE R N* R+N R N+ R+N+ %R 1 2 4 I** 0 3 8 0 2 3 0 6 2 2 4 8 I, some II 0 2 8 0 2 7 0 5 1 3 7 2 II, some III 0 3 1 0 1 9 0 6 2 4 9 6 III 0 5 5 0 8 0 8 7 5 1 2 0 III 1 5 4 3 4 6 8 9 6 1 4 4 III, some P 9 0 5 4 0 1 0 0 1 4 7 1 6 8 P, some III 2 0 4 9 0 1 0 0 4 8 1 9 2 P 0 0 4 3 0 1 0 0 0 9 2 1 6 P 1 0 1 7 1 0 1 0 0 0 . 6 1 0 2 4 0 P 0 0 1 1 8 0 1 0 0 0 1 1 2 6 4 P 0 0 1 2 3 0 1 0 0 0 1 2 NOT SHIFTED 1 0 1 8 0 0 1 0 0 0 . 6 *R = rough eyes (disrupted facet arrangement); R+ = wild-type eyes; N = nicked wings; N+ = wild-type wings. ** I, II, III = 1st, 2nd, 3rd larval instar, respectively; P = prepupae & pupae. m TABLE 33 Eye and wing phenotypes of adult females shifted from 29°C to 20.5°C at different successive intervals. TIME OP SHIFT CULTURE NUMBER CULTURE AGE (HR) DEVELOPMENTAL STAGE 1 24 I, some IIs 2 36 II, s ome I 3 48 II 4 60 III 5 72 III 6 84 III 7 96 P, some III 8 108 P 9 132 P 10 144 P 11 NOT SHIFTED NUMBER OF FLIES IN EACH PHENOTYPIC CLASS R N* R+N R N+ R+N+ %R $N 0 0 53 0 100 0 1 0 16 0 100 6 4 0 51 0 100 7 7 0 64 0 100 10 8 0 100 1 99 7 1 8 0 9 6 50 0 13 0 15 0 46 0 28 0 12 0 70 0 9 0 8 0 52 0 20 0 8 0 71 0 9 0 9 0 50 * See Table 32 for explanation of symbols t—• M I-1 112 occur during the third larval instar, well before the final differentiation of the imaginal discs into the adult organs. In the preceding experiment it was noted that some of the fliesj in cultures shifted during the third larval instar, had only small patches of mutant eye tissue, and that the position and extent of this tissue was related to the time and direction of the shift. To investigate this pattern more closely, eggs collected on petri plates were allowed to develop for approxi-mately 24 hours, at which time they were hatching in large numbers. After the plates had been cleared of all larvae by washing, newly-hatched first-instar larvae were picked from the plates and placed on food in shell vials (100 larvae per vial) which were incubated at 20.5°C or 29°C. Shifts from one tempera-ture to the other were timed from the end of the larva-collection period (i.e. from within 20 minutes after hatching from the egg), and were carried out at shorter intervals during the third lar-val instar. The results show that in the shift-ups, the number of eyes with mutant tissue rose sharply, and the frequency of nicked wings dropped dramatically between 84 and 108 hours, when only third-instar larvae were present (Table 34). In the shift-downs, the number of mutant eyes dropped between 66 and 84 hours, and the frequency of nicked wings rose sharply between 60 and 72 hours, both intervals occurring during the third instar (Table 35). These times coincide fairly well with those shown in Tables 32 and 33, if allowance is made for the duration of the egg phase in the earlier experiment. As many flies with mutant eyes as possible, from vials 5-9 TABLE 34 Eye and wing phenotypes of adult females shifted from 20.5°C to 29°C at different successive intervals (Experiment 2). NUMBER OP FLIES IN TIME OF SHIFT EACH PHENOTYPIC CLASS CULTURE NUMBER HOURS AFTER EGG HATCH DEVELOPMENTAL STAGE R N* R+N R N+ R+N+ 56N 1 0 I* 0 8 0 7 0 53 2 24 I 0 24 0 11 0 69 3 48 II 0 21 0 26 0 45 4 72 II, III 0 21 0 4 0 84 5 84 III 2 19 0 5 8 81 6 96 III 15 8 9 0 75 72 7 108 III 9 0 16 0 100 36 8 120 III, some P 3 0 29 0 100 9 9 132 P, some III 1 0 31 0 100 3 10 144 P 0 0 33 0 100 0 11 168 P 1 0 30 0 100 3 12 192 P 0 0 37 0 100 0 * See Table 32 for explanation of symbols. TABLE 35 Eye and wing phenotypes of N6^V+ adult females shifted from 29°C to 20.5°C at different successive intervals (Experiment 2). TIME OF SHIFT CULTURE NUMBER HOURS AFTER EGG HATCH DEVELOPMENTAL STAGE 1 12 I* 2 24 II 3 36 III 4 43 III 5 48 III 6 60 III 7 66 III 8 72 III, some P 9 84 P, some III 10 96 P NUMBER OF FLIES IN EACH PHENOTYPIC CLASS . N* R+N R N+ R+N+ %R %N 0 0 21 0 100 0 0 0 28 0 100 0 0 0 32 0 100 0 1 0 25 0 100 4 0 0 25 0 100 0 3 0 25 0 100 11 7 0 23 0 100 23 5 7 11 5 57 43 0 18 0 14 0 56 0 13 0 10 0 57 * See Table 32 for explanation of symbols. H H .fc-115 of the shift-ups (Table 34), and vials 6-8 of the shift-downs (Table 35), were examined with a high power dissecting microscope (lOOx), and the position of the mutant tissue in one eye of each fly was drawn on mimeographed eye diagrams of the type shown in Figure 5, along with a note on the wing phenotype of the fly. Each line of the diagram represents five rows of ommatidia, and the orientation of the lines reflects the general orientation of the rows of ommatidia. After being scored in this manner, eyes were then graded according to the following system. Starting from the posterior end, the eye was divided into six regions, each consisting of five vertical rows of ommatidia (see Figure 5). When the boundary between mutant and wild-type tissue lay predominantly in region 1, the fly was scored as 1, and so forth. The results show that the anterior boundary of the mutant tissue in the shift-ups (Table 36) and the posterior boundary in the shift-downs (Table 37), migrated anteriorly in a vertical strip across the eye, with increasing age at the time of shifting. (The absence of shift-down boundaries in region 1 is due to the accidental loss of a 54-hour shift-down culture before it could be scored.) The anterior boundaries in the shift-down series did not migrate, and lay predominantly in region 5 (Table 37). This is generally the anterior limit of mutant tissue seen in all N&H/+ females raised at low temperature. In addition to the correlation of mutant eye tissue with time of shifting, the fre-quency of wing nicking can also be correlated with the position of the mutant eye tissue, as shown in Table 38. Here it can be seen that in the shift-ups, nicking decreased markedly as the FIGURE 5 Diagram used for scoring position of mutant eye tissue boundaries. Anterior end is to the right. 118 TABLE 36 Positions of anterior boundaries of mutant tissue extending from the posterior rim of the eyes of N6-J-V+ females, shift-up experiment 2s. NUMBER OP PLIES WITH BOUNDARY IN GIVEN REGION OF EYE VIAL NUMBER 1** 2 3 4 5 6 5 2 0 0 0 0 6 4 15 4 0 0 7 0 12 12 0 0 8 0 2 15 11 1 9 0 0 0 6 16 * Data were obtained from flies recorded in Table 34. See Figure 5. 119 TABLE 37 Positions of anterior and posterior boundaries of mutant tissue in the centre of the eyes of N g l V + females, shift-down experiment 2*. NUMBER OF FLIES WITH BOUNDARY IN GIVEN REGION OF EYE VIAL NUMBER BOUNDARY 1*» 2 3 4 5 6 6 anterior 0 0 posterior 0 4 7 anterior 0 0 posterior 0 5 8 anterior 0 0 posterior 0 0 0 0 19 5 15 4 1 0 0 1 17 5 3 6 9 0 0 1 10 3 0 8 5 1 * Data were obtained from flies recorded in Table 35. ** See Figure 5. 120 TABLE 38 Correlation of the occurrence of wingtip nicking with position of the boundary of mutant eye tissue. EXPERIMENT WING PHENOTYPE* MUTANT EYE TISSUE BOUNDARY IN REGION SHIPT-UP (anterior boundary) N N 4 2 17 12 4 27 1 16 0 19 SHIFT-DOWN (posterior boundary) N N 0 0 0 9 1 17 4 14 4 12 * N = nicked wingtip(s); N+ = wild-type wingtips. 121 boundary of mutant eye tissue progressed anteriorly from region 2 to region 4, whereas in the shift-downs, wing nicking increas-ed as the boundary shifted from region 3 to 4, although the rise was not as dramatic as the drop in the shift-up series (Table 38). Since developmental synchrony of the larvae was relatively poor in the previous experiment, the method of white prepupa iso-lation (see Materials and Methods) was used in order to time the migration of mutant eye tissue across the eye more precisely. Several batches of eggs were collected in successive 2- to 6-hour intervals, on petri plates containing food, over a 48- and 72-hour period. The 72-hour series was incubated at 20.5°C until the first puparia were detected, at which time all of the plates in this series were shifted to 29°C. Each plate in the 48-hour series was placed at 29°C immediately after collection, and the entire series was shifted to 20.5°C upon the appearance of the first puparia. After the shifts at both temperatures, white prepupae only were collected at defined times from the plates, placed into vials, and allowed to develop into adults. Eyes and wings of emergent adult females were examined with a dissecting microscope, and the eyes were further examined and recorded photographically with a scanning electron microscope. The results, summarized in Figure 6, show that during the 23 hours preceding puparium formation, the longer the interval between a shift-up and puparium formation (i.e. the earlier the shift-up during development), the less mutant eye tissue was found, the mutant area always beginning at the posterior edge of the eye and extending anteriorly (Figure 6a). On the other hand, the longer FIGURE 6 The eye and wing phenotypes of Nsll/+ females shifted from a) 20.5°C to 29°C, and b) 29°C to 20.5°C at different times before puparium formation. Note that the third larval instar lasts about 66 hr. at 20.5°C, and 40 hr. at = wild-type eye facet a r r a n g e m e n t = mutant eye facet arrangement; • = number of flies with wild-type wings; B = number of flies with one or both wings nicked. Note that the anterior rim of the eye is to the left. a . S H I F T - U P u o. > U1H >0 az u X CL in 20-w— fc.Ul ii 10-o •EYE TSP Hi 0 To 20 HOURS FROM SHIFT T O PUPATION —r~ 30 —j— 40 b . SHIFT-DOWN hi Q. >• Uh >0 HZ 11 x 0. K-i i in si 2o^  Q U.U If ii 10-hs EYE TSP- •H ESg ~40" "50 10 20 30 HOURS FROM SHIFT TO PUPATION 124 the interval between a shift-down and puparium formation, the more mutant eye tissue was found, beginning at a point near but not at the anterior edge of the eye and extending posteriorly (Figure 6b). These observations are entirely consistent with the previously observed posterior-to-anterior progression of the boundary between mutant and non-mutant eye tissue (Tables 36, 37). The synchrony obtained in this experiment was much better than in the previous ones, since the positions of the mutant eye tissue boundaries in the adult flies raised from prepupae iso-lated at any given time after a shift, generally differed by only 3-4 rows of facets. This in turn indicates that there was little difference in the developmental rates of different third-instar larvae in these cultures. The eye TSP can be defined as the time (which is about 23 hours at both 20.5°C and 29°C) during which the mutant tissue boundary progresses anteriorly across the eye (Figure 6). It is interesting to note that the eye TSP occurs earlier in the third instar at 29°C than at 20.5°C. From the wing nicking data presented in Figure 6, it can be seen that in the shift-up series the frequency of nicked wings drops about half way through the eye TSP, and conversely, in the shift-down series the frequency of nicked wings rises about half way through the eye TSP. These data, and those in Table 38, indicate that in the shift-ups the wing nicking TSP ends about half way through the eye TSP, and in the shift-downs the wing TSP begins during the eye TSP. In summary, the results of the temperature-shift experi-ell ments with N& /+ females reported here, have shown that: 125 1) the TSPs for both the rough eye and wing nicking phenotypes occur during the third larval instar; 2) the eye facet pattern is affected by temperatures in a vertical wave that proceeds anteriorly from the posterior rim of the eye during the eye TSP; and 3) the wing nicking TSP appears to begin and end during the eye TSP. II. N 1 0Vspl - TSPs for wing nicking, eye facet disruption, tarsal fusion, and bristle disruptions. Plies heterozygous for the ts mutant N -' and the recessive eye mutant spl exhibit a ts expression of wing nicking, eye facet array, and tarsal fusion phenotypes (WELSHONS, personal communi-cation; and see Table 6). Both the eye and wing mutant pheno-types of spl females are enhanced at high temperatures and decreased at low temperatures, and tarsal fusion only occurs at high temperatures. At 28°-29°C, the eyes of N103/spl flies resemble those of Ngll/+ females raised at 20°-22°C (Plate 3), except that facet disarray extends over the entire surface of the eye. At 20°-22°C, the eyes of N1Q3/spl approach wild type, but usually contain small, irregular areas of facet disarray. Since preliminary shift experiments roughly localized the TSPs for eye, wing, and leg phenotypes to the third instar-pre-pupal period, and since increases or decreases in the numbers of certain bristles appeared to be affected by temperature shifts at these stages (data not presented here), the prepupa isolation 126 method already used with was used to time these events more precisely. Eggs from the cross M5/y wa N 1 0 3 $ x spl/Y cr* were collected at 22°C on food in petri plates in successive 12-hour intervals for several days. Half of these plates (shift-up series) were incubated at 20°C and the other half (shift-down series) were shifted to 28°C immediately after collection. Plates were incubated at the respective temperatures until the appear-ance of puparia. White puparia were isolated from the respective cultures during a 12-hour period just before shifting, placed into vials, and incubated further at the same temperatures. Then the larvae and isolated puparia from the shift-up series were all shifted from 20°C to 28°C; those from the shift-down series were all shifted from 28°C to 20°C, and white puparia were collected at defined times. Adult N^3/spl females which emerged from the isolated prepupae were scored for eye, wing, leg, and bristle phenotypes. The results (Figure 7) confirm that the TSPs for all the phenotypes examined occur in the third larval instar. Note that in the eyes, the boundary between strongly mutant tissue and weakly mutant tissue, progresses anteriorly across the eye with increasing larval age at the time of shifting, as was ob-K H served in N /+ females. Furthermore, comparison with the results for (Figure 6) reveals a striking similarity in that the high-temperature eye TSPs of both genotypes end several hours before the low-temperature eye TSPs, with respect to puparium formation. It should be noted here that the partial 103 penetrance of spl in N /spl females at 20°C, precludes precise determination of the limits of the eye TSPs (hence the "approxi-FIGURE 7 The eye, wing, leg, and bristle phenotypes of N103/spl females shifted from a) 20°C to 28°C, and b) 28°C to 20°C at different times prior to or after puparium f o r m a t i o n = eye facet arrangement approaches wild-type; = strongly mutant eye facet arrangement; SI = wingtip nick-ing; • = tarsal fusion; 6'= ocellar bristles; & = dorsocentral bristles; A = anterior scutellar bristles; = vertical bristles. Note that the anterior rim of the eye is to the left. a. S H I F T - U P lil a. Id H >- O ui z ui x o. A P P R O X I M A T E E Y E T S P < > + 2.0 -I— lil D. >• H Q J O H Z o H < J Ui DC Z o z UI J H (0 cc cn + 1.0 0 ;0 . - 1 .0 • 2 . 0 T H I R D I N S T A R P R E P U P A too o g O (/) Z D 5 u-— -J z < CD O CC z < o -{ I h 3 0 20 10 0 10 B E F O R E A F T E R T I M E OF S H I F T (HRS) B E F O R E OR A F T E R PUPARIUM FORMATION b . SHIFT-DOWN -f 2.0 ! ' ! D. >• H Q J 0 H z 0 p < J Id E 0 z lil J t-U) a a + t.o 0 .0 ' 1 .0 — 2.0 o o---o T H I R D I N S T A R T P R E P U P A < U> K < H a. o si 5? 4- + 40 30 . 20 10 B E F O R E T I M E OF S H I F T ( H R S . ) B E F O R E OR A F T E R PUPARIUM FORMATION A F T E R 130 mate" eye TSPs depicted in Figure 7)s but it can definitely be stated that at 28°C the eye TSP ended at least 10 hours before puparium formation. The bristle data show that shift-ups prior to 4 hours before puparium formation result in the appearance of extra vertical, dorsocentral, and anterior postalar bristles, and cause loss of ocellar bristles (Figure 7a). Conversely, shift-downs prior to 10-14 hours before puparium formation markedly restrict the increase or loss of these bristles (Figure 7b). All these phenotypes are seen in both and spl in-dividuals, so the ts expression observed here cannot be definite-ly attributed to one or the other of these mutations. To summarize briefly, the results of the temperature-shift experiments on M^Vspl females have shown that: 1) the eye facet and wing nicking phenotypes of N103/ spl have third-instar TSPs; 2) with respect to puparium formation, the eye TSP of N-^V spl ends several hours earlier at high temperatures than at low temperature, a pattern very similar to that observed in females; and 3) the TSPs for fusion of tarsal segments, appear-ance of extra vertical, dorsocentral and anterior postalar bristles, and loss of ocellar bristles, all occur during the third larval instar. III. OR - radiation-induced rough eye phenocopy. The posterior-to-anterior progression of the boundary between mutant and non-mutant eye tissue in NS11/+ and N^^/spi females shifted at successively later times during the eye TSP, 131 resembles the observations made by BECKER (1957) . He found that a phenocopy of a "rough" eye could be induced by X-irradiation during the third instar-prepupa stages, and that with increasing age at the time of irradiation the irregular arrangement migrat-ed anteriorly across the eye in a vertical band. Since the eye TSPs for both Nsll/+ and N103/spl occur earlier at 29°C than at 20°-22°C (Figures 6, 7), it was asked whether the sensitivity of eye facet arrangement to radiation would also occur earlier at 29°C than at 20.5°C. The results of the experiment described below indicate that no difference exists in the radiation sensi-tive period (RSP) of OR flies irradiated during the third instar and prepupa stages, at 29°C or 20.5°C. For this experiment, two groups of 15 OR females each (inseminated by OR males) were allowed to lay eggs at 20.5°C for several days on petri plates, which were changed every 24 hours (as for the prepupa-isolation temperature-shift experiments). All of the eggs laid by one group of females were transferred to 29°C immediately after collection. The other set of plates was kept at 20.5°C. Before irradiation, white prepupae were isolat-ed from both series at defined times and kept at the respective temperatures until eclosion. At one defined time, all the plates in both series and the isolated pupae and prepupae were irradi-ated for 25 seconds from a Cobalt-60 source delivering approxi-mately 4500 rads per minute. Immediately after radiation the cultures were placed back at their respective temperatures, and isolation of white prepupae at defined times was continued as before. The eyes of adult flies emerging from the isolated pre-132 pupae were examined with a high power dissecting microscope (lOOx), and the position of irregular eye tissue was noted for each eye. Penetrance of the rough eye phenocopy was 100% in recover-ed adult flies irradiated as prepupae or late third instar larvae and, as shown by the data in Table 39, synchrony was fairly good in flies irradiated up to 18 hours before puparium formation. These results, summarized in Figure 8, show that irradiation of OR flies during the larval or prepupal period causes roughly the same pattern of eye facet disturbance at both 29°C and 20.5°C. This indicates that unlike the TSPs for eye facet arrangement in Ngll/+ and N1Q3/spl, the end of the RSP for eye facet arrangement is not displaced at 29°C compared to 20.5°C. IV. Ax 1 6 1 7 2/^ 0 - TSP for lethality; Axl6l72/+ - TSPs for wing vein gapping and loss of ocellar bristles. In the investigation of Ax mutant expression, it was found that expression of the ocellar bristle-loss and wing vein gap phenotypes in Ax16172/+ females was temperature sensitive (Table 19) and that lethality of the combinations of A x ^ ^ ^ with most N mutations was also ts (Table 23). Ax1^172/* females raised at 29°C have significantly fewer ocellar bristles and more gaps in wing veins than females reared at 22°C (Appendix 9), and Ax^"1-^ is lethal or semilethal in combination with N mutations at 29°C but viable at 22°C (Table 23). The temperature-shift experiments to be reported show that the TSP for lethality of Ax16172/!