smellblind: A Gene Required for Drosophila Olfaction. Mary Lilly and John Carlson. Department of Biology, Yale University, New Haven, Connecticut 0651 1.
Copyright 0 1989 by the Genetics Society of America
smellblind: A Gene Required for Drosophila Olfaction Mary Lilly and John Carlson Department of Biology, Yale University, New Haven, Connecticut0651 1 Manuscript received August 24, 1989 Accepted for publication September 21, 1989
ABSTRACT In this article we define and characterize thesmellblind gene (sbl). Weshow that two mutants, sbl and olfDxy,both isolated by virtue of their olfactory phenotypes and analyzed extensively by others withrespecttocourtshipbehavior,containmutations at a singlelocus.Meioticrecombination, duplication, and deficiency mapping are used to localize this gene, sbl, to cytogenetic position 14F615A2-3 on the X chromosome. Mutationsof the locus are shown to produce severe defects notonly in larvalolfactoryresponsetoseveralvolatilechemicals, but also in larvalcontactchemosensory response. Both sbl and olfDxy give a robust response, however, in a newtestoflarval phototactic response, which we describe here. Both alleles are shown to be heat-sensitive lethals. Four additional recessive lethal alleles, two EMS-induced, one dysgenic, and one spontaneous, are also described.
D
ROSOPHILAexhibitsaremarkable ability to detect awide array of volatile chemicals, during both the larval and the adult stages. Larval olfaction is believed to be mediated through the antenno-maxillary complex (BOLWIG1946; SINGH and SINGH 1984), a structure residing at the anterior end of the animal; in the adult, the thirdsegment of the antenna is the primary olfactory organ (BARROWS 1907; VENARD and PICHON1981). Surprisingly little is known about the mechanisms of signal transduction or the means by which the system develops, at either larval or adult stages. Oneapproachto examiningthese processes is the study of genes which affect olfactory response (RODRIGUES and SIDDIQI 1978; SIDDIQI WOODARD et al. 1987; HELFAND and CARLSON 1989; 1989; MCKENNAet al. 1989), an approach which has substantial precedentfromthe study of genes required for visual response (BENZER 1967). and T h e smellblind mutant (sbl) (ACEVES-PINA QUINN1979) was originally isolated in a screen for mutants defective in an olfactory-driven learning paradigm. It was found subsequently to be defective in olfactory response at both the larval and adult stages. T h e mutant has been used in a number of studies in several laboratories to investigate the role of olfactory information in courtship behavior (e.g. TOMPKINS, HALL and HALL 1980;TOMPKINS et al. 1982; MARKOW 1987; TOMPKINS et al. 1983).However, the genetic basis of the sbl mutant has not been defined. It has not been determined, for example, whether the sbl phenotypes result from a mutationin a single gene. Moreover, the nature of the sbl defect has not been characterized; there are few if any data to document The publication costs of this article were partly defrayed by the payment of page charges. This articlemust therefore be hereby marked“advertisement” in accordance with 18 U.S.C. $1734 solely to indicate this fact, Genetics 1 2 4 293-302 (February, 1990)
the olfactory defect or to indicate whether the defect is specific to olfaction. A second olfactory mutant, 01fDx9 (RODRIGUES and SIDDIQI1978; SIDDIQI1987), has also been studied to examine the role of chemosensory information in and HALL courtship behavior (GAILEY,LACAILLADE 1986;JALLON, ANTONYand IWATSUBO 1981). Genetic analysisof this mutant also has been limited. With respect to its phenotype, olfactory response atthe adult stageto several odorants has been characterized, to varying extents, but there is little published documentation of its larval phenotype, either with respect to olfaction or to othersensory stimuli. In this article we define the smellblind locus. We show that sbl and 01fDx9are allelic and that they map to a small region of theX chromosome, between 14F6 and 15A2-3. The larval olfactory phenotype of olfDxy is characterized in some detail, and for both mutants the behavioral specificity of their mutant phenotypes is assessed by measuring two other behaviors. For testing larval visual behavior, a new assay is described. Both mutants are shown to be heat-sensitive lethals, andthe lethality is shown to cosegregate with the olfactory phenotypes. Four additional alleles, all lethal. are also described. MATERIALS AND METHODS Drosophila melanogaster stocks and culture: Canton-S-5 (CS-5) was derived as described elsewhere (HELFAND and CARLSON1989). sbl was obtainedfrom M. HEISENBERG, Institut fur Genetik und Mikrobiologie, Wurzburg. olfDxy, o l f p ’ and oljD“’ weregenerouslyprovided by V. RoDRIGUES and 0. SIDDIQI,Tata Institute, Bombay. 1(1)20137 was obtained from A. SCHALET, Yale University, and 1(1)023was from J. EEKEN,University of Leiden. Duplication and deficiency stocks were obtained from sources indicated in Table 1. The y cv u f car stock was from the Mid-
