UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1971_A1 F68.pdf [ 7.32MB ]
Metadata
JSON: 831-1.0107124.json
JSON-LD: 831-1.0107124-ld.json
RDF/XML (Pretty): 831-1.0107124-rdf.xml
RDF/JSON: 831-1.0107124-rdf.json
Turtle: 831-1.0107124-turtle.txt
N-Triples: 831-1.0107124-rdf-ntriples.txt
Original Record: 831-1.0107124-source.json
Full Text
831-1.0107124-fulltext.txt
Citation
831-1.0107124.ris

Full Text

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  In presenting  this  thesis  an a d v a n c e d d e g r e e at the L i b r a r y I  further  for  agree  scholarly  by h i s of  shall  the U n i v e r s i t y  make  it  written  thesis  of  of  of  Columbia,  British for  for extensive  the  requirements  reference copying of  I agree and this  for  It  financial  is understood gain  Zoology  1971  Columbia  shall  that  not  copying or  for  that  study. thesis  p u r p o s e s may be g r a n t e d by the Head o f my D e p a r t m e n t  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  October  fulfilment  available  permission.  Department  Date  freely  that permission  representatives.  this  in p a r t i a l  or  publication  be a l l o w e d w i t h o u t my  ii  ABSTRACT  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 morphological 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 (N T ), 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 Notchlocus 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 semiF1 lethal allele Ax  both suppressed wing nicking, whereas the two  viable alleles Ax E 2 and AX^172 both enhanced wing nicking. Furthermore, heteroallelic combinations of Ax alleles which affected nicking in different direction, were lethal (Ax E ^/Ax E2 , A x E 1 / A x 1 6 1 7 2 , A x 9 B 2 / A x 1 6 1 7 2 ) , whereas combinations of Ax alleles with similar effects on nicking were viable (Ax E VAx^ B 2 , Ax E 2 / Ax16172). The temperature-shift experiments have revealed an interesting 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 (N 6 Q s l l /N 6 Q S i : L ;Dp 5 1 b 7 ), or the second larval instar  (Ax 16172 /n 264-40  ), or they may be polyphasic, occurring in embryo, larval and pupal stages ( N 2 ^ ~ 1 Q 3 / f a n 0 ) . on the  iv other hand3 the TSPs for all the adult morphological abnormalities examined occur during the third larval instar, including rough eyes and wing nicking ( N 60gll / + >  N 264-103/ s p l ),  leg segment  fusion (^264-103/.^ N 2 6 4 ~ 1 Q 3 /spl), wing vein gapping (Ax l6l 72/+) and disturbance of bristle numbers  ( N 264-103/ s p l j  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 encouragement 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 MATERIALS AND METHODS I. II. III. IV. V. VI. VII. VIII. IX.  Description of strains and mutant stocks. Use of the symbol D£. Incubation temperatures. Procedure for temperature-shift experiments. Preparation of specimens for scanning electron microscopy. Procedure for wing nick counts. Procedure for bristle and wing vein gap counts. Statistical procedures used on nicking, bristle, and vein gap data. Calculation of index of phenotypic expression of bristle and wing vein gap phenotypes.  1 13 13 22 23 24 27 28 28 28 31  RESULTS  33  A.  33  PHENOTYPES OF SELECTED N AND N^ COMBINATIONS I.  Effects of gene dosage on the phenotypes of  n£ and N^O..  33  N1Q3.  36  II.  Effects of gene dosage on the phenotypes of  III.  Effects of gene dosage on the phenotypes of NgU. Effects of gene dosage on the phenotypes of NCO. The phenotypes of Nx/Ny;Dp combinations.  IV. V.  44 58 62  B.  ORIGIN AND MAPPING OF THE ABRUPTEX MUTATIONS  66  C.  PHENOTYPES OF ABRUPTEX MUTATIONS  74  I. II. III.  IV.  AX e 1 . AX E 2 .  74 78  Axl6l72.  80  52  83  Ax?  vii D.  INTERACTIONS OF NOTCH AND ABRUPTEX MUTATIONS I. II.  III.  Viability of N/Ax heterozygotes. The effects of N mutants on the bristle and wing vein phenotypes of Ax mutations. The effects of Ax mutants on the wing nicking phenotypes of N mutations.  89 89 91 98  E. INTERACTIONS BETWEEN DIFFERENT ABRUPTEX MUTATIONS  102  F. DEVELOPMENTAL STUDIES OF SELECTED GENOTYPES  109  I.  N g H / + - TSPs for wing nicking and eye facet disruption. II. N 1Q 3/spl - TSPs for wing nicking, eye facet disruption, tarsal fusion, and bristle disruptions. III. OR - radiation-induced rough eye phenocopy. x v . Ax l6l72/ N 40 _ TSP for lethality; Axl6l72/+ TSPs for wing vein gapping and loss of ocellar bristles. V. Nl03/fano - TSPs for lethality. VI. Ngll/Ngi:L;Dp - TSP for lethality. VII. Summary of temperature shift results. DISCUSSION  109 125 130 132 146 158 161 169  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 TABLE  viii PAGE  1.  Symbols and phenotyplc descriptions of non-Notchlocus mutations used.  16  2.  Symbols and descriptions of Notch-locus alleles used.  18  3.  Symbols and descriptions of chromosome rearrangements 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 N 1Q 3/+ females exhibiting nicking when raised at different temperatures.  38  7.  Viability of N 10 3/N 1Q 3;Dp females in relations to siblings at different temperatures.  43  8.  Number of wings of N 1Q 3/N 1Q 3;Dp females exhibiting nicking when raised at different temperatures.  45  9.  Number of wings of N S H / + 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 Ng 11 /Ng 11 ;Dp and NS1:L/Ngll; Dp/Dp females in relation to their siblings, when raised at different 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 Ax e ^.  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 Ax E 1 at 20.5°C.  75  ix TABLE  PAGE  17.  Counts of bristles in eclosed Ax E1 /Y males raised at 20.5°C.  77  18.  Summary of the bristle and wing vein gap phenotypes of A x E 2 .  79  19.  Summary of the bristle and wing vein gap phenotypes of Ax l 6 l 72.  82  20.  Summary of the bristle and wing vein gap phenotypes 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 combinations of different Ax and N alleles.  90  24.  Summary of the bristle and wing vein gap phenotypes of Ax E2 /N heterozygotes.  92  25.  Summary of the bristle and wing vein gap phenotypes of Ax16172/n heterozygotes.  93  26.  Summary of the bristle and wing vein gap phenotypes of Ax9 B2 /N heterozygotes.  94  27.  Counts of bristles in Ax E1 /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.  30.  Relative viability of A Ax9B2/Axl6l72 at 22°C.  31.  Summary of the bristle and wing vein phenotypes of heterozygous combinations of different Ax alleles at 20-22°C.  107  Eye and wing phenotypes of adult females shifted from 20.5°C to 29°C at different successive intervals.  110  32.  x 9  B 2  / A x 9 B 2  >  Ax9B2/AxE2a  103 and  104  X PAGE  TABLE 33.  Eye and wing phenotypes of adult females shifted from 29°C to 20..5°C at different successive intervals.  Ill  Eye and wing phenotypes of NS 1 1 /* adult females shifted from 20.5°C to 29°C at different successive intervals (Experiment 2).  113  Eye and wing phenotypes of NSJ-1/+ adult females shifted from 29°C to 20.5°C at different successive intervals (Experiment 2).  114  Positions of anterior boundaries of mutant tissue extending from the posterior rim of the eyes of Ngll/+ females, shift-up experiment 2.  118  Positions of anterior and posterior boundaries of mutant tissue in the centre of the eyes of N 8 H / + 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  34.  35.  36.  37.  40.  41.  42.  43.  44.  45.  Data indicating viability of  A x l6l72 / N 40  females relative to their sibs when shifted from 22°C to 29°C at different successive intervals. Data indicating viability of Ax l6l72 /N**° females relative to their sibs when shifted from 29°C to 22°C at different successive intervals. Data indicating viability of  137 138  A X 16172 / N 40  females relative to their sibs when pulsed from 22°C to 29°C and back after 18 hours, at different successive intervals. Data indicating viability of A X 16172 / N 40 females relative to their sibs when pulsed from 29°C to 22°C and back after 24 hours, at different successive intervals. Data indicating viability of N 10 3 / f a no females in relation to their sibs when shifted from 22°C to 29°C at different successive intervals. Data indicating viability of N lc>3 /fa no females in relation to their sibs when shifted from 29°C to 22°C at different successive intervals.  139  140  148 152  xi PAGE  TABLE 46.  47.  48.  49.  50.  51.  52.  Data indicating viability of N 1 Q 3/fa n o females in relation to their sibs when pulsed from 22°C to 29°C and back after 18 hours, at different successive intervals.  155  Data indicating viability of N 103/ fa no  females in relation to their sibs when pulsed from 29°C to 22°C and back after 24 hours, at different successive intervals.  156  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  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  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  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  Expected product proportions of Ax/Ax, Ax/+, and Ax/N, according to activator:repressor model ^Figure 17).  190  LIST OP FIGURES FIGURE  xii PAGE  1.  Genetic map of the Notch locus.  2.  Mating scheme used to synthesize isogenic wildtype strain (OR).  15  3.  The anatomical positions of a) bristles and b) wing veins discussed in the text (LINDSLEY & GRELL 1968).  30  Summary of the effects of relative dosage and of temperature on the eye and wing phenotypes of Ngll-bearing flies.  56  4.  4  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  The eye, wing, leg, and bristle phenotypes of N 1Q 3/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  The eye phenotypes of OR flies irradiated at different times before and after puparium formation, at 20.5°C and 29°C.  135  Viability of AX i6i 72/n40 shifted at different stages of development.  143  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  Relative proportions of viable N 1 Q 3/fa n o 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  Time of death of N 1 Q 3/fa n o females in relation to time of shift from 22°C to 29°C.  154  7.  8.  9. 10.  11.  12.  xiii FIGURE  PAGE  13.  Viability of N S 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 Notchlocus mutant genotypes.  167  Hypothetical molecular models to explain tarsal fusion and enhanced wing nicking in N1(->3/+ females at 29°C.  174  Hypothetical molecular model to explain opposite response to temperature of eye and wing phenotypes of NS 1 1 .  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  15*  16.  19. 20..  "Range of function" model to explain lethality  of A x 9 B 2 / a x E 2  and A x 9 B 2 / A x l 6 l 7 2 .  201  Mating scheme used to replace autosomes of recessive viable Ax stocks with OR autosomes.  205  xiv LIST OP PLATES  PAGE PLATE 1  Wings of a) b) c) +/+;Dg_, d) AxE2/AxE23 and e) OR females, raised at 22°C.  PLATE 2  Wings of N 1Q 3/+ females raised at a) 22°C b) 25°C, cT~29°C.  41  PLATE 3  Scanning electron micrographs of NS 11 /* females raised at a) 20.5°C, and b) 29°C,  47  PLATE 4  Wings of Ca) N C o /+, b) N Co /+;Dp, and o c) NCO/N ;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 heterogeneous 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 serrations 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 references:  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) spl;  Ax E 2 , Ax 5 9 b , A x 5 9 d , NJ'1^, N f l °, and N h 2 1 are to the right of 2)  of N ^ ;  Ax E 1 is to the right of fa no ; 4)  3)  A x 9 B 2 is to the right  A x 9 B 2 and A x 5 9 d are to the left of N ^ ;  between N e l 1 and fa no .  5) fa£ is  N Vc  (M  Tr- — bfi Z et  o 0  N u I I  Z  Z  *!  Y-S  Z  <X PT W  X <  h- 2 o •H  Z  - z Ijj  o J c mci  r?  X <  Z  o c  M co"  Ca  H  a>  <  o  PLATE 1  Wings of a)  b) n S 1 1 / * ,  c) +/+;D£, d) Ax E2 /Ax E2 ,  and e) OR females, raised at 22°C. I8x.  Magnification  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 defective 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 within 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 experimentally . The Abruptex mutations characteristically cause a reduction in the numbers of certain bristles, and interruptions in wing venation (Plate Id).  The original Abruptex allele, Ax 2 8 a , 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 Ax 28a /N individuals (LEFEVRE, RATTY AND HANKS 1953).  The suppression of the Notch phenotype  by Ax 2 8 a , 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 A x 2 8 a was a duplication of the Notch locus with the Abruptex phenotype resulting from a position effect associated 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 28a 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 Ax 2 8 a 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 Ax 2 ^ 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 suppression 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 therefore 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 fa no , and that the wing mutant sites fa no 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^ or A x ^ ^ , also express the fas phenotype, although  with  less so than in fas/faS flies (WELSHONS 1971).  The present  investigation has only concerned certain combinations of recessive 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 fa no 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 morphological 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 temperature-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. ous and fertile at both 20°C and 29°C.  This stock is vigor-  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 rearrangements 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  spa  TM2  SM1  T (2 ;3) e  FM6  SMI +  spa  TM2 >  +  spa >  pol  OREGON R369  pol  pol  SMI  FM6  +  spa  TM2  T ( 2 5 3 )e  Y  '  spa  TM2  SMI  + •  spa  + ' '  +  pol cr*  pol  pol cr*  '  +  >  +  >  o*  16 TABLE 1  Symbols and phenotypic descriptions of non-Notchlocus mutations used.  Name  Symbol  Location  Phenotype  Bar  B  X-57.0  Narrow eye.  Bar of Stone  BS  X-57.0  Extremely narrow eye.  bobbed-lethal  bb-  X-66.0  Recessive lethal in XX females or XO males.  b rown-Vari e gat e d  bwV  2-104.5  Dominant mottled brown eye colour.  carnation  car  X-62.5  Eye colour dark ruby; orange in combination with v.  crossveinless  cv  X-13.7  Wing crossveins missing.  Curly  Cjr  2-6.1  Wings curved upwards; recessive lethal.  deep orange  dor  X-0.3  Orange eye colour.  deep orange-lethal  dorJ  X-0.3  Recessive lethal dor allele.  3-70.7  Black body colour.  3-70.7  Black body colour; allele of e.  X-56.7  Bristles shortened and bent.  Hw  X-0.0  Dominant, extra bristles along wing veins and on head and thorax.  lethal(l)J1  1(1)J1  X-0.0  Recessive lethal; covered by duplications.  miniature-2  m£  X-36.1  Wing size reduced.  ruby  rb  X-7.5  Eye colour ruby; white in combination with w a .  sparkling-poliert  s^ap Q l  4-3.0  Eyes small, glazed.  ebony ebony-sooty forked Hairy wing  17 Name  Symbol  Location  Phenotype  tinylike  tyl  X-36  Small bristles.  Ultrabithorax-130  Ubx 1 3 0  3-58.8  Large haltere size; recessive lethal.  vermilion  v  X-33.0  Eye colour bright scarlet.  white  w  X-1.5  White eye colour.  apricot  w1-  X-1.5  Apricot eye colour; allele of w.  eosin  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  yellow yellow-2  2  2L.  18 TABLE 2  Symbols and descriptions of Notch-locus alleles used.  Allele  Symbol Used  Description  *Abruptex-9B2  Ax 9B2  Bristle loss; wing vein gapping - see Results.  * Abruptex-59d5  Ax 59d  Recessive lethal; bristle loss and wing vein gapping.  *Abruptex-l6l72  Ax16172  Extensive bristle loss and wing vein gapping - see Results.  *Abruptex-El  Ax-El  Recessive lethal; bristle loss and wing vein gapping see Results.  *Abruptex-E2  AxE2  Bristle loss and wing vein gapping - see Results.  facet-glossy  fa®  Irregular eye facet array and glazed eye surface.  facet-notchoid  fano  Nicked wings, thick wing veins; lethal when heterozygous with most N mutations.  *facet-notchoid-E  fanoE  Like fa n o , but milder wing phenotype.  Notch-8  N  Cytologically deficient for several salivary chromosome bands including 3C7. Recessive lethal; nicked wings and thick wing veins.  Notch-264-40  N 40  Phenotypically like N£, but not cytologically deficient.  Notch-264-103  N 103  Not cytologically deficient. Recessive lethal; temperature sensitive wing nicking, fusion of leg segments, and interaction with other alleles - see Results.  Notch-60gll  N  gH  Not cytologically deficient. Recessive lethal; temperature sensitive wing nicking and eye facet disarry - see Results.  19 Allele  Symbol Used  Description  *Notch-70k27  N70k27  Notch-Confluens  N Co  Not cytologically deficient. Recessive lethal; weak wing nicking; strong extra wing vein (Confluens) phenotype in presence of extra N^ loci - see Results.  split  spl  Eyes reduced in size and with irregular facet array; missing or doubled bristles frequent.  Recessive lethal; weak wing nicking, eyes normal; from origin, should also contain Ngll mutant site - see Appendices 3»  •Alleles not described in LINDSLEY AND GRELL (1968).  20 TABLE 3.  Symbols and descriptions of chromosome rearrangements used.  Name  Symbol Used  Markers Carried  Bar of Stone, white-plus Y  B S w + •Y  w + -N + ;B S  Insertion of X chromosome markers into Y chromosome.  Bar of Stone, yellow-plus Y  B S y + •Y  y+-l(l)Jl+;BS  Insertion of X chromosome markers into Y chromosome.  delta-49  dl49,y Hw n£  y, Hw, m£  Inversion in central region of X.  delta-49  dl49»tyl bb 1  bb 1 , tyl  Inversion in central region of X.  Duplication (l;2)51b7  D£  w+, N+  Insertion of X chromosome markers into right arm of chromosome 2.  Description  ^Duplication (l;Y)59k9(4)  59k9(4)  y£, 1(1)J1+  Insertion of X chromosome markers into Y chromosome #.  ^Duplication (l;Y)60dl9(l)  60dl9(1)  xi, k i ) J I +  Insertion of X chromosome markers into Y chromosome §.  D P 6 7g24(l)  y 2 -dor + (inclusive)  Insertion of X chromosome markers into Y chromosome #.  •Duplication (l;Y)67g24(l) First Multiple-6  FM6  •lethal First Multiple-6  B  Multiply inverted X; female sterile.  1(FM6)  Z.> B, 1  FM6 chromosome carrying EMS-induced lethal.  Muller-5  M5  Wa,  Second Multiple-1  SMI  9Z  B  Multiply inverted X. Multiply inverted second chromosome.  21 Name  Symbol Used  ^Translocation (1;Y)2E  T(1;Y)2E  ^Translocation (2;3)e  T(2;3)e  Third Multiple-2  TM2  Markers Carried 1L> K D J i * ; dor+  Insertion of X chromosome markers into Y chromosome (RAYLE & HOAR 1969) Reciprocal translocation between Second and Third chromosomes. Recessive lethal.  Ubx 13 °, e s  Multiply inverted Third chromosome. Compounded X chromosomes.  *Compound X *yellow-whiteforked Compound X  Description  XX,  w f/Y  w, f  Compounded X chromosomes.  * Rearrangements not described in LINDSLEY AND GRELL (1968). # GREEN, personal communication.  II .  22  Use of the symbol Dp .  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).  24 IV.  Procedure for temperature-shift experiments.  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 particularly well-synchronized, so this method was not repeated. Accurate staging of third instar larvae relative to pupation 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 experiment 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 x -  On  °  + n, + 2n„ + 3n + ... 