^0 TABLE 39 Number of rows of disrupted ommatidia in adult OR flies irradiated before or after puparium formation, at 20.5°C and 29°C. NUMBER OF ROWS OF IRREGULAR OMMATIDIA AT ANTERIOR EDGE OF EYE TIME OF RADIATION WITH RESPECT TEMPERATURE TO PUPARIUM FORMATION 20 .5°C 29°C 17 hr. after (pupae) 6.5 hr. after (prepupae) 0.5 hr. after (prepupae) 1.5 hr. before (3rd instar) 6.5 hr. before (3rd instar) 17.5 hr. before (3rd instar) 17 hr. after (pupae) 0.5 hr. after (prepupae) 1.5 hr. before (3rd instar) 6.5 hr. before (3rd instar) 18 hr. before (3rd instar) AVERAGE RANGE (WILD TYPE) 3.5 3-4 7.3 6-9 9.3 8-10 11.8 10-13 13.5 10-16 (WILD TYPE) 7.5 6-9 9.5 7-11 13.5 12-15 * 9-16 NUMBER OF EYES SCORED 14 10 10 16 22 12 6 18 26 18 4 * In the 29°C 18 hr. (before) sample, the 8-11 anterior- rows of ommatidia were wild-type, the next 9-16 rows were rough in a vertical strip in the middle of the eye, and the 7-12 posterior rows were wild-type. (jo u> FIGURE 8 The eye phenotypes of OR flies irradiated at different times before and after puparium formation, at 20.5°C and wild-type facet arrangement;' mutant eye facet arrangement. Note that the anterior rim of the eye is to the left. T I M E OF IRRADIATION B E F O R E OR A F T E R PUPARIUM FORMATION (HOURS) 136 flies occurs during the second larval instar, whereas the TSPs for both the ocellar and wing vein phenotypes of Ax16172/+ females occur in the third instar. The cross M5/wa rb_ £ x Ax-^172/y cs^ was used to gener-ate cultures for shifting which contained both Ax/+ and Ax/N females. Two experiments were performed with cultures establish-ed from 2-hour egglays by approximately 100 inseminated females each. In the first experiment, cultures incubated at 22°C (shift-up) or 29°C (shift-down) were shifted from 22°C to 29°C and vice versa at regular intervals. In the second experiment, cultures incubated at 22°C were shifted to 29°C at regular inter-vals and shifted back to 22°C 18 hours later (pulse-up); cultures incubated at 29°C were shifted to 22°C at regular intervals and shifted back to 29°C 24 hours later (pulse-down). This timing of pulse-shifts was adopted because 18 hours at 29°C is roughly the equivalent, in developmental terms, of 24 hours at 22°C. The results of the shift-up and shift-down series show that shift-ups before or during the second larval instar (Table 40), and shift-downs after the second larval instar (Table 4l), cause significant mortality of Ax16172/n40 females. This indicates that the TSP for Ax/N lethality occurs in the second larval in-star and possibly extends into the early third instar. The results of the pulse-shift experiments show that at no stage of the life cycle do 18-hour pulse-ups significantly reduce viabili-ty (Table 42), whereas in the pulse-down series, 24-hour incuba-tions at 22°C during the second instar rescue most of the Ax/N heterozygotes from pupal death (Table 43). The latter result TABLE 40 Data indicating viability of Ax16172/!^0 females relative to their sibs when shifted from 22°C to 29°C at different successive intervals. NUMBER OF PROGENY TIME OF SHIFT VIABLE ADULTS DEAD PUPAE % SURVIVAL CULTURE CULTURE DEVELOPMENTAL OF Ax/N NUMBER AGE (HR) STAGE M5/Ax $ Ax/N $ M5/Y c^ Ax/N £ FEMALES* 1 24 85 1 75 86 2 2 48 I 87 5 43 75 6 3 72 II 33 18 57 33 35 4 96.5 III, some II 70 79 71 1 99 5 120 III 153 157 130 7 96 6 144 III 135 101 102 10 91 7 168 III, some P 79 81 63 3 97 8 192 P, some III 100 111 104 0 100 9 216 P 169 146 131 4 97 10 240 P 70 69 63 1 99 11 264 P 106 103 113 1 99 12 288 P 64 69 48 0 100 13 NOT SHIFTED UP 66 60 49 1 98 * % survival = viable Ax/N adults * (viable Ax/N adults + dead Ax/N pupae) x 100$. ** E = egg stage. See Table 32 for other symbols. u> TABLE 41 Data indicating viability of females relative to their sibs when shifted from 29°C to 22°C at different successive intervals. NUMBER OF PROGENY TIME OF SHIFT VIABLE ADULTS DEAD PUPAE % SURVIVAL CULTURE NUMBER CULTURE AGE (HR) DEVELOPMENTAL STAGE M5/Ax $ Ax/N $ M5/Y cf Ax/N $ OF Ax/N FEMALES* 1 18 I** 49 45 51 0 100 2 36 II 26 20 28 1 95 3 54 !E X 2 IX Dl 27 17 29 4 81 4 72 H I 59 3 62 64 4 5 90 III3 some P 54 1 36 47 2 6 108 P, some III 30 0 26 33 0 7 127 P 24 0 19 29 0 8 144 P 38 3 21 27 10 9 162 P 32 1 18 30 3 10 180 A, some P 29 0 20 21 0 11 NOT SHIFTED DOWN 102 3 83 109 3 * See Table 40. ** A = adult stage. See Table 32 for other symbols. UJ OO TABLE 42 Data indicating viability of Axl6l72/N females relative to their sibs when pulsed from 22 °C to 29°C and back after 18 hours, at different successive intervals. NUMBER OF PROGENY TIME OF PULSE-UP VIABLE ADULTS DEAD PUPAE % SURVIVAL CULTURE CULTURE NUMBER AGE (HR) DEVELOPMENTAL STAGE (UP) M5/Ax $ Ax/N $ M5/Y cr* Ax/N $ OF Ax/N FEMALES* 1 24-42 Es* 49 50 52 2 96 2 48-66 I 43 60 37 1 98 3 72-90 II 60 68 43 2 97 4 96.5-114.5 III, some II 66 60 42 1 98 5 120-138 III 60 72 57 0 100 6 144-162 III 69 52 77 1 98 7 168-186 P, some III 57 55 32 1 98 8 192-210 P 62 86 60 0 100 9 216-234 P 93 84 68 3 97 10 240-258 P 42 63 57 1 98 11 264-282 P 80 77 60 5 94 * See »* See Table 40. Tables 32, 40 for explanation of symbols • u> V£> TABLE 43 Data indicating viability of Ax16172/n40 females relative to their sibs when pulsed from 29°C to 22°C and back after 24 hours, at different successive intervals. TIME OF PULSE-DOWN CULTURE CULTURE DEVELOPMENTAL NUMBER AGE (HR) STAGE (DOWN) NUMBER OF PROGENY VIABLE ADULTS M5/Ax $ Ax/N $ M5/Y 0* DEAD PUPAE % SURVIVAL OF Ax/N Ax/N $ FEMALES* 1 18-42 I** 44 4 35 38 10 2 36-61 II 50 38 23 11 78 3 54-78 II, III 72 41 56 45 48 4 72-96 III 41 0 43 35 0 5 90-114 III, some P 54 2 37 58 3 6 108-132 P, some III 62 1 40 66 1 7 127-150 P 55 3 35 46 6 8 144-168 P 37 1 30 32 3 9 162-186 P 52 0 47 61 0 * See Table 40. ** See Table 32 for explanation of symbols. -tr O l 4 l confirms the second-instar TSP inferred from the reciprocal shift series (Tables 40, 4l). Furthermore, the pulse-shift results suggest that only a relatively short incubation at 22°C during the second instar is sufficient to prevent death of most Ax/N females, and that 18-hour pulses at 29°C are not sufficient to cause death (Tables 42, 43). The results of the shift-up, shift-down, and pulse-down experiments are summarized in Figure 9-To determine the stage when loss of ocellar bristles and gapping of wing veins are affected by temperature, ocellar bristles and wing vein gaps were counted in the MS/Ax1^172 (Ax/+) female progeny recorded in Tables 40-43. The results show clear-ly that in the shift-up series the frequency of wing vein gaps drops and the ocellar bristle frequency rises during the third larval instar (Figure 10a), and in the shift-down series the wing vein gap frequency rises and the ocellar frequency drops during the third instar (Figure 10b). Furthermore, the wing vein gap and ocellar frequencies become significantly (see footnote to Figure 10) more mutant when cultures are pulsed up during the third instar (Figure 10a), and likewise these phenotypes become significantly less mutant when cultures are pulsed down during the third instar (Figure 10b) (wild-type frequencies for wing vein gaps and ocellars are 0.0 and 2.0, respectively). These results indicate that the TSPs for both the wing vein gap and the ocellar bristle phenotypes of Ax161?2/* occur during the third larval instar. The foregoing results can be summarized as follows: 1) the TSP for lethality of females occurs during the second FIGURE 9 Viability of Ax16172/!^0 shifted at different stages of development. X = shift-up; 0 = shift-down; A = pulse-down. The shift-ups are timed on the 22°C scale (bottom), while shift-downs and pulse-downs are timed on the 29°C scale (top). D E V E L O P M E N T A L S T A G E S AT 29°C ( A P P R O X . ) E G G 1 S T 2 N D P U P A I 1 h HOURS AT 29 C too 1 5 0 100 1 5 0 200 3 0 0 HOURS AT 22 C I \ E G G 1 S T 2 N D D E V E L O P M E N T A L S T A G E S AT 22°C ( A P P R O X . ) FIGURE 10 The number of ocellar bristles and wing vein gaps of Axl6l72/+ females when a) shifted from 22°C to 29°C, or pulsed from 22°C to 29°C for 18 hr., and then back to 22°C, and b) shifted from 29°C to 22°C, or pulsed from 29°C to 22°C for 24 hr., and then back to 29°C. A = wing vein gaps, shift series; A = wing vein gaps, pulse series; o: = ocellar bristles, shift series; = ocellar bristles, pulse series. Note that the points in the pulse series are plotted for the middle of the pulse period. Note also that the drop in ocellar bristle frequency at 155 hr. in the pulse-up series (a), is statistically significant at the 95% level of confidence, and that the other bristle and wing vein gap frequency changes occurring during the third instar (shift-ups pulse-down and shift-down series), are also statistically significant. S H I F T - O R P U L S E - U P APPROXIMATE DEVELOPMENTAL S T A G E S AT 22 C E » ' i " ' Li! : I L 3 . 0 _ o z III D a u DC li z.o_ a. < o uj > 1 .0. r - 2 . 0 8 m r r > m o c m z o -< . 5 HOURS AT 22°C b . S H I F T - O R P U L S E - D O W N APPROXIMATE DEVELOPMENTAL S T A G E S AT 29 C . E I ' I 4.0 3 . 0 — >• o z LU D a Ui a u. CL < e> z LU > 2.0 — 1 .0. 5 0 HOURS AT 29 C \ V \ — -100 1 5 0 2.0 O o m r r > 73 •n XI m o c m z o -< . 1 . 5 146 larval instar, although 18-hour pulses at 29°C during this stage are not sufficient to cause lethality; and 2) the TSPs for en-hancement of wing vein gapping and ocellar bristle loss in Axl6l72^+ females occur during the third larval instar. N1Q3/fano - TSPs for lethality. Unlike most other N/fano combinations, which are lethal under all conditions, N103/fano females die when raised at 29°C but survive at 22°C (Appendix 1). The results of the shift experiments to be reported here show that one or several TSPs for lethality extend from the egg through the larval stage into the prepupal or pupal stages, and that there are three distinct lethal phases in flies reared at 29°C. Moreover, additional observations suggest that at 22°C, whereas most N/Y hemizygotes l n o die as embryos, N /Y embryos may survive into the third larval instar. Eggs of the cross M5/y wf; N 1 0 3 $ x fano/Y d'were collected for shift experiments to determine viability of N~^3/fano females. Each culture was established from the 2-hour egglays of 100 in-seminated females. As described previously for an(j the first experiment consisted of 22°C-29°C (shift-up) and 29°C-22°C (shift-down) shifts, and the second experiment consisted of 18-hour pulses to 29°C at 24-hour intervals in 22°C cultures (pulse-ups), and 24-hour pulses to 22°C at 18-hour intervals in 29°C cultures (pulse-downs). A 147 The results of the shift-up series show that shift-ups before the end of the third larval instar result in low survival (0-7$) of N1Q3/fano females, that after the third instar there is a jump to about 50$ survival, and that successively later shift-ups after pupation allow correspondingly greater survival of N 1 QVfa n o flies (Table 44, Figure 11). Furthermore, when shifted up from the late embryo stage (24-hour egg) onward, death of N1Q3/fano females occurs from the prepupal or early pupal (eye phenotype and sex unscorable due to early death, see Table 4l) to the late pupal stages (Figure 11). It is assumed that most of the unscorable dead pupae resulting from shift-ups before puparium formation were N103/fano females. This is indi-cated by the absence of a temperature-effect on mortality of M5/fano females and M5/Y males and because the sum of the number of verifiable N1Q3/fano flies (surviving adults and dead pupae) and unscorable dead pupae is roughly equal to the number of M5/fano females recovered (Table 44). Inspection of Figure 11 reveals an interesting pattern of lethality: 1) shift-ups from the late embryo to the end of the third larval instar cause death of about half of the N^^/fa110 females in the prepupa or early pupa stage, and about half in the late pupa stage; 2) shift-ups immediately after the third instar permit roughly half of the Nl QVfa n o females to emerge as adults, the remainder dying as late pupae; and 3) progressively later shift-ups after pupation allow proportionately greater survival of N103/fano females. On the other hand, the results of the shift-down series show that shift-downs as early as the first instar (18 hours) do TABLE 44 Data indicating viability of N103/fano females in relation of their sibs when shifted from 22°C to 29°C at different successive intervals. NUMBER OP PROGENY TIME OF SHIFT VIABLE ADULTS DEAD PUPAE /O OUllliV rtXJ OF N103/ fano FEMALES* CULTURE NUMBER CULTURE AGE (HR) DEVELOPMENTAL STAGE M5/fano g NlQ3/fano £ M5/Y a* N 103/fano $ UNSCOR-ABLE 1 24 E** 32 0 28 20 12 0 2 48 I 116 1 88 65 46 1 3 72 II 118 0 115 62 85 0 4 96.5 II, III 96 5 64 45 43 5 5 120 III 112 4 78 55 63 3 6 144 III 62 6 55 . 39 35 7 7 168 P, some III 62 34 57 26 4 53 8 192 P, a few III 115 51 81 56 4 46 9 216 P 159 111 134 32 6 75 10 240 P 133 116 118 22 5 81 11 264 P 80 75 63 4 3 91 12 288 P 68 61 51 0 4 94 13 NOT SHIFTED UP 94 96 80 7 2 91 * % survival = viable N-^Vfa110 adults -r (viable N1Q3/fano adults + dead N103/fano pupae + unscorable dead pupae) x 100% (see text). ** See Tables 32, 40 for explanation of symbols. oo 103 o FIGURE 11 Relative proportions of viable N /fan° adult females, late pupal N"^^/fano female deaths, and early pupal (unscorable) deaths, in cultures shifted from 22°C to 29°C at different successive intervals. = viable N103/fano adults; 1 1 = late pupal N103/fano deaths ;f§§ = unscor-able early pupal deaths. This Figure is based on the data presented in Table 44. I 1 1 1 I -E G G 1 S T 2 N D 3 R D P U P A APPROXIMATE D E V E L O P M E N T A L S T A G E S AT 22°C 151 not permit significant survival of N103/fano females, and that furthermore very few die as pupae (Table 45) (the few which do die as pupae may be attributed to asynchronous egglays). This indicates that incubation of N103/fano females at 29°C during the embryonic stage induces death at some stage prior to puparium formation (possibl even in the egg stage). The results describ-ed above show that one TSP for lethality of N1Q3/fano occurs in the early embryo stage, and another extends through one or more of the larval instars and ends during the pupa stage. These findings are summarized in Figure 12. The results of the pulse-up series show that exposure to 29°C for 18-hour periods at any developmental stage after the late embryo did not cause significant mortality of N103/fano females (Table 46) . Similarly, pulse-downs after the embryo stage did not result in survival of N103/fano females (Table 47). Survival in this case would not be expected, since each pulse-down culture was incubated at 29°C during the embryo stage, and it has already been shown that this results in death before puparium formation (Table 45). As was noted earlier, certain observations have provided evidence that not all N /Y males die as embryos at 22°C. During the experiments reported above, larvae were selected from 22°C and 29°C cultures at the times of shifts and their develop-mental stages determined according to the morphology of their mouthparts and anterior spiracles. In the 22°C, but not in the 29°C series, some of the larvae had yellow mouthparts (about 20%-25% of the lst-2nd instar larvae sampled, fewer of the 3rd TABLE 45 Data indicating viability of N1Q3/fano females in relation to their sibs when shifted from 29°C to 22°C at different successive intervals. NUMBER OF PROGENY TIME OF SHIFT VIABLE ADULTS DEAD PUPAE CULTURE CULTURE DEVELOPMENTAL NUMBER AGE (HR) STAGE M5/fano $ N103/fano £ M5/Y cj* N1Q3/fano $ UNSCORABLE 1 18 Is 31 0 18 3 0 2 36 I, II 63 2 52 2 0 3 54 II, some III 57 0 56 0 0 4 72 III 22 0 31 2 1 5 90 III, some P 47 1 44 0 4 6 108 P, some III 28 0 30 0 2 7 127 P 25 0 31 0 0 8 144 P 17 0 23 0 0 9 162 P 36 0 51 1 0 10 180 A, some P 59 0 47 0 1 11 NOT SHIFTED DOWN 57 0 54 1 0 * See Tables 32, 41 for explanation of symbols. U1 rv> FIGURE 12 Time of death of N103/fano females in relation to time of shift from 22°C to 29°C. Note that the "pupa" stage as used here includes the prepupa stage. Note also that the actual time of death has not been critically ascertained for early embryo shift-ups, but that death does occur before the prepupa stage. u. o 111 H < UJ o < H in j < H Z LlI h Z LLI 0. O _I UJ > tii Q - L A T E E L E M B R Y O -3 - L A T E 3 R D I I N S T A R H Lu X in E A R L Y E M B R Y O E M B R Y O L A R V A P U P A DEVELOPMENTAL STAGE AT T I M E OF DEATH TABLE 46 Data indicating viability of N103/fano females in relation to their sibs when pulsed from 22°C to 29°C and back after 18 hours, at different successive intervals. NUMBER OF PROGENY TIME OF PULSE-UP VIABLE ADULTS TOTAL DEAD PUPAE CULTURE CULTURE DEVELOPMENTAL (NOT SCORED) NUMBER AGE (HR) STAGE (UP) M5/fano % N1Q3/fano ? M5/Y cf> 1 24-42 E* 59 47 50 1 2 48-66 I 61 69 61 7 3 72-90 II 63 34 43 11 4 96.5-114.5 II, III 55 56 61 6 5 120-138 III 56 44 43 4 6 144-162 III 115 77 72 11 7 168-186 P, some III 69 66 53 13 8 192-210 P, (+2 III) 53 44 39 1 9 216-234 P 48 63 40 1 10 240-258 P 66 61 53 4 11 264-282 P 48 53 32 6 * See Tables 32, 40 for explanation of symbols. VJl VJ1 TABLE 47 Data indicating viability of N^-°3/fano females in relation to their sibs when pulsed from 29°C to 22°C and back after 24 hours, at different successive intervals. NUMBER OF PROGENY TIME OF PULSE-DOWN VIABLE ADULTS DEAD PUPAE CULTURE CULTURE DEVELOPMENTAL NUMBER AGE (HR) STAGE (DOWN) M5/fano £ N1U:yfano $ M5/Y. 0* Nlu3/fano UNSCORABLE 1 18-42 I* 39 0 35 1 0 2 36-61 I, II 80 0 52 0 1 3 54-78 II, some III 16 0 36 4 3 4 72-96 III 12 0 21 0 0 5 90-114 III, some P 42 0 56 2 4 6 108-132 P, some III 25 •0 32 0 2 7 127-150 P 18 0 36 0 0 8 144-168 P 38 0 28 1 4 9 162-186 P 35 0 50 0 4 10 180 A, some P 42 0 32 1 0 * See Tables 32, 41 for explanation of symbols. H UI CT\ 157 instars), indicating that they were mutant for the gene y. From the genotypes of the parents used to generate these cultures (M5/X. wfl N 1 0 3 % x fano/Y d* ), these must have been y wf N103/Y males. Recombination between N"^3 and giving y N+/Y males, is unlikely, since these mutations are only 3.0 map units apart (LINDSLEY AND GRELL 1968), and furthermore, M5 suppresses cross-ing over in the X chromosome. The jr larvae had smaller mouth-j . parts and developed more slowly than their y_ (black mouthparts) sibs. Moreover, third instar larvae did not have visible teeth on the mouthhooks, compared to 9-12 teeth in the y^ larvae. No prepupae or pupae with y mouthparts were seen. These results indicate that unlike most other N/Y hemizygotes (POULSON 1939b, 1968), N1Q3/Y males incubated at 22°C survive the embryo stage and may reach the third larval instar before dying. The absence ] Q O of larvae at 29°C suggests that N ~'/Y males do not survive the embryonic stage at this temperature. The preceding results can be summarized as follows: 1) TSPs for lethality of N103/fano females occur in the embryonic stage and also during the larval instars; 2) shift-ups during the early embryo stage cause lethality before puparium formation, whereas later shift-ups cause death after puparium formation; 3) pulse-ups (18-hour periods at 29°C) from the late embryo stage onward, do not cause significant mortality of N103/fano females; and 4) N103/Y hemizygotes die as embryos at 29°C, but at 22°C they may survive into the third instar, dying before puparium formation. VI. NS11/NS1:L;DP - TSP for lethality. 158 As described in the section dealing with gene dosage, the combination has a lethal phenotype which is ts. Females of this genotype survive at 29°C, but at 20°C-22°C they die at some stage prior to puparium formation (Table 11). The temperature-shift experiments reported here show that the TSP for lethality is restricted to the embryo stage, unlike those pre-viously described for Ax16172/!