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Genetic analysis of the gene Additional sex combs and interacting loci Nicholls, Felicity K. M. 1990

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GENETIC ANALYSIS OF THE GENE ADDITIONAL SEX COMBS AND INTERACTING LOCI by FELICITY K.M. NICHOLLS B.Sc, The University of British Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (ZOOLOGY) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1990 (c) Felicity KM. Nicholls, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoology The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: September, 1990 ABSTRACT In order to recover new mutant alleles of the Polycomb group gene Additional sex combs (Asx), mutagenized chromosomes were screened over the putative Asx allele XT129. Thirteen new mutant strains that fail to complement XT129 were recovered. Unexpectedly, the thirteen strains sorted into four complementation groups. Recombination mapping suggests that each complementation group represents a separate locus. The largest group fails to complement a deletion of Asx and maps in the vicinity of 2-72, the published location of Asx. All new mutant strains enhance the phenotype of Polycomb mutant flies and are not allelic to any previously discovered second chromosome Polycomb group genes. Therefore, the new mutants may be considered putative new members of the Polycomb group. This study suggests that Asx belongs to a sub-group of genes displaying intergenic non-complementation. ii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES iv LIST OF FIGURES v ACKNOWLEDGEMENTS .vi INTRODUCTION 1 MATERIALS AND METHODS: 11 MATERIALS AND METHODS 11 CULTURE CONDITIONS: 11 MUTANT STRAINS: 11 MUTAGENESIS: 11 COMPLEMENTATION: 13 ENHANCEMENT BY OTHER SECOND CHROMOSOME POLYCOMB GROUP GENES: 13 RECOMBINATION MAPPING OF NEW MUTANT STRAINS: 14 Method 1) 14 Method 2) 14 CALCULATION OF MAP POSITION AND CONFIDENCE LIMITS: 17 CUTICLE PREPARATION OF EMBRYOS: 17 SCORING OF MUTANT EMBRYOS: 19 RESULTS: 21 GAMMA IRRADIATION SCREEN: 21 COMPLEMENTATION BETWEEN MUTANT STRAINS: 21 RECOMBINATION MAPPING OF MUTANT STRAINS: 25 ENHANCEMENT BY OTHER SECOND CHROMOSOME POLYCOMB GROUP GENES:. 31 EMBRYOLOGY OF THE ASX GROUP: 36 DISCUSSION 49 REFERENCES: 61 iii LIST OF TABLES Table 1. Complementation Data 22 Table 2. Recombination Data from Mapping of Complementing Strains 26 Table 3. Recombination Data from Recombination Mapping of Non-complementing Strains. 28 Table 4. Enhancement of Adult Homeotic Phenotypes of Second Chromosome Polycomb Group Genes by New Mutant Strains 33 Table 5. Enhancement of super sex combs by New Mutant Strains 35 Table 6. Summary of Embryonic Phenotypes ,37 Table 7. Comparison of Mutation Rates of Drosphila Loci 50 iv LIST OF FIGURES Figure 1. Mutagenesis Screen 12 Figure 2. Recombination Mapping Scheme for Complementing Strains 15 Figure 3. Recombination Mapping Scheme for Non-Complementing Strains 16 Figure 4. Calculation of 95% Confidence Intervals 18 Figure 5. Complementation Map 24 Figure 6. Embryology of the Asx Group 43 Figure 7. Head Defects of the Asx Group 45 Figure 8. Embryonic Phenotype of Complementation Group B Mutant 27-97 47 v ACKNOWLEDGEMENTS I gratefully acknowledge my supervisor, Hugh Brock, whose intellectual brilliance and interest in my project has made this work possible. This thesis is dedicated to my fiancee, Terry Estrin, whose love and support has been a constant source of strength for the last three years. Many thanks to my father, for his very helpful financial assistance. To Ian, Melanie, Lisa, Gretchen, Rebecca, Emily and Chris, who are the most wonderful friends a person could ever have. Thanks to Joanie McKeon, who took time away from her project to help with the screen. To Eric Slade for providing major laughs and to Jenny Baird, technical assistant on the screen, for being such a trooper. Thanks also to Marco DiCamillis and Mark Daly, who helped prevent Journal Club from getting too serious. vi INTRODUCTION The early development of the fruit fly, Drosophila melanogaster, is probably the most well understood of any organism. In the early eighties, mutagenesis screens identified numerous mutations upsetting pattern formation in the early embryo (Nusslein-Volhard and Wieschaus, 1980; Wieschaus et al., 1984; Jurgens et al., 1984; Nusslein-Volhard et al., 1984; Perrimon et al., 1984; Schupbach and Wieschaus, 1986). A different class of genes affecting segmentation, the homeotic genes had been discovered earlier (Bridges and Morgan, 1923; Lewis, 1963). How segmentation and homeotic genes work together to establish segmental boundaries and to assign each an appropriate identity is not completely understood. Overall, the events of early development can be divided into three main stages: 1) axis formation; 2) formation of segments; 3) assignment of segment identity (for review see Akam, 1987). The initial polarity of the embryo is achieved through the action of gene products deposited in the egg by the mother's germ line (see Akam, 1987). Evidence of this pre-existing polarity can be seen in the shape of the unfertilized egg. How this initial polarity is established is not completely understood. The establishment of the anterior pole is probably the best understood component of the system. It has been demonstrated that an exponential concentration gradient of an anterior morphogen (the product of the maternal gene bicoid) exists in the anterior two thirds of the egg. As predicted by genetic analysis, multiple binding sites for bicoid protein are located in the upstream regulatory region of the gap gene hunchback. These binding sites activate the gene in a concentration-dependent manner (Struhl et al., 1989; Driever et al., 1989; McDonald and Struhl, 1988; Frohnhofer and Nusslein-Volhard, 1986). 1 INTRODUCTION The posterior system is less well understood. Aside from the formation of pole cells, which will give rise to the germ line, recent studies suggest that the main function of posterior-specific genes is to prevent the persistence of posterior expression of maternal hunchback protein. It remains to be seen which maternal genes, if any, are responsible for turning on the posterior gap genes in their appropriate locations (Review: Manseau and Schupbach, 1989; Struhl, 1989; Irish et al, 1989; Hulskamp et al, 1989/Lehmann and Nusslein-Volhard, 1987). The dorsoventral axis may work on an entirely different system. Ten genes are involved in the translocalization of the putative morphogen dorsal from the cytoplasm to the nucleus. The result is a sharp gradient of nuclear dorsal protein; the protein being almost entirely located in the nucleus at the ventral pole and almost entirely located in the cytoplasm at the dorsal surface (Steward, 1989; Rushlow et al, 1989; Roth et al, 1989; Hashimoto et al. 1988; review: Anderson, 1987). At the syncitial blastoderm stage, the gap genes become active. These genes are active in limited domains of the embryo and may be largely responsible for establishing abdominal segmentation. In situ hybridization reveals that the gap genes are not transcribed in every nucleus of the embryo. Rather, the expression of each is confined to specific and different domains along the anterioposterior axis. Cross-regulatory interactions help sharpen the boundaries between the domains. Recent evidence suggests that these interactions are more complex than previously thought. In the anterior half, gap genes repress each other at the domain borders, whereas some gap genes positively regulate each other in the posterior half (Jackie et al, 1986; Pankratz et al, 1989). A main function of the gap genes is to establish the appropriate pattern of pair rule gene expression. The function is reflected in the fact that all three of the principal gap gene 2 INTRODUCTION proteins display DNA binding capabilities (Rosenberg et al., 1986; Tautz et al., 1987; Nauber et al., 1989). The eight pair-rule genes are initially expressed uniformly in the embryo and then expression coalesces into a series of seven stripes. Each pair-rule stripe is approximately one segment wide, but each pair rule gene is expressed out of register to all the others (see Ingham, 1988 for review). Considerable controversy still exists as to how the gap genes establish pair rule gene expression. Recent findings suggest that the borders of the gap gene domains overlap more extensively than previously thought and that within these regions of overlap the concentrations of the gap proteins vary considerably. Overall, studies are beginning to suggest that each pair rule stripe may be specified independently, and at these regions of gap gene overlap. There is evidence that the gap genes may create stripes by repressing pair rule expression in the interstripe regions (Goto et al., 1989; Stanojevic et al., 1989; Carroll, 1990). The pair rule genes are divided into two classes. Primary pair rule genes: hairy, even-skipped and runt, which are expressed first and are required to establish the correct expression of the remainder of the pair rule genes, the secondary class. Cross-regulatory interactions at the borders of the pair rule stripes help to sharpen the boundaries between the stripes (see Ingham, 1988). One major function of the pair rule genes is to establish expression of the segment polarity genes, principally engrailed and wingless. At the onset of gastrulation, these genes are expressed as fourteen narrow stripes in either the anterior or posterior half of the segmental domains established by the pair rule genes. Correct spatial initiation of these genes is thought to depend on the the specific combinations of pair rule genes expressed in a given cell (Martinez-Arias et al, 1988; DiNardo et al, 1988; Rijsewijk et al, 1987). 