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  POSTERIOR ANTERIOR  CROSS VEINS  31 where n Q , 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 Xri^(x. - x) 1 i=0 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. confidence interval =, v J n-1  t .95  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 increasingly severe expression of the mutant phenotypes.  Index of  expression of bristle phenotypes is given by the formula index =  mutant bristle frequency 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  One hundred six £ Hw  and  .  progeny from the cross N^/dl4_9,  £ 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 ^ D p / D p 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 by WELSHONS (1965).  accord with the rules described  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^/N 40 ;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 w a /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 w a /w a females each. 40 infertility of N  The  /Y;Dp/Dp males is not surprising in view of the  triplication of all the loci (except N^) carried by the duplication.  TABLE 4  Viability of N^°/N1*0;Dp and N^/N210;Dp/Dp females in relation to siblings at different temperatures.  PROGENY FEMALES CROSS*  1  2  * 1. 2.  TEMPERATURE  MALES  1(FM6)/N  l(FM6)/N;Dp**  N/N;DR  N/N;D£;D£  N/Y;D£  N/Y;D£/D£  20.5°C  26  26  20  -  Hj  29°C  74  98  68  20.5°C  53  l6l  72  44  103  4l  29°C  65  157  94  62  168  42  -  69  l(FM6)/wa N 4 0 rb ? x wf, N ^ rb;D£ <?> 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 summarized in Table 5. and  and N ^ combinations are  The genotypes n5/+, N2*0/*, N ^ / N ^ P p ,  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 disruptions frequently occurred in the N^°/Y;Dp, N^°/Y;Dp/Dp, N ^ O / N ^ D p 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 responsible 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  The frequency of wingtip nicking in N 103 _/+ 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  GENOTYPE  NUMBER OP N + LOCI  WING PHENOTYPE*  FEMALES 1  1.0 N  Nlt0/+  1  1.0 N  N 8 /N 4 °;Dp  1  1.0 N  N40/N40.Dp  i  1.0 N  2  .+  N 4 °/N 4 °;Dp/Dp  2  +  N2*0/*; Dp/Dp  3  Co  40 N  /+;Dp  MALES N^°/Y;Dp  1  +  N 4Q /Y;Dp/Dp  2  Co  +/Y;D£  2  Co  * 1.0 N = all individuals have nicked wings; + = wild-type wings; Co = Confluens wings  38 Number of wings of N 1 0 3 /+ females exhibiting nicking when raised at different temperatures.  TABLE 6  TEMPERATURE 20°C  22°C  25°C  29°C  *  CROSS*  % NICKED INDIVIDUALS  MEAN NUMBER OP NICKED WINGTIPS PER FLY (+ 95% CONFIDENCE INTERVAL)  1  74  0.97 ± .12  103  2  80  1.25 ± .09  213  1  71  1.03 ± .14  91  2  82  1.34 ± .09  218  1  100  1.97 ± .05  143  2  100  1.99 ± .02  337  1  100  2.00  90  2  100  2.00  228  1.  OR ? x £ wf N103;Dp. cf  2  M5/2. wf; N 1 0 3 $ x OR err  »  NUMBER OF FLIES EXAMINED  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 N 1Q 3/+ 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 N 4 ( V+ flies, whereas the nick-  ing phenotype in N 1Q 3/+ females raised at 25°C was less pronounced 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 phenotype 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. N 1 Q 3 /fa n o is viable  From these observations, and the fact that at 20°C-25°C (Appendix 1), it is clear that  N 103  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  N 103  is hypomorphic rather than amorphic at these temperatures. other hand, at 29°C the mutant wing expression of  On the females  PLATE 2  Wings of N 1Q 3/+ 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 N 103 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). had normal legs.  At 20°C-25°C- all three genotypes  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 103/ no N fa 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 (N 7 0 k 3 °) with fa no 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  N gll / f a no  heterozygotes had normal  legs (Appendix 1). N 103 /N 103  ;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  n 103 /n 103-  Dp females recovered at 29°C exhibited much more severe wing nicking and tarsal fusion than their 29°C N^Q3/+ sibs. nicking data for the  Wing  103/ 103 ;Dp females raised at the three  TABLE 7  Viability of N 1 0 3 / N 1 0 3 ;Dp females* in relations to siblings, at different temperatures.  FEMALES TEMPERATURE  M5/N;D£  M5/N  . N/N;D£  MALES 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 w a N 1 0 3 $ x ^ wf; N 1Q3 ;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  N103A  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  N 1 0 3 /+ 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 N 1 0 3 /+ flies  have more extensive wing nicking than N®/+ or  females, and  the extent of wing nicking is further increased in N^ 3 /NlQ 3 ;Dp females. III.  Effects of gene dosage on the phenotypes of N g l 1 .  When raised at 25°C or 29°C, N g l l /+ females- resemble typical 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 irregularities (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^ NS 1 1 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  TEMPERATURE  Number of wings of N 1 0 3/N 1 0 3 ;Dp females exhibiting nicking when raised at different temperatures.  10 NICKED  INDIVIDUALS  MEAN NUMBER OP NICKED WINGTIPS PER FLY (± 95$ CONFIDENCE INTERVAL)  NUMBER OP FLIES EXAMINED  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 raised at a) 20.5°C, and b) 29°C. a) 270x, b) 290x.  females Magnifications  ft^vv:  W m  -Vc  • ' • / / ' • ' l ^ 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 reflect 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^ 1 1 phenotypes was examined in the female progeny of the cross w/w ? x wf N S 1 1 rb/Y;Dp o^ (Table 10). contains the  Since the duplication  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 N g l l /+;Dp  flies classed as "R" (Table 10) was not as marked as in their N g l l / + sisters, and 78 of the 108 "R" females were mutant in only one eye.  Nevertheless, the eye roughness of N g l V + ; D p females  is much more extensive than that seen in the occasional +/+;Dp  49 gll  TABLE 9  Number of wings of N /+ females exhibiting nicking when raised at different temperatures.  TEMPERATURE 20° C  22°C  25°C  29° C  *  CROSS*  % NICKED INDIVIDUALS  MEAN NUMBER OP NICKED WINGTIPS PER FLY (± 95$ CONFIDENCE INTERVAL)  NUMBER OF FLIES EXAMINED  1  0  0.00  152  2  1  0.01 ± .02  241  1  2  0.02 ± .02  l6l  2  1  0.01 ± .01  273  1  10  0.11 ± .05  184  2  24  0.28 ± .05  287  1  67  0.85 ± .12  103  2  76  1.19 ± .06  257  1.  OR $ x wf NS 1 1 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 NS 1 1 rb;'D£ c?>  50 TABLE 10  The dosage effect of on wing and eye phenotypes of earing females.  PHENOTYPE  NUMBER IN EACH CLASS GENOTYPE NgH/+  N gll/ + ; D p  EYE COLOUR  N + R*  NR  20.5°C  apricot  179  11  0  0  29°C  apricot  0  0  7  155  20.5°C  wild-type  108  0  115  0  29°C  wild-type  0  0  177  0  TEMPERATURE  N+R+  NR +  *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-sensiell / 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 NS 1 1 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 type of N g l 1 in males. ;Dp females.  decreases the mutant eye pheno-  This is similar to the effect seen in  52 Further studies on the effects of gene dosage on the phenotypes of N^ 1 1 have revealed that the addition of an extra dose of N ^ H affects the eye and wing phenotypes in opposite directions 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 NS 11 /* 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 N gll/ N gll. Dp  females raised at 25°C and 29°C, unlike N g l l /+  females, which are wild type at these temperatures.  In contrast  to the eye phenotypes, expression of the Notch wing phenotype was reduced in N g l l / N g H ; D p compared to  at all tempera-  tures. No thickening could be detected in the wing veins of the N gll /N gll  ;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 N g l l /+ females (Table 9).  At 29°C 22%  (10/42 from experiment 1, and 6/32 from experiment 2, Table 11) of the N ^ V N g l ^ D p females showed wingtip nicking, compared to  TABLE 11  Viability of N s l l /N s l l ;Dp and nS 1 1 /^ 1 1 ;Dp/Dp females in relation to their siblings, when raised at different temperatures. PROGENY FEMALE  CROSS* 1  TEMPERATURE  2  N/N;D£*«  M5/Y;D£  M5/Y  . N/Y.;:Dj  91  1  90  87  92  29°C  53  53  42  29  85  65  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  131  42  24  83  35  113  49  13  37  17  9  57  20.5°C 29°C  *  M5/N  102  20.5°C  1  M5/N;D£  MALE  1.  M5/wf NS 1 1 rb ? x wf; NS 1 1 rb/Y;D£ x?1  2.  M5/wa n S 1 1 rb;D£ ? x wf; NS 1 1 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 considerable reduction in the severity of the mutant eye phenotype seen in N sll /NS i:L ;Dp females.  The eyes of all N ^ / N S 1 1 ; D p / D p  females (cross 3, Table 11) were much less mutant in appearance than those of N g l l / N g u ;Dp females raised at the same temperature, 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 N S H / + , in that they generally had a more mutant facet array and were smaller at all temperatures . 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 N g l l /N s l l ;Dp/Dp females have less severely affected eyes than  N^VnS 1 1 ;Dp females, is consistent  with the other observations on gene dosage. A summary of the main effects of gene dosage and temperature 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 N 6 ^ 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;  sive facet disarray phenotypes:  occasional facet disarray.  extenWing  + = wild-type (no nicks, no thickened wing veins);  .2N, etc., indicates approximate frequency of Notch-winged individuals having a nick in one or both wing tips; Co = Confluens wings.  20 - 22 C  N ^ y N S " ;DP  N  9  25°C  29 °C  20-22 C  +  25°C  29°C  ON  .2 N  sn/N S";DP/DP 9  N g y N  WINGS  EYES  GENOTYPE  g n  9'  +  / y ;  +  .02 N  .1 -.2  N'  ,7-.8  Dp  +  Dp  N g"/+  ;  N ^"/Y  ; Dp  /Dp  Co  Co  Co  N  57 conditions.  Thus, in terms of wing nicking the N ^ ^ allele  behaves more like N^ (i.e., becomes less hypomorphic) at progressively lower temperatures. eye phenotype  On the other hand, in terms of the  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 N g l l /N g l l ;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 N gll/ Y ;Dp males, and therefore would not cause lethality), was excluded by the absence of lethality in the cross l(FM6)/we bb 1 $ x wf. NS 1 1 rb/Y;D£ 0^(165 w^ bb^/Y a", 150 l(FM6)/wa NS 1 1 rb $ , 178 wf_ bbVwf^ NS 1 * 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 Ng 1 1 and viability in this case.  determines the  Test matings of 11 fertile females  raised at 20.5°C (cross 3, Table 11) to w/Y males, yielded no w a /w female or w a /Y male progeny (out of 248 female and 98 male total progeny), confirming that these females were homozygous for Pp_. The lethality of N s l l /N s l l ;Pp females at low temperatures contrasts strikingly with the observed viability of  58 N ^ O / N ^ D p (Table 4) and N 10 3/N 10 3;Dp (Table 7) flies.  Thus,  lethality in the presence of N^ 1 1 is not due to some defect in the N^ allele carried by Dg_, but to the properties of N^ 1 1 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 N s l l /N s l l ;Dp females raised at 20°C-22°C had certain abnormalities, such as sparse thoracic microchaetae and frequently missing ocellar bristles, generally characteristic of Abruptex mutations.  Expression of this phenotype was reduced at 25°C  and was absent at 29°C, which is the same response to temperature observed for the eye phenotype.  This aspect of N^ 1 1 will  be referred to again. IV.  The  Effects of gene dosage on the phenotypes of  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 bb 1 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 N C o /+ females obtained from the cross OR $ x w^ N Co /Y;Dp cr*at 22°C, 42$ had a nick in one or both wingtips.  The non-nicked N C o /+ 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 N C o /+ and N Co /+;Dp wings in Plate 4), like  PLATE ij  Wings of a) N Co /+, b) N Co /+;Dg, and c) N Co /N Co ;Dp females raised at 22°C.  Magnification l8x.  61 that of N Co /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 N C o :N + ratio was determined from the progeny of the cross: at 22°C.  M5/wa N C o $ x w^ NCo/Y;Dp cf ,  The number of N Co /N Co ;Dp females (36 out of 240 off-  spring, with 40 expected) indicates that N Co /N Co ;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 N C o /+ females.  The Confluens phenotype of these females  was even more extreme than that of N Co /+;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 N Co /N Co ;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 N C o indicate a clear functional distinction 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 N C o , 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 N 2 ^ 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 N x /+.  ;Dp females are remarkably similar to those for  The few statistically significant differences are likely  due to differences in genetic background. wingtip nicking of N^^/N^O ;Dp and  The frequency of  N Co/ N 40  ;Dp females from crosses 4 and 5 (Table 12) is also quite similar to the frequency among the respective N x /+ females (note that 8l% of 74 N C o /+ progeny of the cross OR $ x wf^ N C o rb/Y;Dp C*, had nicked wingtips) . Furthermore, the disordered eye facet phenotype seen in females at low temperature was also expressed in the N gll / N 40. D p  females at 20°C-22°C, and the strong wing nicking  and tarsal fusion seen in N 1 0 3/ females at 29°C was expressed +  63 Number of wings of N x /N lt0 ;Dp females exhibiting wing nicking when raised at different temperatures.  TABLE 12  GENOTYPE  CROSS*  TEMPER -ATURE  % NICKED INDIVIDUALS  N1Q3/N40;Dp  1  20°C 22°C 25°C 29°C  82 73 99 100  Ngll/N40.Dp  2  20°C 22°C 25°C 29°C  7 7 38 82  3  20° C 22°C 25°C 29°C  4 5 33 61  4  20.5°C 29°C  9 83  20.5° C  73  Ng^/N^SDP N C o /N 4 o ;Dp  *  5  MEAN NUMBER OP NICKED WINGTIPS PER FLY (± CONFIDENCE INTERVAL)  NUMBER OF FLIES EXAMINED  1.38 + .14 1.22 + .18 1.84 + .07 2.00  104 67 88 72  + .09 + .08 + .18 + .3 0.04 2; .05 0.05 + .05 0.40 + .19 0.8 + .3  59 59 55 22  0.10 0.08 0.55 1.4  mm  -  52 65 55 18 81 86 45  wf; N 1 Q 3 /Y;Dp .<?»  1.  M5/wf N ^ rb $ x  2.  M5/wf;  rb $ x wf; N g l 1 rb/Y;D£ o*  3.  M5/wf;  rb ;D£ ? x wf; N ^ 1 1 rb/Bf_ w^. Y c^  4.  1 (FM6)/wa N S 1 1 rb ? x wf;  5.  l(FM6)/w a N C o rb E - N 7 0 k 2 7 b b 1 £ x wf; N ^ rb/Y;D£..<^  rb/Y;D£ <f  64 in the N 1 0 3 / N ^ Q ; D p females at this temperature. of the  Also, the wings  N Co / N 40  ;Dp females were similar to those of N appearance.  The recessive behaviour of  /+ in  to the wing nicking,  eye, and leg phenotypes of the other N mutants, and the absence of a strong Confluens phenotype in  N Co / N 40  ;Dp females, strongly  supports the assumption that N^^ can be regarded as an amorphic allele.  Observations made on limited numbers of  and  N Co /N 8 ;Dp females raised at 20.5°C (24 and 5 flies from the crosses N8/dl49,y Hw m£ $ x wf^ NS 1 1 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 N C o , 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/+ heterozygotes reflects hypomorphic activity, no wing nicking was observed among 89 N 1 ° 3 /NS 11 ;Dp (22°C), 33 N 1 0 3 /N C o ;Dp (22°C), and 213 NS 1 l/N Co ;Dp (20.5°C)* females, a striking reduction compared to the respective N/+ heterozygotes.  Moreover, the eye  ^Heterologous combinations obtained from the respective crosses: m5/jr  N  M5/y  N103  1 0 3  $  x  w^ N  s 1 1  rb/Y;D£ <?  x wf: N Co /Y;Dp <?  1 (FM6)/wa NS 1 1 rb % x wf N ^ rb/Y;D£ cf9  65 phenotype of N 1 ° 3 /NS 1 1 ;Dp and the Confluens phenotype of N 1 Q 3 / ]vjCo .pp females were intermediate between those of the respective N/+  and  N/+;Djd  combinations, indicating that  N  1 0  3  between N^ and n£ or N*^ in these respects also. wing vein phenotypes of  is intermediate The eye and  N gll / N Co  ;Dp, on the other hand, were essentially identical to those of the respective N/+ heterozygotes, suggesting that N^ 1 1 is amorphic or at least very hypomorphic 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 exemplified by the wing nicking and Confluens phenotypes of  .  66 B.  ORIGIN AND MAPPING OP THE ABRUPTEX MUTATIONS  Pour of the five Ax alleles studied (Ax 51 , Ax E 2 , Ax l 6 l 72 and A x 9 B 2 ) 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 Ax 1 ^ 1 ? 2 and A x 9 B 2 were also EMS-induced (LEFEVREj WELSHONS, personal communications).  Ax E 1 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.  Ax E 2 is not as extreme as Ax E 1 and is  both viable and fertile as a hemi- and homozygote.  The Ax  alleles, Ax 1 ^ 1 ? 2 , A x 9 6 2 and Ax^ 9d , were kindly supplied by Dr. W. J. WELSHONS.  A x l 6 1 7 2 and A x 9 B 2 are viable in the hemi-  zygous and homozygous condition, although homozygous A x 9 B 2 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 genetically within the Notch locus between spl and N C o (WELSHONS 1971). The salivary gland chromosomes of Ax E 1 , Ax E 2 ,  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,  Ax E 1 , Ax E 2 , and Ax9B2 have been mapped genetically at sites within the Notch locus. In the mapping of Ax E 1 , advantage was taken of the facts that Axe-*- is viable and fertile when heterozygous with the nonAbruptex recessive visible mutations in the Notch locus, and that the A x E V n and fa no /N genotypes are lethal.  Thus, in the cross  wi fa no spl rb/w^. Ax E 1 rb + g x wf N ^ rb/B^ w^.Y  , the only  surviving female progeny should be non-disjunctants, "breakthrough" Ax E1 /N and fa no /N females, or fa n o + Ax + crossovers between fa n o and AxE-*-. sented in Table 13.  The results of two such crosses are pre-  It can be seen that there were appreciable p1  numbers of surviving Ax  /N and fa n o spl/N females, which were  very weak and sterile and could easily be recognized phenotypically.  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 Abruptexlike (wing vein gaps) or had nicked wings.  Seven of the excep-  tional females survived long enough to be test-crossed to w a 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  SERIES*  TOTALS  Results of cross for the genetic localization of Ax E1 .  NUMBER OF MALE PROGENY  GENOTYPE OF SURVIVING FEMALE PROGENY Bf.  N/Ax E1  46,986  31  160  17  11,542  25  184  10  56  344  27  58,528  w^ S£l rb  14  w^ rb^  N/fa no spl  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 w a fa no spl rb, w a N2*0 rb, and w^ Ax E 1 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 fa no and Ax E 1 , and position Ax E 1 to the right of fa110.  From these data no decision can  be made as to the position of Ax E 1 with respect to spl, although it would seem that if Ax E 1 is to the right of spl, the two mutants must be extremely closely linked.  This is based on the  fact that 14 crossovers between fa no and AxE-L were recovered, but none between AxE-L and spl.  The frequency of recombination  between fa no and Ax E 1 (Table 13) is 0.05$, which is greater than the map distance between fa no SHONS (1958).  and spl (0.03$) recorded by WEL-  Unfortunately, the present results and the data  of WELSHONS cannot be compared in  order to position Ax E 1 with  respect to spl, since genetic background, temperature and other culture conditions were likely different in the two investigations.  