^0 and N1Q3/fano. In the first experiment, 2-hour egglays from approximately 200 M5/wa N^11 rb females (inseminated by wf; NS11 rb/Y;Dp males) were collected at 20.5°C (shift-up) and 29°C (shift-down), and shifted from one temperature to the other at regular intervals. The results of the shift-up series show that the shift-up during the early embryo stage (0 hour after egg collection) allows sur-vival of N^^/N^ 1 ;Dp females, but that shift-ups during or after the late embryo stage (from 24 hours onward) cause death of this genotype (Table 48). Conversely, shift-downs during the early embryo stage (0 hour) cause death of NS^/NgH;Dp females, where-as shift-downs immediately following the embryo stage (18 hours) or later do not cause lethality (Table 49). These results indi-cate that the TSP for lethality of NSll/Ngll ;Dp females occurs during the embryo stage. In the second experiment, designed to localize the lethal TSP more precisely within the embryo stage, 1-hour egglays from approximately 300 females (see previous experiment) were collect-ed at 22°C and placed at either 20°C (shift-up) or 28°C (shift-TABLE 48 Data indicating viability of N^Vh^-*- ;Dp females in relation to their sibs when shifted from 20.5°C to 29°C at different successive intervals. NUMBER OP PROGENY TIME OF SHIFT MALES FEMALES % SURVIVAI CULTURE NUMBER CULTURE AGE (HR) DEVELOPMENTAL STAGE M5/Y M5/Y;Dg_ N/Y;D]o M5/N M5/N;D£ N/N;D£ OF N/N;Dp FEMALES* 1 0 E** 18 7 50 35 37 46 156 2 24 E 46 24 37 34 38 0 0 3 48 I 37 40 32 41 49 0 0 4 72 II 31 37 34 50 60 0 0 5 96 III 37 32 51 56 41 0 0 6 120 III 37 22 26 20 27 0 0 7 144 III, some P 49 38 31 47 41 0 0 8 168 P, some III 37 44 43 54 54 0 0 9 194 P 41 44 39 39 38 0 0 10 216 P 38 64 50 58 55 1 2 11 NOT SHIFTED UP 162 107 149 138 147 5 3 * % survival = number observed * number expected x 100$, where number expected = total progeny (except NgU/NgJ-^Dp) * 5. ** See Tables 32, 40 for explanations of symbols. M VXl vo TABLE 48 Data indicating viability of NS-^/NgH;Dp females in relation to their sibs when shifted from 29°C to 20.5°C at different successive intervals. NUMBER OF PROGENY TIME OF SHIFT MALES FEMALES % SURVIVAL CULTURE CULTURE DEVELOPMENTAL OF N/N;D£ NUMBER AGE (HR) STAGE M5/Y M5/Y;D£ N/Y;Dp M5/N M5/N;D£ N/N;Dp FEMALES* 1 0 E** 14 9 10 2 10 0 0 2 18 I 24 15 24 14 20 20 103 3 36 II 5 5 7 3 5 3 60 4 54 III 23 8 20 16 12 11 63 5 72 III 17 4 16 14 12 16 127 6 90 III, some P 33 15 35 29 32 31 108 7 106 P, some III 24 12 29 22 31 19 81 8 126 P 21 10 22 16 18 24 138 9 144 P 32 12 31 31 29 18 67 10 187 P 24 11 40 31 24 27 86 11 NOT SHIFTED DOWN 47 21 61 43 66 40 84 * See Table 48. ** See Tables 32, 40 for explanation of symbols. H CT\ o 161 down). Shifts from one temperature to the other were performed hourly during most of the embryo stage. The results of the shift-ups show that shifting from 20°C-28°C before 12 hours does not result in lethality, but that the viability drops markedly in the 12- to 14-hour shift-ups (Table 50). In the shift-down series, death of nearly all Nsll/NSH;Dp females occurs in shifts from 28°C-20°C before 6 hours, but viability rises- markedly in the 6- to 9-hour shift-downs (Table 51). These results, sum-marized in Figure 13, indicate that the TSP for lethality of Ngll/Ngll;Dp females occupies a relatively short interval, occur-ring about the middle of the embryo stage. VII. Summary of temperature-shift results. The findings of the temperature-shift experiments are sum-marized in Figure 14. It can be seen that the TSP for each adult morphological phenotype occurs during the third larval instar, whereas TSPs for lethality occur during several stages of develop-ment, depending on the genotype involved. Although the lethal TSPs appear to be markedly different from one another (Figure 14), there are some similarities worth mentioning. In both cases where the TSP is embryonic (N103/fano and N&H/Ngll;Dp), incubation at the non-permissive temperature causes death at some stage prior to pupation. Furthermore, in both of the larval TSP cases, death occurs after puparium forma-tion, although Ax16172/n40 females all die in late pupal stages, and death of N103/fano females is divided about evenly between TABLE 48 Data indicating viability of NS-^/Ng^-^Dp females in relation to their sibs when shifted from 20°C to 28°C at different successive intervals during the embryo stage. NUMBER OF PROGENY MALES FEMALES % SURVIVAL CULTURE TIME OF SHIFT-UP OF N/N;Dp NUMBER (HOURS) M5/Y M5/Y;D£ N/Y;Dp M5/N M5/N;D£ N/N;Dp FEMALES* 1 3 26 18 45 38 27 27 88 2 4 15 14 27 16 16 19 108 3 5 14 8 22 21 24 10 56 4 6 32 25 29 35 32 35 114 5 7 26 15 29 19 19 24 111 6 8 9 11 23 14 10 12 90 7 9 39 31 41 38 41 30 79 8 10 20 12 32 32 26 27 111 9 11 17 14 16 22 12 15 93 10 12 50 24 40 33 41 13 35 11 13 15 18 21 21 18 2 11 12 14 14 8 16 13 18 1 7 13 15 40 39 31 34 37 0 0 14 16 33 19 27 20 21 0 0 15 17 25 15 23 21 28 0 0 16 NOT SHIFTED 40 53 51 43 54 0 0 * See Table 48. OM ru TABLE 48 Data indicating viability of nS^/nS-*-1;Dp females in relation to their sibs when shifted from 28°C to 20°C at different successive intervals during the embryo stage. NUMBER OF PROGENY MALES FEMALES % SURVIVAL CULTURE TIME OF SHIFT-DOWN OF N/N;D£ NUMBER (HOURS) M5/Y M5/Y;D£ N/Y;D£ M5/N M5/N;D£ N/N;D£ FEMALES* 1 2 48 46 36 34 42 0 0 2 3 47 45 55 39 48 2 4 3 4 37 34 35 38 45 1 3 4 5 37 56 46 37 62 2 4 5 6 37 28 39 15 30 6 20 6 7 51 43 56 37 44 17 37 7 8 71 38 56 43 47 21 41 8 9 30 25 28 33 24 26 93 9 10 40 42 49 44 39 38 89 10 11 46 43 35 33 45 29 72 11 12 35 39 56 56 50 40 85 12 13 37 36 65 45 39 37 83 13 14 44 36 43 52 41 43 100 14 15 65 39 58 66 70 63 106 15 16 50 50 70 44 52 57 107 16 NOT SHIFTED 41 24 35 39 40 33 92 * See Table 48. H (JO FIGURE 13 Viability of Ngll/Ns11;Dp females shifted at different times during the embryo stage. 0 0 shifted from 20°C to 28°C; X- -X shifted from 28°C to 20°C. Note that the arrows indicate the approximate time of egg hatch. FIGURE 14 Temperature sensitive periods for lethality and adult morphological phenotypes of selected Notch-locus mutant genotypes. Note that the TSPs for adult morphology include TSPs for: 1) eye facet arrangement and wing nicking sll eye facet arrangement, wing nicking, tarsal segment fusion, and bristle disturbances (N103/spl); and 3) wing vein gapping and ocellar bristle loss Note that this figure does not indicate the lengths or relative positions of the TSPs during the third instar. >-o J o x Ql dc O h J d Q < N g ' / + N ' ° 3 / s p l A 1 6 1 7 2 / X / + S 1 • 1 1 — r E M B R Y O 1 S T I N S T A R 2 N D I N S T A R 3 R D I N S T A R P R E P U P A A N D P U P A DEVELOPMENTAL STAGE 168 early and late pupal stages (Figure 11). It should be stressed that the data for N103/fano do not rule out the possibility that there are several discrete TSPs for lethality, rather than the single one indicated in Figure 14. 169 DISCUSSION The present investigation has consisted primarily of an examination of the properties of a variety of mutations within the complex Notch locus which exhibit unusual phenotypes. This includes mutations of the Notch type as well as Abruptex alleles, which do not resemble N mutations phenotypically but nevertheless are located within the Notch locus (Figure 1). In addition to the genetic complexities of the locus, the alleles within this region exhibit multiple phenes, which point to an important role in development. The following discussion will deal mainly with the phenotypic responses to alterations of the relative numbers of wild type (N+) and mutant (N) alleles, the phenotypic inter-actions between N and Ax mutations and among different Ax^  alleles, and the developmental studies of certain conditional phenotypes associated with different Notch-locus genotypes. Since the strains used in this investigation were not co-isogenic, and (as noted in Results from time to time) certain minor differ-ences could therefore have resulted from genetic background variability, the discussion of the N dosage and Ax results will be confined to those observations which appear to be generally repeatable in different genetic backgrounds. The present re-sults do not elucidate a complete picture of the function of the Notch locus during development. Nevertheless, the hypothesis that its function is of a regulatory nature, rather than (in a morphological sense) of a structural nature, appears to be a plausible explanation of the data. Moreover, the data do appear 170 to require certain assumptions about the nature of the Notch-locus product and several molecular models are discussed. It should be emphasized that these models are primarily for the purposes of illustration and discussion; the experiments report-ed do not give precise information about the molecular nature of the Notch-locus gene products(s). Ultimately, however, molecular models of the Notch-locus product(s) will have to account for all of the unusual and seemingly contradictory properties of the mutant alleles of this complex locus. As mentioned earlier, point N mutants exist which map as discrete sites within the Notch locus, but are identical to N deficiencies both phenotypically and in their interactions with other mutations within the Notch locus. These alleles can be classed as amorphs (WELSHONS 1965), which by definition produce either a biologically inactive gene product or no gene product at all (MULLER 1932). Since the phenotypic effects of the point mutation are indistinguishable from the effects of which is known to be a deficiency, with respect to embryonic lethality (POULSON 1939b, 1968), wing nicking frequency (Table 5), inter-actions with Notch-locus recessive visibles (LINDSLEY AND GRELL 1968), suppression of Ax phenotypes (Tables 24-26), and reces-siveness to other N alleles in Nx/Ny;Dp combinations (Table 12; also see Table 16), it is reasonable to assume that N ^ is indeed an amorphic allele. By logical extension of this line of reason-ing, N mutations which are not phenotypically like deficiencies, such as N g l 1, NCo, and N 1 0 3, cannot be amorphic. N g l 1 and NCo not only exhibit a milder expression of certain Notch phenotypes 171 than deficiencies, but are also associated with additional pheno-typic changes not caused by deficiencies. Depending on the temperature, the expression of N~^3 may be either milder or more extreme than deficiency of amorphic N mutations. As discussed below, analysis of these three exceptional Notches suggests that the non-amorphic N mutations may be interpreted as hypomorphic, neomorphic, hypermorphic or antimorphic depending upon the parti-cular genotype and phenotype studied. Wing nicking and other typical Notch phenotypes exhibited by heterozygotes for de-ficiency or amorphic N mutations appear to be strictly regulated by gene dosage (WELSHONS 1965; and see Table 5). It follows that those N mutations showing a relatively mild wing nicking phenotype must be hypomorphic to N_ (rather than amorphic) in terms of the product activity whose dosage determines wing nick-ing. This hypothesis is testable, since increasing the number of hypomorphic alleles (and presumably, therefore, the amount of product) should decrease expression of the mutant phenotype, as discussed by MULLER (1932). Thus, the reduced wing nicking (compared to N/+) observed in the N/N;D£ combinations of NS11, NCo, and N 1 0 3 (at low temperatures), as shown in Tables 6, 8, and 12, is precisely the result expected if these three Notches are hypomorphic. It can be concluded that N ^ 1 and and at low temperatures, are hypomorphic to N^ with regard to wing nicking. Consistent with this interpretation is the obser-40 vation that nicking of N , which is assumed to be amorphic, was not markedly reduced in N^Q/N^0;Dp females (Table 5). It should be pointed out that the term "hypomorph" does not necessarily 172 imply that the gene product has diminished activity; the term could equally well describe an allele which produces less pro-duct. However, the analysis to follow indicates that N6^1 and po N cannot be hypomorphic in terms of all phenotypes, and that N ^ 3 is not hypomorphic at 29°C. This in turn suggests that at least in the case of and NCo, "hypomorph" describes the product, rather than the amount of product. Dealing with N 1 0 3 first, it will be recalled that at 29°C, N103/+ females show more intense wing nicking than N /+ or N__/+ (compare Plates la, 2c), and also have fused leg segments. Furthermore, n103/y ;Dp males raised at 29°C have thickened wing veins, unlike males carrying other N mutations. As noted earli-er, the existence of a tarsal fusion phenotype in at least some surviving N70k30/fano heterozygotes (Appendix 4), and in N103/ fano at 25°C (Appendix 1), suggests that the leg phenotype may be associated with a level of N^ product activity intermediate between that of N/+ and N/N. Recently, SHELLENBARGER (1971) has described a ts lethal in the Notch locus which also conditional-ly exhibits leg segment fusion and other adult morphological abnormalities. Reduced N_ product activity, leading to extreme wing nicking and tarsal fusion, could result in 2 9°C N103/+ heterozygotes if the N1^3 allele product either partially inacti-vates, or competes for receptor sites with the N^ product. These alternatives are outlined diagrammatically in Figure 15. Note that the inactivation model (Figure 15a) requires the formation of di- or multimeric complexes of the Notch-locus product, whereas the competition model (Figure 15b) does not necessarily FIGURE 15 Hypothetical molecular models to explain tarsal fusion and enhanced wing nicking in N103/+ females at 29°C. a . 1NACTIVATION OF" N + PRODUCT R E C E P T O R n + F U N C T I O N A L n+ : R E C E P T O R C O M P L E X ( R E D U C E D A M O U N T ) W I L D T Y P E P R O D U C T 175 imply dimer formation, but does require a Notch-product:receptor complex. The formation of both types of complex has been pro-posed by WELSHONS (1965), to account for the array of pleiotropic effects associated with the Notch locus. Formally, both models (a) and (b) (Figure 15) can be classed as antimorphic inter-actions of N ^ 3 with N^, in the sense that the N"1"03 product has an "actively negative value" in terms of the amount of function-ing product (MULLER 1932). A truly amorphic allele, such as 40 the N mutation has been inferred to be, should not form a product which is able to complex with either the product or a receptor site. The observation that at 29°C N^-Q3/NlQ3;Pp females have even more exaggerated wing nicking and tarsal fusion pheno-types than N1Q3/+ females, is entirely consistent with the hypothesis that N"*"^ 3 behaves as an antimorph at 29°C. The tarsal fusion associated with at high temperatures is not always correlated with extreme wing nicking. This follows from the fact that spl females exhibited tarsal fusion but had less extensive wing nicking than N1Q3/+ when raised at 28°C (Figure 7) or 29°C. Without further experiments one cannot say whether this difference is due to extra-locus genetic modifiers in the spl stock or to the spl allele itself. The latter choice appears to be more likely, since fano spl/Y males have a milder wing nicking phenotype than fano/Y males (WELSHONS, personal communication). The possibility that non-Notch-locus modifiers may affect the phenotype of N103/+ or Nl°3/spl, does not necessarily invalidate the hypothesis that 176 N ^ 3 is antimorphlc at 29°C, since an altered "receptor" site (see model b, Figure 15) could formally be described as a genetic modifier. The results of the dosage study of I\fs11 reveal that the eye facet and wing nicking phenotypes respond in opposite directions to both temperature and gene dosage (Figure 4). It has already been suggested that the decreased wing nicking in Ngll/Ngll;Dp females at 22°C indicates that N 6 ^ is hypomorphic in this regard. However, the increased expression of the mutant eye phenotype in such females is not the behaviour expected of a hypomorph. This, and the fact that deficiency N mutations and duplications do not express the eye phenotype seen in females (although +/Y;D£ males do have occasional facet irregularities), suggests that the N^ -*- allele product is functionally altered (i.e. is neomorphic), as opposed to having reduced function, with respect to its role in eye development. Thus it can be assumed that at 20°C-22°C, the product plays an active role in eye develop-ment, either interacting or competing with the N^ product (cf Figure 15) and thereby causing the mutant eye phenotype. A similar conclusion has been reached concerning the mutant spl (WELSHONS 1956b, 1971), whose eye phenotype is qualitatively identical to that of . The observation (Figure 4) that the mutant eye phenotype is expressed with increasing severity in the dosage series - 2 N^ : 1 NSll, 1 N^ : 1 NS11, 1 N^ : 2 NS11 - is consistent with the hypothesis that the NS11 allele product is neomorphic at low temperature. The opposite responses of the eye and wing phenotypes (i.e. 177 when the eye is mutant the wings are more wild type, and vice versa) of N ^ 1 to temperature changes, may be explained if it is assumed that at 29°C, the product is less active, or less product is produced, than at 20°C-22°C. Thus it can be hypothe-sized 1) that at low temperatures, the N g l 1 product is able to participate in both eye and wing development and has sufficient wild-type acitivity to produce predominantly non-nicked wings, but is mutant in that function involved in eye facet arrangement; and 2) that at 29°C, the N s l 1 product is relatively inactive (or less product is produced), and is therefore unable to parti-cipate fully in wing development (causing a predominance of nicked individuals) and (in Ngll/+ females) unable to participate signifi-cantly in eye development, allowing the allele product to direct a normal eye facet arrangement. This hypothesis is illus-trated diagrammatically in Figure 16. The assumption implied in this hypothesis, that normal eye facet arrangement is relatively independent of levels of gene product, whereas wing development is o not, is supported by the facts that N_/+ and +/+;D£ females have normal or near-normal eye facet arrangements, but have nicked (N8/+) or Confluens (+/+;Dg_) wing phenotypes. Since the eyes of ^gll/NgH;Dp females at 29°C are unmistakably mutant (Figure 4), the N s l 1 product must be produced at 29°C, and must compete or interact with the product. This, and the incomplete penetrance of wing nicking in Ngll/+ females at 29°C (Table 9), emphasizes the point that even at 29°C the activity of N6"3"'1' is not totally abolished. It is also noteworthy that two cases of genetic suppression FIGURE 16 Hypothetical molecular model to explain opposite response to temperature of eye and wing phenotypes of Ngl1. a. 22° C N + V W I L D T Y P E P R O D U C T N gn V A C T I V E M U T A N T P R O D U C T 2S°C N + N gn W I L D T Y P E P R O D U C T V I N A C T I V E M U T A N T P R O D U C T V N — K N T T K W I N G W I N G E Y E A C T I V E P A R T I C I P A T I O N O F N =>' 1 I N E Y E A N D W I N G D E V E L O P M E N T R E D U C E D P A R T I C I P A T I O N O F N S 1 ' ' N E Y E • A N D W I N G D E V E L O P M E N T 180 of the NgU eye phenotype, one due to an extra-locus modifier (E-n70^27)a and the other to a mutation within the Notch locus (N70k3°), are both associated with increased wing nicking (Appen-dices 3j 4). Both cases are consistent with the interpretation that mutant N-allele product is removed from competitive activity, either by a reduction in the amount produced, or in the affini-ties which permit the competition or interaction. The lethality of Nsll/Ng1:L;Dp females at 20°C-22°C, but