3 INTRODUCTION The segmentation genes have two major functions. The first, described above, is to divide the embryo up into smaller and smaller segments. The second major function is the regulation of the homeotic genes. The homeotic genes critically depend on the segmentation genes for their expression at the right time and place. The homeotic genes of Drosophila are arranged in two complexes. The bithorax complex consists of three genes, Ultrabithorax, abdominal-A and Abdominal-B (Lewis, 1978; Sanchez-Herrero et al., 1985). Mutations in these genes affect the posterior half of the second thoracic segment, the third thoracic segment and all eight abdominal segments. The Antennapedia complex consists of five homeotic genes, proboscipedia, Deformed, Sex combs reduced, Antennapedia, and labid. Mutations in these genes affect the head, the first thoracic segment and the anterior portion of the second thoracic segment (Wakimoto and Kaufman, 1981; Regulski et al., 1985). In situ hybridization and antibody staining experiments reveal that each homeotic gene is expressed in a complex manner that differs between segments. Each gene tends to be expressed at highest levels in those segments that are particularly affected by mutations in that gene. Not only do mutations in segmentation genes upset this spatial pattern but cross-regulatory interactions bewtween homeotic genes help to define these domains. In general, each of the more posteriorly expressed homeotic genes represses the expression of all the more anterior ones initially active within its own domain (Hafen et al., 1984; Harding et al., 1985; Struhl and White, 1985). Homeotic gene expression within segments is complex. The homeotic genes are expressed during the blastoderm stage when segmentation gene expression is active, but they are also expressed during the development of the peripheral and central nervous systems (Akam, 1985; White and Wilcox, 1984,1985a, 1985b; Beachy et al, 1985; Carroll et 4 INTRODUCTION al., 1988), and during the development of the midgut (Bienz and Tremml, 1988). Analysis of Ultrabithorax gene expression in the central nervous system reveals a complex mosaic pattern of expression, differing from cell to cell. Peifer et al. (1987) have proposed that segment-specific regulatory regions and a variety of different forms of DNA-binding Ultrabithorax proteins resulting from the same transcription unit are responsible for this intrasegmental complexity. Factors that regulate early homeotic gene expression are themselves expressed transiently. By the time of germ band formation, and cross-regulatory interactions have established the domain of each homeotic gene, a new set of controls are established to maintain the pattern of homeotic gene expression. The Polycomb group of genes is required to maintain the repression of many homeotic genes in those segments where each is not normally active. Embryos mutant for two such genes Polycomb and extra sex combs display a normal distribution of Ultrabithorax protein until after germ band establishment, at which time the pattern breaks down, causing Ultrabithorax to become inappropriately expressed at high levels throughout the embryo (Struhl and Akam, 1985; Weeden et al., 1986). The Polycomb group of genes has eleven described members with related phenotypes, including embryonic transformations resembling gain-of-function mutations in the bithorax and antennapedia complexes. The Polycomb group includes Polycomb (Pc) (Lewis, 1978; Duncan and Lewis, 1982), extra sex combs (esc) (Struhl, 1981), Polycomblike (Pel) (Duncan, 1982), super sex combs (sxc) (Ingham, 1984), Additional sex combs (Asx), Posterior sex combs (Psc), Sex combs on midleg (Sem) (Jurgens, 1985), Sex combs extra (See) (Breen and Duncan, 1986),polyhomeotic (ph) (Dura et al. 1985), polycombeotic (pco) (Shearn et al., 1978) and pleiohomeotic (pho) (formerly 1(4)29) (Hochman et al., 1964; Gehring, 1970). 5 INTRODUCTION As a group, the Polycomb genes are diverse. Pc and esc display the strongest embryonic phenotype, with all segments of the embryo resembling the eighth abdominal segment (Lewis, 1978; Struhl, 1981). The rest of the genes generally have a weaker phenotype. Typically an embryonic body segment will partially take on some characteristics of the segment immediately posterior to it. The phenotype of these genes predicts that they interact at some level with the homeotic genes. However, genetic analysis has shown that they do not all interact in the same way. The phenotypes of Pc, esc, Pel, and sxc are all enhanced when heterozygous with three wild type does of the bithorax complex (Duncan and Lewis, 1982; Struhl, 1981; Duncan, 1982; Ingham, 1984). Conversely, the mutant phenotypes of ph, Asx and Psc are suppressed by an extra copy of the complex (Dura et al., 1985; Slade and Brock, unpublished). There is also great variation among the Polycomb group in a number of other characteristics, sxc, esc, ph and pho all show a very strong maternal effect. Pc, Pel, Asx, Scm and See embryos have a stronger phenotype when derived from homozygous mothers, but this effect is rescuable by a wild type paternal contribution. Just as there is variation in the germ line requirement for some of these genes, some, but not all are required later in development, in the imaginal discs (Breen and Duncan, 1986; Duncan and Lewis, 1982; Ingham, 1984; Duncan, 1982 and Slade and Brock, unpublished). Some of the genes have phenotypes other than that of homeotic transformations. For example, some ph embryos show a complete loss of ventral ectoderm, and Psc embryos are twisted and deformed relative to a wild type embryo (Dura et al, 1985; McKeon and Brock, submitted). Recent evidence suggests that the Polycomb group genes may have a more general function in development than the regulation of homeotic genes, ph is required for the growth of ventral epiderm and axonal outgrowth (Dura et al, 1987; Smouse et al, 1988). 6 INTRODUCTION Most of the genes are rquired for the correct spatial expression of engrailed and Ultrabithorax in the central nervous system (McKeon and Brock, submitted) and CNS expression of the two genes is completely suppressed in ph mutant embryos (Dura et al., 1987; Smouse et al., 1988; McKeon and Brock, submitted). Embryos arising from mothers with homozygous sxc, Pel, or pho have segmentation defects (Ingham, 1984; Breen and Duncan, 1986). pco was originally isolated due to its small disc phenotype (Shearn et al., 1978). Despite the many differences between the members of the group, mounting evidence supports the idea that the genes act together to form a regulatory network. The genes show a significant level of genetic interaction. The mutant phenotype of all members of the group except Psc is enhanced when heterozygous with a mutant copy of Pc (Duncan, 1982; Struhl, 1981; Ingham, 1984; Dura et al., 1985; and Campbell and Brock, unpublished). Double and triple homozygous mutant combinations of the phenotypically weak genes Pel, Asx, Psc and Sem display a phenotype equally strong as a Pc or esc mutant (Jurgens, 1985). Recent molecular evidence supports the hypothesis of direct interaction between the genes. Antibodies raised against Pc bind to 60 discrete sites on the polytene chromosomes, including the bithorax and Antennapedia complexes and to Sem, Asx, Psc, sxc, ph, and pco (Zink and Paro, 1989). Why are so many genes required for the maintenance of segment identity? To answer this, a deeper understanding of the molecular nature of determination will be required. One possibility is that all the genes have a similar regulatory DNA-binding function but that many genes are required because each is expressed only transiently. Hence, different Polycomb group genes could bind to the same sites but at different times throughout development. Combinations of Pc group proteins could be required for this enhancer binding 7 INTRODUCTION function. Another possibility is that the Polycomb group genes form a supramolecular structure involved in chromatin changes. Chromatin changes are an important form of stable, heritable gene regulation in other organisms. It is possible that some Pc group proteins associate to permanently change the chromatin conformation of the homeotic genes in such a way as to maintain their determination state. In support of this hypothesis, every other system in Drosophila displaying genetic interaction studies at the molecular level has been proven to involve the physical association of the protein products of the interacting genes to form a functional structure (see discussion). It is probable that a combination of mechanisms are required for the maintenance of determination. To understand the Polycomb group as whole, it is necessary to study each member in detail. With this in mind, I undertook the investigation of the gene Additional sex combs. Asx was first isolated in a second chromosome saturation screen for embryonic lethals, and was localized to cytological division 51AB on the basis of failure to complement a large deletion spanning 51AB (Nusslein-Volhard et al., 1984). The embryonic phenotype consists of posterior directed transformations, generally weaker in character than that seen in Pc embryos. However, when Asx is in double or triple mutant combination with Pel, Psc or Scm, the strength of transformation is equal to that of Pc or esc mutant embryos (Jurgens, 