Nevertheless, it is probably safe to conclude that Ax E 1  maps within the Notch locus, close to or at spl. In order to map AxF  P  with respect to other mutants at the  70 Notch locus, all of the male progeny of wf: fa no spl rb/w* Ax E 2 rb + females were scored for their visible phenotypes.  The  results of two experiments are presented in Table 14.  For  positioning Ax E 2 , the relevant recombinants recovered were the w a fa no Ax E 2 , spl rb, w a fa no spl Ax E 2 , and rb_ classes, which place Ax E 2 to the right of spl (see Appendix 6 for progeny tests When the double crossover classes w a  of these recombinants).  fa no rb and fa no Ax E 2 are included, the genetic map distance between fa no and spl is calculated to be 0.05 unit and between spl and A X e 2 0.01 unit.  This observation, combined with the  fact that spl lies approximately equidistant between fa no and nd in WELSHONS' map (1965) suggests strongly that Ax E 2 lies within the presently defined limits of the Notch locus.  It should be  noted at this point that when linked in the cis position, fa no completely suppresses the wing vein but not the bristle phenotypes of Ax E 2 .  This and other interactions of Ax E 2 with fa no  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 Ax E 2 /  Ax 9 B 2 and Ax 1 ^ 1 72/ Ax 9B2 combinations are lethal, while Ax E 2 / Ax l6l72  females are both viable and fertile.  Thus, in the  cross wa A X E 2 rb/w^ A x 1 6 1 7 2 rb+ $ x w^ Ax 9 B 2 rb/Y CF*, the only surviving female progeny should be Ax* recombinants or nondisjunctants, 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 Ax E2 .  CROSS*  MALE PROGENY GENOTYPES  PERCENT CROSSOVERS  w a fa no S£l rb  8 ,185  8,295  +  8 ,052  8,045  179  130  164  150  3  3  2  7  0  2  1  0  w a fa no s£l +  493  464  + Axe2  609  463  0  1  1  0  17,689  17,560  Ax  e 2  +  w a A x E2 +  + fa no spl rb w a fa no  Ax  +  e 2  + spl rb w a fa no spl  Ax  + + rb  rb  w a fa no rb + fa no  Ax  TOTALS *  e 2  +  e 2  +  0.05s*  0.01***  5.76  fa no spl rb/AxE2 ? x £ wf/Y. <?  1.  w  2  w^ fa no spl rb/AxE2 $ x wf fa no spl rb/Y cfl  .  1.77  Cross 1 consisted of 12 cultures, cross 2 of 18, in halfpint 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 w a fa no rb and + fa no Ax E 2 + Includes double crossover  w a fa no rb  72 regions as observed in the males occurred with normal frequencies. This indicates that Ax E 2 and  A x 16172  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^ 5 2 are reported in Table 15. between  The five Ax-N recombinants recovered place Ax9B2  and  .  The observed frequencies of crossing over  within the Notch locus (0.09% between both N1*0 and Ax$ B 2 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^ 6 2 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 fa n o spl A x e 2 rb stock.  Nevertheless, the available crossover  data and the origin of Ax^  , indicate that Ax9B2  is  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.  CROSS* PROGENY CROSSOVER CLASS  GENOTYPE  NUMBER  + Ax +  3017  %  GENOTYPE ' NUMBER + Ax rb  %  22 59  w a -Ax  w  Ax +  55  1.72  w a Ax rb  28  1.24  Ax-rb  + Ax rb  125  3.91  + Ax +  91  4.02  Ax-N  + + rb  3  0.09  w a + rb  2  0.09  * Male progeny of crosses: 1.  wf; N ^ rb/+Ax9 B2 + ? x wf fa n o spl Ax E 2 rb/Y *f  2.  wf; N £ f _  +/+Ax9b2  rb ? x wf; fa n o spl Ax E 2 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.  Ax  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 postverticals 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 FT in lines 1 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. El that in the Ax  It can be seen  /+ 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 background genotypes are different), the effect of autosomal inserts is a deletion tion of the Notch locus can be measured since N°  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  GENOTYPE  CROSS*  NUMBER OF OCELLARS 0  1  NUMBER OF POSTVERTICALS 2  0  1  2  WING VEIN GAPS** - 1 OR MORE 0  L5  L4 & L5  Ax E1 /+ ?  1  55  34  8  94  3  0  4  89  4  AX E1 /+ $  2  72  16  2  72  9  9  0  75  15  Ax E1 /+;D£ ?  2  1  5  88  32  29  33  46  48  0  Ax E1 /N 8 ;D£ $  3  15  2  2  19  0  0  0  18  1  AX E1 /N 4o ;D£ £ 4  62  10  1  62  8  3  0  73  -  AXE1/Y;D£0^  60  7  1  68  0  0  11  57  -  *  *  4  1.  OR ? x Ax E1 /B s 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 N 2 ^ (line 5) interacts similarly to  N 8 , 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 Ax E1 /N Co ;Dp females from the cross l(FM6)/AxE1 ? x wf^ N C o rb/Y;D£ & exhibited 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 Ax E 1 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 prevented 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. bristle data for  Ax  e : l  /Y  The  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 twothirds, respectively.  In contrast to the drastic reduction in  77 TABLE 17  E1  Counts of bristles in eclosed Ax /Y males* raised at 20.5°C.  NUMBER OP PLIES IN EACH PHENOTYPIC CLASS TYPE OF BRISTLE  0  2  1  MEAN NUMBER OF BRISTLES PER FLY (± 95% CONFIDENCE INTERVAL)  3  4  5  6 1  3.57 ± .16  ORBITALS  0  0  9  32  36  10  OCELLARS  88  0  0  -  -  -  -  0.00  POSTVERTICALS  88  0  0  -  -  -  -  0.00  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  VERTICALS  0.72 ± .14  Data are the pooled observations on Ax E1 /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 , f a no A x E 2 j f a no 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 present in wild-type frequency.  Homozygous AxEl females (which  occasionally eclosed in l(FM6)/AxE1 £ x Ax E1 /B s 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 deformity 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.  Axe2.  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). but to a lesser extent.  Other bristles are also affected,  The results of sample bristle and wing  vein gap counts of hemizygous and homozygous Ax E 2 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-difference 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  TABLE 18  E2 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  Ax E2 /Y cf*  0.66  0.64t  1.00  0.46T  1.00  0.38T  1.00  0.99T  AX E2 /AX E2 $  0.67  0.62t  0.97  0.15TS  1.00  0.l4TS  1.00  0.99  AX E2 /+ %  0.99H  0.95th  1.00  0.95™  1.00  1.00H  1.00  1.00  DORSOCENTRALS  ANTERIOR  SCUTELLARS POSTERIOR  WING VEINS 22°C  29°C  0.97T  0.72  0.39T  1.00  0.97T  0.71  0.54TS  1.00  1.00H  0.88H  0.81™  GENOTYPE  22° C  29°C  22°C  29°C  22° C  Ax E2 /Y a*  1.00  0.89T  1.00  0. 7^T  1.00  Ax E2 /AX E2 £  1.00  0.91T  1.00  0.86TS  AX E2 /+ ?  1.00  0.97™  1.00  1.00H  • 29°C  * 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 formulae 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  Axe2/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 A x ^ 1 ^ 2 hemi- and homozygotes is one of markedly reduced head and thoracic bristle frequencies, and extensive gaps in wing venation. types are much more extreme than those of Ax E2 .  These phenoIn 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 Ax 1 ^ 1 7 2 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 temperature 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 A x 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 GENOTYPE Ax l6l72 /y  #  Ax l6l72 /Ax l6l 7 2 A x 16172 / +  £  %  Ax 16172 /y  j  Axl6l72/Ax16172 A x 16172 / + ^  POSTVERTICALS  VERTICALS  22°C  29°C  22°C  29° C  22° C  29°C  22°C  29°C  0.26  0.53T  0.00  0.03  0.00  0.00  0.97  0.98  0.16s  0.42TS  0.00  0.01  0.00  0.00  0.98  0.99  0.68H  0.70H  0.80H  0.39™  0.43H  0.31H  1.00H  1.00H  DORSOCENTRALS GENOTYPE  OCELLARS  * §  ANTERIOR  SCUTELLARS POSTERIOR  WING VEINS  22° C  29°C  22° C  29°C  22°C  29°C  22°C  29°C  0.76  0.38T  0.35  0.l4T  0.90  T 0.37  0.17  0.14  0.68s  0.20TS  0.56S  0.24TS  0.96  0.49TS  0.44S  0.33 TS  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  Ax l6l72/+  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 heterozygotes, they were in the same direction. can be summarized briefly as follows:  The data for  Ax16172  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) expression of all thecompared mutant phenotypes of l6l72 Ax/+ heterozygotes to homozygotes. Ax IV.  1s  reduced in  Ax9B2.  The characteristic features of Ax 9 B 2 hemi- and homozygotes are marked reductions in the frequencies of ocellar, postvertical, 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  A x 9B2  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 postvertical but not the posterior scutellar frequency (Appendix 10). These differences likely result from genetic background differences, 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 interruptions 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^ B 2 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 interactions 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  Summary of the bristle and wing vein gap phenotypes of A x 9 B 2 * #  TABLE 20  ORBITALS  OCELLARS  POSTVERTICALS  VERTICALS  GENOTYPE  22°C  29°C  22°C  29°C  22°C  29°C  22°C  29°C  Ax5 02 /Y cf  0.99  0.99  0 .00  0.04  0.00  0.22T  0.99  0.99  0.99  0.99  0.01  0.00  0.00  0.20t  0.99  1.00  AX9B2/Ax9B2 AX5B2 /+  ?  0.91H  ?  -  DORSOCENTRALS GENOTYPE A x 9B2 / y  ^  Ax9B2 /Ax 9B2 AX9B2/+  ?  ?  0.06H  0.01  -  ANTERIOR  -  SCUTELLARS POSTERIOR  1.00  -  WING VEINS  22°C  29°C  22°C  29° C  22°C  29°C  22° C  29°C  0.27  0.48t  0.79  0.69  0.92  0.77T  0.78  0.51T  0.35S  0.50t  0.84  0.74  0.99S  0.85T  0.79  0.45T  0.80H  -  1.00H  -  1.00  -  *  See footnotes to Table 18.  #  See Appendix 10 for actual counts of bristles and wing vein gaps.  0.99H  -  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 Ax9B23 0 n the one hand, and Ax E 2 and  AX 16172 5  on the other.  This is particularly  obvious with Ax E 2 and Ax9B2,  since Ax E2 has a fully penetrant  orbital phenotype, with only minor loss of other bristles, whereas  Ax 9B2  has fully penetrant expression of ocellar, postvertical, and dorsocentral phenotypes, with only minor orbital E2 loss. Furthermore, in both Ax and Ax l6l72 than theremales were for instances of females being either more or less mutant 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.  E2 Ax  Ax16172  : :Ax9b2  ORBITALS  XX  XX  0  0CELLARS  0  XX  XX  POSTVERTICALS  0  XX  XX  DORSOCENTRALS  0  X  XX  ANTERIOR SCUTELLARS  0  X  XX  XX  WING VEIN GAPS  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 differences in bristle and wing vein gap frequencies observed in the viable Ax strains.  Ax-E2  0CELLARS  9 > 0»  POSTVERTICALS  ¥ >  ? > a*  9 < o*  9 < o*  9 < a*  9 < a»  9 < <?  POSTERIOR SCUTELLARS  *  9 < tf  (fi  DORSOCENTRALS  WING VEIN GAPS  Ax 9B2  9 > d>*  ORBITALS  ANTERIOR SCUTELLARS  Ax 16172  9 < cr7  ? < o*  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, Ax E 1 , which was originally detected on the basis of its lethality with and  , was lethal when heterozygous with N^^ 1 , N C o ,  but viable with N 1 0 3 at 20°C-22°C.  Death of Ax59d/N  FX  and Ax *VN females occurs mainly in the pupal stages, as is the case with Ax-^^ and Ax E ^ hemizygotes and homozygotes.  In con-  trast to these two mutants, Ax E 2 , A x 1 ^ 7 2 , and Ax^ 6 2 , which survive as hemi- and homozygotes, were viable as Ax/N heterozygotes at 20.5°C-22°C, although there did appear to be some reduced survival of Ax E2 /N lj0 , Ax E 2 /N C o , and A x 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, particularly those involving A x 1 ^ 1 7 2 , were poorly viable, the reduced survival being associated with a corresponding increase in pupal death.  The relatively greater lethality of A x 1 ^ 1 7 2 ^  at 29°C is not paralleled by lethality of Ax-^172 hemizygotes or homozygotes at this temperature.  TABLE 23  90 Summary of the viability of heterozygous combinations of different Ax and N alleles.  NOTCH ABRUPTEX ALLELE  Ax59d  TEMPERATURE  Ax E 1  20-22°C 29°C  Ax E 2  20-22°C 29°C  16172  20-22°C 29°C  Ax  9 6 2  20-22°C 29°C  * L  N ^  • • • N.10.3  .  N®11  -  L  L  _  -  -  L  L  -  L  L  V  L  L  -  -  L  L  -  V  V  V  V  V  V  V  V  V  V  V  V  V  V  V  L  L  L  L  V  V  V  V  V  V  V  V  V  V  R  R  R  V  R  = lethal or semilethal (0-5$ survival in relation to siblings).  •p V^ = reduced viability (5-30$ survival in relation to siblings). V  NC°  L*  20.-22° C 29°C  A x  a!  ALLELE  = viable (greater than 30% survival).  R  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 Ax E 2 , Ax 1 ^ 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 Ax 2 8 a /N 8 heterozygotes (MOHR 1932), and indicates 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 Ax 9 B 2 with  N ^ , and N ^  TABLE 24  E2 Summary of the bristle and wing vein gap phenotypes of Ax /N heterozygotes.*# ORBITALS  GENOTYPE  OCELLARS  POSTVERTICALS  VERTICALS ' 29°C 22°C  22°C  29°C  22° C  29°C  AxE2/N8  0.97 Su  O.78TSU  l.OOSu  O.99SU  1.00  1.00 Su  1.00  0.99  AX E 2 /N 4 °  0.96 Su  0 . 7 9 TSU  1.00su  O.95SU  0.99  1.00 Su  1.00  0.99  AX E 2 /N C o  0.98SU  0.86TSU  1.00 Su  0.99 Su  1.00  1.00 Su  1.00  1.00  A x E2 / n 103  0.68^  0.76 TSu  l.OOSu  O.99SU  1.00  1.00 Su  1.00  1.00  Ax^/N®11  0.95 Su  0.98 Su  0.99  1.00 Su  1.00  1.00 Su  1.00  1.00  22°C  29°C  SCUTELLARS GENOTYPE  DORSOCENTRALS 29°C 22°C  ANTERIOR 22°C 29°C  POSTERIOR 22° C 29°C  WING VEINS 22°C 29 °C  AxE2/n8  1.00  1.00 Su  1.00  O.99SU  1.00  1.00  0.95 Su  0.93 Su  AXE2/N^  1.00  1.00Su  1.00  1.00 Su  0.99  1.00  0.98 Su  0.98 Su  A x E2 / n CO  1.00  1.00Su  1.00  0.97 Su  1.00  1.00  0.99 Su  0.98 Su  AX E 2 /N 1 0 3  1.00  O.99SU  1.00  O.99SU  1.00  1.00  TSu 0.73 SuL 0 ,88  1.00  1.00 Su  1.00  0.99 SU  1.00  0. 99  0,82 SuL 0.93 T S u  Ax  E2  * # T Su L  / N gll  See footnote to Table 18. See Appendix 11 for actual counts of bristles and wing vein gaps. Statistically significant difference between the 29°C and the 22°C frequency. Statistically significant suppression of Ax phenotypes compared to Ax/Ax females. 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  ' POSTVERTICALS  OCELLARS  22° C  29°C  22° C  29° C  '22° C  29°C  AX 1 6 1 7 2 / N 8  0.70 Su  0.68 Su  1.00 Su  1.00 Su  1.00 Su  O.97SU  Ax 16172 /n 40  O.73SU  1.00 Su  Ax 16172 /n CO  0.82 Su  1.00 Su  -  Ax 16172 /n 103  0.57 SuL  0.12sUL  -  Ax l6l72 /N gll  0.63 SuL  O.93SUL  -  GENOTYPE  -  1.00 Su  -  -  0. 99 S u  -  0.60 SuL  -  0.38 SuL  -  VERTICALS 22° C 0.99 1.00 Su 1.00 Su  290 c 0.98 -  -  1.00 Su  -  1.00 Su  -  SCUTELLARS DORSOCENTRALS  POSTERIOR  ANTERIOR  22°C  29°C  22°C  29° C  22° C  A x l6l 7 2 / N 8  1.00 Su  0.99 Su  l.OOSu  0.98Su  O.99SU  A x 161 7 2 / n 40  1.00 Su  O.99SU  Ax 16172 /n CO  1.00 Su  1.00 Su  A x l6l 7 2 / N 103  1.00 Su  0.97 Su  Ax l6l72 /N gll  1.00 Su  0.80 Su L  GENOTYPE  * §  29° C 1.00 Su  • ' WINGVEINS 22°C 0.75 Su  290 c 0.75 Su  O.99SU  -  0.75 Su  -  -  1.00 Su  -  0.76 Su  -  -  0.99  -  0.72 SuL  -  -  1.00 Su  -  0.62 SuL  -  -  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 Ax9 B2 /N heterozygotes. * # ORBITALS  POSTVERTICALS  29°C  22°C  29°C  22°'C  0.99  0.91 te  0.11su  0.17 Su  0.82SU  0.99  0.87te  0.08 Su  0.11 Su  0.99  0.94 te  0.63 Su  A x 9B2 / n 103  0.74e  0.95t  Ax9B2/NgH  0.85e  0.97t  GENOTYPE A x 9B2 / n 8 A x 9B2 / n 40 A x 9B2 / n CO  22°C  OCELLARS  290 c  ' VERTICALS  29 0 c  • 22° C  0.3^  1.00  0.90 TE  0.8l Su  0 . 5 1 TSU  1.00  1.00  o.o6Su  0.84s^  0.64 T S u  1.00  1.00  0.63 Su  0.00T  0.92 Su  0.80 Su  1.00  1.00  0.00  0.02  0.01  0.92 T S u  1.00  1.00  SCUTELLARS DORSOCENTRALS  ANTERIOR  POSTERIOR 22°C  29°C  WING VEINS  GENOTYPE  22°C  29°C  22° C  29°C  22° C  AX 9 B 2 /N 8  0.8l Su  0.58 TSu  1.00 Su  O.99SU  1.00  1.00 Su  0.86 Su 0 .63 TSu  AX9B2/N40  0.82 Su  0.62tSu  1.00 Su  1.00 Su  0.99  1.00 Su  Su 0.93 Su 0 .90  A x 9B2 / n CO  0.75 Su  0.51 TL  1.00 Su  O.99SU  0.99  1.00 Su  TSu 0.89 Su 0 .8l  A x 9B2 / n 103  0.82 Su  1.00 TSu  1.00 Su  1.00 Su  0.98  1.00 Su  0.60E  Ax9132^11  l 0.51 SuL 0.54  0.32E  1.00 TS u  0.99  0.96  0. 68e  * 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 Ax9 B2 /Ax°  29° C  0 #92 TSU  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 Ax E 2 with these N mutants, orbital bristle frequencies xvere significantly reduced at 29°C compared to 22°C, although they were still less mutant than Axe2  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 which both N 1 0 3 and nS-H (Tables 6 , 9 ) . survived at  the temperature at  exhibit stronger Notch phenotypes  Furthermore, the few  29°C,  29°C,  Ax l6l72 /N gll  flies which  also showed reduction of the Abruptex pheno-  types when compared to  22°C  flies (Appendix  The most glar-  12).  ing exception to this (the ocellars of A x  9B2  / n  103  s  Table  26),  can be accounted for by the observation that N 10 3/+ heterozygotes also show drastically reduced numbers of ocellar bristles at  29°C.  Thus, it appears that the exceptional behaviour of N-1-^3 a n d ^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 Ax E 1 /N 1 Q 3 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  Ax  E 1  /N  1 0  3  females  (Table 27) with those from Ax E1 /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 verticals, which were essentially wild type in each case).  It should  be remembered, however, that N 103 does not behave as a deficiency for the Notch locus at 22°C, and that the suppression of Ax E 1 phenotypes by  may reflect the presence of significant levels  of activity in the N 1 Q 3 gene product.  Comparison of the A x E V  N 1 0 3 data (Table 27) with that for Ax E1 /+ (Table 16), shows that  TABLE 27  Counts of bristles in Ax E 1 /N 1 0 3 females* raised at 22°C.  NUMBER OF FLIES IN EACH PHENOTYPIC CLASS  MEAN NUMBER OF BRISTLES PER FLY (± 95$ CONFIDENCE INTERVALS)  TYPE OF BRISTLE  0  1  2  3  4  5  6  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  -  -  DORSOCENTRALS  0  1  33  29  31  -  -  2.96 + .15  ANTERIOR SCUTELLARS  0  1  93  -  -  -  -  1.99 ± .02  POSTERIOR SCUTELLARS  0  0  94  4.00  4.00  •Progeny of the cross M5/£ wf. N 1 0 3 £ x Ax E1 /B S 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 A x ^ phenotypes. Prom the foregoing observations, notwithstanding the exceptions 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 mutations, 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 available Ax/N heterozygotes has revealed that the Ax mutants studied fall into two classes with respect to their effects on the N mutants.  Both Ax E 2 and Ax-^172 enhance wing nicking, whereas  Ax^ B2 and Ax E 1 suppress nicking. Axe2  and  The enhancement of nicking by  Ax 16172  is evident in the frequencies of nicking with N 1 0 3 , N C o , and N^ 1 1 (compare the data in Table 28 with Table 6 for N 1 Q 3 /+, and Table 9 for NS 11 /*; and note that H2% of N C o /+  TABLE 28  Nicking frequencies in wings of Ax/N heterozygotes.* AxE2  N ALLELE  Ax 16172 29°C  22° C  Ax  29°C  22°C  9B2  22 °C  29°C  0.02 ± .01L  1.48 + .17L  0.22 ± .08L  2.00  0.00J  0.94 ± .24  1.63 ± .19H  0.07 ± .04L  2.0  0.07 ± .06  0.00  0.42 + .21  N  2.00  2.00  2.00  N 40  2.00  2.00  2.00  N Co  1.99 ± .02H  2.00H  1.99 ± .02  N103  1.60 + .11H  2.00  Nsl1  0.19 ± .06H  1.97 ± .05H  2.00  H  *  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 enhanceF? ment of the wing nicking phenotype of N mutants by Ax c and A x 16172  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 Ax9 B2 /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  A x El / N 103  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 n 103/ + females at this temperature.  