not at 25°C-29°C (Table 11), can also be explained in terms of the hypothesis outlined above. The fact that the flies of the genotypes N4°/N4Q;Dp (Table 4), N103/N1Q3;Dp (Table 7), NCo/NCo; Dp, and Nx/N^;Dp are all viable, rules out the possibility that the N^ activity of Dg_ itself is insufficient to allow survival. Furthermore, the viability of Nsll/Ns11;Dp/Dp females (Table 11), Ngll/Y ;Dp males, and Ng1:L/N8 ;Dp and Ngll/N^0;Dp females, indi-cates that the 2 N^11 : 1 allele ratio is the factor which determines the lethality of NS11/NS11;Dp females at 20°C-22°C. If, in fact, at 22°C the NS11 product has sufficient activity to + compete with the N_ product, then twice as much defective wild-type product could be sufficient to interfere with N_ activity, pushing it below the threshold necessary for viability. The TSP for lethality of Nsll/Ng1:i-;Dp females is embryonic (Figure 13) and the phenotype associated with the embryonic death of N hemi-zygotes has been well characterized (POULSON 1940). If the lethality in NS^VNg-^Dp females is due to antimorphic (antagon-istic) action by the N&11 product against N^ activity, embryos incubated at 20°C should exhibit disturbances similar to N hemi-181 zygotes, whereas if the lethality results from neomorphic (com-petitive) action, such embryos should have some other phenotype. An examination of the phenotypes of N ^ V N ^ ^ D p embryos incuba-ted at 22°C or 29°C, has not yet been completed, so a conclusion on this point must be postponed. The viability of HS^/N^^Dp females at 25°C-29°C, is entirely consistent with the hypothesis that the N ^ ^ gene product has less antagonistic or competitive activity at higher temperatures (Figure 16). The Abruptex-like phenotype of NS-'-^ /NS1!;Dp females which survive at 20°C-22°C (and those which are incubated at 29°C during the embryo stage and then shifted down, Table 48) will be referred to later, and will not be discussed here except to note that the neomorph hypothesis (Figure l6a) can account for this observation also. Like NCo behaves like a hypomorph in terms of wing nicking but not its other phenotypes. The Confluens phenotype, which is mildly expressed in NCo/+ females, and strongly express-ed in NCo/+;Dp females and NCo/Y;Dp males (WELSHONS 1956a; Plate 4), is also known to result from increased N^ dosage (MORGAN ejt al. 1941; LEFEVRE 1952; WELSHONS 1965). This suggests that NCo is probably hypermorphic compared to in terms of the function responsible for the Confluens phenotype. This hypothesis is supported by the observation that N^°/NCo;Dp females have a much more extreme Confluens phenotype than NCo/+;Dp females (Plate %)• The possibility that the strong Confluens phenotype results from some special interaction between NC° and Dg_ is ruled out by the fact that NCo/N8;Dp_ and NCo/N1|Q;Dp females have essentially 182 identical phenotypes to those of NCo/+ (Table 12). In fact, the reduction of the Confluens phenotype in NCo/Nx;Dp females is pre-dicted by, and therefore lends support to, the hypothesis that NCo is hypermorphic for the Confluens function. It is interesting to speculate on how a molecule such as the product (assuming, for the moment, that the Notch locus does produce a single product) can be hypomorphic in one sense and hypermorphic in another. One possible explanation for this phenomenon is that the N^0 mutation causes a change in a single part of the Notch-locus product responsible for wing control, such that in one cellular milieu it causes wing nicking and in another it causes extra veins. Alternatively, NCo may affect the tertiary structure (folding) of the Notch-locus product, such that a site normally responsible for the completion of wingtip development is altered thereby leading to the wing nicking pheno-type. The same tertiary alterations could also affect a site regulating wing vein production and thereby produce extra veins. Implicit in the latter type of reasoning is the assumption that the Notch-locus product has several active sites, each of which is involved in different aspects of development. The validity of this assumption is supported by inspection of the genetic map of the Notch locus (Figure 1), which reveals that mutations with similar phenotypes generally map at similar sites within the locus (e.g., fa and fa&, fano and fanoE, nd and nd2, and the Ax and Ax-like mutations). As discussed later, this clustering is expecially striking for the Ax mutations. Before discussing the phenotypes of Ax mutations and their 183 interactions with N mutations and each other, the question of whether Ax mutations represent duplications of the Notch locus, as suggested for Ax28a by MORGAN et al. (1941), should be dis-cussed. The duplication hypothesis was based on the observations that Ax28a/N8 females exhibited reduced wing nicking (MOHR 1932) and that there was an extra band in the Notch-locus region of the 28a X in salivary chromosome preparations of Ax (MORGAN et_ al. 1941) . KAUFMAN (personal communication) has examined the banding patterns of salivary chromosomes carrying AxE~*", AxE2, Ax16172, and A x9b2, and none of these mutations are associated with visible duplications or deficiencies. Furthermore, the mutagen EMS was used to induce AxE1 and AxE2 (Appendix 3), Ax 1 6 1 7 2 (WELSHONS, personal communication), and Ax9B2 (LEFEVRE, personal communica-tion). If the fact that EMS induces missense mutations in T4 bacteriophage (KRIEG 1963) is also true in Drosophila, then these four Ax alleles are not likely to be duplications. Finally, genetic recombination tests show that in the presence of Ax alleles, crossing over is not reduced within the Notch locus or in the immediately adjacent regions (Tables 13-15, and see com-ment about Ax"*"^72 mapping, p. 72 ), a criterion which has been used by others to infer the absence of chromosome aberrations (GREEN AND GREEN 1956; CARLSON 1958; WELSHONS AND VON HALLE 1962). It can also be noted that AxE2 and Ax^-1-72 enhance the wing nick-ing effect of N alleles (Table 28), and that AxE1 is usually lethal in combination with N mutations (Table 23). This also tends to rule out the need to postulate that these alleles are duplications. Ax9B2 does suppress wing nicking, but the cyto-184 logical evidence and its mutagenic origin suggest that this allele is not a duplication either. However, the observed cross-over frequency (0.09$) between Ax9B2 and each of two flanking N alleles and NCo) (Table 15) is rather high compared to the total recombination frequencies (0.03$ between the two N's measured directly, and a total of 0.04$ between N^°-spl and spl-N C o) reported by WELSHONS (1958b). On the other hand, the cross-over frequency between fano and spl observed in the present study (Tables 13, 14) was also higher than that reported by WELSHONS (1958a), suggesting that culture and genotypic conditions may at least be partially responsible for the difference. Moreover, as noted in Results, the possibility that genetic modifiers (of N, Ax9B2, and fano spl AxE2) affected the relative viability of non-crossover (Ax9B2) and crossover (Ax+) progeny (Table 15), cannot be discounted. Thus it can be assumed that factors other than an intra-Notch-locus duplication, may account for the apparent increases in N-Ax9B2 recombination. In summary, it appears likely that AxE1, AxE2, Ax16l72 a an(j Ax9b2 are all point mutations within the Notch locus. In the following discussion, therefore, it is assumed that these mutations are not duplications or parti-al duplications of the Notch locus. Like the Notch class of mutations, the Abruptex class of mutations can be readily defined phenotypically. However, as was observed with the N mutations examined, within this group there appear to be allele-specific differences. In fact, on the basis of the patterns of bristle loss (Table 21), sexual dimorphism (Table 22), interaction with N mutations (Table 28), and inter-185 actions among the Ax mutations themselves (Tables 29-31), there are at least two distinct sub-groups of Ax mutations. Neverthe-less, the fact that in terms of the bristle phenotypes, all five N alleles tested interacted similarly (in Ax/N heterozygotes) with all Ax mutations tested (Tables 24-27), emphasizes that the Ax's should be treated as a single class of mutations. Since the Ax mutations behave differently from known N deficiencies, it is obvious that Ax mutations cannot be amorphic. The data seem to indicate that each Ax mutant may affect the dif-ferent functions controlled by the Notch locus in different ways, and furthermore that certain of these functions may be affected in more than one way. The suppression of both the Ax and N 9B2 mutant phenotypes in Ax /N heterozygotes (Tables 26, 28) cannot be attributed to partial intra-cistronic complementation, whereby a hybrid polymer restores some wild-type activity to the gene o product, since in the case of the deficiency, N^ ., there is no N-allele product to participate in polymer formation. If, on the other hand, it is assumed that Ax9B2 is hypermorphic, the in-creased activity on the part of Ax9B2 could suppress the wing nicking of N, and reduced or complete lack of function on the part of the N allele could diminish the bristle loss and wing vein gapping caused by the hypermorphic Ax allele (compare with the suppression of Confluens in N(-'0/Nx;Dp) . This very explana-tion was advanced by MULLER (1932) to explain the reduced ex-pression of N and Ax phenotypes in Ax28a/N8 heterozygotes. By the wing nicking criterion, AxE2 and Ax16172 would be hypomorph-ic, since wing nicking is enhanced by these alleles (Table 28). 186 On the other hand, the suppression of AxE2 and phenotypes by N mutations suggests that these two alleles are hypermorphic, like Ax962. Unfortunately, this model cannot accommodate the observation that Ax phenotypes are suppressed in Ax/+ females compared to Ax/Ax females or Ax/Y males (Tables 16-20), since they should be enhanced if the Ax alleles were truly hypermorphic. Similarly, it can be deduced that the Ax mutations are not (en-tirely) neomorphic so far as their effect on bristle numbers is concerned, since in this case N deficiencies or point mutations should not suppress the Ax mutant phenotypes. From the foregoing discussion it is apparent that some rather special assumptions may be warranted in order to reconcile the seemingly conflicting observations. A way out of the morass described above, which is consis-tent with the observations on the atypical Notches, may be found by combining recent suggestions (BRITTEN AND DAVIDSON 1969; WRIGHT 1970) that the Notch locus is a regulator gene, with the fact that some regulatory loci in bacteria comprise both repres-sor and activator elements (GAREN AND ECHOLS 1962a, b; ENGLESBERG, IRR, POWER AND LEE 1965). If we assume that the Notch locus is this type of regulator gene, or at least that different elements of the wild-type Notch-locus product tend to oppose or balance one another in the developmental processes they influence, then the suppression of Ax phenotypes by both N^ and N alleles could be accommodated. To borrow the regulator gene terminology, we may suppose that the bristle loss caused by Ax mutations is due to a repression of the bristle-forming mechanism. As diagrammed 187. In Figure 173 this could come about a) by mutation of an activa-tor element to a non-functional form (A_), or b) by mutation of a repressor element to a hyper-functional form (R_). It is also possible that a single mutation could affect both types of ele-ments. Thus, according to these models, Ax mutations may be hypo- or amorphic changes (AT) and/or hyper- or antimorphic changes (R ) in the Notch-locus product. Accordingly, an amorph-ic Notch mutation would be represented as A~R~. The hypothesis that mutation of / to Ax causes bristle loss due to increased repression of bristle-forming activity, is consistent with the observation that N deficiencies usually cause increased bristle numbers (MOHR 1932). Thus, mutation of N^ to N could result in reduced repression of bristle-forming activity, leading to the increased bristle numbers. As will be discussed later, the model outlined in Figure 17 can also account for the Axx/Axy inter-actions. This model is not meant to imply that there are two discrete parts to the Notch locus, although, as will be shown later, the genetic evidence does suggest that the left half of the Notch locus is functionally matched or paired with the right half. It is possible, although difficult to prove, that a whole series of mutually antagonistic regulator elements make up the Notch-locus product. In other words, the Notch locus could be an "integrator" gene, as postulated by BRITTEN and DAVIDSON (1969), which controls the transcription of many "producer" (structural) genes. By considering the expected gene products of Ax/Ax, Ax/+, and Ax/N (Table 52), we can see how the models presented in FIGURE 17 Model of Notch locus comprising antagonistic elements. a. A-l | R + | 1 r e p r e s s o r I b r i s t l e l o s s d u e t o l o w e r e d a c t i v a t o r f u n c t i o n A + 1 a c t i v a t o r h y p e r - a c t i v e o r a n t a g o n i s t i c r e p r e s s o r I I b r i s t l e l o s s d u e t o e x c e s s r e p r e s s o r f u n c t i o n A + i v i I I a c t i v a t o r r e p r e s s o r I ' _ i , n o r m a l b r i s t l e d e v e l o p m e n t m e d i a t e d b y b a l a n c e d n o t c h -l o c u s p r o d u c t ( s ) . 190. TABLE 52 Expected product proportions of Ax/Ax, Ax/+, and Ax/N, according to activator:repressor model "(Figure 17) • EXPECTED PRODUCT PROPORTIONS GENOTYPE CASE (a) CASE (b) Ax/Ax 2 R+: 0 A+ 2 RH: 2 A+ Ax/+ 2 R+: 1 A+ 1 RH: 1 R+: 2 A+ Ax/N 1 R+: 0 A+ 1 RH: 1 A+ 191. Figure 17 can explain the phenotypes associated with these geno-types. If we assume that has twice the repressive activity of R^ then the quantitative equivalence of cases (a) and (b) is more apparent (Figure 17, Table 52). It is easy to see from Table 52 that Ax/+ should be phenotypically closer to wild-type than Ax/Ax, since in both cases there is a smaller excess of repressor over activator functions in Ax/+ than in Ax/Ax. There is still an excess of repressor over activator function in the Ax/N product (Table 52), but if we remember that in the model there is less excess repressor in Ax/N than in Ax/Ax, in relation to the rest of the genome, the relatively milder mutant phenotype of Ax/N is not too surprising. Note that according to this model, suppression of Ax phenotypes by N^ occurs by a different mechan-ism tha suppression by N mutants. Also, this model is not inconsistent with the dimer- or complex-formation models based on the N allele-dosage studies (Figures 15, 16). Yet another feature of the models presented in Figure 17 is that they could also account for the phenotypes of N ^ ^ and spl, which have been inferred to be neomorphs on the basis of their responses to alterations of gene dosage (Figure 4) or to modifiers (WELSHONS 1956b, 1971), respectively. Accordingly, so-called neomorphic mutations may actually be mutations which cause intra-gene-product imbalances of the hypo- or hypermorphic variety, as opposed to true hypo- and hypermorphs, which by definition affect the synthesis or function of the whole gene product. As noted earlier, the Abruptexes tend to map in one region 192. of the Notch locus. Inspection of Figure 1 shows that those Ax alleles which have been mapped with any degree of precision are situated in the right half of the genetic map. Furthermore, by viture of its phenotype of sparse thoracic microchaetae (WELSHONS 1965) and its larval-pupal lethal phase (WRIGHT 1970), the lethal mutation 1(1)NB, which maps in this region (Figure 1), can also be regarded as Abruptex-like. It has already been noted that under certain conditions, N s l 1 (which maps near the right limit of the Notch locus (Figure 1)) manifests Abruptex-like character-2 istics. Moreover, nd , which also maps near the right limit (Figure 1), expresses both N-like (wing-nicking) and Ax-like (vein gapping) phenotypes (WELSHONS, personal communication). Thus it becomes increasingly apparent that non-amorphic mutations which affect similar developmental processes are likely to be positioned at similar sites within the Notch locus. The separa-tion of fano and nd, which have similar phenotypes, appears to contradict this generalization. However, as the following dis-cussion will show, this exception can be reconciled with the generalization that different regions within the Notch locus are specific in terms of their developmental function. As stated earlier, the Notch locus appears to consist of two parts which are functionally related to one another. This is inferred from a correlation of the genetic positions of a number of non-amorphic alleles within the locus, with their inter-allelic complementation pattern. All heteroallelic combinations among the recessive visible mutations, fag, spl, fano, and nd, except fanQ/nd, exhibit complementation (i.e., are non mutant in appear-193. ance) (WELSHONS 1965). Recently, it has been reported that Ax59b and do not complement fas, in that the eyes of Ax/fag heterozygotes are rough, although not glossy (WELSHONS 1971). Moreover, the Ax-like allele, 1(1)NB, allows complete pseudodomi-nant expression of fa®, the l(l)NB/faS heterozygote having eyes which are both rough and glossy, unlike the other (N-like) 1(1)N alleles, which allow only partial pseudodominance of fas (WELSHONS 1965). If the genetic map of the Notch locus (Figure 1) is fit-ted to these patterns of complementation, such that noncomple-menting alleles are situated opposite one another, a spiral genetic map is obtained (Figure 18). This pattern is strikingly reminiscent of the spiral genetic map generated by congruence of the linear genetic map with the circular complementation map of the ad-8 locus in Neurospora crassa (KAPULER AND BERNSTEIN 1963) . Assuming that the correlation of the genetic map with the complementation pattern (Figure 18) is not fortuitous, several interpretations of its significance are possible. One possibility is that during the evolution of Drosophila, an intra-band tandem repeat of genetic material occurred (BAUER 1943), and that while maintaining similar roles in development, the two halves evolved divergently. It is conceivable, for example, that specific func-tions originally common to both halves were selectively lost in one or the other region, thereby leading to the apparent division of functions now observed. Another possibility is that the tertiary structure of the Notch locus product has a spiral con-figuration. This interpretation has been given to the spiral correlation of the genetic and complementation maps of the ad-8 FIGURE 18 Correlation of the genetic map positions and complementation pattern of certain Notch-locus mutations. Alleles positioned opposite one another are non-complementary. The position of 1(1)NB with respect to has not been determined. 