1985). It was later demonstrated that Asx has a maternal effect, but that the effect is rescuable zygotically by a wild type paternal contribution (Breen and Duncan, 1986). Asx adults show many of the homeotic transformations typical of the group. The most commonly seen transformation in adults is that of the fourth abdominal segment into the fifth, resembling the Misciidestral pigmentation mutation of the bithorax complex. Less often, the i first abdominal segment is transformed into the second, and rarely, sex combs are found on the second leg. This phenotype depends greatly on genetic background and culture 8 INTRODUCTION conditions. Frequently, defects in the abdominal tergites of odd numbered segments are seen. The penetrance of the above homeotic transformations is decreased when an extra wild type copy of the bithorax complex is introduced to the genome (Slade and Brock, unpublished). Recent molecular evidence confirms the role of Asx in the regulation of the bithorax complex. Initial distribution of Ultrabithorax in Asx embryos is normal but becomes ectopic in both epidermis and CNS following germ band extension (McKeon and Brock, submitted). engrailed distribution is also ectopic in both epidermis and CNS of Asx mutants (McKeon and Brock, submitted). The penetrance of these defects is not complete, but these studies only examine the role of zygotic Asx expression in the regulation of these genes and the maternal contribution could turn out to be critically important. In this study, I employed ionizing radiation in a screen to induce mutations in Asx. Since most previously existing alleles were ethyl-methylsulfonate (EMS)-induced and were few in number, it was necessary to generate new alleles of different types in order to study the gene further (Nusslein-Volhard et al., 1984; Jurgens, 1985). In addition, since the gene was only roughly localized to cytological region 51AB on the basis of its failure to complement a large deletion in the region Df(2R)L+R48 (Nusslein-Volhard et al, 1984), it was my aim to generate chromosomal rearrangements with breakpoints within the gene in the hopes of more precisely defining its location. In a F2 radiation screen for new mutants of Asx, thirteen new alleles were isolated. This rate is nearly an order of magnitude greater than that expected. The thirteen strains were crossed to ensure allelism. Unexpectedly, the radiation-induced alleles fell into four complementation groups. All of the groups fail to complement Asx^^^ (referred to subsequently as XT129). Preliminary recombination mapping suggests, and subsequent mapping has confirmed the recovery of eight new alleles of Asx. The remaining three INTRODUCTION complementation groups map at separate locations on the second chromosome. The results are consistent with the hypothesis that Asx and at least three previously undescribed genes display intergenic non-complementation with XT129. 10 MATERIALS AND METHODS: CULTURE CONDITIONS: Flies were grown in half pint milk bottles or 8 dram shell vials on a sucrose-cornmeal-agar medium, seeded with baker's yeast. Tegosept (methyl-p-hydroxybenzoate) was included in food as a mould inhibitor. Bottle crosses were performed at 22°C, pair matings were maintained at 25°C. MUTANT STRAINS: The following second chromosome stocks were used in the radiation screen, cinnabar (cn) brown (bw) was obtained from the Bowling Green stock center. DTS911Cyo pr cn: DTS91 is a temperature sensitive mutation, lethal at 29°C. Cyo purple (pr) cn is a standard second chromosome balancer, dominantly producing curled wings. This strain was supplied by T. Grigliatti. AsxP^^^pr cn/Cyo pr cn was recovered in a screen for embryonic lethals upsetting cuticle pattern (Nusslein-Volhard et al., 1984). This was kindly supplied by Gerd Jurgens. MUTAGENESIS: The scheme is diagrammed in Fig. 1. Two batches of cn bw males were irradiated, the first batch with 4,000 rads and the second with 4,500 rads from a ^ Co source. The irradiated males were crossed to DTS911 Cyo females in bottles, cn bw/Cyo pr cn F l males were collected and pair mated to 3 - 5 Asx^^^pr cn/Cyo pr cn virgins. F2 progeny were screened for lack of straight-winged flies in the vial, thus indicating that the lesion on the mutagenized chromosome was located within Asx. 11 c n b w D T S 9 1 C y O F 1 JLJD t U U C y O A „ „ XT129 _ S-JS C O . C y O Figure 1. Mutagenesis Screen. 12 MATERIALS AND METHODS COMPLEMENTATION: Roughly 10 males of each newly recovered strain were crossed to 5 - 10 females of every other strain in 8 dram shell vials in a complete complementation matrix. The flies were supplied with fresh medium every 3-4 days, 3 times. Between 70 and 200 progeny were scored for each cross. Failure to complement was defined as the complete absence of trans heterozygotes among the F l progeny. Trans heterozygotes display white eyes (due to the homozygous cn bw condition) and straight wings. Reciprocal crosses were not performed. ENHANCEMENT BY OTHER SECOND CHROMOSOME POLYCOMB GROUP GENES: Approximately 10 females from each newly induced strain were mated to 5 -10 esc, Pel, Psc, or sxc males, in 8 dram shell vials. Matings were supplied with fresh medium every 3-4 days, three times. Transheterozygotes were distinguished by lack of balancer markers and by appropriate eye colour. Between 15 - 20 transheterozygous males were scored on the basis of increased penetrance and expressivity of the following homeotic transformations: sex combs on the 2nd and 3rd thoracic legs; 4th abdominal segment transformed into the 5th, or the 5th into the 4th; 1st abdominal segment transformed into the 2nd; lack of humeral bristles and leg structures growing in the place of the antenna. The two most common homeotic transformations were the presence of extra sex combs and the transformation of A4 to A5. The sex comb phenotype was assigned a "1" if 3 or fewer sex comb teeth appeared on the mesothoracic (T2) leg. "2" was assigned if 3 - 6 teeth were present on the T2 leg or if 3 or fewer teeth were present on both the T2 and T3 legs. "3" was assigned if more than 6 sex comb teeth were present on the T2 leg or more than 3 teeth on both the T2 and T3 legs. The A4 to A5 phenotype was scored as follows. The degree of 13 MATERIALS AND METHODS transformation of A4 to A5 was calculated as the degree of ectopic pigmentation of A4. Accordingly, "1" was assigned if only 1 to 3 separate, small patches of pigment were present. "2" was assigned if about half of the tergite was pigmented. "3" was assigned if A4 was nearly or completely black. A l to A2 transformations were not noted and hence are not included in the overall calculation. Antennapedia transformations were rare, and are not included in the overall calculation, but are noted in the text where they occur. Similarly, loss of the humerus was specific to certain strains and unusual, and hence will only be noted in the text where it occurs. RECOMBINATION MAPPING OF NEW MUTANT STRAINS: Method 1) This scheme is diagrammed in Fig. 2.. 50 virgins of one complementing strain were collected and mated in bottles to 10 - 25 males of another complementing strain. F l trans heterozygous virgins were recovered and mated to XT129. More than 1,000 F2 progeny were scored for the presence of straight wings. The two tester strains were deemed separable by recombination if straight-winged flies were present. Method 2) This scheme is diagrammed in Fig. 3. Approximately 50 S Sp BI bw^ virgins were mated to 10 - 25 males of each strain. 25 - 50 F l trans heterozygous virgins were collected per strain and mated to 10 - 25 XT 129 males. Only straight winged F2 progeny were scored for recombination between the markers. The recombination interval 14 A X T 1 2 9 F 1 B 9 x C y O Figure 2. Recombination Mapping Scheme for Complementing Strains. 15 • e s t e r S S D BI b w ° P C y^> x C y O S S D BI h w D X T 1 2 9 F1 t e s t e r x C y O Figure 3. Recombination Mapping Scheme for Non-Complementing Strains. 16 MATERIALS AND METHODS containing reciprocal classes of recombinant flies is the interval in which the locus was located. CALCULATION OF MAP POSITION AND CONFIDENCE LIMITS: The map position was determined as follows: The recombination interval containing reciprocal recombinant classes is the interval containing the gene. The ratio of one reciprocal recombinant class to the other class was taken as the distance of the gene along chromosome between the flanking markers. Knowing the map positions of the flanking markers then allows an estimate of the map position of the gene. This position is approximate because of the large distance between the markers and because of differential recombination rates along the chromosome arm. See Fig. 4 for equation of calculation of 95% confidence limits. CUTICLE PREPARATION OF EMBRYOS: Flies were allowed to lay overnight on 2% agar laying trays brushed with a solution of yeast, ethanol and acetic acid (5% each). The parents were removed, and the trays were allowed to age a further 24 hours, after being supplied with a small knob of a thick yeast paste to attract hatched, heterozygous first instar larvae. Since embryos hatch at 22 hours post-fertilization, unhatched embryos remaining on the plate after 24 hours could be assumed to be homozygous mutant embryos or unfertilized eggs. Embryos were transferred to a closable screw cap basket. The baskets were transferred to a 50% bleach solution for 5 min. to dechorionate. The baskets were then rinsed three times in embryo wash (2.4% NaCl; 0.03% Triton-X 100) and rinsed one time in water and blotted on paper towels. Next the baskets 17 Figure 4. Calculation of 95% Confidence Intervals. The 95% confidence interval is calculated by the above equation, where p= the frequency of those crossovers between the flanking markers which are between the left marker and the gene being mapped; q= the frequency of those crossovers between flanking markers which are between the right marker and the locus (1 - p); N= the total number of crossovers examined; M= the distance in map units between the flanking markers (O'Brien, 1978). 