The suppression of wing  nicking has generally been observed when Ax E1 /N heterozygotes have been available, including the  breakthroughs re-  covered in the mapping of Ax E1 (Table 13).  The facts that Ax E 1  and Ax9b2  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 nicking by an Abruptex mutant. are two Inclasses summary, ofthe Abruptex foregoing mutation: observations 1) those indicate which that enhance there  101 the wing nicking of Notch mutants (AxE2 and A those which suppress wing nicking Ax 2 ^ a ).  >  and 2)  and Ax9 B 2 1 as well as  It has already been noted that Ax^ B2 differs qualita-  tively from A x E 2 and Ax 1 6 1 ? 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 combinations 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 Ax E ^ and Ax59d with the viable mutations Ax E 2 and  Ax161725  are lethal (as are heterozygotes).  This type of lethal interaction is reminiscent  of the lethality of most N mutants with fa no , and is hence perhaps not unexpected.  Surprisingly, however, the viable mutation  Ax 9 B 2 is lethal when heterozygous with the other two viable Abruptexes (AxE2 and A x 1 6 1 7 2 ) , while both Ax E 1 /Ax 9 B 2 ^xl6l72  are  viable,  and  Ax  E2  /  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 Ax 9 6 2 / A x E 2 and A x 9 B 2 / A x l 6 1 7 2 has been confirmed at both 22°C and 29°C (Table 30).  However, it can be seen in the control cross  (M5/Ax 9 B 2 $ x Ax 9B2 /Y cP ) that Ax 9 6 2 homo- and hemizygotes exhibit significant lethality (also pupal) at 29°C.  This cross  was started by crossing M5/M5 females to stock Ax 9 B 2 /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 autosomal temperature-sensitive modifier of Ax in the Ax 9 B 2 stock, which kills Ax 9 B 2 individuals at 29°C.  103 TABLE 29  AxE1  Summary of viability of various heterozygous combinations of Ax alleles at 22°C.  AxEl  Ax59d  Ax 9 6 2  L*  L  V  L  L  L  -  L  L  V  L  L  V  V  Ax^ Ax 9 5 2 Ax E 2 A x 16172  * L = lethal V = viable  A X  E 2  A x ^  v  2  104 TABLE 30  Relative viability of Ax 9 B 2 /Ax 9 B 2 , Ax 9 B 2 /Ax E 2 , and Ax 9 B 2 /Ax l 6 l 7 2 at 22°C,  PROGENY GENOTYPES'* FEMALES Axx  AX E 2  Ax16172  Ax 9 B 2  MALES  TEMPERATURE  M5/Axx  Ax 9 B 2 /Ax x  M5/.Y  ; Ax 9 B 2 /Y  22°C  124  0  100  122  29°C  131  0  101  121  22°C  86  0  76  94  29°0  136  0  76  98  22° 0  101  96  95  128  29° 0  151  23  85  38  * Progeny of the cross M5/AX 962 £ 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  on  wing nicking could not be determined, since none of the Ax^^/N combinations tested were viable. has not yet been checked.  The viability of  It is also noteworthy that the viable  combinations of the Notch-suppressing Ax's investigated in the present study ( A x 9 B 2 / A x 9 B 2 a female-sterile.  anc j  A x e 1 / A x 9 b 2 ) are essentially  In other experiments, furthermore, it has been  observed that Ax E VAx E 1 ;Dp females are sterile, although they lay many eggs.  This is similar to the observation that the combina-  tion Ax 5 9 b /Ax59d ;Dp j s 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 A x 9 B 2 / A  heterozygotes examined were recovered among progeny of the mapping 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 9B2  F?  /kx^"1,  cross were considered to be Ax  since no extreme Ax  females with wild-type eye colour were recovered).  The results  1 (Table show that the overall mutant appearance of surviving i 31) TT Oindividuals Ax•p */Ax is generally more extremely Abruptex than "p "1  is the case for Ax  IT O  males or Ax^^ males or females (compare  Table 31 with Tables 17 and 18).  Similarly, the Ax E 2 /Ax 9 B 2  females which managed to eclose were much more extremely mutant than either homozygote.  Comparison of the Ax E 2 /Ax 9 B 2 bristle  data (Table 31) with those for Ax E 2 and Ax 9 B 2 homozygotes (Tables 18, 20), shows that this is particularly obvious in the frequencies of the orbitals, verticals, dorsocentrals, and scutellars.  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 homozygotes.  In addition to the above observations, it was noted  that the meso- and meta-thoracic legs of Ax E 1 /Ax E 2 , Ax E 2 /Ax 9 B 2 , and  Ax E1 /Ax 16172  females were often deformed, the thoracic microchaetae 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 Ax E 2 /Ax l 6 l 7 2 and AxE1/Ax9B2 were intermediate between the respective 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  GENOTYPE  Summary of the bristle and wing vein phenotypes of heterozygous combinations of different Ax alleles at 20-22°C *#. ORBITALS  OCELLARS  POSTVERTICALS  VERTICALS  Ax E1 /Ax E2  0.01e  0.00  0.00  1.00  Ax E 1 /Ax 9 B 2  0.56  0.00  0.00  1.00  Ax E 2 /Ax 1 6 1 ? 2  0.451  0.00  0.00  0.99  AX E 2 /AX 9 B 2  0.00E  0.00  0.00  0.55E  GENOTYPE  DORSOCENTRALS  SCUTELLARS ANTERIOR POSTERIOR  Ax E1 /Ax E2  0 .08  0.00  0.98  AX E 1 /AX 9 B 2  0.301  0.071  0.871  AX E2 /Ax !6172  0.80 1  0.891  1.00  Ax E 2 /AX 9 B 2  0 .00E  0.43E  0.79E  WING VEINS  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 Ax E1 /Ax9 B2 females, but it was noted that the vein gapping was also intermediate between Ax E1 /Y males and Ax9 B2 /Ax9 B2 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  (Ax E1 /Ax E2 , Ax E1 /Ax 1 6l7 2 3 Ax E2 /Ax9 B2 ) all exhibit more extreme mutant phenotypes than those of the individual homo- or hemizygotes, whereas the viable genotypes (Ax E1 /Ax9 B2 and Ax E2 /Axl6l7 2 ) 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 complementation, having more extreme mutant phenotypes than either Ax homozygote alone and resulting in lethality, and 2) heterozygous 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 Ax E 1 , Ax E 2 , Ax l6l72 s  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 instar 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 collection, 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 NS 1 1  TABLE 32  CULTURE NUMBER  Eye and wing phenotypes of N s l l /+ adult females shifted from 20.5°C to 29°C at different successive intervals.  TIME OF SHIFT CULTURE DEVELOPMENTAL AGE (HR) STAGE  NUMBER OF FLIES IN EACH PHENOTYPIC CLASS R N*  R+N  R N+  R+N+  %R  I**  0  38  0  23  0  62  48  I, some II  0  28  0  27  0  51  3  72  II, some III  0  31  0  19  0  62  4  96  III  0  55  0  8  0  87  5  120  III  1  54  3  4  6  89  6  144  III, some P  9  0  54  0  100  14  7  168  P, some III  2  0  49  0  100  4  8  192  P  0  0  43  0  100  0  9  216  P  1  0  171  0  100  0.6  10  240  P  0  0  118  0  100  0  11  264  P  0  0  123  0  100  0  1  0  180  0  100  0.6  1  24  2  12  NOT SHIFTED  *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  CULTURE NUMBER  Eye and wing phenotypes of adult females shifted from 29°C to 20.5°C at different successive intervals.  TIME OP SHIFT CULTURE DEVELOPMENTAL AGE (HR) STAGE  NUMBER OF FLIES IN EACH PHENOTYPIC CLASS R N*  R+N  R N+  R+N+  %R  $N  1  24  I, some IIs  0  0  53  0  100  0  2  36  II, s ome I  1  0  16  0  100  6  3  48  II  4  0  51  0  100  7  4  60  III  7  0  64  0  100  10  5  72  III  8  0  100  1  99  7  6  84  III  1  8  0  9  6  50  7  96  0  13  0  15  0  46  8  108  P  0  28  0  12  0  70  9  132  P  0  9  0  8  0  52  10  144  P  0  20  0  8  0  71  0  9  0  9  0  50  11  P, some III  NOT SHIFTED  * See Table 32 for explanation of symbols t—• M1 I-  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 approximately 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 larval 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  CULTURE NUMBER  Eye and wing phenotypes of adult females shifted from 20.5°C to 29°C at different successive intervals (Experiment 2).  TIME OF SHIFT HOURS AFTER DEVELOPMENTAL EGG HATCH STAGE  NUMBER OP FLIES IN EACH PHENOTYPIC CLASS R N*  R+N  R N+  R+N+  I*  0  8  0  7  0  53  56N  1  0  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  CULTURE NUMBER  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 HOURS AFTER DEVELOPMENTAL STAGE EGG HATCH  NUMBER OF FLIES IN EACH PHENOTYPIC CLASS . N* R + N  R N+  R+N+  %R  %N  0  0  21  0  100  0  II  0  0  28  0  100  0  36  III  0  0  32  0  100  0  4  43  III  1  0  25  0  100  4  5  48  III  0  0  25  0  100  0  6  60  III  3  0  25  0  100  11  7  66  III  7  0  23  0  100  23  8  72  III, some P  5  7  11  5  57  43  9  84  P, some III  0  18  0  14  0  56  10  96  P  0  13  0  10  0  57  1  12  2  24  3  I*  * 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 frequency 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 2 s .  NUMBER OP PLIES WITH BOUNDARY IN GIVEN REGION OF EYE VIAL NUMBER  1**  2  3  4  5  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  6  * 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  0  0  19  5  posterior  0  4  15  4  1  0  anterior  0  0  0  1  17  5  posterior  0  5  3  6  9  0  anterior  0  0  0  1  10  3  posterior  0  0  0  8  5  1  7  8  * Data were obtained from flies recorded in Table 35. ** See Figure 5.  120  TABLE 38  EXPERIMENT  Correlation of the occurrence of wingtip nicking with position of the boundary of mutant eye tissue.  MUTANT EYE TISSUE BOUNDARY IN REGION  WING PHENOTYPE*  SHIPT-UP (anterior boundary)  N  4  17  4  1  0  N  2  12  27  16  19  SHIFT-DOWN (posterior boundary)  N  0  0  1  4  4  N  0  9  17  14  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 increased 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 isolation (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 72hour 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 N s l l /+ 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; • B  = number of flies with wild-type wings;  = number of flies with one or both wings nicked.  the anterior rim of the eye is to the left.  Note that  a.  SHIFT-UP  u U1H >0 az u X CL o. >  •EYE  in w—  TSP  20-  fc.Ul  10-  ii o  Hi 0 HOURS  b.  To FROM  SHIFT  TO  —j—  —r~ 30  20  40  PUPATION  SHIFT-DOWN  hi Q.  >•  Uh >0 HZ 1 x11 0.  Ki i  in si 2o^ Q  EYE  TSP-  •H  U.U  10If ii  hs  10 HOURS  FROM  SHIFT  20 TO PUPATION  30  ESg  ~40"  "50  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 isolated 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& ments with Nell /+ 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 0 V s p l - 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 communication; 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 N 103 /spl flies  resemble those of N g l l /+ 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 N 1Q3 /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-prepupal 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 more precisely.  was used to time these events  Eggs from the cross M5/y w a N 1 0 3 $ x spl/Y cr*  were collected at 22°C on food in petri plates in successive 12hour 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 appearance 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 obK 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 TSPs, with respect to puparium formation. It should beeye 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 N 103 /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=  eye facet arrangement approaches wild-type;  = strongly mutant eye facet arrangement; ing;  •  = tarsal fusion; 6'= ocellar bristles; & = dorsocentral  bristles; A bristles.  SI = wingtip nick-  = anterior scutellar bristles;  = vertical  Note that the anterior rim of the eye is to the left.  a. S H I F T - U P <  A P P R O X I M A T E  E Y E  T S P  >  lil a. Id H >- O ui z ui x o.  + 2.0 -I—  lil D. > • H Q J + 1.0  O H Z  o  H < J Ui DC Z  o  z UI J H (0 cc cn  0;0.  -  1 .0  T H I R D  P R E P U P A  I N S T A R  • 2.0  too og O (/) Z D 5 u— -J z < CD O CC z <  o -{  I  h  30  20  10  0  B E F O R E  T I M E OF S H I F T (HRS) FORMATION  10 A F T E R  B E F O R E OR A F T E R  PUPARIUM  b.  SHIFT-DOWN  -f 2.0  + D . >• !'!  t.o  H Q J 0 H z 0 p  o  0.0  o--  <  J I Ed  -o  ' 1 .0 0 z l il J t U)  a a  — 2.0  T H I R D  I N S T A R  P R E P U P A  T  < U > K < H  a. o  si  5?  40  30 .  BEFORE  420  T I M E OF S H I F T ( H R S . ) PUPARIUM FORMATION  + 10 B E F O R E OR A F T E R  AFTER  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 definitely attributed to one or the other of these mutations. To summarize briefly, the results of the temperature-shift experiments on M ^ V s p l females have shown that:  1) the eye  facet and wing nicking phenotypes of N 1 0 3 / 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, appearance 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 migrated anteriorly across the eye in a vertical band.  Since the eye  TSPs for both N s l l /+ and N 103 /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 sensitive 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. kept at 20.5°C.  The other set of plates was  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 irradiated for 25 seconds from a Cobalt-60 source delivering approximately 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 recovered 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 N g l l /+ and N 1Q 3/spl, the end of the RSP for eye facet arrangement is not displaced at 29°C compared to 20.5°C. IV.  A x 1 6 1 7 2 / ^ 0 - TSP for lethality; Ax l 6 l 7 2 /+ - 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).  Ax 1 ^ 1 7 2 /* 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 Ax 1 6 1 7 2 /!^ 0  TABLE 39  TEMPERATURE 20 .5°C  29°C  Number of rows of disrupted ommatidia in adult OR flies irradiated before or after puparium formation, at 20.5°C and 29°C.  TIME OF RADIATION WITH RESPECT TO PUPARIUM FORMATION  NUMBER OF ROWS OF IRREGULAR OMMATIDIA AT ANTERIOR EDGE OF EYE AVERAGE  RANGE  (WILD TYPE)  17 hr. after (pupae)  NUMBER OF EYES SCORED 14  6.5 hr. after (prepupae)  3.5  3-4  10  0.5 hr. after (prepupae)  7.3  6-9  10  1.5 hr. before (3rd instar)  9.3  8-10  16  6.5 hr. before (3rd instar)  11.8  10-13  22  17.5 hr. before (3rd instar)  13.5  10-16  12  6  (WILD TYPE)  17 hr. after (pupae) 0.5 hr. after (prepupae)  7.5  6-9  18  1.5 hr. before (3rd instar)  9.5  7-11  26  6.5 hr. before (3rd instar)  13.5  12-15  18  9-16  4  18 hr. before (3rd instar)  *  * 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. eye is to the left.  Note that the anterior rim of the  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  Ax 16172 /+  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 intervals 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  Ax 16172 /n 40  females.  This indicates  that the TSP for Ax/N lethality occurs in the second larval instar 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 viability (Table 42), whereas in the pulse-down series, 24-hour incubations at 22°C during the second instar rescue most of the Ax/N heterozygotes from pupal death (Table 43).  The latter result  Data indicating viability of Ax 1 6 1 7 2 /!^ 0 females relative to their sibs when shifted from 22°C to 29°C at different successive intervals.  TABLE 40  CULTURE NUMBER  TIME OF SHIFT CULTURE DEVELOPMENTAL AGE (HR) STAGE  NUMBER OF PROGENY VIABLE ADULTS M5/Ax $  DEAD PUPAE Ax/N £  % SURVIVAL OF Ax/N FEMALES*  Ax/N $  M5/Y c^  85  1  75  86  2  1  24  2  48  I  87  5  43  75  6  3  72  II  33  18  57  33  35  4  96.5  70  79  71  1  99  III, some II  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  NOT SHIFTED UP  66  60  49  1  98  13  * % 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  CULTURE NUMBER  Data indicating viability of females relative to their sibs when shifted from 29°C to 22°C at different successive intervals.  TIME OF SHIFT DEVELOPMENTAL CULTURE AGE (HR) STAGE I**  NUMBER OF PROGENY VIABLE ADULTS M5/Ax $  Ax/N $  M5/Y  cf  DEAD PUPAE Ax/N $  % SURVIVAL OF Ax/N FEMALES*  49  45  51  0  100  II  26  20  28  1  95  !E X 2 IX Dl  27  17  29  4  81  59  3  62  64  4  III3 some P  54  1  36  47  2  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  102  3  83  109  3  1  18  2  36  3  54  4  72  5  90  6  11  HI  NOT SHIFTED DOWN  * See Table 40. ** A = adult stage.  See Table 32 for other symbols. UJ OO  TABLE 42  CULTURE NUMBER  Data indicating viability of Ax l 6 l 7 2 /N females relative to their sibs when pulsed from 22 °C to 29°C and back after 18 hours, at different successive intervals.  TIME OF PULSE-UP CULTURE DEVELOPMENTAL AGE (HR) STAGE (UP)  NUMBER OF PROGENY VIABLE ADULTS M5/Ax $  DEAD PUPAE  Ax/N $  M5/Y cr*  Ax/N $  % SURVIVAL 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  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  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  III, some II  P, some III  * See Table 40. »* See Tables 32, 40 for explanation of symbols  •  u> V£>  TABLE 43  CULTURE NUMBER  Data indicating viability of  Ax 16172 /n 40  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 DEVELOPMENTAL AGE (HR) STAGE (DOWN) I**  NUMBER OF PROGENY VIABLE ADULTS Ax/N $  M5/Y 0*  Ax/N $  % SURVIVAL OF Ax/N FEMALES*  44  4  35  38  10  50  38  23  11  78  M5/Ax $  DEAD PUPAE  1  18-42  2  36-61  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  II  * See Table 40. ** See Table 32 for explanation of symbols.  -tr  O  l4l 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 9To 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/Ax 1 ^ 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 Ax 1 6 1 ? 2 /* occur during the third larval instar. The foregoing results can be summarized as follows: TSP for lethality of  1) the  females occurs during the second  Viability of Ax 1 6 1 7 2 /!^ 0 shifted at different  FIGURE 9  stages of development. down; A  = pulse-down.  X = shift-up; 0 = shift-  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 . ) EGG  I  1ST  1  P U P A  2ND  h  HOURS AT 29 C  HOURS AT 22 C  I  EGG  \  1ST  too  100  150  150  2ND  DEVELOPMENTAL S T A G E S AT 22°C ( A P P R O X . )  200  300  FIGURE 10  The number of ocellar bristles and wing vein gaps of Ax l 6 l 7 2 /+ 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 shift series; A  = wing vein gaps,  = 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.  OR  SH IFT -  PULSE-UP  APPROXIMATE DEVELOPMENTAL S T A G E S AT 22 C E  » '  i "  '  Li!  :  I  L r- 2.0  8 m r r >  3.0_  m  o z  o  c m z o -<  III D a u DC li z.o_  .5  a. <  o  uj >  1 .0. HOURS AT 22°C  b.  SHIFT-  OR  PULSE-DOWN  APPROXIMATE DEVELOPMENTAL S T A G E S AT 29 C . E  4.0  I ' I  2.0  O o m  r r > 73  •n XI m  3.0 —  >•  o c m z o -<  o z  LU D a Ui a 2.0 — u. C <L e>  .1 .5  \  \  — -  z  L >U 1 .0. 50  HOURS AT 29 C  100  150  V  146 larval instar, although 18-hour pulses at 29°C during this stage are not sufficient to cause lethality; and 2) the TSPs for enhancement of wing vein gapping and ocellar bristle loss in Ax l6l72^ +  females occur during the third larval instar. N 1 Q 3/fa n o - TSPs for lethality.  Unlike most other N/fa no combinations, which are lethal under all conditions, 103 no N /fa 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 no 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 fa no /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 inseminated females.  As described previously for  an(j  the first experiment consisted of 22°C-29°C (shiftup) 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$) of N 1 Q 3 /fa n o females, that after the third instar there is a jump to about 50$ survival, and that successively later shift-ups after pupation allow correspondingly greater survival of N 1 Q V f a n o flies (Table 44, Figure 11).  