196. locus in crassa (KAPULER AND BERNSTEIN 1963), although CRICK and ORGEL (1964) strongly attacked this interpretation. There have been several reported cases of circular complementation maps in Drosophila (CARLSON 1961; SUZUKI AND PROCUNIER 1969) and other organisms (FINCIiAM AND DAY 1965), and it may be that as more com-plex genetic systems are analyzed, many more examples will be discovered. In fact, SHELLENBARGER (1969) has found that the complementation pattern of 17 EMS-induced lethals within the Notch locus fits a circular map. It would be very interesting indeed to see whether the genetic positions of these lethals are correlated with the complementation map, and whether this pattern fits the spiral map presented in Figure 18. Throughout the preceding discussion it has generally been implied or assumed that the Notch locus produces a single gene product. This has also been the opinion of other investigators, who based their conclusions on the non-complementation (lethality) of all heteroallelic N mutant combinations tested (WELSHONS 1965) and the similarity of the embryonic abnormalities in hemi-, homo-, and heterozygotes of various lethal N alleles (POULSON 1968). Further support for a single Notch-locus product comes from con-sideration of the interactions of fano, spl and AxE2 (Appendices 6, 7)> The genotype fano spl AxE2/+ + + expresses a rough eye phenotype which is virtually indistinguishable from that of spl/ spl, whereas the genotypes fano spl +/+ + AxE2, + spl +/+ + AxE2, and fano + +/+ + AxE2 do not have rough eyes. The genotype fa110 + +/+ spl AxE2 has a mild rough eye phenotype which overlaps wild type, but this is no more extreme than the phenotype of + + +/+ 197. spl AxE2. (Plies of the genotype fa110 + AxE2/+ spl + have not been examined yet.) The phenotypic differences between the cis and trans configurations indicates that the fully penetrant spl phenotype of fano spl AxE2/+ + + is not due to an additive effect of the three mutant alleles acting independently, but to the presence of the three mutant sites in the same product molecule. In addition to the enhancement of the spl mutant eye pheno-type by AxE2, the coupling of fano to AxE2 completely suppresses the wing vein gap phenotype of AxE2 and significantly suppresses an extreme bristle-loss phenotype associated with the coupling of spl to A x E 2 (Appendix 7). This enhancement of one phenotype and suppression of the others is rather unusual, especially since spl separates the fano and AxE2 mutant sites (Table 14, Figure 1). However, examination of the spiral map (Figure 18) suggests the following explanation. If we assume that the enhancement of the spl eye phenotype is due to an extension of the effects of the fano lesion to the right of the mutant site (Figure 1), the sup-F? / F? pression of the spl Ax bristle phenotype (and the k-xr wing vein phenotype) may be due to a comparable effect to the left of fano, influencing the sites which are complementary to the spl-Ax region (i.e., the fa^ region, see Figure 18). Admittedly this is not the only possibility, since effects of the fano mutation on the tertiary folding of a Notch-locus product molecule containing the spl and AxE2 mutant sites, could equally well account for the observations. Nevertheless, the interactions of these three mutant alleles suggest at least that the region of the Notch locus spanning the fano-AxE2 mutant sites produces a single 198. molecular product. Furthermore, these observations Indicate that fano is not entirely a hypomorphic allele, as was suggested by WELSHONS (1965) on the basis of N/fano lethality, since in this event we would not expect enhancement of spl. WELSHONS (1971) has recently reported that the coupling of an amorphic (N) allele to spl results in inactivation of the spl mutant function. One of the phenomena underlying the differences between the two groups of Abruptexes is the unusual system of lethal inter-actions among the Ajc alleles. It is not difficult to conceive of a lethal/non-lethal heterozygote causing lethality, such as AxE1/AxS2, Ax EVAx l 6 l72, Ax59d/AxE2, a n d Ax59d/Axl6l72 (Table 29), in the same way that N/fano heterozygotes are usually lethal. HOUSE (1959a) has also reported a lethal interaction between Ax2^a and the lethal allele Ax^. However, the observation that certain heteroallelic combinations of viable Ax alleles (Ax9B2/ A x e 2 and A x 9 b 2 / A x 1 ^ 1 7 2 ) result in lethality (Tables 2 9 , 30) is an entirely different matter. At first sight, one might postulate that this kind of interaction, or negative complementation, indi-cates that the products of Ax9B2 and (or Ax16172) are mutual-ly antagonistic and that the resulting inactivation of these gene products is responsible for the lethality. However, the observa-tion that the lethal Axx/Ax^ combinations exhibit severe bristle loss phenotypes (Table 31) > and the fact that relatively severe hypomorphic situations, like the viable and semilethal N/fano genotypes, exhibit bristle disturbances in the opposite direction, tend to discount the Ax-product inactivation theory. One plaus-ible alternative is that the two groups of Ax_'s affect generally 199. different sets of functions (possibly overlapping one another to some extent). In the case of the viable alleles, these regula-tory upsets would not be sufficient to cause homozygous lethality, but combinations of regulator mutations whose range of effects differ might affect enough functions to cause lethality. This type of model is illustrated in Figure 19, and is consistent with the different phenotypic pattern expressed by Ax 9 B 2 compared to the other two alleles (Table 21). Another alternative is that the two groups of Ax's differ fundamentally in terms of the pri-mary lesion in the Notch-locus product. For instance, if we refer to the models presented in Figure 17, the heterozygous com-binations of an A~-type Ax (model a) and an RH-type Ax (model b) would result in the product ratio of 1 R^ : 1 : 1 which might easily exhibit a more extremely mutant phenotype than either homozygous A~/A~ or RH/RH (see Table 52). Either of the above models could account for the observed lethal patterns and also accommodate the viability of A X E 1 / A X 9 B 2 (Table 29). Whether or not Ax59d/Ax9B2 is lethal has little bearing on the validity of these models, since if lethality occurred it could be attributed either to effects on functions not affected by Ax 9 B 2 (Figure 19 model), or simply to being more severely mutant than Ax^ -1-(either model). From the data presently available, therefore, it is not possible to distinguish between these types of models, if indeed either is the correct one, and a test of these ideas probably must await the development of methods to characterize the Notch-locus product biochemically. Although lethality of the Ax9B2/AxE2 type has not been re-F I G U R E 19 "Range of function" model to explain lethality of Ax9B2/AxE2 and A x 9 B 2 / A x 1 6 1 7 2 . The presence of an arrow in a space indicates that the given genotype is mutant in the function represented by that space. G E N O T Y P E H Y P O T H E T I C A L F U N C T I O N S A F F E C T E D D E V E L O P M E N T A L R E S U L T 2 f u n c t i o n s a f f e c t e d ; > g e n o t y p e s u r v i v e s . o J LU - J J < o m LlI H Ul X 9 b 2 / . i ix / A x 9 b 2 / , 1 6 1 7 2 A x / Ax A x " / A * " " ' 2 I ^ | ^  I 1 I I ^ I 1 | 1 I 1 I I I I 3 f u n c t i o n s a f f e c t e d ! g e n o t y p e d i e s . 2 f u n c t i o n s a f f e c t e d ; > g e n o t y p e s u r v i v e s 202. ported before, two cases of synthetic lethality involving an Ax allele have been reported. In one case, combining the recessive A lethal allele Ax with the third-chromosome mutant Hairless (H) results in lethality of ;H/+ females, while in the second case Ax28a/Y;H/+ and Ax28a/Ax28a;H/+ are lethal above 26°C (HOUSE 1959a). This is most interesting, since H, which is itself a recessive lethal, has a bristle loss and wing vein gapping pheno-type similar to that of Ax (GOWEN 1933). Moreover, H, which by itself has no effect on wing vein L2, enhances the gapping of L2 caused by Ax28a/Ax28a (HOUSE 1955), H/+ and Ax28a/+ enhance one oanother in double heterozygotes, and N°/+;H/+ females have re-duced expression of both the N and H phenotypes, an effect resembling that seen in Ax28a/N8 flies (HOUSE 1959a). The re-semblance between H effects and Ax effects appears to be more than a chance similarity. The Ax28a-like interactions between H and o N strengthen this contention, and show the dual involvement of both and H^ activity in normal development. The observed interactions of H, Ax, and N support the hypothesis that (in con-trast to the Ax/+ situation, which must be a case of dilution of Ax mutant product by Nf^  product) the reduced expression of Ax in Ax/N heterozygotes reflects lowered Ax activity in relation to the rest of the genome (Table 52). The existence of Notch-locus modifiers at other loci is not surprising at all. In fact, the existence of such "modifiers" is predicted by the hypothesis that the Notch locus is a regulatory gene which influences several developmental systems. According to this hypothesis, modifiers of Notch-locus alleles might reflect 203. changes in the response potential of developmental systems under Notch-locus control, or they could be mutations in other regulator genes. Besides the interaction with H, many cases of modifiers of N mutant alleles have been reported, a few of which are sup-pressors (MORGAN 1919) and enhancers (Appendices 2-4) of wing nicking, an enhancer of S£l (WELSHONS 1956b; VON HALLE 1965), and modifiers which cause head deformities in the presence of N (HILLMAN 1961). Consideration of the results of the present in-vestigation, therefore, must be tempered by the realization that genetic modifiers of the alleles studied could have, and very likely did, influence observations especially of the quantitative type, as was noted from time to time in Results. Nevertheless, the results of the investigations of N allele dosage effects and Ax interactions with N and one another, seem to be sufficiently uniform for the purposes of the present discussion. It can also be stated that preliminary results from AxE2, Ax1^172, and Ax952 strains made co-isogenic for OR autosomes* (Figure 20), confirm both the lethality of Ax9B2/AxE2 and Ax 9 B 2/Ax l 6 1 7 2 and the mor-phological differences between Ax952 and AxE2. The sexual dimorphisms frequently encountered in the pheno-types of the Ax mutations (Table 22) cannot be so easily dis-missed as being due to genetic background variation, since these differences appeared in sibling males and females reared under *Probably for part of the X chromosome also, since the autosomal inversions SMI and TM2 would tend to increase recombination between the Ax and X chromosomes. FIGURE 20 Mating scheme used.to replace autosomes of recessive viable Ax_ stocks with OR autosomes. +° = chromosome from OR stock. 206. identical conditions. Since the Notch locus is sex-linked, in-complete or aberrant dosage compensation of the Ax mutant pro-ducts is one possible explanation. By definition N_ is dosage compensated, since one dose of in males produces the same phenotype as two doses in females (STERN I960). The fact that a single dose of in N/+ females leads to a mutant phenotype does not reflect defective dosage compensation of N^ (since dosage compensation concerns male-female differences), but indicates that N^ is "haplo-insufficient" in females (MOHR 1932). The in-terpretation that defective dosage compensation of Ax accounts for the sexual dimorphism of mutant phenotypes may be weakened by the fact that certain autosomal mutants also exhibit sexual di-morphism. For example, the fourth-chromosome mutant cubitus-interruptus (ci), which causes wing vein gaps, and its dominant allele, ciD, are significantly less mutant in females than in males (HOUSE AND EBERSOLE 1971). It is possible that sexual physiological differences could explain dimorphisms such as these. On the other hand, LUCCHESI (personal communication), on the basis of autosomal and sex-linked enzyme specific activities in triploid and intersex (2X;3A) females, has suggested that sex-linked dos-age compensation is a special case of "diploid" regulation, which normally operates in autosomes as well as the sex chromosomes. Thus, the phenotypic sex differences of the Ax and ci alleles could both result from defective dosage regulation of the mutant gene products. The results of the temperature-shift experiments with several different Notch-locus genotypes, show that the presence 207. of the N^ gene product is necessary for normal development at several stages of the Drosophila life cycle. The most striking observation is that the TSPs for the adult morphological pheno-types all occur during the third larval instar, whereas TSPs for lethality occur during several stages of development, depending on the genotype involved. The TSPs for the eye facet, wing nicking, bristle disturb-ance, wing vein gapping, and leg segment fusion phenotypes, all occur prior to pupation (in the third instar), which is the stage when the actual differentiation of the adult morphological struc-tures takes place. This suggests that during the third larval instar, activity of the Notch locus affects determination of the pattern of differentiation of the cells in imaginal discs respon-sible for the adult eyes, wings, legs, and epidermis. Recently, SHELLENBARGER (1971) reported that heat shocks of larvae homo-zygous for the ts Notch-locus lethal allele, K p N ^ - 1 , induce mutant adult phenotypes such as eye- and headlessness, rough eye, small wing, notched wing, and leg segment fusion, although he found that heat shocks during the pupa stage also cause some adult morphological abnormalities. Recently, it has been report-ed that a ts allele (ss a^ a) at the spineless locus, which is definitely a differentiation pattern-determining gene, also has a third instar TSP (for conversion of antennal to leg structures) (GRIGLIATTI AND SUZUKI 1971). Ultimately, in order to understand the significance of pattern-determination TSPs, we must know the molecular properties of the gene products of loci such as N+ and 208. The restriction of TSPs for morphological traits to the third larval instar contrasts with the TSPs for lethality, which occur at several stages of development. This provides an inter-esting comparison with the pattern of TSPs for lethality and visible phenotypes observed for the sex-linked ts lethal mutation l(l)E6ts (GRIGLIATTI AND SUZUKI 1970). In the case of l(l)E6ts, the lethal TSP occurred at the end of the third instar, whereas TSPs for deposition of pteridine pigments were embryonic-second instar (Malphigian tubules) or late pupal (testis and eye). The biphasic nature of the pigment TSPs of 1(1 )E 6 t s is not surprising, since the early TSP is for larval tissue and the late TSP for adult tissue, whereas the Notch-locus morphological TSPs were all for adult phenotypes. Another contrast is that the TSPs for the mutant pigment deposition of l(l)E6ts occurred at about the same time as pigment deposition normally takes place, whereas the TSPs for Notch-locus-mediated morphological traits occur well before the differentiation of the structures affected. This suggests that these particular developmental functions of the N^ locus t s occur before the actual differentiative processes, while 1(1)E6 may more closely affect differentiation. This is consistent with the hypothesis that the Notch locus is a regulator gene. The polarized progression of eye facet arrangement seen in both Ngll/+ (Figure 6) and N1Q3/spl (Figure 7) females shifted at progressively later stages during the third instar, suggests that a wave of determination of ommatidial organization originates in that portion of the eye disc destined to form the posterior edge of the adult eye, and then progresses anteriorly. These observa-209. tions and conclusions are very similar to those of BECKER (1957) who found that a rough-eye phenocopy could be induced in wild-type flies by X-irradiation during the third instar-prepupal stages3 and that the irregular arrangement migrated anteriorly across the eye in a vertical band with increasing larval age at the time of irradiation. BECKER'S results differ from the pre-sent temperature-shift results, since X-ray sensitivity occurred during both larval and prepupal stages (BECKER 1957), while the TSPs for eye facet arrangement end before the prepupa stage (Figures 6, 7). Moreover, in both Ngll/+ and N103/spl the 29°C eye TSPs end earlier with respect to puparium formation than at 20°C-22°C (Figures 6, 7). In order to determine whether the sensitivity of eye facet arrangement to radiation also occurs earlier at 29°C, I repeated BECKER'S experiments with OR flies incubated at 20.5°C and 29°C. The results (Figure 8) confirm that the RSP for eye facet arrangement extends into the prepupa stage, and also show that there is no difference in the RSP at these two temperatures. The temporal differences between the stages of sensitivity to radiation and temperature shifts suggest that radiation may affect eye development at a different level of complexity than the effects of temperature on the Notch-locus primary gene product. Further to the question of eye differentiation, KURODA (1970) has reported that a posterior-to-anterior gradient of om-matidial differentiation occurs in the eye discs of mature third-instar Oregon-R larvae, cultured in vitro. This is also a later developmental stage than the beginning of the TSPs reported here. 210. Furthermore, he observed that irradiation of mature third-instar discs immediately after isolation, preferentially inhibited the organization of ommatidium-forming cells in the anterior part of the eye disc, an observation consistent with the results of BECKER (1957) and those reported here (Figure 8). The observa-tion (KURODA 1970) that visible ommatidial precursor-cell-cluster formation occurs later than the TSPs reported here, is further evidence that the Notch-locus role in the development of adult structures is pre-differentiative, or else occurs at a very early stage of differentiation. In passing, it can be noted that posterior-to-anterior pro-gression of eye facet differentiation has been observed in the mosquito Aedes aegypti as well as other insects (WHITE 1961), thereby suggesting that this pattern of development may be a general one so far as compound eyes are concerned. Indeed, polar-ized determination of other adult structures in insects may also be the rule, since a proximal-to-distal progression of conversion of aristal to leg, or leg to aristal segments, has been observed w i t h ssalt0a (GRIGLIATTI AND SUZUKI 1971). It would be of interest in this regard to see whether tarsal segment fusion in N10^/+ heterozygotes also proceeds in a proximal-to-distal direction. This was not examined for in the N 1 0Vspl temperature shift ex-periment (Figure 7). The several different lethal TSPs observed in the present experiments (Figure 14) indicate that mutation at the Notch locus can affect vital functions at several discrete developmental stages. Moreover, the fact that the TSPs for lethality differ 211. from those for adult morphological phenotypes suggests that the vital processes affected may be only loosely related to the morphological processes (cf GRIGLIATTI AND SUZUKI 1970). The monophasic lethal TSPs of Axl6l72/N40 (Figure 9) and Ngll/Nsll;Dp (Figure 13), may indicate that these genotypes are each defective in only one vital process, whereas the length of the TSP (or TSPs) and heterogeneity of kill-periods (Figure 12) of N~^3/fano may be interpreted in several ways. Although the N103/fano shift data (Tables 44-47) do not allow a critical decision as to whether the TSP is monophasic or polyphasic, re-sults recently reported by SHELLENBARGER (1971) suggest that it may be polyphasic. He found that peak sensitivity of K D N ^ 5 - 1 homozygotes to temperature occurred during the embryo, first-second instar, and prepupal stages. TARASOFF and SUZUKI (1970) described sex-linked lethal mutations in D. melanogaster with both monophasic and polyphasic TSPs and proposed that polyphasic TSPs may reflect: 1) repetitive gene activation and inactivation; 2) tissue-specific activation and inactivation of a gene; or 3) repetitive use of a gene product which is synthesized only once during development. Any one or a combination of these hypotheses could explain the polyphasic TSP of l(l)Nts~1 (and N 1 Q 3/fa n o, if it is polyphasic), but suggestion (2) is particularly attractive, considering the wide array of pleiotropic effects associated with Notch-locus mutations and since position-effect variegation of certain Notch-locus recessive mutants (COHEN 1962) suggests that the Notch locus can function autonomously in different tissues. The observation that death of N103/fano females occurs at 212. three distinct developmental stages, depending on their stage when shifted to 29°C (Figure 12), further indicates that the TSP may be polyphasic, or at least that more than one developmental event is vitally affected. This pattern of lethality is similar to that observed in l(l)Nts"1, in which embryonic heat treatments result in death before puparium formation and later treatments cause death just before or at the emergence of the adult (SHELLEN-BARGER 1971). It should be pointed out that the bimodal lethal phase (early pupal or late pupal) of N103/fano females shifted up during the larval stages (Figure 11) could result from genetic background heterogeneity. Such modification of the lethal phase has been reported for unconditional lethals (HADORN 1961), and recently for a conditional lethal (SUZUKI 1970). Nevertheless, I no this does not affect the conclusion that N /fano probably has more than one TSP for lethality. As discussed earlier, the monophasic embryonic TSP for lethality of N ^ ^ / n S 1 1 ;Dp females may or may not result from em-bryonic disturbances similar to those of N homozygotes, depending on whether is antimorphic or neomorphic in this instance. l6l72 The monophasic second instar TSP for lethality of Ax / is very interesting, since most monophasic ts lethals in Drosophila have TSPs in either the embryo or late third instar-pupal stages (SUZUKI 1970). Incubation of Ax l 6 l 7 2/N 4° at 29°C during the TSP does not prevent the determination or differentia-tion of imaginal discs, since death occurs in late pupae or partially-eclosed adults. However, the fact that A x 1 ^ 7 2 hemi-40 and homozygotes are not ts lethal, and the assumption that N is 213. amorphic, suggest that Ax16172/n40 larvae do not possess suf-ficient product activity when incubated at 29°C during the TSP. Thus, ts lethality of this genotype may reflect a hypomorphic gene-product activity. Other than this, the data do not suggest the nature of the defect responsible for inviability, although we can assume that the function affected is regulatory rather than structural, since the TSP is so far removed from the differentia-tive (pupal) stage. LITERATURE CITED 214. BAUER, H. (1943), Eine neue Mutation, facet-notchoid, bei Drosophila melanogaster und die Deutung des notches-Effekts. Z. ind. Abst. Vererb. 