18 MATERIALS AND METHODS were added to 8 ml of 0.1 M PIPES, ph 6.9; 2 mM MgS04 m 0^ m l orange cap plastic tubes. 10 ml of heptane were added to the tube which was then shaken vigorously for 1 minute. 2 ml 37% formaldehyde was then added to the tube which was then shaken on a rotating shaker for 20 minutes at 250 RPM. The baskets were blotted dry and transferred to 1:1 heptane methanol solution in a 50 ml tube and set to shake at 200 RPM for 10 minutes on a rotating shaker. The baskets were transferred to fresh 50 ml tubes containing 90% methanol and vortexed briefly, two times and then blotted and left overnight in 4:1 acetic acid:glycerol at 60 degrees. Embryos were transferred from the baskets to a small drop on a slide of Hoyer's Mountant (for recipe, see Roberts, 1986) using a fine paint brush. A coverslip was placed over top and the slide allowed to sit on a slide warmer at 45°C overnight. SCORING OF MUTANT EMBRYOS: Embryos were scored according to the following numerical system. Head defects, due to non-completion of head involution, were considered mild and given a numerical value of "1" if defects were confined to a slight shortening of the cephalopharyngeal apparatus (CPA), and slight splaying of the cirri. If the CPA were visibly shortened, and the cirri splayed flat out at the tip of the head, the embryo was assigned a "2". The defects were considered most severe and given a numerical value of "3" if the CPA were condensed, exploded or splintered. Once all the values for all the embryos scored for a given strain were averaged, an intermediate value of "2" could result if there was much variability between severity of defects among embryos. Homeotic transformations of the thorax were considered mild ("1") if 3 - 5 posterior pointing abdominal denticles appeared in the ventral setal belts (VSB's) of the third thoracic 19 MATERIALS AND METHODS segment (T3). The transformations were considered moderate ("2") if the abdominal denticles appeared in both T2 and T3. Transformations were deemed severe ("3") if anteriorly pointing abdominal denticles, arranged such that the T2 and T3 VSB's became trapezoidal rather than linear in shape, and if anterior pointing abdominal denticles appeared in the first thoracic segment. Homeotic transformations of the abdomen were classified as follows: Transformations were mild if 3 - 5 extra abdominal denticles appeared in Al, such that the VSB approached a trapezoid in shape, and if A7 became rectangular, suggesting a transformation toward A8 ("1"). Defects were moderate if more posteriorly directed denticles, forming a trapezoid, appeared in Al and if A6 and A7 were rectangular ("2"). Defects were severe ("3") if A4 or A5, A6 and A7 were rectangular, and if Al was trapezoidal and contained anteriorly pointing denticles, suggesting a transformation to a VSB posterior to A2. Segmentation defects were observed in some of the embryos. These defects consisted of loss of part or all of a VSB, often in alternating segments; fusion of all or parts of adjacent VSB's, or loss of denticles in the midline of VSB's. These defects were scored as present or absent. No attempt was made to score the penetrance of the embryonic phenotypes. 20 RESULTS: GAMMA IRRADIATION SCREEN: In order to recover new alleles of Asx, cn bw chromosomes were irradiated with a source and crossed to DTS911Cyo to obtain balanced, mutagenized chromosomes. These males were mated individually to XT129 females, and scored for the absence of unbalanced flies, indicating failure to complement XT129 (see Fig. 1). Out of 6,720 individual pair matings established, 1,671 of the matings yielded no progeny. Thirteen new mutant strains that fail to complement XT129 were recovered. This corresponds to a mutation rate of 0.19%. COMPLEMENTATION BETWEEN MUTANT STRAINS: Because the mutation rate of chromosomes that failed to complement XT129 was high, it raised the possibility that mutations in more than one locus might fail to complement XT129. Alternatively, Asx might be a very large locus or unusually radiation sensitive. To begin to distinguish between these possibilities, complementation mapping was performed with every newly induced mutation, and with the existing Asx alleles in our laboratory. The data are shown in Table 1. A complementation map derived from these data is shown in Fig. 5. The largest complementation group was designated A and has 12 members. B has three members and C and D have one member each. While it is interesting that four complementation groups were recovered from a single screen, it is not possible to determine from these data whether Asx is one locus displaying intragenic complementation or whether several loci have been ^ / recovered. 21 RESULTS Table 1. Complementation Data. This table represents a complete complementation matrix for all newly induced alleles and previously existing Asx alleles. The numbers shown on the left of the slash for each cross are the number of balanced, heterozygous progeny recovered from the cross. The numbers to the right of the slash depict the number of unbalanced, trans heterozygous progeny recovered. 22 COMPLEMENTATION GROUP A Trix Dl 26-108 26-42 6-167 25-150 4-130 27-124 27-72 27-108 2-54 IIF51 32-37 6-44 27-97 25-149 24-164 XT129 A Trix 73/0 99/0 63/0 119/0 72/0 81/0 95/0 69/0 127/0 100/0 139/0 82/28 72/40 60/26 83/49 58/19 69/0 Dl 104/0 67/0 66/0 78/0 76/0 126/0 113/0 84/0 65/0 109/0 70/47 52/30 77/29 80/28 100/43 108/0 26-108 97/0 80/0 113/0 157/0 5110 154/0 123/0 73/0 174/0 141/63 91/45 65/27 78/28 58/19 99/0 26-42 174/0 77/0 143/0 88/0 72/0 97/0 120/0 96/0 159/61 67/39 54/14 91/27 65/10 79/0 6-167 131/0 131/0 167/0 143/0 137/0 91/0 123/0 74/39 63/55 69/35 45/26 75/31 178/0 25-150 119/0 89/0 50/0 78/0 86/0 96/0 77/37 70/39 58/28 109/28 69/39 111/0 4-130 68/0 7610 84/0 96/0 109/0 132/70 160/88 50/23 90/70 73/32 92/0 27-124 101/0 93/0 82/0 122/0 62/38 68/35 80/37 60/32 53/16 145/0 27-72 111/0 99/0 90/0 79/31 80/39 75/31 61/29 88/24 84/0 27-108 120/0 131/0 57/31 87/43 69/35 57/36 94/20 73/0 2-54 78/0 73/37 89/41 75/30 48/40 64/24 85/0 IIF51 129/58 55/28 45/30 75/46 130/71 76/0 B 32-37 6-44 27-97 £ 25-149 101/0 98/0 171/47 142/49 112/0 180/0 149/51 68/12 76/0 79/44 90/35 60/0 144/99 89/0 fl D 24-164 40/25 75/0 XT129  A B C D Df(2R)Trix 27-97 25-149 24-164 Asx 6-44 A s x I 1 F 5 1 32-37 26- 108 27- 124 4-130 27-108 26- 42 27- 72 25-150 6-167 1 (2)54 Figure 5. Complementation Map 24 RESULTS However, the following evidence demonstrates that the A group contains at least two genes. 1(2)54 is a P element- induced lethal recovered by Cooley et al (1988). This strain contains a single P element located at position 42B1-2 (H. Brock, personal communication). 1(2)54 revertants, which have been demonstrated cytologically to have lost the P element at 41B1-2 are viable with group A mutants. Thus, the P element at this site in L(2)54 is responsible for its failure to complement XT129 (Campbell and Brock, personal communication). Therefore, not all members of the A group are allelic. One outstanding problem from this analysis is the location of XT129. Since it fails to complement every new mutation, it cannot be localized on the basis of these data. The XT129 chromosome was examined cytologically, and has a large inversion from region 48C to 51C. However, it is not yet known if either of these inversion breakpoints are associated with the XT129 phenotype. This problem is discussed below. RECOMBINATION MAPPING OF MUTANT STRAINS: Because members of the A, B, C and D complementation groups were heterozygous viable, yet all mutations die over XT129, it was possible to carry out the cross shown in Fig. 2 to see if mutations in different complementation groups were separable by recombination. Note that because the mutant chromosomes lack useful flanking markers, it is not possible to determine the orientation of the genes. One member of the A and two members of B as well as the mutations defining the C and D groups were crossed. The results are shown in Table 2. The results clearly show that each mutation tested is separable by recombination from every other. These results strongly support the interpretation that each of the A, B, C and D complementation groups is a separate locus. As argued above, the A group is divisible into at least two loci, showing that mutations in at least 5 loci fail to complement XT129. 25 COMPLEMENTING STRAINS: A; B + + CyO or CyO A B + + 6 _ A 4/24- i64 1,046 15 6-44/IIF51 1,046 78 32-37/24-164 945 8 32-37/25-149 895 287 25-149/24-164 1,125 377 IIF51/25-149 1,065 . 192 24-164/IIF51 1,376 88 Table 2. Recombination Data from Mapping of Complementing Strains. 