Furthermore, when  shifted up from the late embryo stage (24-hour egg) onward, death of N 1 Q 3 /fa n o 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  N 103 / f a no  females.  This is indi-  cated by the absence of a temperature-effect on mortality of M5/fa no females and M5/Y males and because the sum of the number of verifiable N 1 Q 3 /fa n o 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^^/fa 110 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 N l Q V f a 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 N 1 0 3 /fa n o 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  CULTURE NUMBER  Data indicating viability of  N 103 /fa no  females in relation of their sibs when shifted from 22°C to 29°C at different successive intervals. TIME OF SHIFT DEVELOPMENTAL CULTURE AGE (HR) STAGE  1 2  24  E**  48  I  3  72  II  4 5  96.5 120  6  144  7  168  P, some III  8  192  9  NUMBER OP PROGENY VIABLE ADULTS DEAD PUPAE M5/fano g N l Q 3 /fa n o £ M5/Y a* N 1 0 3 /fa n o  /O OUllliV rtXJ OF N 1 0 3/ UNSCOR- fano $ ABLE FEMALES*  20  12  0  65 62  46 85  1 0  78  45 55  43 63  5 3  55 .  39  35  7  34  57  26  4  53  115  51  81  56  4  46  P  159  111  134  32  6  75  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  94  96  80  7  2  91  32 116 118  1 0  II, III III  96 112  5 4  64  III  62  6  62  P, a few III  216  10  13  NOT SHIFTED UP  0  28 88 115  * % survival = viable N-^Vfa 110 adults -r (viable N 1 Q 3/fa n o adults + dead  N 103/ fa no  pupae  + unscorable dead pupae) x 100% (see text). ** See Tables 32, 40 for explanation of symbols. oo  103 FIGURE 11  Relative proportions of viable N  o  /fa n ° 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  N 1 0 3 /fa n o adults; 1 1 = late pupal N 1 0 3 /fa n o deaths ;f§§ = unscorable early pupal deaths. presented in Table 44.  This Figure is based on the data  I  EGG  1  1ST  1  2ND  1  3RD  I-  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 N 103/ fa no 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 N 1 0 3 /fa n o 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 N 1 Q 3 /fa n o 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 N 1 0 3 /fa n o females (Table 46) . Similarly, pulse-downs after the embryo stage did not result in survival of N 1 0 3 /fa n o females (Table 47). Survival in this case would not be expected, since each pulsedown 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 developmental 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  Data indicating viability of N 1Q 3/fa no females in relation to their sibs when shifted from 29°C to 22°C at different successive intervals.  TABLE 45  CULTURE NUMBER  TIME OF SHIFT CULTURE DEVELOPMENTAL AGE (HR) STAGE  NUMBER OF PROGENY VIABLE ADULTS M5/fano $  N 1 0 3 /fa n o £  DEAD PUPAE  M5/Y cj* N 1 Q 3/fa n o $ UNSCORABLE  1  18  Is  31  0  18  3  0  2  36  I, II  63  2  52  2  0  3  54  57  0  56  0  0  4  72  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  57  0  54  1  0  11  II, some III III  NOT SHIFTED DOWN  * See Tables 32, 41 for explanation of symbols. U1 rv>  FIGURE 12  Time of death of N 1 0 3 /fa n o 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 <  U J o  < H E3 L in I j H < Lu H X  L A T E E M B R Y O L A T E 3RD I N S T A R  Z in LlI  h  Z LLI  E A R L Y E M B R Y O  0. O  _IJ U > tii Q  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  CULTURE NUMBER  Data indicating viability of N 103/ fa no  females in relation to their sibs when pulsed from 22°C to 29°C and back after 18 hours, at different successive intervals. TIME OF PULSE-UP CULTURE DEVELOPMENTAL AGE (HR) STAGE (UP)  NUMBER OF PROGENY VIABLE ADULTS M5/fano %  N 1 Q 3 /fa n o ?  M5/Y cf>  TOTAL DEAD PUPAE (NOT SCORED)  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  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.  TABLE 47  CULTURE NUMBER  TIME OF PULSE-DOWN CULTURE DEVELOPMENTAL AGE (HR) STAGE (DOWN)  1  18-42  2  36-61  3  54-78  4  72-96  5  90-114  6  I*  NUMBER OF PROGENY VIABLE ADULTS M5/fano £  N 1 U : yfa n o $  DEAD PUPAE  M5/Y. 0* N l u 3/fa n o  UNSCORABLE  39  0  35  1  0  I, II  80  0  52  0  1  II, some III  16  0  36  4  3  12  0  21  0  0  III, some P  42  0  56  2  4  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  A, some P  42  0  32  1  0  10  180  III  * 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 fa no /Y d* ), these must have been y wf N 1 0 3 /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 crossing over in the X chromosome.  The jr larvae had smaller mouthj.  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. prepupae or pupae with y mouthparts were seen.  No  These results  indicate that unlike most other N/Y hemizygotes (POULSON 1939b, 1968), N 1 Q 3 /Y males incubated at 22°C survive the embryo stage and may reach the third larval instar before dying. ] Q O of  larvae at 29°C suggests that N  The absence  ~'/Y males do not survive  the embryonic stage at this temperature. The preceding results can be summarized as follows: 1) TSPs for lethality of N 1 0 3 /fa n o 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 N 1 0 3 /fa n o females; and 4) N 1 0 3 /Y hemizygotes die as embryos at 29°C, but at 22°C they may survive into the third instar, dying before puparium formation.  158 VI.  N S 1 1 /NS 1 : L ;DP - TSP for lethality.  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 previously described for Ax 1 6 1 7 2 /!^ 0 and N 1 Q 3/fa n o . In the first experiment, 2-hour egglays from approximately 200 M5/wa N^ 1 1 rb females (inseminated by wf; NS 1 1 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 survival 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, whereas 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 collected at 22°C and placed at either 20°C (shift-up) or 28°C (shift-  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.  TABLE 48  CULTURE NUMBER  TIME OF SHIFT CULTURE DEVELOPMENTAL STAGE AGE (HR)  MALES M5/Y  NUMBER OP PROGENY FEMALES  M5/Y;Dg_ N/Y;D]o  M5/N  % SURVIVAI OF N/N;Dp M5/N;D£ N/N;D£ FEMALES*  E**  18  7  50  35  37  46  156  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  162  107  149  138  147  5  3  1  0  2  11  NOT SHIFTED UP  * % 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  CULTURE NUMBER  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.  TIME OF SHIFT CULTURE DEVELOPMENTAL AGE (HR) STAGE  1  0  2  MALES M5/Y  NUMBER OF PROGENY FEMALES  M5/Y;D£ N/Y;Dp  M5/N  % SURVIVAL OF N/N;D£ M5/N;D£ N/N;Dp FEMALES*  E**  14  9  10  2  10  0  0  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  21  61  43  66  40  84  11  NOT SHIFTED DOWN  47  * 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 N s l l /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, occurring about the middle of the embryo stage. VII.  Summary of temperature-shift results.  The findings of the temperature-shift experiments are summarized 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 development, 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 (N 103 /fa no  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 formation, although  Ax 16172 /n 40  females all die in late pupal stages,  and death of N 103/ fa no females is divided about evenly between  TABLE 48  CULTURE NUMBER  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.  TIME OF SHIFT-UP (HOURS)  1 2  3 4  3 4  5 6  5 6  7 8  7  9  8  10 11  9 10 11  MALES  NUMBER OF PROGENY FEMALES  M5/Y  M5/Y;D£  N/Y;Dp  M5/N  26  18 14  45  38 16  27 16  21  24  15 14 32 26 9 39 20  25  29  35  32  15 11  29  19 14  12  111 90  38  19 10 41  30  79  35 24  56 114  31  23 41  12 14  32 16  32 22  26 12  27 15  111  24  40  33 21  41  13 2  35 11 7 0  13  15  18  21  12  14  14  8  16  13 14  15 16  40  39  33  15  17  * See Table 48.  88 108  8  12  NOT SHIFTED  27 19 10  27 22  17 50  16  % SURVIVAL OF N/N;Dp M5/N;D£ N/N;Dp FEMALES*  18  93  18  31  13 34  37  1 0  19  27  20  21  0  0  25  15  23  21  28  0  0  40  53  51  43  54  0  0 OM ru  TABLE 48  CULTURE NUMBER  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.  TIME OF SHIFT-DOWN (HOURS)  MALES M5/Y  M5/Y;D£  NUMBER OF PROGENY FEMALES N/Y;D£  M5/N  M5/N;D£  N/N;D£  % SURVIVAL OF N/N;D£ FEMALES*  1  2  48  46  36  34  42  0  0  2  3 4  47  55  39 38  48  2  4  37  45 34  1  5 6  37  56  45 62  2  3 4  37 51  28  30 44  6  20  17 21  37 41  26  93 89 72  3 4 5 6  7 8  43 38  35 46 39 56 56  9 10  9 10 11  71 30 40 46  11  12  35  39  49 35 56  12  13 14  37 44  36 36  15 16  65 50 41  7 8  13 14 15 16  NOT SHIFTED  * See Table 48.  25 42  28  37 15 37 43 33 44  47 24  38  33 56  39 45 50  29 40  65 43  45 52  39 41  37 43  83 100  39  58  66  70  63  106  50  70  44  52  57  107  24  35  39  40  33  92  43  85  H (JO  FIGURE 13  Viability of N g l l /N s 1 1 ;Dp females shifted at different times during the embryo stage. 0 X-  0  shifted from 20°C to 28°C;  -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. include TSPs for: sll  1)  Note that the TSPs for adult morphology eye facet arrangement and wing nicking  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  Ng'/+  o x  Ql dc  O N '°3/spl h J d Q <  AX  16172/ /  +  S  •  1  E M B R Y O  1  1ST  I N S T A R  1 —  2ND  I N S T A R  r  3RD  I N S T A R  DEVELOPMENTAL  STAGE  P R E P U P A  A N D  P U P A  168 early and late pupal stages (Figure 11).  It should be stressed  that the data for N 103/ fa no  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 interactions 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 differences 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 Notchlocus 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 reported 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). mutation  Since the phenotypic effects of the point  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), interactions with Notch-locus recessive visibles (LINDSLEY AND GRELL 1968), suppression of Ax phenotypes (Tables 24-26), and recessiveness 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 , N C o , and N 1 0 3 , cannot be amorphic.  N g l 1 and N C o  not only exhibit a milder expression of certain Notch phenotypes  171 than deficiencies, but are also associated with additional phenotypic 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 particular genotype and phenotype studied.  Wing nicking and other  typical Notch phenotypes exhibited by heterozygotes for deficiency 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 nicking.  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 NS 1 1 , N C o , 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 obser40  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 product.  However, the analysis to follow indicates that N 6 ^ 1 and  po N  cannot be hypomorphic in terms of all phenotypes, and that  N ^ 3 is not hypomorphic at 29°C. least in the case of  This in turn suggests that at  and N C o , "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, N 1 0 3 /+ females show more intense wing nicking than N /+ or N__/+ (compare Plates la, 2c), and also have fused leg segments. Furthermore,  n 103/ 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  N 70k30 /fa no  heterozygotes (Appendix 4), and in N 1 0 3 / fa no 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 conditionally 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 N 1 0 3 /+ heterozygotes if the N 1 ^ 3 allele product either partially inactivates, or competes for receptor sites with the N^ product. alternatives are outlined diagrammatically in Figure 15.  These  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 N 1 0 3 /+ females at 29°C.  a.  1NACTIVATION OF" N +  PRODUCT  R E C E P T O R  F U N C T I O N A L  n+ :  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 interactions 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 functioning  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 receptor site.  product or a  The observation that at 29°C N^-Q3/NlQ3;Pp females  have even more exaggerated wing nicking and tarsal fusion phenotypes than N 1Q 3/+ 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. follows from the fact that  This  spl females exhibited tarsal  fusion but had less extensive wing nicking than N 1 Q 3/ + 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 fa no spl/Y males have a milder wing nicking phenotype than fa no /Y males (WELSHONS, personal communication).  The possibility that non-  Notch-locus modifiers may affect the phenotype of N 10 3/+ or Nl°3/spl, does not necessarily invalidate the hypothesis that  N^  3  is  176 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. and the fact that deficiency N mutations and not express the eye phenotype seen in  This,  duplications do 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. 20°C-22°C, the  Thus it can be assumed that at  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 NS 1 1 , 1 N^ : 2 NS 1 1 - is consistent with the hypothesis that the NS 1 1 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. sized  Thus it can be hypothe-  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 less  2) that at 29°C, the N s l 1 product is relatively inactive (or product is produced), and is therefore unable to parti-  cipate fully in wing development (causing a predominance of nicked individuals) and (in N g l l /+ females) unable to participate significantly in eye development, allowing the direct a normal eye facet arrangement. trated diagrammatically in Figure 16.  allele product to This hypothesis is illusThe 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 N g l 1 .  a.  2S°C  22° C  N +  gn  N  N  N +  V  V  W I L D T Y P E P R O D U C T  A C T I V E M U T A N T P R O D U C T  gn  V I N A C T I V E M U T A N T P R O D U C T  W I L D T Y P E P R O D U C T  V N — K  W I N G  W I N G  A C T I V E A N D  N T T K  P A R T I C I P A T I O N  W I N G  D E V E L O P M E N T  O F  N  E Y E  =>'  1  I N  E Y E  R E D U C E D • A N D  W I N G  P A R T I C I P A T I O N D E V E L O P M E N T  O F  N  S  1  '  ' N  E Y E  180 of the N g U eye phenotype, one due to an extra-locus modifier (E-n70^27)a and the other to a mutation within the Notch locus (N 70k3 °), are both associated with increased wing nicking (Appendices 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 affinities which permit the competition or interaction. The lethality of N sll /Ng 1: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 N 4 °/N 4Q ;Dp (Table 4), N 1 0 3 /N 1 Q 3;Dp (Table 7), N C o /N C o ; 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 N sll /N s11 ;Dp/Dp females (Table 11), N gll/ Y  ;Dp males, and N g 1:L /N 8 ;Dp and N gll /N^ 0 ;Dp females, indi-  cates that the 2 N^ 1 1 : 1  allele ratio is the factor which  determines the lethality of NS 11 /NS 11 ;Dp females at 20°C-22°C. If, in fact, at 22°C the NS 1 1 product has sufficient activity to +  compete with the N_ product, then twice as much defective wildtype 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 hemizygotes has been well characterized (POULSON 1940).  If the  lethality in NS^VNg-^Dp females is due to antimorphic (antagonistic) action by the N& 1 1 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 (competitive) action, such embryos should have some other phenotype. An examination of the phenotypes of N ^ V N ^ ^ D p embryos incubated at 22°C or 29°C, has not yet been completed, so a conclusion on this point must be postponed.  The viability of H S ^ / N ^ ^ D p  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  N C o behaves like a hypomorph in terms of wing  nicking but not its other phenotypes.  The Confluens phenotype,  which is mildly expressed in N Co /+ females, and strongly expressed in N Co /+;Dp females and N Co /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 N C o  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 N Co /+;Dp females (Plate %)• The possibility that the strong Confluens phenotype results from some special interaction between N C ° 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 N C o /+ (Table 12).  In fact, the  reduction of the Confluens phenotype in N Co /N x ;Dp females is predicted by, and therefore lends support to, the hypothesis that N C o 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, N C o 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 phenotype.  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&, fa no and fa n o E , nd and nd 2 , 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 Ax 2 8 a by MORGAN et al. (1941), should be discussed.  The duplication hypothesis was based on the observations  that Ax 2 8 a /N 8 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~*", Ax E 2 ,  Ax 16172,  and A x 9 b 2 , and none of these mutations are associated with visible duplications or deficiencies.  Furthermore, the mutagen EMS was  used to induce Ax E 1 and Ax E 2 (Appendix 3), A x 1 6 1 7 2 (WELSHONS, personal communication), and Ax 9 B 2 (LEFEVRE, personal communication).  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 comment 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 Ax E 2 and Ax^-1-72 enhance the wing nicking effect of N alleles (Table 28), and that Ax E 1 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.  Ax 9 B 2 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 Ax 9 B 2 and each of two flanking N alleles  and N C o ) (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 splN C o ) reported by WELSHONS (1958b).  On the other hand, the cross-  over frequency between fa no 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, Ax 9 B 2 , and fa no spl Ax E 2 ) affected the relative viability of noncrossover (Ax 9 B 2 ) 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-Ax 9 B 2 recombination. likely that Ax E 1 , Ax E 2 , Ax16l72a within the Notch locus.  an( j  In summary, it appears Ax 9 b 2 are all point mutations  In the following discussion, therefore,  it is assumed that these mutations are not duplications or partial 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 different 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 o to the gene product, since in the case of the deficiency, N^., there is no Nallele product to participate in polymer formation.  If, on the  other hand, it is assumed that Ax 9 B 2 is hypermorphic, the increased activity on the part of Ax 9 B 2 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 explanation was advanced by MULLER (1932) to explain the reduced expression of N and Ax phenotypes in Ax 2 8 a /N 8 heterozygotes. the wing nicking criterion, Ax E 2 and  By  A x 16172  would be hypomorphic, since wing nicking is enhanced by these alleles (Table 28).  186 On the other hand, the suppression of Ax E 2 and  phenotypes  by N mutations suggests that these two alleles are hypermorphic, like Ax 9 6 2 .  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 (entirely) 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 consistent 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 repressor 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 activator 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 elements.  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~. that mutation of /  The hypothesis  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 Ax x /Ax y interactions.  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 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  a c t i v a t o r  I  I  b r i s t l e l o s s d u e t o 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  I '  e x c e s s  r e p r e s s o r  _  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+  Ax/N  1 R+: 0 A+  1 RH: 1 R+: 2 A +  1 RH: 1 A +  191. Figure 17 can explain the phenotypes associated with these genotypes.  