8l: 374-390. (cited in WELSHONS 1958a). BECKER, H. J. 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(1939b), Effects of Notch deficiencies. Droso-phila Info. Serv. 12_: 64-65. POULSON, D. F. (1940), The effects of certain X-chromosome deficiencies on the embryonic development of Drosophila melanogaster. J. Exp. Zool. 83: 271-325. 220. POULSON, D. F. (1968), The embryogenetic function of the Notch locus in Drosophila melanogaster. Proc. Twelfth Int. Cong. Genetics, Tokyo 1: 143. PRATT, L. R. (1971)3 Developmental and Genetic Analysis of a Purported New Class of Sex-linked Mutations in Drosophila melanogaster. Master's Thesis, University of British Columbia, Vancouver. RAYLE, R. E., and D. I. HOAR. (1969), Gene order and cytological localization of several X-linked mutants of Drosophila melanogaster. Drosophila Info. Serv. 44: 94. SHELLENBARGER, D. L. (1969), Genetic structure of the 3C region of Drosophila melanogaster. Master's Thesis, University of Iowa, Ames. SHELLENBARGER, D. L. (1971), A temperature-sensitive Notch mutant of Drosophila melanogaster. Genetics 68: 561-562. 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Serv. 30: 79. WELSHONS, W. J. (1956b), Dosage experiments with split mutants in the presence of an enhancer of split. Drosophila Info. Serv. 30: 157-158. 222. WELSHONS, W. J. (1958a), A preliminary investigation of pseudo-allelism at the Notch locus of Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S. 44: 254-258. WELSHONS, W. J. (1958b), The analysis of a pseudoallelic reces-sive lethal system at the Notch locus of Drosophila melanogaster. Cold Spring Harbor Symp. Quant. Biol. 23: 171-176. WELSHONS, W. J. (1965), Analysis of a gene in Drosophila. Science 150: 1122-1129. WELSHONS, W. J. (1971), Genetic basis for two types of recessive lethality at the Notch locus of Drosophila. Genetics 68: 259-268. WELSHONS, W. J., and E. S. VON HALLE. (1962), Pseudoallelism at the Notch locus in Drosophila. Genetics 4_7: 743-759-WELSHONS, W. J., E. S. VON HALLE, and B. J. SCANDLYN. (1963), Notch pseudoalleles in Drosophila melanogaster. Proc. Eleventh Int. Cong. Genetics, The Hague 1: 1-2. WHITE, R. H. (1961), Analysis of the development of the compound eye in the mosquito, Aedes aegypti. J. Exp. Zool. 148: 223-239• 223. WRIGHT, T. R. F. (1970), The genetics of embryogenesis in Drosophila. Advances in Genetics 15.: 261-395. 224 APPENDIX 1 Interactions of N g l 1 and N 1 0 3 with faS and fano Since N g l 1 was known to have a ts eye phenotype (WELSHONS AND VON HALLE 1962), and N 1 0 3 to have ts interaction with spl, and ts wing and leg phenotypes (WELSHONS, personal communication), a check on the interaction of these N alleles with fag and fano was made. To investigate the interactions with fag, the following crosses were performed at 20°C, 22°C, 25°C, and 29°C: faS/faS ? x w* NS11 rb/B£ w^'Y d* , and faS/faS ? x 2. wf. N1Q3;Cy D£ Bw^ & At all temperatures the N/fa£ progeny of both crosses had eyes with disarrayed ommatidia and the glazed appearance of fag hemi- or homozygotes. No difference between the roughness of N/fa£ and that of fas alone, could be detected in any of the heterozygotes excepting NSll/faS at 20°C and 22°C. In the latter cases, the roughness was no greater than one would expect from the combination of the NgH/+ and fas phenotypes. The pseudo-dominant expression of fag observed with and N 1 0 3, is con-sistent with that reported for fa with these two N alleles (LINDSLEY AND GRELL 1968). Both Ngll/fano and N103/fano were found to be relatively viable at low temperatures, but lethal at 29°C. The crosses used, and the results obtained, appear below. 225. Cross 1: w^ fano/wa fano ^ x / nS 1 1 rb/B^ w^-Y cJ* Results: PROGENY Results: Results Temperature B+ <j> <j> Bs cfd* 20° C 31 121 22°C 3 11 25°C 16 113 29°C 0 47 Ngll rb j x wa fano & PROGENY Temperature B 9 9 B+ 9 9 B <3»(3» 20°C 266 40 239 22°C 109 38 62 25°C 133 35 121 29°C 80 0 55 ra N 1 0 3 £ x w a fano PROGENY Temperature B 9 B+ 9 $ B o»o" 20°C 244 194 204 22°C 64 83 71 25°C 138 86 129 29°C 71 0 69 Significant pupal lethality was seen in the 25°C and 29°C cultures involving N^H, although this was not sufficient to ac-count for all the expected female progeny. For example, in 226. cross 2, 28 dead pupae were counted in the 29°C culture. Of the 24 which had developed to sufficiently advanced stages, 23 were Ngll/fano females, many of which had drastically reduced head and eye sizes on one or both sides. Other morphological phenotypes were not examined. No significant pupal lethality was observed in the 20°C or 22°C N s l 1 cultures, nor in any of the N 1 0 3 cultures. Furthermore, the data show that at the three lower temperatures, N103/fano was much more viable than Ngll/fano# Temperature-shift studies of the N103/fano lethality are reported in Results (part F, section V). The wings of both N103/fano and NS11/fano adults were deep-ly serrated at the tips and along both edges (N-*-Q3/fano being more extreme in this regard), had extremely thickened wing veins, and frequently contained large bubbles. In addition, N1Q3/fano females raised at 25°C had fused tarsal segments, while those raised at lower temperatures only occasionally had fused tarsi (1 out of 24 examined at 22°C) . The legs of the NS1]-/fano females which survived, did not show any tarsal fusion. 227 APPENDIX 1 Removal of E- n 7 0 j and bb^ from the chromosomes, and mapping of E^ N70j In the course of the first experiments designed to study the effects of gene dosage on N^0, it became apparent that the initial wa rb chromosome contained a bobbed-lethal (bb1) allele, since all attempts to make this chromosome homozygous failed, and the cross l(FM6)/we bb1 ? x w^ N ^ rb/Y;Dg_ cf yielded no non-Bar-eyed female progeny. In order to remove the bb-1-mutant from the wf^  rb bb1 chromosome, recombinants between N C o and the flanking eye colour mutations were obtained from wa N C o rb bb V + + + + females, the wild-type X chromosome having a f o come from the OR stock. Two strains were established, w^ N , and NCo rb, from which the bb1 had been removed, as indicated by the following results of the cross 1 ( F M 6 ) / x $ x In(l)dl49,tyl bb-^ /Y (cf^cfwere not counted). FEMALE PARENTAL $ X-CHROMOSOME PROGENY a Co r b b bl NCo r b wa NCo PHENOTYPE — gg. ^ B eyes 1^3 65 129 B^ eyes 0 Ik 133 In the course of routine checks it was discovered that the wa chromosome was associated with a significantly lower fre-quency of wing nicking than the N^0 rb and w^ N^0 rb bb^ chromo-somes, suggesting that an enhancer of notching present in the original stock is not present in the strain. From the data 228. tabulated below, it can be seen that the N C o rb strain is not significantly different from the original strain in terms of wing nicking, whereas the frequency of individuals with nicked wingtips is halved in the wf; N C o stock. NO. OP FEMALES CROSS GENOTYPE wf; rb btr + + + + N C o rb + + w a N C o NOT % NICKED NICKED NICKED FEMALES OR % x wf; N ^ rb bb1/Y;Dp (f 60 OR % x N£^ rjb/Y;D£ cr> 95 OR ° x wf; NCo/Y;Dp o^ 42 14 17 58 81 85 42 The enhancer of nicking, designated E-N 7^, was localized genetically in the following manner. NCo-containing female progeny from the cross: + rb + + + + + cv v f car $ . x ^ + + c v v f car , were scored for wingtip nicking and for the markers cv, v, f, and car. The results are tabulated below. RECESSIVE MARKERS 0 WINGS N 1 WING N 2 WINGS N % N INDIVIDUALS + + + + + 71 133 124 -<] CO + + + + car 4 14 19 895S + + + f car 28 46 64 80$? + + V f car 18 54 81 88$ + cv V f car 21 21 10 60% Z + + + + 1 5 11 W + + + f + 1 0 3 — 229. RECESSIVE MARKERS 0 WINGS N 1 WING N 2 WINGS N % N INDIVIDUALS + + V f + 1 8 6 93% + cv V f + 3 2 1 50% + + V + + 5 14 10 83% + cv V + + 6 5 7 67% + cv + + + 3 4 0 57% L + + f car 0 1 1 -+ cv + + car 0 1 0 -y. + V f + 0 0 1 -L + V + + 0 0 1 -It can be seen that all the genotypes of which appreciable numbers were available, exhibited wingtip nicking in 78% or more of individuals, except each class resulting from a crossover between N^0 and cv;, and containing c_v, which had wingtip nicking frequencies ranging from 50% to 67%. This suggests that E-N7QtJ' is situated to the left of cv. Although the following cannot be regarded as conclusive, additional observations made on some of the progeny listed above, suggest that E-N70j is located closer to rb than to cv. The evi-dence is as follows. During the course of scoring this cross, it was noticed that some of the N C o cv v f car females had darker eyes than others of this genotype, and that these females had a lower incidence of wing nicking than the lighter-eyed females. Of 37 females examined in this regard, 13 had darker eyes and 24 had the lighter eyes. Only 4 of the darker-eyed flies exhibited nicking, whereas 18 of the lighter-eyed females were nicked. If 230. it is assumed that the darker-eyed females have the genotype + N C o + cv v f car/y + + cv v f car, and the lighter-eyed females + N C o rb cv v f car/y + + cv v f car, these data suggest that E-N7Qj' is situated close to rb. This assumption is supported by the observations that: 1) all ^ N^ cv v f car females examined (their genotype presumably being + + + cv v f car/y + + cv v f car, since interference is essentially complete in the y-rb region) had the darker eye colour, and 2) all N C O cv+ v f car females examined (presumably + N C O rb + v f car/y + + cv v f car) had the lighter eye colour. 231 APPENDIX 1 Screening procedures used to isolate new Notch-locus mutations Procedure No. 1, diagrammed on the next page, was designed to screen for new mutations which were lethal when heterozygous with N. OR males which had been fed 0.0125 M EMS in 1% sucrose for 24 hours, were mated to compound-X (XX/Y) females of the geno-type shown. Individual male progeny of this cross, raised at room temperature (20°C-22°C), were each mated to 2 1(FM6)/1(I)J1 and 2 XX,^ w jr^ 'Y females in the same vial. Those vials which produced both and ^ female progeny, but no males, were saved as putative lethals covered by the duplication w+ • Y. The 1(FM6)/+X females from the vials scored as putative lethals, were mated individually to wf^  rb/BS w+•Y males. Progeny of this cross were scored for the absence or presence of females, indicating the respective presence or absence of a Notch-locus lethal failing to complement with . From approximately 2600 chromosomes tested in this manner, 32 putative Notch-locus lethals were recovered. Of these, 1 was not lethal, 19 were lethal but not covered by the duplication, 11 were lethals covered by the N+ duplication, but were not lethal when heterozygous with and 1 was a Notch-locus lethal. This lethal, originally designat-ed 1(1)NE^, was later renamed Ax5-1- when it was found to be an Abruptex allele (see Table 13 for mapping of AxE1). Procedure No. 1 >L w £ili w^-Y $ x +/Y c? EMS I 1(PM6)/1(1)J1 ? ) ) x +*/Bs w+.Y (individual or» ) 1(FM6)/+» •J ' ? l(l)Jl/+» 1(FM6)/BS w^-Y 1(1)J1/BS w^-Y XX,y w f;B^ _ w^-Y (y+ females) (males die) (y females) +*/Bs w+-Y (males die if +* carries a lethal) l(FM6)/+» $ x N ^ rb_/B^ _ cf l(FM6)/wa N 4 0 rb +*/wa N110 rb 1(FM6)/BS w + - Y +*/Bs w + - Y (B females live) (B females die if carries N-locus lethal) (males die) (males live if +* carries lethal covered by duplication) r\j o j ro 233. Procedure No. 2 (not diagrammed) was used to screen for visible, recessive viable mutations in the Notch locus. OR males which had been fed 0.005 M EMS in 1% sucrose, were mated to XX/Y females. Male progeny of this cross, raised at room temperature, were screened directly for visible phenotypes affecting wing nicking, wing venation, or eye facet arrangement. Of a total of 4686 male progeny examined 111 were saved for further testing. These were mated individually to virgin 5CX/Y females. Of these 19 were sterile, 67 did not yield mutant progeny, 3 were autosomal dominants, 7 were lethals, and 15 were X-linked visibles. Of the last group, two proved to be in the Notch locus. On the basis of subsequent tests, these were designated AxE2 and fanoE, re-spectively (see Appendix 4 for mapping of fanoE, and Table 14 for mapping of AxE2). 234. Procedure No. 3, diagrammed on the next page, was designed to screen for 1) revertants of N s l 1 to and 2) forward muta-tions of N s l 1 to N a m o rP h (either as the result of mutation at the N^"1, site, or at a site cis to N ^ 1 ) . It can also be used to screen for extra-locus modifiers of Notch, as the results show. wa NS11 rb/Bs w*»Y males which had been fed 0.0125 M EMS in 1% sucrose for 24 hours, were mated to dl49/wa N*^ rb females (8 half-pint bottles, 20 $ $ x 4-5 c^cfeach). The eyes of female progeny, raised at 20.5°C, were scored for white eye colour (putative revertants) or non-mutant eye facet pattern, N, or modifier mutations). Of 3008 female progeny examined, none were white-eyed, but 11 red-eyed flies with wild-type or nearly wild-type eye facet patterns, were saved for further testing. Of these 2 were sterile, 5 did not breed true, 1 was probably non-disjunctant for the maternal X chromosomes (there also appeared to be non-disjuction of the w^/Y chromosomes in the treated male parents) and was discarded, and 2 bred true and were saved. Stocks established from these 2 putative revertant females were provisionally named Re 7 0 k 2 7 and Re70k30> respectively. Sub-sequent tests indicated that the partial eye revertant Re70^27 was a sex-linked, recessive lethal, dominant enhancer of wing nicking (Appendix 4), so this mutation was re-named E-N 7^ 2 7. The full eye revertant R e ^ ^ O is phenotypically a notch mutation, and I have so far been unable to separate it from the N5"1"1 site (Ap-pendix 4). Re7°k30 has therefore been re-named N7Qk30. Note that where N7^^30 j_s referred to elsewhere in this report, it is taken to include the mutant site. 235. Procedure No. 3 EMS I dl49,y Hw m^/wf. N ^ rb $ x w^ NS11 rb/B^ w^-Y cf* 20 .5°C dl49 ,y Hw m£/wf: N g l 1 rb £ wf. N ^ rb/wf. NS11 rb f Score eyes of red-eyed females for facet pattern. Wild-type eyes indicate NgU.»N+, N^il-'Nj o r extra locus modifier. Females die unless NSll mutation has been induced (score for appearance of white-eyed females). Re-test putative mutants further by mating to dl49,y Hw m^ males. 236 APPENDIX 1 Description and mapping of fanoE, E-N?0k27, and N?0k30 fanoE The new mutation fanoE was so named, on the basis of its phenotype and, as described below, the lack of recombination between fano and fanoE. fanoE has thickenings at the ends of the wing veins, and occasional nicks in the wingtips. Expression of both phenotypes is mild compared to fano, and heterozygotes of fanoE/fano are intermediate in appearance. fanoE/spl, fanoE/fa, and fanoE/faS have wild-type wings and eyes. N8/fanoE and N1*0/ fanoE are lethal, and N s l l/fa n o E is semilethal at 20.5°C (148 male and 8 female progeny were recovered from the cross fanoE/ y_ fanoE ^ x wf N g l 1 rb/B^ w^«Y a*) and lethal at 29°C. In order to map fanoE with respect to fa110, two series were run of the cross fanoE cv/wf fano spl rb £ x wf N^f rb/B^ w^-Y &r . Series 1 consisted of 24 cultures, each with 25 pairs of parents per 1/2-pint bottle, transferred to fresh bottles for a total of 6 broods, each of which lasted 3 days except that in 8 cultures, one brood lasted 7-8 days, and in 14 cultures there were 1 or 2 5-6 day broods. Series 2 consisted of 9 cultures, each with 5 pairs of parents per 1/4-pint bottle, transferred every 3-4 days for a total of 5 broods. Male and female progeny of these cross-es were counted. The results, tabulated below, show that no fano-fanoE crossovers (N^V+ females) were recovered, although 22 237. N^O/fa1*0 spl and 6 N4°/fanoE breakthroughs survived and 41,830 males were counted. This indicates that fa n o E is very closely linked to fano in the Notch locus. TOTAL FEMALES SERIES MALES b£ N/fano spl N/fanoE N/+ 1 38,119 35 20 5 0 2 3,711 8 2 1 0 TOTAL 41,830 43 22 6 0 II. E-N 7° k 2 7 E_N70k27 (abbreviated E-N k 2 7) maps to the left of w^ (see below), is a recessive lethal, and in cis or trans combination with NS-1-1 causes a marked reduction in the 20-22°C rough eye phenotype of N g l 1 and an increased frequency of wing nicking. With practice, E-N k 2 7 NSH/+ + can readily be distinguished from Ng13-/+ and from wild-type, at 20-22°C. From the cross E-N k 2 7 w a NSll/dl49 £ x OR <f , 7% (10/146) of E-Nk27 wa Ngll/+ + + females raised at 20.5°C, and 27% (84/316) raised at 22°C, had nicked wings. E-N k 2 7/N C o females also are more strongly nicked than N /+. Lethality of E-N k 2 7 occurs during the pupa stage, and is preceded by the formation of prominent melanotic tumors, which become visible during the third instar. Both the lethality and tumor formation are absent in males carrying a duplication of the dor+ region, but E-N k 2 7 and dor may not be allelic, since E-Nk27/ dor has wild-type eyes and E-Nk27/dor1 survive (see below). 238. E-Nk2? was mapped to the left of wf^  by the cross E-Nk2? w a Ngll rb/+ + + + ^ x w^ rb_ cf1 . Eggs were collected for 3 days In 10 half-pint bottles (5 pairs of parents per bottle), and progeny were raised at 22°C. The recombination frequencies, calculated from the female data tabulated below, are: 1.60* (E-Nk2?