26 RESULTS This is a minimum estimate. Nothing in the data exclude the possibility that either the A or B groups could be further subdivided since only 1 to 2 members of a complementation group were tested. In principle, the data presented in Table 2 could be used to generate relative map positions. However, as noted above, the absence of flanking markers makes it impossible to detemine the absolute order. Second, it is not known if any of these mutant chromosomes have rearrangements that suppress recombination. Third, the data provide no internal estimate of the relative viability of double mutant, unbalanced heterozygotes. For these reasons, a map is not derived here. As discussed below, data obtained subsequently in the laboratory by D. Sinclair support the observations described above. To determine the map position of each new mutation induced, preliminary recombination mapping was performed. As described in the Materials and Methods, males of each strain, were crossed to females marked with S Sp BI bw^. Recombination occurs in the heterozygous females, which were crossed to XT129. Any chromosome containing the newly induced mutation will die. Thus only one crosover class should be recovered in each interval except the one containing the mutation. Because time for the recombination experiment was limited, it was decided to examine the A complementation group mutations in the most detail, although an attempt was made to map the mutations that fall in the B, C and D complementation groups. However, probably because recombinants had less viability than the parental class, and the B, C and D crosses were scored after the A crosses, insufficient number of recombinant chromosomes were recovered for the B, C and D groups to reliably interpret the data. Therefore, I here present only the recombination data for the A group (see Table 3). 27 RESULTS Table 3. Recombination Data from Recombination Mapping of Non-complementing Strains. Numbers in brackets represent flies recovered in unexpected intervals (see text). Calculation of 95% confidence intervals were performed as described in Fig. 4. 28 STRAIN PARENTALS +SpBlbwD S+++ ++BlbwD AsxD 1 410 113 (1) 125 A s x I I F 5 1 472 84 (34) 71 4-130 313 89 (6) 105 25- 150 212 30 (10) 46 26- 108 262 89 0 101 26- 42 276 81 0 97 6-167 193 51 0 65 27- 72 248 69 (2) 72 27-124 410 88 (21) 78 27-108 252 111 (1) 125 MAP CONFIDENCE SSp-H- -H-fbw SSpBl+ POSITION LIMITS (2) 121 126 78.5 1.56 (29) 62 158 68.1 1.5 (1) 66 104 73.5 1.9 (2) 51 70 74.5 1.85 (2) 96 130 75.0 1.6 (2) 105 142 75.5 1.9 (2) 66 103 73.5 1.9 (3) 72 96 75.5 1.9 (26) 66 96 74.5 1.9 (2) 132 127 80.0 1.52 RESULTS It can be seen that the number of flies recovered was variable. A group mutations recovered by us and others mapped between 68 and 80 centimorgans on chromosome 2R. The previously determined map position of Asx is 2-72 (Jurgens, 1985). The 95% confidence limits for each calculation are shown. Mutations that map to each extreme have non-overlapping . confidence limits, suggesting that the A group consists of several loci. However, I prefer to interpret the data more conservatively for the following reasons. First, the dominant marker chromosome has only one mutation on chromosome 2R, and the distance between BI and bw^ is 50 map units. The A group mutations are nearly equidistant from the markers, which is too far away to allow precise mapping, partly because of the possibility of undetected double crossovers. The results suggest that undetected double crossovers may have been a significant problem. Small, but significant numbers of the reciprocal recombinant classes in those intervals not containing the lesion were obtained for some mutants. Progeny testing of those flies was performed by crossing males to XT129 females. This revealed that this recombinant class did not possess a lethal gene and were therefore not a result of "leakage" of the lethal effect of XT129. The simplest interpretation is that this class results from double crossover events and suggests that the risk of undetected double crossovers within the interval containing the lesion is significant. Second, as reported in Lindsley and Zimm (1987), BI has a slight tendency to suppress recombination. Third, it may be that some of the mutant chromosomes have rearrangements that themselves repress recombination. Therefore, my data suggest that the newly induced A group mutations, plus previously described Asx mutations fall into one locus. However, the possibility that the A group contains more than one locus near the middle of chromosome 2R cannot be ruled out, especially since 1(2)54, another A group mutation, maps to 42B1-2. 30 RESULTS In an attempt to localize the B,C, and D groups as well as to detemine if any of the A group alleles possessed visible rearrangments, cytological preparations of each newly induced and previously existing As* strain were analyzed. Other than a confirmation of the previously described deletion at 51A in the Df(2R)Trix strain, no visible mutations for any of the newly induced strains could be discerned. ENHANCEMENT BY OTHER SECOND CHROMOSOME POLYCOMB GROUP GENES: The results presented above suggest that mutations in at least five loci that fail to complement XT129 have been recovered. In addition to Asx, there are four other Pc group genes on the second chromosome: Pel, Psc, sxc and esc. It is therefore possible that some or all of the newly induced mutations could be allelic to these previously described Pc group members. If so, crossing the new mutations to these genes would be expected to result in failure of complementation. If the new mutations are not allelic to the previously described Pc group members, the same experiment could be used to detemine whether they are indeed members of the Pc group. As discussed in the Introduction, trans heterozygotes of Pc group mutations show a stronger homeotic phenotype than that seen in any single member. If the new mutations are members of the Pc group, or interact with the Pc group, then trans heterozygotes of the new mutations and Pc group genes should exhibit enhanced homeotic transformations. To test these two hypotheses, I crossed every newly induced mutation to Pel, Psc, esc and sxc. I also crossed every gene to Pc, because it is the best characterized member of the Pc group. Crosses were performed as described in Materials and Methods, and were scored for homeotic transformations in the abdomen, thorax and head. Briefly, degree of transformation was assigned a numberical value. The results are reported as the average 31 RESULTS score for each character examined, for each cross. The results are shown in Table 4. It can be seen immediately that trans heterozygotes for every new mutation and every existing Pc group mutation on the second chromosome survived. Therefore, none of the new mutations are allelic to a previously described Pc group gene. Trans heterozygotes of the mutant strains and Pc all show enhancement of homeotic phenotypes, especially A group mutations and XT129. If enhancement of the Pc phenotype is taken as evidence for membership in the Pc group, then every new mutation must be considered a potential member of the Pc group. Enhancement of homeotic phenotypes by second chromosome members of the Pc group yielded more variable results. None showed enhancement by Psc or esc. This may be because these genes are predominantly maternal in effect. In contrast, the homeotic phenotypes of most genes were enhanced by Pel. These transformations were almost exclusively restricted to the transformation of the 4th abdominal segment to the 5th, and to the presence of sex combs of the meso- and metathoracic legs. Some trans heterozygotes of mutant 26-108 and Pel displayed a strong antenna to leg transformation. Little consistent difference in the enhancement of the Pel phenotype could be detected among members of different complementation groups. However, these data further support the contention that the newly isolated mutations are members of the Pc group. The enhancement phenotypes of the new mutations with sxc are of particular interest, and are presented in Table 5. Little or no enhancement of adult phenotype is observed for trans heterozygotes of sxc and the B, C, and D group mutations. Rather, these trans heterozygotes survive better than the singly mutant balanced sibs. The one exception is mutant 27-97, which exhibits a loss of the humerus in nearly 50% of the trans heterozygotes. 32 RESULTS Table 4. Enhancement of Adult Homeotic Phenotypes of Second Chromosome Polycomb Group Genes by New Mutant Strains. N= the number of trans heterozygous flies examined. P= the penetrance of homeotic transformations among trans heterozygotes, expressed as a frequency. E= The expressivity of defects examined. (See Materials and Methods for the derivation of this score). An asterisk (*) indicates that sex combs ectopically located on the 2nd and 3rd thoracic legs were found. 33 3 w4 Pc Pe l STRAIN N P E N XT129 15 0.8 1.1* 29 0.9 4 .4* Irix. 20 0.8 4 .0* 21 1.0 3.9* 27-108 20 0.8 1.9* 14 0.9 3.2 4-130 20 1.0 3.6* 15 1.0 4 .6* 27-124 25 0.8 0 .9 * 17 0.9 2 .9* 6-167 18 0.6 2 .2* 15 1.0 3.7* 26-42 17 0.9 2 .0* 15 0.9 4 .5* 25-150 15 0.9 1.9* 15 0.9 4 .3* 27-72 31 0.2 2 .6* 20 0.6 3.5* 26-108 17 1.0 3.2* 25 1.0 4 .0* IIF51 19 0.7 2 .8* 17 0.9 3.6* 1(2)54 16 0.9 3.6* 21 0.9 3.1* 27-97 15 0.1 1.0 19 0.1 1.4* 6-44 18 0.2 1.7* 17 0.1 1.0* 32-37 14 0.1 1.0* 18 0.3 1.6* 25-149 20 0.2 1.7* 15 0.1 1.9* 24-164 19 0.1 1.0* 14 0.1 2 .0* Psc° esc WILD TYPE N P E N P E P 14 0.7 1.2* 34 0.3 1.0 0.02 13 0.9 1.8 24 0.8 1.6 0.01 23 0.2 2.6* 21 0.8 1.4 0.03 18 0.4 1.0* 18 0 0 0 20 0 0 23 0.4 1.3 0.02 24 0 0 23 0.4 1.4 0.01 20 0.5 1.0 20 0.5 1.4 0 10 0.2 1.5* 20 0.7 1.4 0.01 20 0.2 1.3 34 0.3 •1.1 0.05 16 0.5 2.3* 18 1.0 1.4 0.04 21 0.3 1.0 19 0.1 1.0 0.02 24 0.3 1.0 18 0 0 0.01 22 0.7 1.7 13 0.2 1.3 0.01 22 0 0 12 0.4 1.0 0.04 26 0 0 18 0.7 1.0 0.08 33 0 0 26 0.3 1.5 0.01 10 0 0 14 0.1 1.0 0.02 NUMBER NUMBER NUMBER TRANS FREQ. HOMEOTIC FREQ. TRANS STRAIN SCORED HETEROZYGOTES HETEROZYGOTES TRANSFORMATION HETEROZYGOTES XT129 91 90 Df(2R)Trix 50 4 27-108 96 88 4-130 74 70 27-124 130 110 6-167 130 108 26- 42 126 118 25- 150 146 134 27- 72 86 80 26- 108 102 94 IIF51 126 116 1(2)54 171 117 27- 97 129 66 6-44 67 37 32-37 97 62 25-149 82 50 24-164 81 46 1 1.0 0.01 46 1.0 11.0 8 1.0 0.09 4 1.0 0.06 20 0.1 0.18 22 1.0 0.2 8 1.0 0.07 12 0.8 0.08 6 1.0 0.08 9 1.0 0.09 10 1.0 0.09 54 0.6 0.32 63 0.7 0.48 30 0.01 0.44 35 0.04 0.36 32 0.02 0.39 35 0.02 0.43 Table 5. Enhancement of super sex combs by New Mutant Strains. 