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 mechanism 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)N B , 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 character2  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 fa n o 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, fa g , spl, fa n o , and nd, except fa nQ /nd, exhibit complementation (i.e., are non mutant in appear-  193. ance) (WELSHONS 1965). Ax59b and  Recently, it has been reported that  do not complement fa s , in that the eyes of Ax/fa g  heterozygotes are rough, although not glossy (WELSHONS 1971). Moreover, the Ax-like allele, 1(1)N B , allows complete pseudodominant 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 fa s (WELSHONS 1965).  If the genetic map of the Notch locus (Figure 1) is fit-  ted to these patterns of complementation, such that noncomplementing 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 configuration.  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 complex 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 consideration of the interactions of fa n o , spl and Ax E 2 (Appendices 6, 7)>  The genotype fa n o spl Ax E 2 /+ + + expresses a rough eye  phenotype which is virtually indistinguishable from that of spl/ spl, whereas the genotypes fa n o spl +/+ + Ax E 2 , + spl +/+ + Ax E 2 , and fa n o + +/+ + Ax E 2 do not have rough eyes.  The genotype fa110  + +/+ spl Ax E 2 has a mild rough eye phenotype which overlaps wild type, but this is no more extreme than the phenotype of + + +/+  197. spl Ax E 2 .  (Plies of the genotype fa110 + Ax E 2 /+ spl + have not  been examined yet.)  The phenotypic differences between the cis  and trans configurations indicates that the fully penetrant spl phenotype of fa n o spl Ax E 2 /+ + + 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 phenotype by Ax E 2 , the coupling of fa n o to Ax E 2 completely suppresses the wing vein gap phenotype of Ax E 2 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 fa n o and Ax E 2 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 fa n o lesion to the right of the mutant site (Figure 1), the suppression of the spl Ax  F?  /  F?  bristle phenotype (and the k-xr  wing  vein phenotype) may be due to a comparable effect to the left of fa n o , 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 fa n o mutation on the tertiary folding of a Notch-locus product molecule containing the spl and Ax E 2 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 fa n o -Ax E 2 mutant sites produces a single  198. molecular product.  Furthermore, these observations Indicate that  fa n o is not entirely a hypomorphic allele, as was suggested by WELSHONS (1965) on the basis of N/fa n o 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 interactions among the Ajc alleles.  It is not difficult to conceive of  a lethal/non-lethal heterozygote causing lethality, such as Ax E1 /AxS2, A x E V A x l 6 l 7 2 , Ax59d / A x E2,  and  A x 59d / A x l6l72  ( T a b l e 29),  in the same way that N/fa n o heterozygotes are usually lethal. HOUSE (1959a) has also reported a lethal interaction between Ax 2 ^ 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 , entirely different matter.  30) is an  At first sight, one might postulate  that this kind of interaction, or negative complementation, indicates that the products of A x 9 B 2 and  (or  A x 16172)  are mutually antagonistic and that the resulting inactivation of these gene products is responsible for the lethality.  However, the observa-  tion that the lethal Ax x /Ax^ combinations exhibit severe bristle loss phenotypes (Table 31) > and the fact that relatively severe hypomorphic situations, like the viable and semilethal N/fa n o 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 A x 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 primary lesion in the Notch-locus product.  For instance, if we  refer to the models presented in Figure 17, the heterozygous combinations of an A~-type Ax (model a) and an R H -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 R H /R H (see Table 52).  Either of the above  models could account for the observed lethal patterns and also accommodate the viability of  AX  E 1  /AX9B2  (Table 29).  Whether or  not A x 5 9 d / A x 9 B 2 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 A x 9 B 2 (Figure 19 model), or simply to (either model).  being more severely mutant than Ax^-1-  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 A x 9 B 2 / A x E 2 type has not been re-  FIGURE  19  "Range of function" model to explain lethality of Ax 9 B 2 /Ax E 2 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.  HYPOTHETICAL FUNCTIONS AFFECTED  GENOTYPE  DEVELOPMENTAL RESULT  2 >  o  J LU -J J <  o  ix  9b2  /  .  /  Ax  f u n c t i o n s  g e n o t y p e  i  I ^ | ^ I1 I  3  f u n c t i o n s  g e n o t y p e  Ax  9b2  /  ,  / Ax  a f f e c t e d ; s u r v i v e s .  a f f e c t e d !  d i e s .  16172  I ^ I1 |1 I  m  LlI  H Ul  X  2  Ax"  /A*""'  2  I  1  I  I  I  >  f u n c t i o n s  g e n o t y p e  a f f e c t e d ; 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 Ax 28a /Y;H/+ and Ax 2 8 a /Ax 2 8 a ;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 phenotype 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  A x 28a / A x 28a  (HOUSE 1955), H/+o and Ax 2 8 a /+ enhance one  another in double heterozygotes, and N°/+;H/+ females have reduced expression of both the N and H phenotypes, an effect resembling that seen in A x 2 8 a / N 8 flies (HOUSE 1959a).  The re-  semblance between H effects and Ax effects appears to be more than a o chance similarity. N  The Ax 28a -like interactions between H and  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 contrast 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 suppressors (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 Ax E 2 , A x 1 ^ 1 7 2 , and A x 9 5 2 strains made co-isogenic for OR autosomes* (Figure 20), confirm both the lethality of Ax 9 B 2 /Ax E 2 and A x 9 B 2 / A x l 6 1 7 2 and the morphological differences between A x 9 5 2 and Ax E 2 . The sexual dimorphisms frequently encountered in the phenotypes of the Ax mutations (Table 22) cannot be so easily dismissed 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.  identical conditions.  206. Since the Notch locus is sex-linked, in-  complete or aberrant dosage compensation of the Ax mutant products is one possible explanation. compensated, since one dose of  By definition N_ is dosage in males produces the same  phenotype as two doses in females (STERN I960). single dose of  The fact that a  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 dimorphism.  For example, the fourth-chromosome mutant cubitus-  interruptus (ci), which causes wing vein gaps, and its dominant allele, ci D , 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 dosage 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 phenotypes 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 disturbance, 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 structures 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 responsible for the adult eyes, wings, legs, and epidermis.  Recently,  SHELLENBARGER (1971) reported that heat shocks of larvae homozygous 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 ( s s 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)E6 ts (GRIGLIATTI AND SUZUKI 1970).  In the case of l(l)E6 ts ,  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)E6 ts 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 ts 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 N g l l / + (Figure 6) and N 1 Q 3 /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 wildtype 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 N g l l / + and N 1 0 3 /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 ommatidial differentiation occurs in the eye discs of mature thirdinstar 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 progression 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 with  ss alt0a (GRIGLIATTI AND SUZUKI 1971).  It would be of interest  in this regard to see whether tarsal segment fusion in N 1 0 ^/+ heterozygotes also proceeds in a proximal-to-distal direction. This was not examined for in the N 1 0 V s p l temperature shift experiment (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  A x l6l 7 2 / N 40  (Figure 9) and N g l l / N s l l ; D p (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 /fa n o may be interpreted in several ways.  Although the  N 1 0 3 / f a n o shift data (Tables 44-47) do not allow a critical decision as to whether the TSP is monophasic or polyphasic, results 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, firstsecond 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)N t s ~ 1 (and N 1 Q 3 / f a 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 tissues. The observation that death of N 103 / f ain no different 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)N t s " 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 (SHELLENBARGER 1971).  It should be pointed out that the bimodal lethal  phase (early pupal or late pupal) of  N 103 / f a no  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). I no this does not affect the conclusion that N  Nevertheless,  /fa n o 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 embryonic 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 instarpupal stages (SUZUKI 1970).  Incubation of A x l 6 l 7 2 / N 4 ° at 29°C  during the TSP does not prevent the determination or differentiation of imaginal discs, since death occurs in late pupae or 72 partially-eclosed adults. the the fact that A x 1 ^that hemiand homozygotes are not ts However, lethal, and assumption N 40 is  213. amorphic, suggest that  A x 16172 / n 40  larvae do not possess sufficient 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 differentiative (pupal) stage.  214. LITERATURE CITED 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.  (1957), Uber Rontgenmosaikflecken und Defektmuta-  tionen am Auge von Drosophila und die Entwicklungsphysiologie des Auges.  BODENSTEIN, D.  Z. ind. Abst. Vererb., 88: 333-373.  (1950), The postembryonic development of  Drosophila.  Biology of Drosophila, ed. M. DEMEREC (Hafner  Publ. Co.), 275-367.  BRIDGES, C. B., and K. S. BREHME. Drosophila melanogaster.  Carnegie Inst. Wash. Publ. 552.  BRITTEN, R. J., and E. H. DAVIDSON. higher cells: CARLSON, E. A.  a theory.  (1944), The Mutants of  (1969), Gene regulation for  Science 165.: 349-357.  (1959), Allelism, complementation, and pseudo-  allelism at the dumpy locus in Drosophila melanogaster. Genetics 4_4: 348-373CARLSON, E. A.  (1961), Limitations of geometrical models for  complementation mapping of allelic series. 788-790.  Nature 191:  215. COHEN, J.  (1962), Position-effect variegation at several closely  linked loci in Drosophila melanogaster.  Genetics 4_7:  647-659.  CRICK, F. H. C., and L. E. ORGEL. allelic complementation.  DEMEREC, M.  (1964), The theory of inter-  J. Mol. Biol. 8_: 161-165.  (1939), The nature of changes in the white-Notch  region of the X-chromosome of Drosophila melanogaster. Proc. Seventh Int. Genetical Cong. (Edinburgh), 99-103.  DRIVER, E. C.  (1931)j Temperature and gene expression in  Drosophila.  J. Exp. Zool. 59.: 1-28.  ENGLESBERG, E., J. IRR, J. POWER, and N. LEE.  (1965), Positive  control of enzyme synthesis by gene C in the L-arabinose system.  J. Bacteriol. 90: 946-957-  EPSTEIN, R. H., A. BOLLE, C. M. STEINBERG, E. KELLENBERGER, E. BOY DE LA TOUR, R. CHEVALLEY, R. S. EDGAR, M. SUSMAN, G. H. DENHARDT, and A. LIELAUSIS.  (1963), Physiological  studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol.  FINCHAM, J. R. S., and P. R. DAY. Bartholomew Press, Dorking).  28: 375-394.  (1965), Fungal Genetics. (The  216. POSTER, G. G., and D. T. SUZUKI.  (1970), Temperature-sensitive  mutations in Drosophila melanogaster, IV.  A mutation  affecting eye facet arrangement in a polarized manner. Proc. Natl. Acad. Sci. U.S. 67: 738-745.  GAREN, A., and H. ECHOLS.  (1962a), Genetic control of induction  of alkaline phosphatase synthesis in E. coli. Proc. Natl. Acad. Sci. U.S. 48: 1398-1402.  GAREN, A., and H. ECHOLS.  (1962b), Properties of two regulating  genes for alkaline phosphatase.  GERSH, E. S.  J. Bacteriol. 83.: 297-300.  (1965), A new locus in the white-notch region of the  Drosophila melanogaster X-chromosome. Genetics 51: 477-480.  GOWEN, J. W.  (1933), Constitutional effects of the Hairless  gene in diploid and triploid Drosophila.  Am. Nat. 67:  178-180.  GREEN, M. M., and K. C. GREEN.  (1956), A cytogenetic analysis  of the lozenge pseudoalleles in Drosophila.  Z_. ind. Abst.  Vererb. 8 7 : 708-721. GRIGLIATTI, T., and D. T. SUZUKI.  (1970), Temperature-sensitive  mutations in Drosophila melanogaster, V.  A mutation affect-  ing concentration of pteridines. Proc. Natl. Acad. Sci. U.S. 67:  1101-1108.  217. GRIGLIATTI, T., and D. T. SUZUKI.  (1971), Temperature-sensitive  mutations in Drosophila melanogaster, VIII. mutant, ss al|0a .  HADORN, E.  The homeotic  Proc. Natl. Acad. Sci. U.S. 68: 1307-1311.  (1961), Developmental Genetics and Lethal Factors  (Wiley, New York).  HILLMAN, R.  (1961), A genetically controlled head abnormality  in Drosophila melanogaster. genetic analysis.  HOUSE, V. L.  Origin, description, and  Genetics 46: 1395-1409.  (1955), Observations on vein-forming potentialities  in the Drosophila wing.  HOUSE, V. L.  I.  Anat. Rec. 122: 471.  (1959), A comparison of gene expression at the  Hairless and Abruptex loci in Drosophila melanogaster. Anat. Rec. 134: 581-582.  HOUSE, V. L., and L. A. EBERSOLE.  (1971), The effect of genetic  background and temperature variation on the expression of the ci D allele in Drosophila melanogaster.  Genetics 68:  s29.  KAPULER, A.M., and H. BERNSTEIN.  (1963), A molecular model for  an enzyme based on a correlation between the genetic and complementation maps of the locus specifying the enzyme. J. Mol. Biol. 6: 443-451.  218. KRIEG, D. R.  (1963), Ethyl methanesulfonate-induced reversion  of bacteriophage T4 rll mutants.  KURODA, Y.  Genetics 48: 561-580.  (1970), Differentiation of ommatidium-forming cells  of Drosophila melanogaster in organ culture.  Exp. Cell  Res. 59: 429-439.  LEPEVRE, G., JR.  (1952), Dp (1; 2R)w 5 1 b 7. Drosophila Info. Serv.  26: 66.  LEPEVRE, G., JR., P. J. RATTY, JR., and G. D. HANKS.  (1953),  Frequency of Notch mutations induced in normal, duplicated and inverted X-chromosomes of Drosophila melanogaster• Genetics 38: 345-359.  LINDSLEY, D. L., and E. H. GRELL.  (1968), Genetic Variations  of Drosophila melanogaster.  Carnegie Inst. Wash. Publ.  No. 627.  LOOMIS, W. P., JR.  (1969), Temperature-sensitive mutants of  Dictyostelium discoideum.  MOHR, 0. L.  J. Bacteriol. 99.: 65-69.  (1932), On the potency of mutant genes and wild-type  allelomorphs. 190-212.  Proc• Sixth Int. Cong. Genetics, Ithaca 1:  219. MORGAN, T. H.  (1919), Contributions to the genetics of Drosophila  melanogaster.  IV.  character "notch".  A demonstration of genes modifying the Carnegie Inst. Wash. Publ. 278: 343-388.  MORGAN, T. H., J. SCHULTZ, and V. CURRY.  (1941), Investigations  on the constitution of the germinal material in relation to heredity.  MULLER, H. J.  Yearbook, Carnegie Inst. Wash. 40: 282-287.  (1932), Further studies on the nature and causes  of gene mutations.  Proc . Sixth Int. Cong. Genetics,  Ithaca 1: 213-255.  POULSON, D. F.  (1937)» Chromosomal deficiencies and the embryonic  development of Drosophila melanogaster.  Proc. Natl. Acad.  Sci. U.S. 23: 133-137-  POULSON, D. F.  (1939a), The developmental effects of a series  of Notch deficiencies in the X-chromosome of Drosophila melanogaster.  Proc. Seventh Int. Genetical Cong.  (Edinburgh), 240-241.  POULSON, D. F.  (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.  SLYZINSKA, H.  Genetics 68: 561-562.  (1938), Salivary chromosome analysis of the white-  facet region of Drosophila melanogaster.  Genetics 23:  291-299. SPIEGEL, M. R.  (1961), Schaum's Outline of Theory and Problems  of Statistics (Schaum, New York), p. 344.  221. STERN, C.  (I960), Dosage compensation - development of a concept  and new facts.  SUZUKI3 D. T.  Canad. J. Genet. Cytol. 2_: 105-118.  (1970), Temperature-sensitive mutations in  Drosophila melanogaster.  SUZUKI, D. T., and D. PROCUNIER.  Science 170: 695-706.  (1969), Temperature-sensitive  mutations in Drosophila melanogaster, III. Dominant lethals and semilethals on chromosome 2.  Proc. Natl. Acad. Sci.  U.S. 62: 369-376.  TARASOFP, M., and D. T. SUZUKI.  (1970), Temperature-sensitive  mutations in Drosophila melanogaster, VI. Temperature effects on development of sex-linked recessive lethals. Devel. Biol. 23: 492-509.  VON HALLE, E. S.  (1965), Localization of E-spl.  Drosophila  Info. Serv. 40: 60.  WELSHONS, W. J. (1956a),  : Notch-Confluens.  Drosophila  Info. Serv. 30: 79.  WELSHONS, W. J.  (1956b), Dosage experiments with split mutants  in the presence of an enhancer of split. Serv. 30: 157-158.  Drosophila Info.  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. the Notch locus in Drosophila.  (1962), Pseudoallelism at Genetics 4_7: 743-759-  WELSHONS, W. J., E. S. VON HALLE, and B. J. SCANDLYN. Notch pseudoalleles in Drosophila melanogaster.  (1963), 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. 223-239•  J. Exp. Zool. 148:  223. WRIGHT, T. R. F. Drosophila.  (1970), The genetics of embryogenesis in Advances in Genetics 15.: 261-395.  224 APPENDIX 1 Interactions of N g l 1 and N 1 0 3 with faS and fa no  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 fa g and fa n o was made. To investigate the interactions with fa g , the following crosses were performed at 20°C, 22°C, 25°C, and 29°C: faS/faS ? x w* NS 1 1 rb/B£ w^'Y d* , and faS/faS ? x 2. wf. N 1 Q 3 ;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 fa s alone, could be detected in any of the heterozygotes excepting NSll/faS a t 20°C and 22°C.  In the latter  cases, the roughness was no greater than one would expect from the combination of the N g H / + and fa s phenotypes. dominant expression of fag observed with  The pseudo-  and N 1 0 3 , is con-  sistent with that reported for fa with these two N alleles (LINDSLEY AND GRELL 1968). Both N g l l / f a n o and N 1 0 3 / f a n o were found to be relatively viable at low temperatures, but lethal at 29°C. and the results obtained, appear below.  The crosses used,  225. Cross 1:  n S 1 1 rb/B^ w^-Y cJ*  w^ fa n o /w a fa n o ^ x /  Results:  PROGENY  Temperature  B  +  <j> <j>  B s cfd*  20° C  31  121  22°C  3  11  25°C  16  113  29°C  0  47  N gll r b  j  x wa  f a no  &  Results:  PROGENY  Temperature  B  99  99  B+  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 fa n o Results  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 account 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 N gll / f a no  females, many of which had drastically reduced head and eye sizes on one or both sides. were not examined.  