-wa), 1.51* (wa-NS1:i), and 4.17* (NSi:L-rb) . GENOTYPE NO. OP PROGENY FEMALES MALES Non-crossovers E-N wa N®11 rb Single cross-overs E-N-wa + + + + E-N + + + + w a NSll rb 1039 1067 0 1033 (not distinguishable from non-crossovers) 18 0 Single cross-overs w a-NgH Single cross-overs NgU-rb E-N wf; + + + + N g U rb E-N w a N g U + + + + rb 19 0 15 0 45 0 49 48 TOTALS 2252 1081 The following results of pair-matings in vials (M5/E-N?0k iva £ x Duplication see below), indicate that E-N?0k is situated in the dor region. Note that p-DTS is a dor-*- allele (PRATT 1970). 239. X/Dg/Y MALE PARENTS y59b z/pp60dl9(l) jr dor/T(l;Y)2E p-DTS rb/Dp67g24(l) PROGENY LARVAL MELA-NOTIC M5/X % X/E-N $ M5/Y cr* E-N/Dp-Y cf* TUMORS D f ( 1 ) s c 8wa/Dp59k9(4) 45 14 32 11 49 13 43 18 12 23 12 26 17 present present absent absent w a NS 1 1 rb/Bs w+« Y 39 49 28 present III. N?0k30 N70k30/+ females have a wild-type eye facet arrangement at 22° C, and enhanced wing nicking compared to Ngll/+. Lethality of ^70kl8/fano j[_s variable, depending on the genetic background (see below). Those that die, do so in the late pupal stage (20-22°C). This is unlike N g l l/fa n o, some of which survive to adult-hood, the rest apparently dying before pupation (Appendix 1). Controlled experiments have not been done, so it cannot be ascer-tained whether the difference between Ng"^/fano and N70k30/fano is due to genetic background differences, or to the Ngll^N70k30 mutational change. Eighty-seven percent (134/154) of the N 7 Q k 3°/+ females raised at 22°C (wf; N 7 0 k3° rb/dl49,y H w ^ ? x OR cf ), had one or both wingtips nicked. Viability of N 7 Q k 3° with fano was investigated in the fol-lowing crosses and results. 240. Cross 1. wf n70*30 rb/dl49,y Hw m£ ? x wf fano cf Progeny: 151 dl49/fan0 $ , 23 N70k30/fano £ , 108 dl49/Y o^ Cross 2. wf fano/wa fa110 ? x wf N7°k30/Bs w^-Y <f Progeny: 233 fano/Bs w^-Y cf, 1 N70k30/fano £ . Surviving N/fano females have deeply serrated wings and very thick veins. The lone female survivor of cross (2) had fused tarsal segments (other N/fan0 females not examined). The results of the mapping cross wf n 7 ° ^ 3 0 rb/+ + + $ x wf rb tf 3 reported below, indicate that the mutation responsible for conversion of N6"1""1" to N^^3^., is situated within the Notch locus, at or very close to the N g l 1 site. Eggs were collected in 3 day broods (6 broods) in 10 half-pint bottles (5 pairs per bottle), progeny were raised at 22°C, and females were scored for eye colour and facet pattern. Among 27,671 female progeny counted, wf-N and N-rb recombination was standard (1.42$ and 4.95$, re-spectively), but no confirmed crossovers between ^70^30 ancj jjgll were recovered. One white-eyed (wf rb) female ivas recovered with eyes similar to those of but when backcrossed to w a rb males, this exception only produced 3 female progeny, none of which were 427 APPENDIX 1 Tests of exceptional female progeny from A x ^ mapping crosses (see Table 13) A. s£l rb $ $ The results of individual matings of 11 of the spl rb females, all of which had notched wings, to w^ spl rb males, are reported below. Of the other females, two were sterile and one was not tested because she was not virgin. PROGENY * ¥ + No. spl rb wa spl rb wa N spl rb N spl rb spl rb wa spl 1 13 0 0 0 8 1 2 10 1 9 0 13 0 3 11 0 10 0 17 0 4 27 0 19 1 15 0 5 14 1 17 0 14 0 6 20 0 12 0 12 0 7 29 2 20 0 29 1 8 38 0 32 2 34 1 9 23 0 25 0 30 0 10 41 0 32 0 41 0 11 36 2 32 1 31 0 These results indicate that the spl rb exceptions were all true crossovers between fano and spl, rather than resulting from 242. phenocopy-like events. The absence of N females in the progeny of female No. 1 probably resulted from selective death of this class due to late scoring of this vial. B. w^ rb^ £ $ The results of crosses of the rb* female exceptions (note that only No. 5 was definitely virgin when discovered), to w a spl rb males, are reported below. 1 2 PARENTAL No. Parental wings 3 4 5 PROGENY Phenotypes" Sex notched notched abruptex notched notched g d» I ? o* I ? (f I a* I $ o* I Eye Eye colour facets Wings + + Ax, + 24 0 10 5 0 5 3 0 3 6 0 6 29 0 3 w a + Ax, + 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 rb + Ax, + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 „a w rb spl + 3 0 0 2 0 1 1 0 2 0 1 8 3 0 6 w a spl + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 rb spl + 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 wa rb spl N 5 1 3 3 1 4 0 1 2 3 0 2 6 2 2 w a spl N 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 rb spl N 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 + + N/Ax 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 The "I" progeny sex class comprised intersexes. These in-dividuals possessed sexcombs and were either male-like or female-like with respect to their genitalia and body markings. All flies of this class were sterile, and the flies themselves were 243. weak and usually died earlier than the true males and females. The presence of intersexes among their progeny suggests that the w+ rb+ exceptional females were triploids, a conclusion which is supported by the results of further tests, made on the F-j_ female progeny. The tests of the different classes of F^ females will be considered separately. The results of progeny tests of F-^  virgin females with wild-type eye facets and Ax or Ax* wing veins (crossed to wfj; spl rb cf^ cf7), are presented in the following table. The data indicate that F-j_ females No. 1-1, 1-5, and 2-5 were triploids. Females 1-1 and 1-5 carried AxE^ and either spl rb_ or wf; fano spl rb, and furthermore the paucity of male progeny suggests (although not conclusively) that they also carried . The split notched, and split non-notched daughters of female no. 2-5, and the fact that 2-5 herself was apricot, and neither notched nor Ax-like, suggests that this female had the genotype wf; rb/wa AxE1/ wf. S£l rb. Females number 1-3, 2-3, 3-1, 3-2, 4-2, 4-3, 4-5, 4-7-5-1 and 5-2 were diploids with the genotype wf; fano spl rb/AxE1, and females number 2-1 and 2-7 were diploids with the genotype w a spl rb/AxE1. The results of crosses of F]_ virgin split-eyed normal-winged females to wf; spl rb males are presented below,_(top of p. 247). These data indicate that F^ females 1-2, 1-7, 2-2 and 2-6 were diploids with the genotype wf; fano spl rb/wa spl rb. The small number of "white split notched" males from female 1-7 likely resulted from overcrowding and late scoring of the vial. Progeny tests of F-, $ £ with + eye facets and Ax or Ax+ wing veins. P 2 PHENOTYPE px 2 No. FX % Pheno. c2 Sex White split White split notched Apricot split Apricot split notched Ruby split Ruby split notched Ax Ax+ Apricot Ax;Ax+ Ruby Ax;Ax+ 1-1 ($ ( 9 0 0 0 0 0 8 1 0 w+ rb+ Ax+ N+ (cf I 1 0 0 0 0 0 0 0 0 \ (I 2 0 0 0 0 0 2 0 0 1-3 w+ rb+ ($ 17 0 2 0 0 0 17 2 1 Ax+ N+ ( (cf 0 21 0 0 0 0 0 0 0 1-5 w + Ax+ (9 6 0 0 0 0 0 6 0 0 N+ rb+ ( (cf 0 0 0 0 0 0 0 0 0 2-1 w+ Ax (9 42 0 0 0 0 0 39 0 0 rb + ( (<? 34 0 2 0 0 0 0 0 0 2-3 w+ Ax (9 31 0 2 0 1 0 53 2 0 rb+ ( («J» 2 26 0 3 1 2 0 0 0 2-5 w a N+ (? 7 1 0 0 1 0 0 6 0 rb+ Ax+( (cf / 0 0 0 0 0 0 0 0 0 (I 1 0 0 0 0 0 2 0 0 rv> . e r 4=" P2 PHENOTYPE ? ! ? No. Pi $ Pheno. P2 Sex White split White split notched Apricot split Apricot split notched Ruby split Ruby split notched Ax Ax+ Apricot Ax;Ax+ Ruby Ax;Ax+ 2-7 w+ Ax (9 25 0 1 0 0 0 20 0 0 rb+ ( (d> 28 0 0 0 0 0 0 0 0 3-1 w+ Ax (9 23 0 1 0 0 0 28 2 0 rb+ ( (<* 1 25 0 0 0 1 0 0 0 3-2 w+ Ax (9 23 0 2 0 0 0 33 1 0 rb+ ( (<? 2 26 0 1 0 0 0 0 0 4-2 w + N+ (9 24 0 3 0 2 0 42 0 0 Ax+ rb C cs» 1 21 0 1 0 1 0 0 0 4-3 Ax (9 ( 41 0 1 0 0 0 49 0 1 (c? 3 26 0 2 0 0 0 0 0 4-5 Ax (? ( 41 0 0 0 1 0 33 1 0 (c? 3 18 0 0 0 1 0 1 0 4-7 Ax (9 r 17 0 2 0 1 0 29 1 1 \ 0 18 0 1 0 0 0 0 0 IV) VJ1 F 2 PHENOTYPE Pi ? No. Px ? Pheno. f2 Sex White split White split notched Apricot split Apricot split notched Ruby split Ruby split notched Ax Ax+ Apricot Ax;Ax+ Ruby Ax;Ax+ 5-1 Ax (? ( (of 12 0 2 0 1 0 15 3 0 2 20 0 1 0 0 0 0 0 5-2 Ax (? ( (cf 16 0 1 0 0 0 23 1 1 1 19 0 0 0 0 0 0 0 ro -t cr* F 2 PROGENY 247. Fx No . ? £ d* o* white white white white split split split split notched notched 1-2 49 0 16 18 1-7 56 0 23 3 2-2 17 0 14 12 2-6 54 0 27 10 Results of progeny tests of white split notched females to w a spl rb males are presented below. f 2 PROGENY F-]_ ? No. white split white split notched ? o» I ? &> I 1-4 1 0 0 0 0 0 1-6 2 0 0 0 3 0 2-4 65 0 0 0 65 0 2-8 54 2 0 1 57 0 4-1 7 4 1 5 1 1 4-6 20 6 5 4 1 0 These data indicate that F-^  females 2-4, 2-8 and 1-6 were diploids with genotype wf; fano spl rb/wa fano spl rb. This indi-cates that parental females 1 and 2 were non-virgin, both having been inseminated by their wf; fano spl rb brothers. Females 4-1, 248. and 4-6, on the other hand, were triploids, which, judging from their origin and the phenotypes of their progeny, must have had the genotype rb/wf; fano spl rb/wf. sgl rb . It has already been inferred that the presence of intersexes + + among their progeny suggests that the original w_ rb female exceptions were triploids. Careful consideration of the pheno-types of the original females, and those of the succeeding two test generations, confirms this hypothesis for at least four of the exceptions. Notwithstanding the known non-virginity of females 1 and 2, the data indicate that females 1, 2, 4, and 5 all contained the chromosomes fano spl rb, wa N^Q rb, and w+ Axgl rb+, and that female 3 had at least wf^  fano spl rb and w + A x e 1 rb+. APPENDIX 10 F P Tests of exceptional progeny from Ax1^ mapping crosses 249 Series 1 Male progeny whose phenotypes suggested they were recombin-ants for fano, spl, or AxE2 were mated to XX/Y females to establish stocks for further testing. The phenotypes of the original males are described below (see footnote to Table 14 for the crosses used). PHENOTYPE Recombinant No. (Culture-Brood) 1-3 2-3 5-7 7-4 8 - 2 8-4 8 - 8 EYES WINGS apricot apricot ruby, split + ruby, split ruby apricot notched notched + notched + + notched Recombinant 8-8 was sterile. The fertile recombinants all bred true, the progeny male phenotypes being the same as those of the respective parental males. Because certain of the bristle phenotypes of the apricot notched stocks resembled those of AxE2, several individuals from each of the fertile notched stocks, and from the ruby split stock 8-2 were mated to AxE2/AxE2 virgin females, along with a control cross of AxE2/AxE2 ff x fan0 spl c^ 1. 250. The presence or absence of wing vein gaps in the progeny of these crosses was scored, and the results are tabulated below. The figures presented are the pooled data from progeny of up to 24 fertile individual matings of males from a given stock to AxE2/ A x e 2 females. PROGENY Recombinant No. ? ? No gaps 1 or more No gaps 1 or more gaps gaps 1-3 73 977 0 1079 2-3 65 1041 0 1023 7-4 23 631 0 607 8-2 554 231 0 66 3 CONTROL 561 184 0 755 These data suggest that 1-3, 2-3, and 7-4 all contained the E2 mutant Ax , whereas 8-2 did not, and that therefore their geno-types were: w^ fano AxE2, fano AxE2, fano AxE2, and s£l rb, respectively, although the nature of the control cross does not p? eliminate the possibility that'spl suppresses the Ax wing vein phenotype. It will later be shown that this is not the case. These data indicated that AxE2 was to the right of fa110, and from the single rb recombinant, probably to the right of spl. Unfor-tunately, these stocks were all accidentally lost before further tests could be made, but the data from the next series confirm FP the inferred map position of Ax . 251 Series 2 Male progeny whose phenotypes indicated that they were F 2 recombinant for fa110, spl, or Ax , were mated to XX/Y females to establish a stock of the recombinant chromosome. The pheno-types of these original males are recorded below. PHENOTYPE Recombinant No. EYES WINGS 1-3 apricot notched 1-4 apricot notched 2-4 ruby, split + 3-4 ruby, split + 4-3 apricot notched 5-3 ruby, split + 9-4 ruby, split + 10-4 apricot, extreme split notched 12-1 apricot, extreme split notched 12-5 ruby, split + 13-3 white notched 15-4 ruby, split + 17-3 ruby, split + The "extreme split'1 recombinants 10-4 and 12-1 had very narrow eyes with only a few facets present in an amorphous, glazed matrix of eye tissue. In each case except for male 1-4, which was sterile, the phenotypes of the respective male progeny (when mated to XX/Y females) were identical to those of the original recombinants. In addition to the male recombinants, the following female recombinants from experiment 2 were recovered. Recombinant No. 252. PHENOTYPE EYES WINGS 1-2 apricot notched 5-5 ruby, split + 7-2 ruby, split + 7-3 apricot notched 14-3 ruby, split + 18-2 apricot notched The phenotypes of the male progeny of the recombinant females are tabulated beloxtf. PHENOTYPES OF PROGENY Recombinant No. white apricot white apricot white ruby split split split split notched notched notched notched 1-2 21 1 3 17 0 0 5-5 21 0 0 0 0 ,28 7-2 23 0 0 0 0 28 7-3 14 1 0 25 0 0 14-3 23 0 0 0 1 13 18-2 11 0 0 22 0 0 As was observed with each original recombinant (both sexes), the Abruptex wing vein gap phenotype was not present in any of their progeny. However, the bristle phenotypes of the "apricot notched", and "apricot split notched" males were similar to those •p p of Ax /Y males, while the "white notched", and "split ruby" males were more like wild-type, although even these had bristle defects. Consequently, in order to determine whether AxE2 was present in any of these recombinants, males from most of the K> 2 F? recombinant cultures were mated to virgin Ax /Ax females, and 253. the female progeny were inspected for wing vein gaps and for the number of anterior orbital bristles present. The results of these tests are reported below. $ PROGENY PHENOTYPES No. of flies with No. of flies with given PARENTAL REC. No. of anterior orbitals <? PHENOTYPE # 0 gaps 1 or more gaps 0 1 2 apricot notched 1-2 1 51 52 0 0 apricot notched 1-3 0 40 40 0 0 apricot notched 4-3 0 53 53 0 0 apricot notched 7-3 0 34 34 0 0 apricot notched 18-2 1 40 41 0 0 white notched 13-3 45 2 15 21 11 apricot 10-4 split notched 3 74 77 0 0 apricot 12-1 split notched 22 154 176 0 0 ruby split 3-4 33 7 35 4 1 ruby split 5-3 18 24 26 15 1 ruby split 5-5 27 17 27 14 3 ruby split 7-2 15 36 23 19 9 ruby split 9-4 31 13 37 6 1 ruby split 12-4 28 16 26 16 2 ruby split 14-3 20 20 32 6 2 ruby split 15-4 29 12 25 12 4 ruby split 17-3 25 39 46 14 4 254. The high frequency of occurrence of wing vein gaps, and the absence of orbitals in the apricot notched, and apricot split F? notched heterozygotes with Ax , indicate that both recombinant F ? classes contain the Ax mutant site, and have the genotypes T? 0 wa fano AxE2 and wf_ fano spl Ax , respectively. Conversely, the lower frequencies of wing vein gaps, and presence of some flies possessing anterior orbitals, in the heterozygotes of white notched, and ruby split, with AxE2, indicates that AxE2 is not present in these recombinants, which must have the genotypes wa fano rb and spl rb, respectively. These data place A x E 2 to the right of spl. This conclusion was subsequently confirmed F? when the recombinant genotype spl A x ^ was recovered as a single rp o male in the progeny of the cross wf^  fano spl Ax /+ + + + $ x OR <? , the female parents of which were the progeny of a cross F 2 between OR females and recombinant stock 12-1. All spl Ax individuals established from this recombinant, have the charac-F P teristic Ax wing phenotype, have a rough eye phenotype inter-TT O mediate between that of fano spl Ax and spl, and have markedly E2 reduced bristle frequencies compared to spl or Ax alone. APPENDIX 10 255 Interactions of fano, spl, and Ax; E2 The phenotypes of various combinations of fano, spl, and AxE2 indicate that: 1) coupling of spl and AxE2 results in extreme expression of the spl eye phenotype, extreme bristle loss, and near-normal expression of the AxE2 wing vein gap phenotype; 2) coupling of fano to spl AxE2 results in further enhancement of the spl eye phenotype, suppression of bristle loss compared to spl AxE2, and complete suppression of wing vein gapping; 3) coup-ling of fano to AxE2 does not cause a mutant eye phenotype, possibly slight enhancement of bristle loss compared to AxE2, and complete suppression of wing vein gapping. The eye and wing phenotypes~of various combinations of alleles at 20°C-22°C are summarized below in tabular form. The rough eye and wing vein gap phenotypes (where expressed) are enhanced at 29°C. Note that the spl eye phenotype is normally completely recessive in spl/+. GENOTYPE fano spl A x E 2 / Y <? spl A x e 2 / Y <? fano A x E 2 / Y <? EYES very narrow, glazed, few or no discrete ommatidia intermediate between fano spl A x e 2 and spl in size; discrete ommatidia but strongly spl wild type WINGS nicked, thick veins, no vein gaps vein gaps like A x e 2 nicked, thick veins, no vein gaps fano spl A x e 2 / + + + % about same size and rough- wild type ness as spl/spl spl AxE2/+ + 2 spl AxE2/fano $ fano spl/AxE2 £ occasional roughness, overlapping wild type; slightly smaller than +. like sjol AxE2/+ + wild type 256. occasional vein gaps Bristle counts of various combinations of fano, spl, Ax occasional vein gaps E 2 and +, are tabulated on the following pages. The following crosses were used to generate the genotypes listed in the table. L I N E 1 2 3 6 , 7 8 , 9 10,11 12,13 C R O S S X X / Y $ x fano spl A x e 2 / Y <3 X X / Y ? x spl A x e 2 / Y cf X X / Y ? x w ^ fano A x E 2 / Y 8 OR ? x fano spl A x E 2 / Y <7 OR 9 x spl A x E 2 / Y cf AxE2/AxE2 ? x fano spl rb/Y d* OR $ x fano spl rb/Y d» OR x wa fa n o A x E 2 / Y & O R B I T A L S O C E L L A R S P O S T V E R T I C A L S L I N E G E N O T Y P E T E M P . 0 1 2 3 4 5 6 0 1 2 0 1 2 1 fano spl A x E 2 / Y 20 .5°C 0 15 43 1 0 0 0 48 10 1 10 25 24 2 spl A x E 2 / Y 20 .5°C 39 6 0 0 0 0 0 45 0 0 43 2 0 3 fano A x E 2 / Y 20.5°C 0 0 2 11 39 0 0 3 8 41 0 0 52 4 ) fano spl A x e 2 / + 20 .5°C 0 0 29 57 l4l 36 10 7 78 h -j co 1 10 262 ) + + h -j co 5 ) 29°C 0 1 7 30 58 1 0 97 0 0 10 44 43 6 ) 20 .5°C 0 1 50 77 38 9 0 73 68 34 5 42 128 ) spl A x e 2 / + + 7 ) 29°C 3 5 56 15 7 0 0 86 0 0 52 29 5 8 ) 20 .5°C 0 0 0 0 74 12 2 0 0 88 0 0 88 ) fano spl +/+ + A x e 2 9 ) 29°C 0 0 0 3 55 4 0 43 16 3 0 11 51 10 ) fano spl/+ + 20 .5°C 0 0 0 0 0 3 118 0 3 118 0 0 121 ) 11 ) 29°C 0 0 0 0 19 44 40 63 34 6 0 9 94 12 ) fano A x e 2 / + + 20.5°C 0 0 0 0 0 14 104 0 0 118 0 0 118 ) 13 ) 29°C 0 0 0 0 6 34 78 0 5 113 0 0 118 ro ui LINE 0 DORSOCENTRALS l — r SCUTELLARS TOTAL (lines 1,2 not WING VEIN GAPS 1 6 16 21 13 3 0 8 1 19 2 I F 3 15 4 1 59 0 0 0 2 28 13 3 0 0 27 14 4 0 0 (gaps in L5 (100*); many in L4 ANTERIOR POSTERIOR 3 0 1 2 0 1 2 52 0 0 0 4 0 0 0 5 268 9 88 176 0 2 271 272 0 0 0 5 0 3 19 33 41 89 7 0 2 9 85 97 0 0 0 6 0 0 0 2 173 19 48 108 0 1 174 156 (+18 with 1 or more gaps) 7 0 6 32 40 8 77 9 0 20 39 27 28 (+57 with 1 or more gaps) 8 0 0 0 0 88 0 0 88 0 0 88 86 1 1 0 9 0 0 2 20 40 6 28 28 0 0 62 32 14 16 0 10 0 0 0 0 121 0 0 121 0 0 121 119 0 0 0 11 0 0 0 2 101 1 18 84 0 0 103 102 0 0 0 12 0 0 0 1 117 0 1 117 0 0 118 115 0 0 0 13 0 0 0 7 111 0 0 118 0 1 117 118 0 0 0 ro ui CO 259 APPENDIX 10 Counts of bristles and wing vein gaps in AxE2 and OR flies Data in lines 1-7 were taken from progeny of stock cultures ( A x E 2 / A x E 2 £ x A x e 2 / Y d* ), raised in uncrowded conditions. Lines o F2 8,9 were obtained from progeny of OR + x Ax /Y c? . Lines 10-12 were obtained from progeny of stock OR cultures, reared in un-crowded conditions (females raised at 29°C were not examined in detail, but there were no obvious differences compared to males). Note that males and females in lines 2,3 are sibs; males and females in lines 4-7 are sibs; females in lines 8,9 are sibs, males and females in lines 10,11 are sibs. The data presented in this and the following appendices are the number of flies with a given number of bristles of a particu-lar type (or wing vein gaps, or nicked wings), followed by x ± one-sided 95$ confidence intervals for the mean. See Methods and Materials for explanation of x and confidence intervals. ORBITALS LINE GENOTYPE TEMP. 0 2 3 4 5 6 7 8 9 12 AX E 2/Y AX E 2/Y A E2 E2 Ax /Ax AX E 2/Y A X E 2 / A X E 2 AX E 2/Y A x E 2 / A X E 2 A X E 2 / H-A X E 2 / + 10 OR 11 OR OR d* 20.5°C cf 9 ? 