35 RESULTS In contrast, two types of enhancement behaviour are seen among group A alleles. All the newly induced Asx alleles (4-130,27-124,25-150,27-72, 26-108, 27-108, 26-42) show markedly decreased viability as trans heterozygotes with sxc. While the expected survival rate of trans heterozygotes is 33%, survival of 10% or less was observed. Surviving trans heterozygotes show homeotic transformations of A4 to A5, as well as sex combs on the meso-and metathoracic legs. The opposite result is seen in trans heterozygotes of Df(2R)Trix and sxc. Trans heterozygotes show markedly increased viability relative to their heterozygous siblings (R. Campbell, personal communication). As noted above, the A group mutation 1(2)54 has a P element inserted at 42B1-2, very close to the published location of sxc at 41C5-6. Normal viability and no phenotypic enhancement was seen among trans heterozygous progeny of a cross between these two strains was found in this study. XT129 shows nearly complete non-complementation with sxc. EMBRYOLOGY OF THE ASX GROUP: Embryonic cuticle preparations were made of each mutant strain to determine phenotypic similarities and differences between the Asx Group genes. The head, thorax and abdomen of each embryo were scored independently and given a numerical value reflecting the severity of the homeotic transformation or defect. The values given in Table 6 are an average of all embryos scored. All embryos display the same general phenotype of weak, posteriorly directed transformations of the body segments. Strain-specific variations and exceptions to this general phenotype will be described below. In general, head involution is incomplete, such that the cephalopharyngeal apparatus (CPA) is located more anteriorly, and the mouth 36 RESULTS Table 6. Summary of Embryonic Phenotypes. 37 NUMBER STRAIN SCORED HEAD THORAX XT129 Df (2R)Trix 27-108 26- 108 27- 72 26- 42 25-150 6-167 4-130 27- 124 IIF51 1(2)54 74 32 52 30 21 32 24 50 39 41 19 30 1.5 0.7 2.4 2.2 2.4 2.9 2.1 2.3 2.9 2.4 1.2 1.2 0.2 1.4 2.8 2.0 2.6 2.6 1.5 2.0 2.1 2.3 0 0.1 32-37 6-44 27-97 0 0 24 0 0 2.8 0 0 0 25-149 53 2.3 1.7 24-164 24 1.0 SEGMENTAL ABDOMEN DEFECTS COMMENTS  1.4 + K e i l i n ' s organs reduced or missing. 1.8 + 2.1 + 1.7 + 2.2 2.7 1.1 + 2.4 1.8 -1.0 + 0 - K e i l i n ' s organs reduced or missing. 0 0 Not embryonic l e t h a l . 0 Not embryonic l e t h a l . 2.9 + Mouth hooks at posterior end. 1.4 + 0 + 90% have no v i s i b l e phenotype. Remaining 10%-reduced K e i l i n ' s organs. RESULTS hooks more laterally, than in wild type embryos (Fig. 6). The ventral setal belt (VSB) of the first thoracic segment medially contains what appears to be T2 or T3 denticles as well as abdominal-type denticles (these are distinguished from TI denticles by their triangular rather than hair-like shape, and by their darker colour). The anterior-most rows of T2 and T3 denticles contain abdominal-type denticles which point anteriorly, suggesting a transformation at least as far posterior as A2. The Keilin's organs of T2 and T3 can be reduced or absent in some of the Asx Group genes (Fig. 7c, e). The VSB of Al is transformed toward A3, A4 or A5, as evidenced by the change in its shape from a narrow strip of 3 rows of denticles, to a VSB trapezoidal in shape due to the addition of 1-3 narrow rows of anterior pointing denticles at its anterior margin. The VSB's of A2, A3 and A4 are transformed toward A6 or A7 and the VSB's of A5 through A7 are transformed to A8, as evidenced by their alteration from a trapezoidally shaped belt to a rectangular one. The phenotypes of Df(2R)Trix and Asx^l have already been published (Breen and Duncan, 1986). As noted above, Df(2R)Trix consists of a small deletion uncovering the published region of Asx. Trix, therefore, can be considered an amorphic, loss of function allele of Asx. The phenotypes of both Df(2R)Trix and Asx^l fit the general description given above (Fig. 6b). Analysis of Df(2R)Trix embryos in our laboratory reveals that, compared to the newly isolated Asx alleles, the head phenotype of Df(2R)Trix is relatively weak (Fig 7b). While condensation and splintering of the CPA is commonly seen among the new Asx alleles, and in other members of the Asx Group, the head defects of Trix are confined to a mild shortening of the lateral graten and splaying of the cirri (Fig. 6c). The defects are barely detectable in some embryos. I here report a new finding for the Asx gene: even-skipped defects were observed in approximately 10% of Trix embryos (Fig.6f). 39 RESULTS The newly induced Asx alleles are a fairly uniform group. The head defects of some alleles, especially mutants 4-130 and 26-42 are very strong (Fig 6d). The CPA's of some embryos of these strains are reduced to condensed blobs of chitinous material with few distinguishable features (Fig 7d). While all embryos of some of the new alleles have consistently strong head defects (mutants 26-108,4-130, 27-124, 27-72 and 26-42), other alleles display slightly more variable head defects, ranging from slight shortening of the lateral graten to complete condensation and splintering of the CPA (mutants 6-167 and 25-150). Thoracic and abdominal transformations in the new alleles are similar in strength to those observed in previous work. Briefly, thoracic transformation is variable in all new alleles, ranging from the presence of one to three abdominal denticles ectopically located in T3, to many anteriorly-pointing denticles present in both T2 and T3, resulting in an abdominal-like trapezoidal VSB. All new alleles show variation in posterior directed abdominal transformations. As many segments as A5, A6 and A7 may be transformed to A8, or A7 may be exclusively transformed. Al is nearly always transformed to A2 or posterior (Fig. 7d). Three of the new alleles display segmentation defects. 5% of mutant 25-150 and 26-108 embryos show even-skipped segmentation defects, and 10% of mutant 27-108 embryos display odd-skipped defects (Fig 6f). Analysis of the previously isolated AsxH^l allele reveals a markedly different embryonic phenotype (Fig. 6g). All arrested embryos of this strain have a nearly identical phenotype. All display mild to moderate head defects, confined to shortened lateral graten. Surprisingly, no thoracic or abdominal homeotic transformations were found. In addition, while some reduction or absence of Keilin's organs has been reported for some Trix embryos, all Keilin's organs of all mutant embryos are reduced or missing in T3 and reduced in T2 of 40 RESULTS (Fig 7e). It is possible that the lack of homeotic transformations could be accounted for is regarded as a very weak hypomorph. Even so, why a weak hypomorph should possess a strong Keilin's organ phenotype is unclear. The phenotype of 1(2)54 is quite unlike Asx. The only detectable phenotype observed in arrested embryos were mild head defects consisting of very slight shortening of the lateral graten and slight splaying of the cirri. No homeotic transformations were noted (Fig. 6h). Two members of group B, mutants 32-37 and 6-44 are not embryonic lethal. The third member of the group, mutant 27-97, has an unusual phenotype, which differs dramatically from the Asx phenotype. The body is shortened and tubby (Fig 8a). The head defects are so severe that generally a large hole is all that remains of the head, and the CPA is missing (Fig 8b). Where the CPA is present, it consists of an amorphous blob at the anterior-most tip. Thoracic transformations are entirely absent, but all abdominal segments are transformed into A8. Interestingly, in many embryos of this strain, chitinous structures resembling mouth hooks are present at the posterior-most tip. The filtzkorper, which normally exit the body at the rear end, are relocated anteriorly, exiting between A7 and A8 (Fig 8b). Segmentation defects, particularly at the midline, are frequently seen in these embryos 90% of mutant 24-164 embryos have no visible phenotype. Very mild head defects are seen in the remaining 10%. These defects usually consist of a very mild shortening of the CPA, causing the head to become slightly bent. No homeotic transformations of thoracic or abdominal VSB's were observed. In addition to the head defects, these embryos display even-skipped segmentation defects. The Keilin's organs of T2 and T3 are reduced or absent. Apart from the very low penetrance, and predominance of segmentation defects among embryos with a mutant phenotype, these embryos most closely resemble Asx^^* embryos (Fig 6e). 41 RESULTS All aspects of the standard Asx phenotype are present in mutant 25-149 embyros, but are generally weaker and more variable. Head defects are consistently present but are variable in that they range from very mild to moderate in strength. Thoracic and abdominal transformations are also weak to moderate (Fig 6i, 7f). In spite of its lethality with four complementation groups, XT129 has a moderately weak embryonic phenotype. A wide range of severity of head defects are seen, from very slight shortening of the lateral graten and splaying of the cirri, to approximately 10% of embryos that possess a severely condensed, splintered of exploded C P A Very mild thoracic transformations are observed, usually consisting of one to two abdominal-type denticles in the VSB of T3 (Fig 7c). No abdominal phenotype was found. 