Other morphological phenotypes  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, 103/ no was much more viable than N fa Temperature-shift studies of the N 1 0 3 / f a n o  N gll / f a no #  lethality are reported in Results (part F, section V). The wings of both N 1 0 3 / f a n o and NS 1 1 /fa n o adults were deeply 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, N 1 Q 3 / f a n o  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^N 70j In the course of the first experiments designed to study the effects of gene dosage on N^ 0 , it became apparent that the initial w a  rb chromosome contained a bobbed-lethal (bb1)  allele, since all attempts to make this chromosome homozygous failed, and the cross l(FM6)/we bb 1 ? x w^ N ^ rb/Y;Dg_ cf yielded no  non-Bar-eyed female progeny.  In order to remove the bb-1-  rb bb 1 chromosome, recombinants between  mutant from the wf^  N C o and the flanking eye colour mutations were obtained from w a N C o rb bb V + + + + females, the wild-type X chromosome having a  come from the OR stock.  fo  Two strains were established, w^ N  ,  and N C o rb, from which the bb 1 had been removed, as indicated by the following results of the cross bb-^/Y  1(FM6)/x  $ x In(l)dl49,tyl  (cf^cfwere not counted). FEMALE PROGENY PHENOTYPE B eyes B^ eyes  a  $ X-CHROMOSOME CoPARENTAL rb bbl N Co r b wa — gg.  N Co  ^  1^3  65  129  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^ chromosomes, 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 NOT % NICKED NICKED NICKED FEMALES  CROSS  GENOTYPE  OR % x wf; N ^ rb bb1/Y;Dp (f  60  14  81  N C o rb + +  OR % x N£^ rjb/Y;D£ cr>  95  17  85  wa NCo  OR ° x wf; N Co /Y;Dp o^  42  58  42  wf; + +  rb btr + +  The enhancer of nicking, designated E - N 7 ^ , was localized genetically in the following manner. N Co -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 and car.  , cv, v, f,  The results are tabulated below.  RECESSIVE MARKERS  1 WING N  2 WINGS N  71  133  124  0 WINGS N  % N INDIVIDUALS  + + +  + +  + +  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%  + + +  1  5  11  W  +  1  0  3  Z  +  + +  f  +  -<] CO  + +  —  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  + cv  V  + +  6  5  7  67%  +  3  4  0  57%  car  0  1  1  -  + cv  + + car  0  1  0  -  y. +  V  f  +  0  0  1  -  L +  V  + +  0  0  1  -  + cv + + L  +  +  f  83%  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- 70j N is located closer to rb than to cv. dence is as follows.  The evi-  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-N 7 Q j ' is situated close to rb. the observations that:  1) all ^  This assumption is supported by 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 genotype 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.  which produced both  and ^  Those vials  female progeny, but no males, were  saved as putative lethals covered by the  w + • Y.  duplication  The 1(FM6)/+X females from the vials scored as putative lethals, were mated individually to wf^  rb/BS w + •Y males.  this cross were scored for the absence or presence of  Progeny 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 Ax E1 ).  Procedure No. 1  EMS I  >L  w £ili w^-Y $  x  1(PM6)/1(1)J1 ? ) ) x •J 1(FM6)/+»  +/Y c?  +*/B s w+.Y  (individual or» )  '  l(l)Jl/+»  1(FM6)/B  (y+ females)  S  w^-Y  ? 1(1)J1/B  S  w^-Y  XX,y w f;B^_ w^-Y (y females)  (males die)  l(FM6)/+» $  x  l(FM6)/w a N 4 0 rb  +*/w a N110 rb  (B females live)  (B females die if carries Nlocus lethal)  N ^ rb_/B^_ 1(FM6)/BS w + - Y (males die)  +*/B s w + -Y (males die if +* carries a lethal)  cf +*/B s w + - Y (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. last group, two proved to be in the Notch locus.  Of the  On the basis  of subsequent tests, these were designated Ax E 2 and f a n o E , respectively (see Appendix 4 for mapping of f a n o E , and Table 14 for mapping of Ax E 2 ).  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 r P 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. w a N S 1 1 rb/B s 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,  or modifier mutations).  N,  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 nondisjunctant 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 R e 7 0 k 2 7 and Re 7 0 k 30 > respectively. Subsequent tests indicated that the partial eye revertant R e 7 0 ^ 2 7 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 i s phenotypically a notch mutation, and I have so far been unable to separate it from the N5"1"1 site (Appendix 4).  Re 7 ° k 30 has therefore been re-named N 7 Q k 30.  Note that  where N 7 ^^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^ NS 1 1 rb/B^ w^-Y cf*  20 .5°C  dl49 ,y Hw m£/wf: N g l 1 rb £  wf. N ^ rb/wf. N S 1 1 rb f  Score eyes of red-eyed  Females die unless  females for facet pattern.  NSll  Wild-type eyes indicate  been induced (score for  N g U . » N + , N^il-'Nj  appearance of white-  or  locus modifier.  extra  mutation has  eyed females).  Re-test putative mutants further by mating to dl49,y Hw m^ males.  236 APPENDIX  1  Description and mapping of fa n o E , E-N? 0 k 2 7, and N? 0k 30  fanoE The new mutation fa n o E was so named, on the basis of its phenotype and, as described below, the lack of recombination between fa n o and fa n o E .  f a n o E has thickenings at the ends of the  wing veins, and occasional nicks in the wingtips.  Expression of  both phenotypes is mild compared to fa n o , and heterozygotes of fa n o E /fa n o are intermediate in appearance. and fa noE /faS have wild-type wings and eyes.  fa noE /spl, fa n o E /fa, N 8 /fa n o E and N1*0/  f a n o E are lethal, and N s l l / f a n o E is semilethal at 20.5°C (148 male and 8 female progeny were recovered from the cross  fa n o E /  y_ f a n o E ^ x wf N g l 1 rb/B^ w^«Y a*) and lethal at 29°C. In order to map fa n o E with respect to fa110, two series were run of the cross fa n o E cv/wf fa n o 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. es were counted.  Male and female progeny of these cross-  The results, tabulated below, show that no  fa n o -fa n o E crossovers (N^V+ females) were recovered, although 22  237. N^O/fa1*0 spl and 6 N 4 ° / f a n o E breakthroughs survived and 41,830 males were counted.  This indicates that f a n o E is very closely  linked to fa n o in the Notch locus.  SERIES  TOTAL MALES  b£  1  38,119  35  20  2  3,711  8  2  41,830  43  22  TOTAL II.  FEMALES N/fa n o spl N/fanoE  N/+  5  0 1  6  0 0  E-N7°k27 E _ N 70k27  (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 N S H / + + 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-N k 2 7 w a  N gll/ +  + + females  raised at 20.5°C, and 27% (84/316) raised at 22°C, had nicked wings. N  /+.  E-N  k27  / N C o females also are more strongly nicked than  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 - N k 2 7 / dor has wild-type eyes and E-N k 2 7 /dor 1 survive (see below).  238. E-N k 2 ? was mapped to the left of wf^ by the cross E-N k 2 ? w a N gll  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-N k2 ?-w a ), 1.51* (wa-NS1:i), and 4.17* (NSi:L-rb) .  NO. OP PROGENY FEMALES MALES  GENOTYPE  Non-crossovers  E-N w a N ® 1 1 rb  + +  +  +  Single crossovers E-N-w a  E-N +  +  +  Single crossovers wa-NgH  E-N wf; +  Single crossovers NgU-rb  E-N w a N g U +  +  +  +  w a NSll rb  +  +  +  N g U rb  +  TOTALS  rb  1039  0  1067  1033  (not distinguishable from non-crossovers) 18 0  19  0  15  0  45  0  49  48  2252  1081  The following results of pair-matings in vials (M5/E-N?0k iva £ x Duplication  see below), indicate that E-N? 0 k is  situated in the dor region. (PRATT 1970).  Note that p-DTS is a dor-*- allele  239. LARVAL MELANOTIC M5/Y cr* E-N/Dp-Y cf* TUMORS  PROGENY  X/Dg/Y MALE PARENTS  M5/X %  D f ( 1 ) s c 8 w a / D p 59k9(4)  X/E-N $  present  45  49  14  13  12  jr dor/T(l;Y)2E  32  43  23  26  absent  p-DTS rb/Dp 6 7g24(l)  11  18  12  17  absent  w a N S 1 1 rb/B s w + « Y  39  49  28  y 59b  z/pp60dl9(l)  III.  present  present  N?0k30 N 70k30 / +  females have a wild-type eye facet arrangement at 22° C, and enhanced wing nicking compared to N g l l / + .  Lethality of  ^70kl8/f a no j[_s variable, depending on the genetic background (see below). 22°C).  Those that die, do so in the late pupal stage (20-  This is unlike N g l l / f a 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 ascertained whether the difference between N g "^/fa n o and is due to genetic background differences, or to the  N 70k30 / f a no 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 k 3 ° rb/dl49,y H w ^ ?  x OR cf ), had one or  both wingtips nicked. Viability of N 7 Q k 3 ° with fa n o was investigated in the following crosses and results.  240. rb/dl49,y Hw m£ ? x wf fa n o cf  Cross 1.  wf  Progeny:  151 dl49/fa n0 $ , 23 N70k30/f a no £ , 108 dl49/Y o^  Cross 2.  wf fa n o /w a fa110 ? x wf N7° k 30/B s w^-Y <f  Progeny:  233 fa n o /B s w^-Y cf, 1 N7 0 k 30/f a n o £ .  n70*30  Surviving N/fa no females have deeply serrated wings and very thick veins.  The lone female survivor of cross (2) had fused  tarsal segments (other N/fa n0 females not examined). The results of the mapping cross wf  n7°^30  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$, respectively), but no confirmed crossovers between ^70^30 were recovered.  anc j  jjgll  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  w a spl rb  w a N spl rb  N spl rb  spl rb w a 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 fa n o and spl, rather than resulting from  phenocopy-like events.  242. 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. PARENTAL  1  No. Parental wings  PROGENY Phenotypes"  Sex  2  4  3  notched  notched  abruptex  g  ?  ?  d» I  o* I  5  notched  (f I  notched  a* I  $  o* I  Eye Eye colour facets Wings  +  +  Ax, +  wa  +  Ax, +  0 0  rb  +  Ax, +  „a w rb  spl  wa  5 0  5  3  0  3  6  0  6  29 0  3  0  1 0  0  0  0  0  0  0  0  0 0  1  0 0  0  0 0  0  0  0  0  0  0  0  0 0  3  +  3 0  0  2 0  1  1  0  2  0  1  8  3 0  6  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  w a rb  spl  N  5 1  3  3 1  4  0  1  2  3  0  2  6 2  2  wa  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  0 0  0  0 0  1  0  0  0  0  0  0  1 0  0  +  +  N/Ax  24 0 10  The "I" progeny sex class comprised intersexes.  These in-  dividuals possessed sexcombs and were either male-like or femalelike 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 wildtype 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. 1-1 and 1-5 carried Ax E ^ and either  Females  spl rb_ or wf; fa n o 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; wf. S£l rb.  rb/w a Ax E 1 /  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; fa n o spl rb/Ax E1 , and females number 2-1 and 2-7 were diploids with the genotype w a spl rb/Ax E1 . 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; fa n o spl rb/w a 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  1-1  2-1  2-3  2-5  Ruby  Ax +  Ax;Ax+  Ax;Ax+  Apricot split  Apricot split notched  Ruby split  Ruby split notched  9  0  0  0  0  0  8  1  0  1  0  0  0  0  0  0  0  0  2  0  0  0  0  0  2  0  0  w + rb + ($ Ax + N + ( (cf  17  0  2  0  0  0  17  2  1  0  21  0  0  0  0  0  0  0  w + Ax+ (9 N+ rb+ ( (cf  6  0  0  0  0  0  6  0  0  0  0  0  0  0  0  0  0  0  42  0  0  0  0  0  39  0  0  34  0  2  0  0  0  0  0  0  31  0  2  0  1  0  53  2  0  («J»  2  26  0  3  1  2  0  0  0  w a N + (? rb+ Ax + ( (cf /  7  1  0  0  1  0  0  6  0  0  0  0  0  0  0  0  0  0  (I  1  0  0  0  0  0  2  0  0  ($  w+ rb ( Ax+ N+ (cf +  (I  1-5  Apricot  White split notched  I \  1-3  Ax  White split  %  px 2 F X No. c2 Pheno. Sex  w + Ax rb +  (9  w + Ax rb+  (9  ( (<?  (  r.ve>r 4="  P 2 PHENOTYPE ? ! ? Pi $ P 2 No. Pheno. Sex 2-7  3-1  3-2  4-2  4-3  4-5  4-7  w + Ax rb+  White split  White split notched  Apricot split  Apricot split notched  Ruby split  Ruby split notched  Ax  Apricot  Ax +  Ax;Ax+  Ax;Ax+  Ruby  (9  25  0  1  0  0  0  20  0  0  (d>  28  0  0  0  0  0  0  0  0  (9  23  0  1  0  0  0  28  2  0  (<*  1  25  0  0  0  1  0  0  0  (9  23  0  2  0  0  0  33  1  0  (<?  2  26  0  1  0  0  0  0  0  w + N + (9 Ax + rb C cs»  24  0  3  0  2  0  42  0  0  1  21  0  1  0  1  0  0  0  Ax  41  0  1  0  0  0  49  0  1  (c?  3  26  0  2  0  0  0  0  0  (? (  41  0  0  0  1  0  33  1  0  (c?  3  18  0  0  0  1  0  1  0  17  0  2  0  1  0  29  1  1  0  18  0  1  0  0  0  0  0  w + Ax rb+ w + Ax rb+  Ax  Ax  (  (  (  (9 (  (9 r \  IV)  VJ1  F2  Pi ? P x ? f 2 No. Pheno. Sex 5-1  Ax  (? (  (of  5-2  Ax  (? (  (cf  White split  PHENOTYPE  White split notched  Apricot split  Apricot split notched  Ruby split  12  0  2  0  1  0  15  3  0  2  20  0  1  0  0  0  0  0  16  0  1  0  0  0  23  1  1  1  19  0  0  0  0  0  0  0  Ruby Ax split notched Ax +  Apricot  Ruby  Ax;Ax+  Ax;Ax+  ro cr* -t  F2  Fx  No .  247.  PROGENY  ? £  d* o*  white split  white split notched  white split  white split 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  4-6  20  4  1 6  5  5  1 4  1  1  0  These data indicate that F-^ females 2-4, 2-8 and 1-6 were diploids with genotype wf; fa n o spl rb/w a fa n o spl rb.  This indi-  cates that parental females 1 and 2 were non-virgin, both having been inseminated by their wf; fa n o 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; fa n o spl rb/wf. sgl rb .  It has already been inferred that the presence of intersexes + + among their progeny suggests that the original w_ rb exceptions were triploids.  female  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  fa n o spl rb, w a N^Q rb, and w +  Ax g l rb + , and that female 3 had at least wf^ fa n o spl rb and w + A x e 1 rb + .  249 APPENDIX  10  FP Tests of exceptional progeny from Ax 1 ^ mapping crosses  Series 1 Male progeny whose phenotypes suggested they were recombinants for fa n o , spl, or Ax E 2 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). Recombinant No. (Culture-Brood)  PHENOTYPE  EYES  WINGS  1-3  apricot  notched  2-3  apricot  5-7  ruby, split +  notched +  7-4 8-2  8-4 8-8  ruby, split  notched + +  ruby apricot  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 Ax E 2 , several individuals from each of the fertile notched stocks, and from the ruby split stock 8-2 were mated to Ax E 2 /Ax E 2 virgin females, along with a control cross of Ax E 2 /Ax E 2 ff x fa n 0 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 Ax E 2 / A x e 2 females. PROGENY  ??  Recombinant No.  No gaps 1 or more No gaps gaps  1 or more 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^ fa n o Ax E 2 ,  fa n o A x E 2 , fa n o Ax E 2 , and s£l rb,  respectively, although the nature of the control cross p? does not eliminate the possibility that'spl suppresses the Ax phenotype.  wing vein  It will later be shown that this is not the case.  These data indicated that Ax E 2 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 the inferred map position of AxFP .  251  Series 2 Male progeny whose phenotypes indicated that they were F2 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. Recombinant No.  PHENOTYPE  EYES  WINGS  1-3  apricot  notched  1-4  apricot  notched  2-4  ruby, split  +  3-4  ruby, split  +  4-3  apricot  5-3  ruby, split  +  9-4  ruby, split  +  notched  10-4  apricot, extreme split  notched  12-1  apricot, extreme split  notched  12-5  ruby, split  13-3  white  15-4  ruby, split  +  17-3  ruby, split  +  +  notched  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.  252. EYES  Recombinant No.  PHENOTYPE  WINGS  1-2  apricot  5-5  ruby, split  +  7-2  ruby, split  +  7-3  apricot  14-3  ruby, split  18-2  apricot  notched  notched + notched  The phenotypes of the male progeny of the recombinant females are tabulated beloxtf. Recombinant No.  PHENOTYPES OF PROGENY white apricot white apricot white split split split notched notched notched notched  ruby split  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 Ax E 2 was  present in any of these recombinants, males from most of the recombinant cultures were mated to virgin AxK> 2/Ax F? 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.  PARENTAL REC. <? PHENOTYPE #  $ PROGENY PHENOTYPES No. of flies with No. of flies with given No. of anterior orbitals 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  w a fa n o Ax E 2 and wf_ fa n o 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 A x E 2 , indicates that Ax E 2 is not present in these recombinants, which must have the genotypes w a fa n o rb and spl rb, respectively.  These data place A x E 2 to  the right of spl. This conclusion was subsequently confirmed when the recombinant genotype spl A xF? ^ was recovered as a single male in the progeny of the cross wf^ fa n o spl Ax  rp o  /+ + + + $ x  OR <? , the female parents of which were the progeny of a cross F2 between OR females and recombinant stock 12-1. All spl Ax individuals established from this recombinant, have the characFP teristic Ax wing phenotype, have a rough eye phenotype interTT O mediate between that of fa n o spl Ax and spl, and have markedly reduced bristle frequencies compared to spl or AxE2 alone.  255  APPENDIX 10  Interactions of fa n o , spl, and Ax;E2 The phenotypes of various combinations of fa n o , spl, and Ax E 2 indicate that:  1) coupling of spl and Ax E 2 results in  extreme expression of the spl eye phenotype, extreme bristle loss, and near-normal expression of the Ax E 2 wing vein gap phenotype; 2) coupling of fa no to spl Ax E 2 results in further enhancement of the spl eye phenotype, suppression of bristle loss compared to spl Ax E 2 , and complete suppression of wing vein gapping; 3) coupling of fa n o to Ax E 2 does not cause a mutant eye phenotype, possibly slight enhancement of bristle loss compared to Ax E 2 , 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 fa no spl  spl A x  fa no  e 2  Ax  Ax  /Y  E 2  fa n o spl  /Y  Ax  EYES E 2  /Y  very narrow, glazed, few or no discrete ommatidia  <?  intermediate between fa n o spl A x e 2 and spl in size; discrete ommatidia but strongly spl  <?  wild type  <?  e 2  /+  + +  %  WINGS nicked, thick veins, no vein gaps vein gaps like  Ax  e 2  nicked, thick veins, no vein gaps  about same size and rough- wild type ness as spl/spl  256. spl Ax E 2 /+ + 2  occasional roughness, overlapping wild type; slightly smaller than +.  spl Ax E 2 /fa n o $  like sjol Ax E 2 /+ +  fa n o spl/Ax E2 £  wild type  occasional vein gaps  occasional vein gaps  Bristle counts of various combinations of fa n o , spl, AxE 2 and +, are tabulated on the following pages.  The following  crosses were used to generate the genotypes listed in the table. LINE  CROSS  1  XX/Y  $ x fa n o spl A x e 2 / Y <3  2  XX/Y  ? x spl A x  3  XX/Y  ? x w ^ fa n o A x E 2 / Y  e 2  /Y  cf 8  OR ? x fa n o spl A x E 2 / Y <7 6,7  OR 9 x spl A x  8,9  Ax E 2 /Ax E 2 ? x fa n o spl rb/Y d*  E 2  /Y  cf  10,11  OR $ x fa n o spl rb/Y d»  1 2 ,13  OR  x wa f a n o A x E 2 / Y  &  LINE  GENOTYPE  1  fa n o spl  2  spl  3  fa n o  5 6 7 8 9  )  )  ) )  )  Ax  E 2  e 2  /+  e 2  /+  )  )  )  )  11  )  12  )  )  )  )  fa  no  +  fa n o spl/+ +  fa n o  Ax  e 2  /+  +  3  4  OCELLARS  6  5  0  1  POSTVERTICALS  2  0  1  2  15  43  1  0  0  0  48  10  1  10  25  24  20 .5°C  39  6  0  0  0  0  0  45  0  0  43  2  0  20.5°C  0  0  2  11  39  0  0  3  8  41  0  0  52  20 .5°C + + 29°C  0  0  29  57 l4l  36  10  7  78  0  1  7  30  58  1  0  97  0  0  10  20 .5°C  0  1  50  77  38  9  0  73  68  34  5  29°C  3  5  56  15  7  0  0  86  0  0  52  29  5  20 .5°C  0  0  0  0  74  12  2  0  0  88  0  0  88  29°C  0  0  0  3  55  4  0  43  16  3  0  11  51  20 .5°C  0  0  0  0  0  3 118  0  3 118  0  0  121  29°C  0  0  0  0  19  20.5°C  0  0  0  0  29°C  0  0  0  0  +  spl +/+  2  0  /Y  Ax  ORBITALS  1  20 .5°C  /Y  /Y  fa n o spl  spl A x  E 2  )  10  13  E 2  Ax  0  h -j co  4  Ax  TEMP.  