9 20 .5°C 20.5°C cf 22°C j 22°C 0* 29°C 29°C 20 .5°C 29°C <3* 22°C £ 22°C cP 29°C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 3 0 0 0 0 1 2 46 67 2 163 1 165 25 138 30 86 0 0 0 0 1 4 0 0 1 0 0 2 0 0 0 0 0 0 0 0 5 144 27 75 4.00 + .06 3.97 ± .04 3.99 ± .02 4.01 ± .02 3.82 ± .05 3.70 ± .08 5.95 ± .04 5.67 ± .09 0 109 6 . 0 0 0 139 6.00 4 138 5.97 ± .03 ro CT\ o 0 OCELLARS POSTVERTICALS VERTICALS LINE 0 x x 2 3 4 5 6 7 8 9 10 11 12 0 1 1 73 1.99 ± .03 1 47 1.98 + .04 4 64 1.91 ± .07 0 2 163 1.99 ± .02 0 12 156 1.93 ± .04 0 2 72 1.97 ± .04 0 0 44 2.00 0 5 64 1.93 ± .06 0 2 163 1.99 ± .02 0 0 1 167 1.99 ± .01 0 52 74 39 0.92 ± .10 77 50 38 0.76 ± .10 0 87 29 3 0.29 ± .08 90 25 4 0.28 ± .08 0 0 0 150 2.00 0 0 12 93 1.89 ± .06 0 0 3 106 1.97 ± .03 0 0 3 136 1.98 ± .02 0 0 1 141 1.99 ± .01 0 0 150 2.00 0 1 105 1.99 ± .02 0 0 109 2.00 0 2 137 1.99 ± .02 0 0 142 2.00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 165 4.00 1 167 3.99 ± .01 6 159 3.96 + .03 4 115 3.97 ± .03 0 150 4.00 1 105 3.99 ± .02 0 0 109 4.00 139 4.00 0 142 4.00 ru o\ 0 0 0 DORSOCENTRALS ANTERIOR SCUTELLARS POSTERIOR SCUTELLARS LINE 0 x x 2 3 4 5 6 7 8 9 10 11 12 0 0 0 0 0 0 0 0 0 0 1 73 3.99 ± .03 0 0 0 47 4.00 0 0 1 68 3.99 ± .03 0 0 2 163 3.98 ± .02 0 0 0 168 4.00 0 74 2 .00 0 74 2.00 0 0 0 0 0 0 0 0 0 0 0 0 165 2.00 0 168 2.00 0 3 12 43 107 3 . 5 4 + .09 1 0 6 2 8 7 1 . 4 8 ± . 0 8 0 0 5 32 82 3.65 ± .09 6 23 90 1.71 ± .09 0 150 4.00 2 10 94 3.87 i .07 0 109 4.00 0 139 4.00 0 142 4.00 0 0 150 2.00 0 0 106 2.00 0 0 109 2.00 0 0 139 2.00 0 0 142 2.00 0 0 165 2.00 0 0 168 2.00 0 10 149 1.94 + .03 0 7 112 1.94 ± .04 0 0 150 2.00 0 0 106 2.00 0 0 109 2.00 0 0 139 2.00 0 1 141 1.99 ± .01 ro CT\ ro WING VEIN GAPS LINE 0 x 2 3 4 5 6 7 8 9 0 0 133 18 12 0 0 0 0 130 23 13 0 0 0 0 9 16 40 36 42 0 0 15 15 73 6 1 6 3 38 4 9 0 0 0 0 17 21 62 3 2 0 0 0 0 2.26 ± .08 0 0 2.30 + .08 6 7 4.85 ± .19 1 0 3.69 ± .14 0 0 0.91 ± .12 0 0 1.54 + .15 10 109 0 0 0 0 0 0 0 0 0.00 11 139 0 0 0 0 0 0 0 0 0.00 12 142 0 0 0 0 0 0 0 0 0.00 ru <T\ U) 264 APPENDIX 10 Counts of bristles and wing vein gaps in Ax^-*-72 flies Data in lines 1-6 were taken from progeny of stock cultures x Ax16172^y J1 ) a raised in uncrowded condi-tions . Data in lines 7,8 were obtained from the cross: OR $ x Axl6l72/y d . Note that males and females in lines 1,2 are sibs; males and females in lines 3-6 are sibs; females in lines 7,8 are sibs. See Methods and Materials for explanation of x and confidence intervals, and see Appendix 8. ORBITALS LINE GENOTYPE TEMP. 0 1 1 Axl6l72/Y 3 4 5 6 7 8 Ax l 6 l 7 2/Ax l 6 1 7 2 ? 22°C 16172 Ax /Y a* 29°C AX 1 6 1 7 2/AX 1 6 1 7 2 ? 29°C A x l 6 l 7 2 / + Axl6l72/+ ^ 22°C £ 29°C 5 6 cf 20.5°C 16 33 50 10 0 0 Axl6l72/Axl6l72 20.5°C 26 35 22 5 0 0 Axl6l72/Y ^ 2 2o c 23 61 77 21 2 0 0 0 0 54 59 34 5 1 0 0 1 21 33 42 0 0 0 2 32 22 7 0 0 0 0 9 95 15 3 0 0 0 0 73 13 1 x 1.50 + .14 1.07 ± .16 1.55 ± .11 0.95 ± .12 3.20 ± .10 2.54 + .10 4.10 ± .09 4.17 ± .08 ro CTi ui 2 0 OCELLARS POSTVERTTCALS VERTICALS LINE 0 1 2 x 0 1 2 x 0 1 2 4 ' x 1 109 0 0 0.00 109 0 0 0.00 0 0 1 20 88 3.80 + .07 2 88 0 0 0.00 88 0 0 0.00 0 0 0 9 79 3.90 + .06 3 183 1 0 0.01 ± .01 184 0 0 0 . 0 0 0 0 2 21 161 3.86 + .05 4 153 0 0 0 . 0 0 153 0 0 0 . 0 0 0 0 1 12 140 3.91 + .05 5 92 5 0 0.052 ±.026 97 0 0 0 . 0 0 0 0 1 6 90 3.92 + .04 6 62 1 0 0.016 ±.017 63 0 0 0 . 0 0 0 0 0 4 59 3.94 + .03 7 5 40 77 1.59 ± .09 43 54 25 0.85 ± .11 0 0 0 0 122 4.00 8 35 37 15 0.77 ± .14 44 33 10 0.61 ± .13 0 0 0 0 87 4.00 rv> o> cr> DORSOCENTRALS ANTERIOR SCUTELLARS POSTERIOR SCUTELLARS LINE 0 1 2 3 4 X 0 1 2 X 0 1 2 X 1 0 5 47 38 19 2.65 + .14 44 48 12 0.75 + .12 1 11 97 1.88 + .06 2 2 9 44 25 8 2.32 + .16 22 38 28 1.07 + .14 0 10 78 1.89 ± .06 3 0 2 45 83 54 3.03 + .10 79 81 24 0.70 + .09 4 29 151 1.80 ± .06 1 9 51 53 39 2.78 + .13 35 65 52 1.11 + .10 2 10 l4l 1.91 ± .05 5 15 31 39 9 3 1.53 + .12 73 22 2 0.27 + .06 39 45 13 0.73 ± .08 6 25 30 7 1 0 0.79 + .09 34 27 2 0.49 + .07 19 27 17 0.97 ± .10 7 0 0 0 0 122 4.00 0 0 122 2.00 0 0 122 2.00 8 0 0 0 5 82 3.94 + .05 2 6 79 1.89 + .07 0 0 87 2.00 ro o> —j W I N G V E I N G A P S L I N E 0 1 2 3 4 5 6 7 8 x 1 0 0 0 0 0 2 18 18 64 7.41 ± .15 2 0 0 0 0 5 3 6 4 4 5.95 ± .54 3 0 0 0 0 11 23 36 32 63 6.68 ± .17 4 0 0 0 2 82 22 17 1 0 4.46 ± .12 5 0 0 0 0 2 4 26 34 27 6.86 ± .17 6 0 0 0 0 9 17 29 0 0 5.36 ± .18 7 0 0 91 24 6 0 0 0 0 2.30 ± .09 8 0 0 12 30 45 0 0 0 0 3.38 ± .13 ro cr\ oo 269 APPENDIX 10 QR? Counts of bristles, wing vein gaps and wing nicking in Ax^ flies Data were obtained from progeny of the following crosses. LINE CROSS 1,2 l(FM6)/Ax9B2 £ x Ax9B2/Y $ 3-6 M5/Ax^B2 $ x Ax9B2/y ( s e e Table 30) 7 O R $ x A x 9 B 2 / Y & Note that Ax^B2/Y males were obtained from stock cultures (XX,^ w f / Y $ x A x ^ B 2 / Y ). Female parents (lines 1,2) were obtained from the cross l(FM6)/AxE1 ? (AxE1 stock) x stock Ax9B2/Y <7 . Female parents (lines 3-6) were obtained from the cross M5/M5 ^ x stock A x 9 b 2 / Y <7 . Males and females in lines 1,2 are sibs; males and females in lines 3-6 are sibs. See Methods and Mater-ials for explanation of x and confidence intervals, and see Appendix 8. ORBITALS LINE GENOTYPE TEMP. 0 3 ^ 5 6 9B2 1 Ax /Y cf 20.5°C 0 0 0 0 2 87 5-98 ± .03 2 A X 9 B 2/AX 9 B 2 20.5°C 0 0 3 75 5.96 ± .04 3 AX9B2/Y cf 22°C 0 0 0 0 0 1 1 1 1 7 5 . 9 1 ± . 0 5 4 A X 9 B 2 / A X 9 B 2 9 22°C 0 0 0 0 0 3 93 5.97 ± . 0 3 5 AX9B2/Y cf 29°C 3 36 5.92 + .08 6 AX9B2/AX9B2 £ 29°C 0 0 0 0 0 1 22 5-96 ± .08 7 Ax9B2/+ 9 22°C 0 0 0 1 12 31 63 5.46 + .12 ro o O C E L L A R S P O S T V E R T I C A L S V E R T I C A L S L I N E 0 1 2 x 0 1 2 x 0 1 2 3 4 x 1 88 1 0 0.01 ± .02 89 0 0 0.00 0 0 0 1 88 3.99 ± .02 2 75 2 1 0.05 ± .06 70 3 5 0.17 ± .10 0 0 0 2 76 3.97 ± .03 3 128 0 0 0.00 128 0 0 0.00 0 0 0 9 119 3.93 ± .04 4 95 1 0 0.01 + .02 96 0 0 0.00 0 0 0 4 92 3.96 ± .04 5 36 3 0 0.08 ± .08 26 9 4 0.44 ± .19 0 0 0 1 38 3.97 ± .05 6 23 0 0 0.0 17 3 3 0.39 ± .27 0 0 0 0 23 4.0 7 95 12 0 0.11 ± .06 105 2 0 0.02 + .03 0 0 0 0 107 4.00 ro M D O R S O C E N T R A L S A N T E R I O R S C U T E L L A R S P O S T E R I O R S C U T E L L A R S L I N E 0 1 2 3 4 x 0 1 2 x 0 1 2 x 1 8 30 50 1 0 1.49 ± .13 0 13 76 1.85 ± .07 0 2 87 1.98 ± .03 2 2 2 72 2 0 1.95 ± .08 0 7 71 1.91 ± .06 1 3 74 1.94 ± .06 3 28 60 40 0 0 1.09 ± .11 7 40 81 1.58 ± .09 1 19 108 1.84 ± .06 13 33 50 0 0 1.39 ± .13 4 24 68 1.67 ± .10 0 2 94 1.98 ± .03 5 0 10 21 7 0 1.92 ± .19 5 14 19 1.37 ± .20 3 12 24 1.54 ± .18 6 0 2 19 2 0 2.00 ± .16 2 8 13 1.48 ± .25 0 7 16 1.70 ± .18 7 0 0 25 37 45 3.19 ± .13 0 0 107 2.00 0 0 107 2.00 ro -a ro L I N E 0 W I N G V E I N G A P S x W I N G N I C K I N G 3 5 6 16 26 62 20 3 0 0 0 0 1.75 ± .14 127 0 0 0.00 9 20 58 6 0 0 0 0 0 1.66 ± .13 93 0 0 0.00 0 2 4 7 11 8 3 1 0 3.89 ± .41 35 1 0 0.03 ± .05 0 0 3 0 1 4.44 ± .65 19 1 0 0.05 ± .09 102 5 0 0 0 0 0 0 0 0.05 ± .04 107 0 0 0.00 ru —5 0 274 APPENDIX 10 Counts of bristles, wing vein gaps, and wing nicking in F2 Ax /N heterozygotes The heterozygotes examined were generated by the following crosses . LINE CROSS 1,2 M5/N8 $ x A x E 2 / Y 3,4 M5/wa N 4 0 rb $ x AxE2/Y $ 5,6 M 5 / y w a N 1 0 3 $ x A x E 2 / Y c? 7,8 l(FM6)/wa N®11 rb £ x AxE2/Y £ 9,10 M5/wa N^ll rb $ x AxE2/Y <? 11,12 AxE2/AxE2 $ x wa N g l l rb/BS w+• 13,14 M5/wa N C o £ x A x E 2 / Y d* Note that the figures in Table 24 were calculated from F2 results of the crosses of M5/N females to Ax /Y males. See Methods and Materials for explanation of x and confidence inter-vals, and see Appendix 8. ORBITALS LINE GENOTYPE TEMP. 0 1 2 3 4 5 6 X 1 AXE2/N8 22°C 0 0 0 0 3 25 158 5.83 ± .05 2 29°C 0 0 0 4 24 23 11 4.66 + .18 3 A x E W ° 22° C 0 0 0 0 3 18 88 5.78 ± .08 4 29°C 0 0 0 1 15 3 9 4.71 ± .32 5 AX E 2/N 1 0 3 22°C 0 0 0 5 171 12 2 4.06 ± .05 6 29°C 0 0 0 3 18 16 5 4.55 ± .21 7 20.5°C 0 0 0 0 4 13 107 5.83 ± .07 8 29°C 0 0 0 0 1 8 70 5.87 ± .07 9 AxE2/Ngll 22° C 0 0 0 0 7 33 117 5.70 ± .08 10 29°C 0 0 0 0 0 5 36 5.88 ± .09 11 20.5° 0 0 0 0 1 23 138 5.85 ± .05 12 29°C 0 0 0 0 0 5 28 5.85 ± .11 13 A X E 2 / N C ° 22°C 0 0 0 0 3 10 103 5.86 ± .07 14 29°C 0 0 0 1 4 14 10 5.14 ± .26 O C E L L A R S P O S T V E R T I C A L S V E R T I C A L S L I N E 0 1 2 X 0 1 2 x 0 1 2 3 4 1 0 0 186 2.00 0 2 184 1.99 ± .02 0 0 0 0 186 4.00 2 0 1 63 1.98 ± .03 0 0 64 2.00 0 0 0 2 62 3.97 3 0 0 109 2.00 0 2 107 1.98 + .02 0 0 0 0 109 4.00 4 0 3 25 1.89 ± .11 0 0 28 2.00 0 0 0 1 27 3.96 5 0 0 190 2.00 0 0 190 2.00 0 0 0 0 190 4.00 6 0 1 41 1.98 ± .04 0 0 42 2.00 0 0 0 0 42 4.00 7 4 8 112 1.87 ± .07 0 0 124 2.00 - - - - - -8 0 2 77 1.97 ± .03 0 1 78 1.99 ± .02 - - - - - -9 0 5 152 1.97 ± .03 0 2 155 1.99 ± .02 0 0 0 0 157 4.00 10 0 0 41 2.00 0 0 41 2.00 0 0 0 0 41 4.00 11 3 14 145 1.88 ± .05 0 0 162 2.00 - - - - - -12 0 0 33 2.00 0 0 33 2.00 - - - - - -13 0 0 116 2.00 0 0 116 2.00 0 0 0 0 116 4.00 14 0 1 28 1.97 ± .06 0 0 29 2.00 0 0 0 0 29 4.00 / D O R S O C E N T R A L S A N T E R I O R S C U T E L L A R S P O S T E R I O R S C U T E L L A R S L I N E 0 1 2 3 4 X 0 1 2 X 0 1 2 X 1 0 0 0 0 186 4.00 0 1 185 1.99 + .01 0 1 185 1.99 ± .01 2 0 0 0 1 63 3.98 ±. .03 0 1 63 1.98 + .03 'o 0 64 2.00 3 0 0 0 0 109 4.00 0 1 108 1.99 ± .02 0 3 106 1.97 ± .03 4 0 0 0 0 28 4.00 0 0 28 2.00 0 0 28 2.00 5 0 0 0 0 190 4.00 0 0 190 2.00 0 0 190 2.00 6 0 0 0 2 40 3.95 ± .06 0 1 41 1.98 ± .04 0 0 42 2.00 7 0 0 0 0 124 4 . 0 0 0 3 121 1.98 ± .03 1 2 121 1.97 ± .04 8 0 0 0 22 57 3.72 + .09 0 3 76 1.96 + .04 1 4 74 1.92 ± .06 9 0 0 0 0 157 4 . 0 0 0 1 156 1.99 ± .01 0 1 156 1.99 ± .01 10 0 0 0 1 40 3.98 ± .05 0 1 40 1.98 ± .05 0 1 40 1.98 ± .05 11 0 0 0 2 160 3.99 ± .02 1 0 161 1.99 ± .02 0 1 161 1.99 ± .01 12 0 0 0 6 27 3.82 ± .12 0 0 33 2.00 0 0 33 2.00 13 0 0 0 0 116 4 . 0 0 0 1 115 1.99 ± .02 0 0 116 2.00 ro —4 14 0 0 0 0 29 4 . 0 0 0 2 27 1.93 ± .09 0 0 29 2.00 WING VEIN GAPS WING NICKING LINE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 125 36 75 22 0 9 26 23 93 23 1 18 9 6 1 0 6 23 3 8 1 2 22 12 4 1 96 6 8 x 1 2 x 86 5 0 2 0 0 0 0 6 0 0 2 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.38 + .10 0 0.58 ± .19 0 0 . 16 ± .09 0 0.13 ± .16 0 0 0 0 0 2.08 + .05 12 0 1.00 + .41 0 0 1.44 + .12 112 0 0.58 ± .30 0 0 0 . 0 8 ± . 0 5 0 0 0.19 ± .20 0 0 178 2.00 0 60 2.00 0 0 22 0 21 1 1 0 95 2.00 25 2.00 82 1.60 ± .11 28 2.00 2 0.19 ± .06 34 1.97 ± .05 106 1.99 ± .02 26 2.00 ro oc 6 4 5 7 0 279 APPENDIX 10 Counts of bristles, wing vein gaps, and wing nicking in Ax1^172/^ heterozygotes The heterozygotes examined were generated by the following crosses . LINE CROSS 1,2 M5/N8 $ x A x i 6 i72/ Y d" 3,4 M5/wa N 4 0 rb £ x A x i 6 i7 2/Y J 5 dl49,y Hw m2/wa N 4 0 rb $ x Axl6l72/Y <? 6 M5/y wa N 1 0 3 $ x A x i 6 i72/ y $ 7,8 1(FM6)/wa NS11 rb £ x Axl6l72/Y g 9 M5/wa NS11 rb $ x Axl6l72/Y tf 10,11 M5/wa N C o $ x Axl6l72/Y ^ Note that the figures in Table 25 were calculated from results of the crosses of M5/N females to AxE2/Y males. See Methods and Materials for explanation of x and confidence intervals, and see Appendix 8. O R B I T A L S L I N E G E N O T Y P E T E M P . 0 1 2 3 4 5 6 X 1 A X 1 6 1 7 2 / N 8 22°C 0 0 0 1 158 28 6 4.20 ± .06 2 29°C 0 0 0 6 15 9 0 4.10 + .23 3 22°C 0 0 0 2 112 37 13 4.37 ± .09 4 A x 1 6 1 7 V ° 29°C 0 0 0 0 0 1 0 -5 20 .5°C 0 0 0 1 134 21 1 4.14 ± .05 6 Ax16172/n103 22°C 1 1 10 9 41 0 0 3.42 ± .20 7 20.5°C 0 0 0 9 130 9 0 4.00 + .05 8 AX 1 6 1 7 2/N«" 29°C 0 0 0 0 1 2 0 4.7 9 22°C 0 0 2 21 83 0 0 3.76 ± .08 10 A X 1 6 1 7 2 / N C O 22°C 0 0 0 2 40 49 34 4.92 ± .12 11 29°C 0 0 0 0 1 0 0 — ro oc o O C E L L A R S P O S T V E R T I C A L S V E R T I C A L S LINE 0 1 2 X 0 1 2 X 0 1 2 3 4 X 1 0 1 192 1.99 ± .01 0 0 193 2.00 0 0 0 3 190 3.98 ± .02 2 0 0 30 2.00 0 2 28 1.93 ± .08 0 0 0 3 27 3.90 ± .10 3 0 0 164 2.00 0 2 162 1.99 ± .02 0 0 0 1 163 3.99 ± .01 0 0 1 - 0 0 1 - 0 0 0 0 1 -5 0 2 161 1.99 ± .02 0 4 159 1.98 ± .02 0 0 0 0 163 4.00 6 50 10 2 0.23 ± .11 15 20 27 1.19 ± .18 0 0 0 0 62 4.00 7 5 19 124 1.80 + .07 62 59 25 0.75 ± .10 0 0 0 1 147 3.99 ± .01 8 0 2 1 1.3 0 0 3 2.0 0 0 0 0 3 4.0 9 3 9 94 1.86 ± .07 47 39 20 0.75 ± .13 0 0 0 0 106 4.00 10 0 1 124 1.99 ± .02 0 2 123 1.98 + .02 0 0 0 0 125 4.00 11 0 0 1 0 0 1 0 0 0 0 1 ro oo h-1 D O R S O C E N T R A L S A N T E R I O R S C U T E L L A R S P O S T E R I O R S C U T E L L A R S L I N E 0 1 2 3 4 X 0 1 2 X 0 1 2 X 1 0 0 0 0 193 4.00 0 0 193 2.00 0 2 191 1.99 ± .02 2 0 0 0 1 29. 3.97 ± .06 0 1 29 1.97 ± .06 0 0 30 2.00 3 0 0 0 1 163 3.99 ± .01 0 2 162 1.99 ± .02 0 2 162 1.99 ± .02 4 0 0 0 0 1 - 0 0 1 - 0 0 1 -5 0 0 0 0 163 4.00 0 1 162 1.99 ± .01 0 0 163 2.00 6 2 3 20 18 19 2.79 ± .23 0 4 58 1.94 ± .06 0 1 61 1.98 + .03 7 0 0 4 32 112 3.73 ± .07 4 26 118 1.77 ± .07 0 0 148 2.00 8 0 0 0 1 2 3.7 0 3 2.0 0 0 3 2.0 9 0 0 10 21 75 3.61 ± .11 8 26 72 1.60 ± .11 0 0 106 2.00 10 0 0 0 2 123 3.98 ± .02 0 0 125 2.00 0 0 125 2.00 11 0 0 0 0 1 0 0 1 0 0 1 ro ex ro W I N G V E I N G A P S W I N G N I C K I N G L I N E 1 2 2 0 1 2 __3 0 0 167 0 5 6 7 8 x 1 x 1 27 2 108 0 0 0 0 0 0 0 0 0 2.03 ± .03 0 2.00 ± .09 0 0 0 184 2.00 0 30 2.00 0 0 0 0 0 1.98 ± .06 0 0 133 2.00 4 - - - - - - - - - - 0 0 1 -5 - - - - - - - - - - 0 0 163 2.00 6 0 0 30 7 1 0 0 0 0 2.24 + .14 4 6 28 1.63 + .19 7 0 0 0 8 28 39 59 0 0 5.11 i .14 129 3 2 0.05 + .04 8 1 2 0 0 0 0 0 0 0 0.7 0 0 3 2.0 9 0 0 25 9 23 2 1 0 0 3.08 ± .23 56 4 0 0.07 + .06 10 0 9 93 0 0 0 0 0 0 1.91 ± .05 0 1 115 1.99 + .02 11 mm mm _ _ _ M. _ ro oc u> 4 0 3 284 APPENDIX 10 9 B 2 Counts of bristles, wing vein gaps, and wing nicking in Ax /N heterozygotes The heterozygotes examined were generated by the following crosses. LINE CROSS 1 , 2 M 5 / N $ x A X 9 B 2 / Y 3 dl49,y Hw mf/wf N ^ rb $ x Ax9B2/Y <? 4,5 M5/wf N^. rb ? x A x 9 B 2 / Y & 6 , 7 M5/y w f N 1 Q 3 $ x A X 9 B 2 / Y c f 8,9 1(FM6)/wa NS11 rb $ x A x 9 B 2 / Y d* 10,11 M 5 / w f N S 1 1 rb ? x A x 9 B 2 / Y J1 12,13 M 5 / w a N C o $ x A x 9 B 2 / y ^ Note that the figures in Table 26 were calculated from results of the crosses of M5/N females to A x 9 b 2 / Y males. See Methods and Materials for explanation of x and confidence intervals, and see Appendix 8. ORBITALS LINE GENOTYPE TEMP. 0 1 2 3 4 5 6 X 1 9B2 8 22°C 0 0 0 0 0 3 112 5.97 ± .03 Ax /N 2 29°C 0 0 0 1 5 23 37 5.45 ± .15 3 20.5°C 0 0 0 0 1 3 207 5.98 ± .02 4 A x 9 B V ° 22°C 0 0 0 0 0 6 95 5.94 ± .04 5 29°C 0 0 0 0 12 20 25 5.23 ± .18 6 Ax9B2/n103 22°C 0 0 0 0 93 28 18 4.46 ± .10 7 29°C 0 0 0 0 0 2 5 5.7 ± .4 8 20.5°C 0 0 0 0 5 34 83 5.64 ± .09 9 „ 9B2/Mgll Ax /N 29°C 0 0 0 0 1 4 15 5.70 ± .23 10 22°C 0 0 0 4 27 36 49 5.12 ± .14 11 29°C 0 0 0 0 0 4 21 5.84 ± .13 12 AX9B2/NCo 22°C 0 0 0 0 0 3 78 5.96 ± .04 13 29°C 0 0 0 1 0 11 24 5.61 ± .19 ro oc VJl OCELLARS POSTVERTICALS LINE 0 1 2 X 0 1 2 X 1 91 23 1 0.22 + .07 7 27 81 1.64 ± .10 2 47 16 3 0.33 ± .12 31 25 10 0.68 ± .16 3 160 41 10 0.29 ± .07 5 24 182 1.84 + .05 4 86 14 1 0.16 ± .07 8 22 71 1.62 ± .11 5 46 10 1 0.21 + .11 16 24 17 1.02 ± .18 6 25 53 61 1.26 + .11 5 12 122 1.84 ± .07 7 7 0 0 0.0 0 3 4 1.6 ± .5 8 121 1 0 0.01 + .02 119 3 0 0.02 ± .03 9 19 1 0 0.05 ± .09 0 3 17 1.85 + .15 10 116 0 0 0.00 115 0 1 0.02 + .03 11 24 1 0 0.04 ± .07 0 4 21 1.84 ± .13 12 15 31 35 1.25 ± .14 4 19 58 1.67 ± .11 13 31 4 0 0.11 ± .10 4 18 14 1.28 ± .19 VERTICALS 0 1 2 3 4 X 0 0 0 2 113 3.98 + .02 0 0 2 22 42 3.61 + .12 0 0 0 0 211 4.00 0 0 0 0 101 4.00 0 0 0 0 57 4.00 0 0 0 0 139 4.00 0 0 0 0 7 4.00 0 0 0 0 122 4.00 0 0 0 0 20 4.00 0 0 0 1 115 3.99 ± .02 0 0 0 0 25 4.00 0 0 0 0 81 4.00 0 0 0 0 36 4.00 rv a a DORSOCENTRALS ANTERIOR SCUTELLARS POSTERIOR SCUTELLARS LINE 0 1 2 3 4 X 0 1 2 X 0 1 2 X 1 0 0 26 35 54 3.24 + .13 0 0 115 2.00 0 0 115 2.00 2 0 0 47 16 3 2.33 ± .12 0 1 65 1.98 ± .03 0 0 66 2.00 3 0 2 30 84 95 3.29 ± .09 0 1 210 2.00 ± .01 1 1 209 1.99 ± .02 4 0 0 18 37 46 3.28 ± .13 0 1 100 1.99 ± .02 0 2 99 1.98 ± .03 5 0 0 36 16 5 2.46 + .15 0 0 57 2.00 0 0 57 2.00 6 0 0 34 33 72 3.27 ± .12 0 0 139 2.00 1 5 133 1.95 ± .04 7 0 0 0 0 7 4.0 0 0 7 2.0 0 0 7 2.0 8 1 13 105 3 0 1.90 ± .06 90 25 7 0.32 ± .09 0 3 119 1.98 ± .03 9 0 1 15 2 2 2.25 ± .29 0 0 20 2.00 0 0 20 2.00 10 0 5 104 7 0 2.02 ± .05 63 32 21 0.64 + .12 0 3 113 1.97 ± .03 11 0 2 19 3 1 2.12 + .21 0 0 25 2.00 0 2 23 1.92 ± .10 12 0 0 27 26 28 3.01 ± .16 0 0 81 2.00 0 2 79 1.98 ± .03 13 0 0 35 1 0 2.03 ± .05 0 1 35 1.97 ± .05 0 0 36 2.00 ro CO WING VEIN GAPS WING NICKING LINE 0 1 2 3 4 5 6 7 8 X 0 1 2 X 1 40 23 51 1 0 0 0 0 0 1.11 + .15 113 2 0 0.02 ± .01 2 3 12 13 10 12 7 6 0 0 2.97 + .37 11 11 42 1.48 + .17 3 62 68 80 0 0 0 0 0 0 1.09 + .10 177 23 10 0.20 ± .06 62 19 16 0 0 0 0 0 0 0.53 + .13 78 18 2 0.22 + .08 5 24 18 12 1 0 0 0 0 0 0.82 + .20 0 0 57 2.00 6 0 1 38 36 61 2 0 0 0 3.18 + .13 129 8 1 0 . 0 7 2 ±.o4: 7 2 0 0 0 0 0 0 0 0 - 0 0 7 2.0 8 0 0 32 19 11 2 2 0 0 2.83 + .16 66 0 0 0.00 9 6 3 5 4 0 1 0 0 0 1.58 + .59 11 5 3 0.58 ± .34 10 0 2 35 10 9 0 0 0 0 2.46 + .19 56 0 0 0.00 11 13 7 4 0 0 0 0 0 0 0.63 + .28 15 8 1 0.42 ± .21 12 33 20 24 0 0 0 0 0 0 0.88 ± .17 80 0 0 0.00 13 2 13 20 0 0 0 0 0 0 1.51 + .18 13 12 11 0.94 ± .24 ro cc CO 289 APPENDIX 10 Counts of bristles and wing vein gaps in heteroallelic Axx/Axy combinations The data on the following pages were obtained from the following crosses. CROSS l ( F M 6 ) / A x E 1 ? x A x e 2 / Y c? Ax E 2/Ax E2 ? x A x E 1 / B S w+.Y <? LINE 1,3 2 4 5,6 7 8 9 l(FM6)/AxE1 $ x Axl6172/Y $ l(FM6)/AxE1 $ x A x 9 B 2 / Y d* Ax16172/Ax16172 ? x A xE2 / Y # AxE2/AxE2 $ x Axl6l72/Y ^ wf AxE2 rb/+ Ax 1 6 1 7 2 + $ x wf Ax9B2 rb/Y c? The wings of all the genotypes except AxE2/Ax1^172 (lines 7,8) were too deformed to score accurately for wing vein gaps. The data for AxE2/Axl6172 females are tabulated below. NUMBER OF FLIES WITH GIVEN NUMBER OF WING VEIN GAPS LINE 0 1 2 3 4 5 6 7 8 x 7 0 0 0 0 4 1 11 7 2 1 4.56 ± .20 8 0 0 1 3 35 15 8 3 1 4.59 ± .23 See Methods and Materials for explanation of x and confid-ence intervals, and see Appendix 8. ORBITALS LINE GENOTYPE TEMP. 0 1 2 3 4 5 6 X 1 20.5°C 13 0 0 0 0 0 0 ) 2 A X E 1 / A X E 2 20 .5°C 10 0 0 0 0 0 0 ) ) N 0.04 ± .07 3 20 .5°C 2 1 0 0 0 0 0 ) ) 4 A x E 1/AX 1 6 17 2 20.5°C 2 0 0 0 0 0 0 -5 AX E 1/AX9 B 2 20.5°C 0 0 17 43 50 6 0 3.39 ± .10 6 20.5°C 0 0 20 35 33 10 0 3.34 ± .16 7 Ax E 2/Ax l 6 l7 2 20.5°C 0 4 24 26 12 0 0 2.70 ± .18 8 20 .5°C 0 6 21 30 11 0 0 2.68 + .18 9 AX E 2/AX9 B 2 22° C 28 0 0 0 0 0 0 0.0 ro vo o 0 C E L L A R S P O S T V E R T I C A L S V E R T I C A L S LINE 0 1 2 X 0 1 2 X 0 1 2 3 4 X 1 13 0 0 13 0 0 0 0 0 0 13) 2 10 0 0 0.0 10 0 0 0.0 0 0 0 0 10? 4.0 3 3 0 0 3 0 0 0 0 0 0 3? 4 2 0 0 - 2 0 0 - 0 0 0 1 1 -5 116 0 0 0.00 116 0 0 0.00 0 0 0 0 116 4.00 6 98 0 0 0.00 98 0 0 0.00 0 0 0 0 98 4.00 7 66 0 0 0.00 66 0 0 0.00 - - - - - -8 68 0 0 0.00 68 0 0 0.00 0 0 1 1 66 3-96 ± 9 28 0 0 0.0 28 0 0 0.0 1 6 10 8 3 2.21 ± vo DORSOCENTRALS ANTERIOR SCUTELLARS POSTERIOR SCUTELLARS LINE 0 1 2 3 4 x 0 1 2 x 0 1 2 x 1 1 1 2 0 0 0 13 0 0 0 0 13) \ 2 6 3 1 0 0 0 . 3 1 ± .19 1 0 0 0 0 . 0 0 1 ) 9) \ 1 . 9 6 ± .07 3 2 1 0 0 0 3 0 0 0 0 ; 3) 2 0 0 0 0 - 2 0 0 - 2 0 0 -5 30 37 49 0 0 1.16 ± .13 97 16 3 0.19 ± .07 4 28 84 1.69 ± .09 6 21 35 42 0 0 1.21 + .14 90 8 0 0.08 ± .05 1 21 76 1.77 ± .08 7 0 0 26 26 14 2.82 + .16 1 16 49 1.73 ± .10 0 1 65 1.98 ± .03 8 0 1 6 15 46 3.56 ± .15 0 11 57 1.84 ± .09 0 0 68 2.00 9 28 0 0 0 0 0.0 16 11 1 0.46 ± .19 2 8 18 1.57 ± .21 ro vo ru 

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