30 to 40% of the embryos are missing the T2 and T3 Keilin's organs (Fig 6c). 42 RESULTS Figure 6. Embryology of the Asx Group. A, wild type; b, Df(2R)Trix; c,XT129; d, 27-124 (representative of all new Asx alleles recovered); e, 24-164 (complementation group D); f, 26-108, showing segmentation defects; g, , 1(2)54; i, 25-149 (Complementation group C). See text for description. 43 RESULTS Figure 7. Head Defects of the Asx Group. A, wild type; b, Df(2R)Trix; c, XT129; d, 27-124; e, AsxIIF51; f, 25-149 (Complementation group C). Arrow heads point out abdominal denticles ectopically located in the thorax. V= ventral pits, K= Keilin's organs. These are pointed out where present. 45 RESULTS Figure 8. Embryonic Phenotype of Complementation Group B Mutant 27-97. Arrow heads point out the hole that takes the place of the head (a), and chitinous structures resembling mouth hooks at posterior end (b). 47 DISCUSSION Thirteen mutant lines that fail to complement XT129 were recovered in a gamma irradiation screen for new alleles of Asx. New mutant alleles were isolated at a rate of 0.19%. Comparison with the mutation rates of both "housekeeping" and developmentally important genes indicates that the obtained mutation rate is approximatley an order of magnitude higher than expected (see Table 7). Although relatively rare , such high mutation rates for a single locus are not unprecedented in Drosophila. For example, an X-ray screen for revertants of Antennapedia yielded mutation rates as high as 0.24% (Hazelrigg and Kaufman, 1983). The Antennapedia gene is, however, unusual in its very large size of over 102 kb (Bermingham and Scott, 1988). Complementation and recombination mapping has partly revealed the cause of the high mutation rate. The new mutant lines fall into four complementation groups. The largest group, A, fails to complement a deletion at 51A which uncovers the Asx locus. Recombination mapping in this study indicated, and further studies in our laboratory have confirmed, that the new mutants belonging to group A all map to 2-72, the published location of Asx (D.Sinclair, personal communication). Also belonging to group A is 1(2)54, a P element mutation isolated in a separate screen. Cytological examination and reversion of this allele show that the lesion is located within a gene at cytological position 42B1-2 (H. Brock, personal communication). Preliminary studies in this thesis revealed that mutants of complementation groups B, C, and D are separable from each other by recombination. This result has been confirmed subsequently in our laboratory and some of the map postions are now known. Group B mutants map to 2L at position 32F2-33A2. The position of the group C mutant is unknown, but lies very close to Asx (see below). The group D 49 DISCUSSION Table 7. Comparison of Mutation Rates of Drosphila Loci. 50 GENE CHROMOSOMES DOSE (R) SCREENED Alcohol dehydrogenase 3,000 86,900 Chaoptin 4,000 10,261 v e s t i g i a l 4,000 425,000 pink 4,000 6,730 s n a i l 4,000 13,700 Dorsal 4,000 22,000 torpedo 4,000 10,366 hairy 3,500 15,980 Polycomblike 4,200 7,800 MUTANTS RECOVERED RATE (%) REFERENCE 19 0.022 Aaron, 1979. 2 0.02 Van Vactor et a l . , 1988. 126 0.029 Lasko and Pardue, 1988. 2 0.029 Kemphues et a l . , 1983. 5 0.036 Grau et^ a l . , 1984. 6 0.028 Steward and Nusslein-Volhard, 5 0.048 Price £t a l . , 1989. 3 0.018 Ingham et a l . , 1983. 1 0.012 Duncan, 1982. DISCUSSION mutant is located at postion 37F6-38A1. (D. Sinclair, personal communication). What is the nature of the relationship between complementation groups and loci? The answer to this question is complex and for reasons discussed below, not yet completely understood. The A complementation group is interpreted to consist of mutations in one gene, with the exception of 1(2)54. Because only one mutation of 1(2)54 is in existence, and therefore the phenotype of the deletion is unknown, it is presently not possible to speculate on the nature of the 1(2)54 locus. However, because this mutation is lethal in combination with every Asx allele, the product of 1(2)54 could either be very similar to the Asx product in structure and/or function, or could interact directly with the product of Asx. It is possible that the Asx gene could be a complex locus. It is certainly likely to be large. The mutation rate specifically for the Asx locus in this screen is 0.12%. This is significantly higher than the average mutation rate for Drosophila loci of between 0.02 and 0.03%. Complexity could further account for the variety of embryonic phenotypes seen among the various Asx alleles. These range from a complete absence of homeotic transformations, loss of Keilin's organs and mild head defects to a phenotype of severe head defects, moderately strong homeotic transformations, segmentation defects and the Keilin's organs present and intact. These different phenotypes manifest themselves not so much as a graded continuum of strength but as presence or absence of components of the phenotype. The embryonic phenotypes of the Asx alleles allow some possible predictions about which strains are loss of function mutations and which are gain of function mutations. Df(2R)Trix, which deletes the entire locus, and can therefore safely be classified as a loss of function allele, displays very weak head defects, reduction of the Keilin's organs, and moderate homeotic transformations. In contrast, the newly induced Asx strains have very 52 DISCUSSION strong head defects, moderate to moderately strong homeotic transformations and normal Keilin's organs. It is not unreasonable to postulate, on the basis of phenotype alone, that at least some of the new Asx alleles are gain of function mutations. Gain of function in these alleles explains the difference in complementation between Asx alleles and sxc. Whereas Df(2R)Trix shows no interaction with sxc, all the new alleles of Asx display a marked decrease in viability when trans heterozygous with sxc. While it is odd that only gain of function alleles of Asx should be recovered in this screen when loss of function alleles are also lethal with XT129, this explanation nevertheless provides the best model to account for the data. The embryonic phenotype of As^^l j s unusual and it is difficult to make predictions about the nature of the mutation on the basis of its appearance. AsxIIF51 embryos lack homeotic transformations, show head defects and the reduction or absence or Keilin's organs. It is possible that this allele is a very weak hypomorph. The concept of gain- or loss or function can also be used to explain the interactions between the B and D complementation groups and XT129. As mentioned above, the B group has been mapped to postion 32F2-33A2. Previously existing deletions this location nevertheless complement XT129 (D. Sinclair, personal communication). The simplest explanation of this behaviour is that the newly obtained strains are gain of function mutations in which the product of the B gene interacts with the product of XT129. Mutants 27-97 and 32-37 fail to complement the embryonic polarity gene spalt. However, a deletion uncovering the spalt locus complements XT129. Therefore 27-97 and 32-37 are gain of function alleles of spalt (D. Sinclair, personal communication). Interestingly, 6-44 is not uncovered by the deletion that uncovers spalt, but is uncovered by a larger deletion 53 DISCUSSION that includes the smaller deletion. Therefore, 6-44 is not allelic to 32-37 and 27-97, and hence, both the A and B complementation groups consist of two loci. The same argument can be made for the D complementation group. Pre-existing deletions of 37F6, the mapped region of mutant 24-164, also complements XT129. Hence, it is likely that mutant 24-164 is also a gain of function mutation (D.Sinclair, personal communication). No previously characterized mutations in 37F-38 fail to complement 24-164 (D. Sinclair, personal communication). No predictions about the nature of the C complementation group mutant can be made at present. Recombination mapping placed the location of 25-149 close to 2-72, the location of Asx. Three lines of evidence suggest that it is not an Asx allele. First, I have shown that the A and C complementation groups are separable by recombination. Second, it is not likely to be a hypomorph of Asx because it is completely viable when trans heterozygous with Trix. Third, although the penetrance of the embryonic phenotype is not very high, arrested embryos that are visibly mutant display a relatively strong phenotype. Since every other Asx allele is lethal in combination with Df(2R)Trix, it is most likely that 25-149 is not an Asx allele. Fine scale recombination mapping will be required to resolve this problem. Should the outcome of such mapping continue to confirm the alleleism of mutant 25-149 and Asx, their complementation might be explained on the basis of intragenic complementation with a complex locus (see above). The outstanding problem of this analysis is the nature of the XT129 mutation. Since it fails to complement five genes, it must possess unique properties. However, at present it is unknown whether it is a mutation in any of the six genes identified in this study or whether it represents a seventh mutant