Ax  e 2  44  1  10 262 44  43  42 128  40  63  34  6  0  9  94  0  14 104  0  0  118  0  0  118  6  34  0  5 113  0  0  118  78  ro  ui  1  6  16  21  13  2  28  13  3  0  WING VEIN GAPS  SCUTELLARS  DORSOCENTRALS l — r  LINE 0  TOTAL (lines 1,2 not 3  0 8  2 1 19 I F  0  27  14  4  4 1  0  0  59  0  0  0  (gaps in L5 (100*); many in L4  POSTERIOR 0 2 1  ANTERIOR 0 2 1  3  3 15  52  0  0  0  88 176  0  2 271 272  0  0  0  0  2  9  0  0  0  48 108  0  1 174 156  (+18 with 1 or more gaps)  9  0  20  39  27  28  (+57 with 1 or more gaps)  0  0  88  0  0  88  86  1  1  0  40  6  28  28  0  0  62  32  14  16  0  0  0 121  0  0 121  0  0 121 119  0  0  0  0  0  2 101  1  84  0  0 103 102  0  0  0  0  0  0  1 117  0  1 117  0  0 118 115  0  0  0  0  0  0  7 111  0  0 118  0  1 117 118  0  0  0  4  0  0  0  5 268  9  5  0  3  19  41  89  6  0  0  0  2 173  19  7  0  6  32  40  8  77  8  0  0  0  0  88  9  0  0  2  20  10  0  0  11  0  12 13  33  7  18  85  97  ro CO  ui  259 APPENDIX  10  Counts of bristles and wing vein gaps in Ax E 2 and OR flies Data in lines 1-7 were taken from progeny of stock cultures (AxE2/AxE2  £  x  Axe2/Y  d*  ), raised in uncrowded conditions. o  8,9 were obtained from progeny of OR +  Lines  F2 x Ax  /Y c? . Lines 10-12  were obtained from progeny of stock OR cultures, reared in uncrowded 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 particular 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  TEMP.  GENOTYPE  0  AXE2/Y  d*  20.5°C  2  AXE2/Y  cf  20 .5°C  0  0  0  1  46  1  0  4.00 + .06  3  A E2/AxE2 Ax  9  20.5°C  0  0  0  2  67  0  0  3.97 ± .04  4  AXE2/Y  cf  22°C  0  0  0  2  163  0  0  3.99 ± .02  5  AXE2/AXE2  j  22°C  0  0  0  1  165  2  0  4.01 ± .02  6  AXE2/Y  0*  29°C  0  0  2  25  138  0  0  3.82 ± .05  7  AxE2/AXE2  ?  29°C  0  0  3  30  86  0  0  3.70 ± .08  8  A X  E 2  /  H-  9  20 .5°C  0  0  0  0  1  5  144  5.95 ± .04  9  A X  E 2  /  +  29°C  0  0  0  0  4  27  75  5.67 ± .09  10  OR  <3*  22°C  0  0  0  0  0  0  109  6.00  11  OR  £  22°C  0  0  0  0  0  0  139  6.00  12  OR  cP  29°C  0  4  138  5.97 ± .03 ro CT\ o  POSTVERTICALS  OCELLARS LINE  x  0  VERTICALS  x  1  73  1.99 ± .03  0  2  72  1.97 ± .04  2  0  1  47  1.98 + .04  0  0  44  2.00  3  1  4  64  1.91 ± .07  0  5  64  1.93 ± .06  4  0  2  163  1.99 ± .02  0  2  163  1.99 ± .02  0  0  0  0  165  4.00  5  0  12  156  1.93 ± .04  0  1  167  1.99 ± .01  0  0  0  1  167  3.99 ± .01  6  52  74  39  0.92 ± .10  77  50  38  0.76 ± .10  0  0  0  6  159  3.96 + .03  7  87  29  3  0.29 ± .08  90  25  4  0.28 ± .08  0  0  0  4  115  3.97 ± .03  8  0  0  150  2.00  0  0  150  2.00  0  0  0  0  150  4.00  9  0  12  93  1.89 ± .06  0  1  105  1.99 ± .02  0  0  0  1  105  3.99 ± .02  10  0  3  106  1.97 ± .03  0  0  109  2.00  0  0  0  0  109  4.00  11  0  3  136  1.98 ± .02  0  2  137  1.99 ± .02  0  0  0  0  139  4.00  12  0  1  141  1.99 ± .01  0  0  142  2.00  0  0  0  0  142  4.00 ru  o\  DORSOCENTRALS  ANTERIOR SCUTELLARS  LINE 0  x  POSTERIOR SCUTELLARS  x  0  0  1  73  3.99 ± .03  0  74  2.00  0  74  2.00  2  0  0  0  0  47  4.00  3  0  0  0  1  68  3.99 ± .03  4  0  0  0  2  163  3.98 ± .02  0  0  165  2.00  0  0  165  2.00  5  0  0  0  0  168  4.00  0  0  168  2.00  0  0  168  2.00  6  0  3  12  43  107  3.54  62  87  1.48  .08  0  10  149  1.94 + .03  7  0  0  5  32  82  1.71 ± .09  0  7  112  1.94 ± .04  8  0  0  0  0  150  9  0  0  2  10  94  10  0  0  0  0  11  0  0  0  0  0  12  + .09  10  ±  3.65 ± .09  6  23  90  4.00  0  0  150  2.00  0  0  150  2.00  3.87 i .07  0  0  106  2.00  0  0  106  2.00  109  4.00  0  0  109  2.00  0  0  109  2.00  0  139  4.00  0  0  139  2.00  0  0  139  2.00  0  142  4.00  0  0  142  2.00  0  1  141  1.99 ± .01 ro CT\ ro  WING VEIN GAPS LINE  x  0  2  3 4  0  0  133  18  12  0  0  0  0  2.26 ± .08  5  0  0  130  23  13  0  0  0  0  2.30 + .08  6  0  0  9  16  40  36  42  6  7  4.85 ± .19  7  0  0  15  15  73  6  1  1  0  3.69 ± .14  8  63  38  49  0  0  0  0  0  0  0.91 ± .12  9  17  21  62  3  2  0  0  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 U)  <T\  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 Ax 16172^y  J1 ) a raised in uncrowded condi-  tions . Data in lines 7,8 were obtained from the cross:  OR $ x  A x l6l72 / 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 1 2 3  GENOTYPE Axl6l72/Y  TEMP.  0  1  cf 20.5°C  16  33  50  10  20.5°C  26  35  22  23  61  54  Ax l6l72 /Ax l6l72  A x l6l72 / Y  ^  22oc  4  Axl6l72/Axl6172  ?  5  Ax 16172 /Y  a* 29°C  6  AX16172/AX16172  7  A x  8  Axl6l72/+  l 6 l 7 2  /  +  22°C  5  6  0  0  0  1.50 + .14  5  0  0  0  1.07 ± .16  77  21  2  0  0  1.55 ± .11  59  34  5  1  0  0  0.95 ± .12  1  21  33  42  0  0  3.20 ± .10  32  22  7  0  0  2.54 + .10  x  29°C  0  2  ^  22°C  0  0  0  9  95  15  3  4.10 ± .09  £  29°C  0  0  0  0  73  13  1  4.17 ± .08  ?  ro  CTi  ui  4 '  0  1  2  0.00  0  0  1  20  88  3.80  +  .07  0  0.00  0  0  0  9  79  3.90  +  .06  0  0  0.00  0  0  2  21  161  3.86  +  .05  153  0  0  0.00  0  0  1  12  140  3.91  +  .05  0.052 ±.026  97  0  0  0.00  0  0  1  6  90  3.92  +  .04  0.016 ±.017  63  0  0  0.00  0  0  0  4  59  3.94  +  .03  77  1.59 ± .09  43  54  25  0.85 ± .11  0  0  0  0  122  4.00  15  0.77 ± .14  44  33  10  0.61 ± .13  0  0  0  0  87  4.00  2  1  VERTICALS  POSTVERTTCALS  OCELLARS 0  2  1  x  LINE  0  1  109  0  0  0.00  109  0  0  2  88  0  0  0.00  88  0  3  183  1  0  0.01 ± .01  184  4  153  0  0  0.00  5  92  5  0  6  62  1  0  7  5  40  8  35  37  x  x  rv> cr> o>  ANTERIOR SCUTELLARS  DORSOCENTRALS LINE  0  1  2  3  4  X  0  1  2  POSTERIOR SCUTELLARS 0  X  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  2  6  79  1.89  0  0  87  2.00  +  .05  +  .07  ro  o> —j  LINE  1  0  2  3  WING  VEIN  GAPS  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)/Ax 9B2 £ x Ax9B2/Y $  3-6  M5/Ax^ B2 $ x Ax9B2/ y  7  OR  $  x Ax9B2/Y  ( s e e Table 30)  &  Note that Ax^ B2 /Y males were obtained from stock cultures (XX,^ w  f/Y  $ x  Ax^B2/Y  ).  Female parents (lines 1,2) were obtained  from the cross l(FM6)/AxE1 ? (Ax E1 stock) x stock Ax 9 B 2 /Y <7 . Female parents (lines 3-6) were obtained from the cross M5/M5 ^ x stock  Ax9  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  20.5°C  0  20.5°C  0  3  ^  5  1  9B2 Ax /Y  2  AX9B2/AX9B2  3  AX 9 B 2 /Y  cf  22°C  0  0  0  0  0  4  AX9B2/AX9B2  9  22°C  0  0  0  0  0  5  AX 9 B 2 /Y  cf  29°C  6  AX 9 B 2 /AX 9 B 2  £  29°C  0  0  0  0  7  Ax9B2/+  9  22°C  0  0  0  1  cf  0  0  6  0  2  87  5-98 ± .03  0  3  75  5.96 ± .04  11  117  5.91  ±  .05  3  93  5.97 ± . 0 3  3  36  5.92 + .08  0  1  22  5-96 ± .08  12  31  63  5.46 + .12  ro  o  OCELLARS LINE  0  2  1  POSTVERTICALS x  0  1  2  x  VERTICALS  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  ANTERIOR  DORSOCENTRALS LINE  0  1  2  4  3  x  0  SCUTELLARS  1  2  x  POSTERIOR  0  SCUTELLARS  2  1  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  WING V E I N LINE  x  0  16  26  9  20  5  0  2  6  0  0  102  5  3  WING  GAPS  62  20  58 4  3  6  7  0  0 0  11  0 0  8  0 0  3  1 3  0  0  0  0  0  0  0  1.75 ± .14  127  0  0  0.00  0  1.66 ± .13  93  0  0  0.00  0  3.89 ± .41  35  1  0  0.03 ± .05  4.44 ± .65  19  1  0  0.05 ± .09  0.05 ± .04  107  0  0  0.00  0 0  NICKING  0  1  ru  —5  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/N 8 $ x  3,4  M5/w a N 4 0 rb $ x Ax E2 /Y $  5,6  M5/y  7,8  l(FM6)/wa N® 1 1 rb £ x Ax E2 /Y £  9,10  M5/w a N^ll  w  a  N  A x  1 0 3  E 2  $  / Y  x  A x  E 2  / Y  c?  rb $ x Ax E2 /Y <?  11,12  Ax E 2 /Ax E 2 $ x w a  13,14  M5/wa N C o £  x  A x  N  E 2  g  l l  / Y  rb/BS w + • 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 intervals, and see Appendix 8.  ORBITALS 6  1  2  3  22°C  0  0  0  0  3  25  158  5.83 ± .05  29°C  0  0  0  4  24  23  11  4.66 + .18  22° C  0  0  0  0  3  18  88  5.78 ± .08  29°C  0  0  0  1  15  3  9  4.71 ± .32  22°C  0  0  0  5  171  12  2  4.06 ± .05  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  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  22°C  0  0  0  0  3  10  103  5.86 ± .07  29°C  0  0  0  1  4  14  10  5.14 ± .26  1 2  AX E 2 /N 8  3 A  x  E  W  6  9  AXE2/N103  A x E2 / N gll  13 A X  14  E 2  / N  5  X  °  4 5  TEMP.  4  0  GENOTYPE  LINE  C  °  POSTVERTICALS  OCELLARS  2  VERTICALS  0  1  2  3  4  1.99 ± .02  0  0  0  0  186  4.00  2.00  0  0  0  2  62  3.97  1.98 + .02  0  0  0  0  109  4.00  28  2.00  0  0  0  1  27  3.96  0  190  2.00  0  0  0  0  190  4.00  0  0  42  2.00  0  0  0  0  42  4.00  1.87 ± .07  0  0  124  2.00  -  -  -  -  -  -  77  1.97 ± .03  0  1  78  1.99 ± .02  -  -  -  -  -  -  5  152  1.97 ± .03  0  2  155  1.99 ± .02  0  0  0  0  157  4.00  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  LINE  0  1  0  1  2  1  0  0  186  2.00  0  2  184  2  0  1  63  1.98 ± .03  0  0  64  3  0  0  109  2.00  0  2  107  4  0  3  25  1.89 ± .11  0  0  5  0  0  190  2.00  0  6  0  1  41  1.98 ± .04  7  4  8  112  8  0  2  9  0  10  X  x  /  DORSOCENTRALS LINE  0  1  2  3  ANTERIOR  4  1  0  0  0  0  186  2  0  0  0  1  63  3  0  0  0  0  4  0  0  0  5  0  0  6  0  7  X  0  SCUTELLARS  2  1  X  POSTERIOR  0  1  SCUTELLARS  2  X  4.00  0  1  185  1.99 + .01  0  1  185  3.98 ±. .03  0  1  63  1.98 + .03  'o  0  64  109  4.00  0  1  108  1.99 ± .02  0  3  106  0  28  4.00  0  0  28  2.00  0  0  28  2.00  0  0  190  4.00  0  0  190  2.00  0  0  190  2.00  0  0  2  40  3.95 ± .06  0  1  41  1.98 ± .04  0  0  42  2.00  0  0  0  0  124  4.00  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  4.00  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  4.00  0  1  115  1.99 ± .02  0  0  116  2.00  14  0  0  0  0  29  4.00  0  2  27  1.93 ± .09  0  0  29  2.00  1.99 ± .01 2.00 1.97 ± .03  ro —4  WING VEIN GAPS 4  6  WING NICKING 8  0  2  LINE  0  1  2  1  125  18  22  0  0  0  0  0  0  0.38 + .10  0  0  178  2.00  2  36  9  12  0  0  0  0  0  0  0.58 ± .19  0  0  60  2.00  3  75  6  4  0  0  0  0  0  0  0 . 1 6 ± .09  0  0  95  2.00  4  22  1  1  0  0  0  0  0  0  0.13 ± .16  0  0  25  2.00  5  0  0  96  6  1  0  0  0  0  2.08 + .05  12  22  82  1.60 ± .11  6  9  6  6  0  1  0  0  0  0  1.00 + .41  0  0  28  2.00  9  26  23  86  0  0  0  0  0  0  1.44 + .12  112  21  2  0.19 ± .06  10  23  3  5  2  0  0  0  0  0  0.58 ± .30  0  1  34  1.97 ± .05  93  8  0  0  0  0  0  0  0  0.08 ± .05  0  1  106  1.99 ± .02  ro  2.00  oc  5  7  x  1  x  7  8  11 12 13 14  23  1  2  0  0  0  0  0  0  0.19 ± .20  0  0  26  279 APPENDIX 10 Counts of bristles, wing vein gaps, and wing nicking in A x 1 ^ 1 7 2 / ^ heterozygotes The heterozygotes examined were generated by the following crosses . LINE  CROSS  1,2  M5/N 8 $ x A x i 6 i 7 2 / Y d"  3,4  M5/w a N 4 0 rb £ x A x i 6 i 7 2 / Y  5  dl49,y Hw m 2 /w a N 4 0 rb $ x Ax l 6 l 7 2 /Y <?  6  M5/y w a N 1 0 3 $ x A x i 6 i 7 2 / y  7,8  1(FM6)/wa N S 1 1 rb £ x Ax l 6 l 72/ Y  9  M5/w a N S 1 1 rb $ x Ax l 6 l 7 2 /Y tf  10,11  M5/w a N C o $ x Ax l 6 l 72/ Y  J  $ g  ^  Note that the figures in Table 25 were calculated from results of the crosses of M5/N females to Ax E 2 /Y males.  See Methods and  Materials for explanation of x and confidence intervals, and see Appendix 8.  ORBITALS  0  1  2  3  4  22°C  0  0  0  1  2  29°C  0  0  0  3  22°C  0  0  29°C  0  20 .5°C  LINE  GENOTYPE  1 A X  4  A  x  1 6 1 7 2  1  6  1  / N  7  V  °  A x 16172 / n 103  7 8  AX16172/N«"  9 10 A X  11  1 6 1 7 2  / N  6  158  28  6  4.20 ± .06  6  15  9  0  4.10 + .23  0  2  112  37  13  4.37 ± .09  0  0  0  0  0  0  0  1  134  21  1  4.14 ± .05  22°C  1  1  10  9  41  0  0  3.42 ± .20  20.5°C  0  0  0  9  130  9  0  4.00 + .05  29°C  0  0  0  0  1  2  0  4.7  22°C  0  0  2  21  83  0  0  3.76 ± .08  22°C  0  0  0  2  40  49  34  4.92 ± .12  29°C  0  0  0  0  0  0  X  8  5  6  5  TEMP.  C O  1  1  0  -  —  ro  oc o  OCELLARS  LINE  0  POSTVERTICALS  2  1  X  0  0  1  2  3  4  2.00  0  0  0  3  190  3.98 ± .02  2  1  VERTICALS  X  X  1.99 ± .01  0  0  193  30  2.00  0  2  28  1.93 ± .08  0  0  0  3  27  3.90 ± .10  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  8  0  2  1  0  0  3  2.0  0  0  0  0  3  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  1  0  1  192  2  0  0  3  0  1.3  -  -  3.99 ± .01 4.0  ro oo h-1  DORSOCENTRALS LINE  0  1  2  3  ANTERIOR  4  1  0  0  0  0  2  0  0  0  1  3  0  0  0  1  163  4  0  0  0  0  1  5  0  0  0  0  163  6  2  3  20  18  7  0  0  4  8  0  0  9  0  10 11  X  2  1  X  0  0  193  29. 3.97 ± .06  0  1  3.99 ± .01  0  -  POSTERIOR  0  1  SCUTELLARS  2  X  2.00  0  2  191  29  1.97 ± .06  0  0  30  2  162  1.99 ± .02  0  2  162  0  0  1  0  0  1  4.00  0  1  162  1.99 ± .01  0  0  163  19  2.79 ± .23  0  4  58  1.94 ± .06  0  1  61  32  112  3.73 ± .07  4  26  118  1.77 ± .07  0  0  148  0  1  2  3.7  0  2.0  0  0  3  0  10  21  75  3.61 ± .11  8  26  72  1.60 ± .11  0  0  106  2.00  0  0  0  2  123  3.98 ± .02  0  0  125  2.00  0  0  125  2.00  0  0  0  0  1  0  0  1  0  0  1  193  4.00  0  SCUTELLARS  3  -  1.99 ± .02 2.00 1.99 ± .02 -  2.00 1.98 + .03 2.00 2.0  ro  ex ro  LINE  0  1  2  __3  WING  VEIN  GAPS  4  5  6  WING  7  8  0  x  NICKING  x  1  1  0  0  167  0  0  0  0  0  2.03 ± .03  0  0  184  2.00  2  0  1  27  0  0  0  0  0  2.00 ± .09  0  0  30  2.00  3  2  2  108  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 u>  oc  284 APPENDIX 10 9B2  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  M5/N  3  dl49,y Hw mf/wf N ^ rb $ x Ax9B2/Y <?  4,5  M5/wf N^. rb ? x  6,7  M5/y  8,9  1(FM6)/wa NS 1 1 rb $ x  $  AX  x  9 B 2  $ x  wf N1Q3  10,11  M5/wf N S 1 1  12,13  M5/wa  NCo  rb $  /Y  Ax  9  B 2  AX9  B 2  /Y  &  /Y  cf  Ax  9 B 2  ? x Ax9B2/Y  x Ax9B2/y  /Y  d*  J1  ^  Note that the figures in Table 26 were calculated from results of the crosses of M5/N females to  Ax  9 b 2  /Y  males.  See Methods  and Materials for explanation of x and confidence intervals, and see Appendix 8.  ORBITALS 0  1  2  3  4  5  6  22°C  0  0  0  0  0  3  112  5.97 ± .03  29°C  0  0  0  1  5  23  37  5.45 ± .15  20.5°C  0  0  0  0  1  3  207  5.98 ± .02  22°C  0  0  0  0  0  6  95  5.94 ± .04  29°C  0  0  0  0  12  20  25  5.23 ± .18  22°C  0  0  0  0  93  28  18  4.46 ± .10  7  29°C  0  0  0  0  0  2  5  8  20.5°C  0  0  0  0  5  34  83  5.64 ± .09  29°C  0  0  0  0  1  4  15  5.70 ± .23  22°C  0  0  0  4  27  36  49  5.12 ± .14  29°C  0  0  0  0  0  4  21  5.84 ± .13  22°C  0  0  0  0  0  3  78  5.96 ± .04  29°C  0  0  0  1  0  11  24  5.61 ± .19  GENOTYPE  LINE 1 2  9B2 8 Ax /N  3 4  Ax  9 B  V°  5 6  9 10  A x 9B2 / n 103  „ 9B2 /M gll Ax /N  11 12 13  AX9B2/NCo  TEMP.  X  5.7  ± .4  ro oc  VJl  OCELLARS 2  VERTICALS  POSTVERTICALS 1  2  0  1  2  7  27  81  1.64 ± .10  0  0  0  2  113  3.98 + .02  0.33 ± .12  31  25  10  0.68 ± .16  0  0  2  22  42  3.61 + .12  10  0.29 ± .07  5  24  182  1.84 + .05  0  0  0  0  211  4.00  14  1  0.16 ± .07  8  22  71  1.62 ± .11  0  0  0  0  101  4.00  46  10  1  0.21 + .11  16  24  17  1.02 ± .18  0  0  0  0  57  4.00  6  25  53  61  1.26 + .11  5  12  122  1.84 ± .07  0  0  0  0  139  4.00  7  7  0  0  0.0  0  3  4  1.6 ±  0  0  0  0  7  4.00  8  121  1  0  0.01 + .02  119  3  0  0.02 ± .03  0  0  0  0  122  4.00  9  19  1  0  0.05 ± .09  0  3  17  1.85 + .15  0  0  0  0  20  4.00  10  116  0  0  0.00  115  0  1  0.02 + .03  0  0  0  1  115  11  24  1  0  0.04 ± .07  0  4  21  1.84 ± .13  0  0  0  0  25  4.00  12  15  31  35  1.25 ± .14  4  19  58  1.67 ± .11  0  0  0  0  81  4.00  13  31  4  0  0.11 ± .10  4  18  14  1.28 ± .19  0  0  0  0  36  4.00  LINE  0  1  X  1  91  23  1  0.22 + .07  2  47  16  3  3  160  41  4  86  5  0  X  .5  4  3  X  3.99 ± .02  rv a a  DORSOCENTRALS LINE  0  1  2  3  4  ANTERIOR SCUTELLARS X  0  2  1  X  1  0  0  26  35  54  3.24 + .13  0  0  115  2  0  0  47  16  3  2.33 ± .12  0  1  3  0  2  30  84  95  3.29 ± .09  0  4  0  0  18  37  46  3.28 ± .13  5  0  0  36  16  5  6  0  0  34  33  72  7  0  0  0  0  8  1  13  105  9  0  1  10  0  11  POSTERIOR SCUTELLARS 0  2  1  X  2.00  0  0  115  2.00  65  1.98 ± .03  0  0  66  2.00  1  210  2.00 ± .01  1  1  209  1.99 ± .02  0  1  100  1.99 ± .02  0  2  99  1.98 ± .03  2.46 + .15  0  0  57  2.00  0  0  57  2.00  3.27 ± .12  0  0  139  2.00  1  5  133  7  4.0  0  0  7  2.0  0  0  7  3  0  1.90 ± .06  90  25  7  0.32 ± .09  0  3  119  15  2  2  2.25 ± .29  0  0  20  2.00  0  0  20  5  104  7  0  2.02 ± .05  63  32  21  0.64 + .12  0  3  113  1.97 ± .03  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  1.95 ± .04 2.0 1.98 ± .03 2.00  ro CO  WING VEIN GAPS 4  LINE  0  1  2  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  6  0  1  38  36  61  2  0  0  0  3.18  +  .13  129  8  1  0.072  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  +  13  12  11  3  6  WING NICKING  5  8  7  X  0  -  .18  1  2  X  2.00 ±.o4:  0.94 ± .24 ro cc  CO  289  APPENDIX 10 Counts of bristles and wing vein gaps in heteroallelic Ax x /Ax y combinations  The data on the following pages were obtained from the following crosses. LINE  CROSS  1,3  l(FM6)/AxE1  2  A x E 2 / A x E 2 ? x A x E 1 / B S w+.Y <?  4  l(FM6)/AxE1 $ x Ax l6 172/ Y  5,6  l(FM6)/AxE1 $ x  ? x Ax  Ax  e 2  /Y  9 B 2  /Y  c?  d*  Ax 16172 /Ax 16172 ? x A x E 2 / Y  7  $  #  8  Ax E 2 /Ax E 2 $ x Axl6l72 /Y  9  wf Ax E 2 rb/+ A x 1 6 1 7 2 + $ x wf A x 9 B 2  ^  rb/Y c? The wings of all the genotypes except Ax E 2 /Ax 1 ^ 1 7 2 (lines 7,8) were too deformed to score accurately for wing vein gaps. The data for A x E 2 / A x l 6 1 7 2 females are tabulated below. NUMBER OF FLIES WITH GIVEN NUMBER OF WING VEIN GAPS 0  1  2  3  7  0  0  0  0  8  0  0  1  3  LINE  4  5  6  7  8  41  11  7  2  1  4.56 ± .20  35  15  8  3  1  4.59 ± .23  x  See Methods and Materials for explanation of x and confidence intervals, and see Appendix 8.  ORBITALS GENOTYPE  LINE 1  2  AX  E 1  /AX  E 2  3  4  5 6  7  AxE1/AX16172  AXE1/AX9B2  AxE2/Axl6l72  8  9  AXE2/AX9B2  TEMP.  0  1  2  3  4  5  6  20.5°C  13  0  0  0  0  0  0  20 .5°C  10  0  0  0  0  0  0  20 .5°C  2  1  0  0  0  0  0  20.5°C  2  0  0  0  0  0  0  20.5°C  0  0  17  43  50  6  0  3.39 ± .10  20.5°C  0  0  20  35  33  10  0  3.34 ± .16  20.5°C  0  4  24  26  12  0  0  2.70 ± .18  20 .5°C  0  6  21  30  11  0  0  2.68 + .18  28  0  0  0  0  0  0  0.0  22° C  X  ) ) ) N  )  0.04 ± .07  )  -  ro vo o  POSTVERTICALS  0CELLARS  LINE  0  1  2  1  13  0  0  2  10  0  0  3  3  0  0  4  2  0  0  5  116  0  0  6  98  0  7  66  8 9  VERTICALS  0  1  2  3  4  0  0  0  0  13)  0  0  0  0  10? 4.0  0  0  0  0  3?  -  0  0  0  1  1  0  0.00  0  0  0  0  116  4.00  0  0  0.00  0  0  0  0  98  4.00  66  0  0  0.00  -  -  -  -  -  0.00  68  0  0  0.00  0  0  1  1  66  3-96 ±  0.0  28  0  0  0.0  1  6  10  8  3  2.21 ±  0  1  2  13  0  0  10  0  0  3  0  0  2  0  0  0.00  116  0  0  0.00  98  0  0  0.00  68  0  0  28  0  0  X  0.0  -  X  0.0  X  -  -  vo  DORSOCENTRALS LINE 1  0  2  1  11  ANTERIOR SCUTELLARS  4  3  0  x  2  0  0  0  2  1  x  13  0  0  10  0  0  3  0  0  2  0  0  0.0  POSTERIOR SCUTELLARS 0  2  1  x  0  0  0  1  0  0  13) \ ) 9) \ ; 3)  2  0  0  1.96  ±  2  6  3  1  0  0  3  2  1  0  0  0  2  0  0  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  16  11  1  0.46 ± .19  2  8  18  1.57 ± .21  0.31  ±  .19  -  0.0  -  .07  -  ro vo  ru  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0107124/manifest

Comment

Related Items