locus. Neither breakpoint of the large inversion characteristic 54 DISCUSSION of this strain corresponds to any of the identified genes, or to any other known developmentally important gene. The possibilty cannot be ruled out that the XT129 chromosome possesses a mutation in each of the five genes. In that event, the observed complementation behaviour would be explained on the basis of allelism, not intergenic complementation. The likelihood that the XT129 chromosome contains multiple mutations in Pc group genes is slim for the following reasons. First, the statistical probability of a chromosome containing lesions in five genes that enhance Polycomb group phenotypes by chance alone is extremely small. Second, if several Pc group mutations coexist on the same chromosome, it is reasonable to expect that the genes would enhance each other's phenotype (as is characteristic for the group). The XT129 embryonic phenotype is however suprisingly weak, and is certainly weaker than most Asac alleles. However, at present, the possibility cannot be eliminated that XT129 has more than one mutation and that at least some of the complementation groups recovered might not interact with Asx. The possibility of second-site lesions on the XT129 chromosome can be ruled out as follows. A wild type arm 2L could be attached by recombination to the XT129 2R and the resultant second chromosome crossed to the group B and D mutants. If the newly constructed chromosome still fails to complement groups B and D, then the hypothesis of second site mutations may be ruled out. For the reasons given above, the remainder of the discussion will be based on the idea that at least some of the mutations interact with XT129. Arguing from the standpoint that this is true, the fact remains that XT129 has special properties. It is possible that it is a unique allele of Asx. XT129 is lethal in combination with a deletion of Asx but not with deletions of the B or D loci. On the other hand, none of the other gain of function alleles of Asx isolated here show this behaviour. It is also possible that XT129 is a mutation in a sixth, 55 DISCUSSION unknown locus. It is possible but unlikely that XT129 is an allele of sxc, as evidenced by its near lethality with sxc when trans heterozygous. Also, given that XT129 is a unique single mutation, an interesting result of this analysis is that complementation groups B and D are gain of function alleles, displaying a direct product interaction with the product of XT129. At present, the nature of the interaction with the group C gene is unknown. The non-complementation of XT129 with B and D group genes could be used in a reversion screen to localize XT129. If XT129 chromosomes were irradiated, and crossed to mutant 24-164, for example, all trans heterozygotes would die, except those which had lost the XT129 mutation. If the reversion resulted from chromosome rearrangement, the visible lesion would localize the site of the original XT129 mutation. While intergenic non-complementation is a relatively rare occurrence, such a behaviour is not inconsistent with already known properties of the Polycomb group. First, one criterion for membership in the Polycomb group is that trans heterozygous adults of any member in combination with Pc display stronger homeotic transformations than those seen in single mutants. This suggest a certain minimal level of interaction. Second, ph is lethal in trans heterozygous combination with Psc (C.-T. Wu, personal communication). Third, some Polycomb group gene embryos which normally display weak homeotic transformations become severely mutant in double or triple combinations, with a phenotype approaching or exceeding that seen in Pc or esc embryos (Jurgens, 1985). Given, therefore, the estimated number of the Polycomb group genes (more than 60; Jurgens, 1985) and the predominance of genetic interaction within the group, it is not inconsistent with previous data that a subgroup of closely interacting genes should exist. In summary, then, I believe I have identified at least five loci that display genetic interaction with XT129. The major unresolved problems of this analysis are 1) where is the 56 DISCUSSION J XT129 lesion located? 2) Is XT129 the only mutation on the chromosome that interacts with the newly induced mutations? 3) What is the nature of the XT129 mutation? All of the new mutants showed enhancement of adult transformations by Pc and can therefore be regarded as potential new members of the Polycomb group. Pel also enhanced the phenotype of the new mutants, in much the same manner as Pc. Psc and esc had little effect on the mutants. Little consistent difference in enhancement behaviour could be distinguished between genes, although the enhancement by Pc and Pel was slightly more marked among A complementation group alleles and XT129. It is very surprising, in this light, that mutants 27-97 and 32-37 are alleles of spalt. This suggests that XT129 regulates spalt, or that somehow the products of spalt and XT129 interact. Further investigation will be required to distinguish between these possibilities. Of interest was the enhancement behaviour of sxc. While sxc did not interact with B, C, or D group alleles, three types of enhancement behaviour was seen for the A complementation group genes. Df(2R)Trix was not enhanced by sxc. In contrast, all of the remaining new Asx alleles displayed a partial failure of complementation with sxc. As discussed above, a possible explanation for this behaviour is that sxc only interacts with gain of function alleles of Asx, while showing no interaction with the loss of function Trix allele. Since Asx and sxc are clearly separate genes, a gain of function, product/product interaction is a logical explanation for this phenomenon. This hypothesis also lends further support to the argument that XT129 is a unique, strong, gain of function allele of Asx. Segmentation defects were found in Trix embryos, as well as in embryos of three of the new Asx alleles. This is the first report of segmentation defects in Asx embryos. All of the complementation groups also possess segmentation defects. Such defects are not unique among the Polycomb group. Segmentation disruption has been reported for Pel, pho and sxc 57 DISCUSSION (Breen and Duncan, 1986; Ingham, 1984). Although the penetrance of defects is low, their presence nevertheless suggest a role for some Polycomb group genes in the establishment, or more likely, maintenance of segmentation, in addition to their role in homeotic gene determination maintenance. Molecular evidence may confirm this hypothesis. The Polycomb group gene ph is required for the correct expression of ftz and eve in the embryonic CNS (Smouse et al., 1988). Since interaction of a Pc group gene with segmentation genes has been demonstrated, and segmentation defects are manifest in Asx and most of the genes it interacts with, it is not unreasonable to suggest that the Asx sub-group may have some role in segmentation. The mechanism of action of the Polycomb group genes has proved difficult to elucidate, and investigation of this sub-group of genes could aid in our understanding of how the group as a whole functions. Some level of genetic interaction is not only common between members of the Polycomb group, it is a defining characteristic. That some members should display a closer, direct interaction is not surprising. The fact remains nevertheless that intergenic non-complementation is a relatively rare event in Drosophila. Only four other examples of direct interaction are known in Drosophila. 1) Certain neurogenic loci, notably Notch, Delta and Enhancer of Split (Shepard et al., 1989); 2) Modifiers of position effect variegation (Locke et al., 1988; 3) Muscle genes (Homyk and Emerson, 1988; Mogami and Hotta, 1981) and 4) Sperm flagellum alpha-tubulin genes (Regan and Fuller, 1988). Examples exist in other organisms such as muscle gene interaction in Caenorhabditis elegans (Epstein et al., 1986; Park and Horvitz, 1986), tubulin genes in Saccharomyces cerevisiae (Stearns and Botstein, 1988) and bacteriophage T4 assembly (Wood and Crowther, 1983; Berget and King, 1983). 58 DISCUSSION Where the molecular basis of the above examples is understood, the interaction seems to invariably consist of protein-protein association to form a supramolecular structure. It is not understood why these particular examples of structure formation should be so sensitive to dosage, when other supramolecular structures have methods to overcome dose sensitivity such as gene duplication, alternate pathways, feedback regulation or similar proteins that can stand in for the loss of others. A regulatory pathway is another possible cause of direct gene interaction. Further, recent evidence suggests that regulatory proteins tend to aggregate together (O'Connor et al., 1988; Hoey and Levine, 1989; Desplan and O'Farrell, 1988). It is possible that combinations of physically associating genes may be required to properly regulate gene expression. At least some Polycomb group genes probably have such a role. Pc has been demonstrated to bind many specific target genes, including the Bx-C and Antp-C as well as other Pc gourp genes, on salivary gland chromosomes (Zink and Paro, 1989). If as suggested by the above examples the products of the Asx group form a physically associating structure, what type of structure might that be? One hypothesis is that the determination of the Bx-C is maintained by changes in chromatin structure (Peifer et al. 1987). 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