ource
294
M. Lilly and J. Carlson TABLE 1 Chromosomal aberrationsused in the cytogenetic localization of the sbl phenotypes
Reference Aberration breakpoints X chromosome
Df(1)132
Df(1)r"'
984)
Df(lP7 15A6 ~p(1;4)r+ft Tp(1;31f7'b ~p(1;2)~+75~
Df(1)r""
14B17-C1;15A2-3 KULKARNI, STEINLAUF AND HALL(1 988) 14D1-2;15D1-2 GANETZKY (1 14C7-D 1 ;14E3-F 1 GANETZKY (1 984) 14F6; JARRY ( 1 979) AND ROY (1 980) CRAYMER 13F;16A2 15A4; 16C2 CRAYMER AND ROY(1 980) AND ROY (1 980) CRAYMER 14B13f ;15A9
America Drosophila Stock Center, Bowling Green, and attached-X females were from a C ( I ) A y/shi" stock obtained Yale University. from D. KANKEL, Cultures were grown in half-pint milk bottles with cardboard stoppers or in cylindrical vials with cotton stoppers. Drosophila were kept on cornmeal-molasses-agar medium (DOANE1967), supplemented with dry active yeast. Animals were grown in 25" incubators in the dark unless specified otherwise. For the experiment shown in Figure 8, eggs were collected on apple juice plates, which consisted of 25% v/v apple juice (Shure Fine), 2.5% agarose (FMC Seakem LE), and 0.25%v/v ethyl acetate (Fluka). Olfactory assay: The larval olfactory assay, described in detail in MONTE et al. 1989, was based on those of ACEVESPINAand QUINN (1979) and RODRIGUES (1980). After a 24h egglay, culture bottles or vials were cleared of adults and allowed to incubate an additional 3-5 days at 25". Larvae were then removed from the culture by adding a solution of 15% sucrose and agitating the medium with a brush. The larvae, which float to the top of the dense sucrose solution, were then tranferred by means of atruncated Pasteur pipette to a beaker containing distilled water. The larvae quickly settled to the bottom, after which the water was poured off and replaced with fresh water. After a second, similar wash, the larvae were collected with a brush onto a spatula, and approximately 100 were placedonto thecenter of the assay plate. The assay plate was a 100 mm X 10 mm plastic Petri plate (Falcon) containing 10 mlof 1.1% agarose (FMC Seakem LE agarose). Two filter discs (Schleicher & Schuell 740-E, 1/4 inch) were placed diametrically opposed to each other at opposite edges of the plate. After placing the larvae in the center of the plate with a brush, 25 pI of odorant was added to one disc and an equal volume of diluent was added to the other. Odorantswere from Fluka, were of the highest purity available, and were diluted in double-distilled water. The cover was then replaced on the plate and the animals were allowed to migrate for 5 min. Although a number of tests failed to reveal significant external cues, in most experiments odorant was placed on alternate sides of the plate in alternate assays as a precautionary measure. At the end of the test period, the number of animals on each half of the plate was counted. A response index (RI) (RODRIGUES and SIDDIQI1978) was calculated as described in the legend to Figure 1. Experimental errors refer toSEM. Contact chemosensory assay: The contact chemosensory (1979). The assaywas modified from that of TOMPKINS assay plate was a sectioned plastic Petri plate (100 mm X 15 mm; Macalaster Bicknell)containing four quadrants. 10 ml of 1 % agarose was poured into each oftwo diametrically opposed quadrants; 10 mlof 1% agarose containing a defined concentration ofNaClwas poured into each of the other two quadrants. These volumeswerealmost large
J. Hall A. Schalet A. Schalet
Indiana Stock Center A. Schalet Indiana Stock Center A. Schalet
enough to fill the quadrants. Shortly after the agarose had hardened, 12 mlof 1% agarose maintained at55" was distributed so as to provide an even surface over the entire plate. The plates were used 1.5-2hr later, aperiod designed to allow the plates to equilibrate to room temperature and to allow NaCl to diffuse through the thin superficial layer of 1% agarose to the surface of the plate. Larvaewere prepared as described for the olfactoryassay, and 100 animals were placedin the center of the plate with a brush. The plate was covered, and the larvae wereallowed to migrate. After 5 min, the number of animals on each quadrant was counted, and a response index was determined as described in the legend to Figure 4. Experimental errors refer to SEM. Phototaxis assay: The phototaxis assay was analogous to the contact chemosensory assay. 10 ml of 1% agarose containing dyes was placed ineach of two diametrically opposed "dark" quadrants (Figure 6). This agarose was dyed by adding 1 ml each of red, green, and blue food coloring (Durkee) to 100 ml of 1% agarose maintained at 55". into the other two, "clear," quadrants was poured 10 ml of 1 % agarose. To obtain a dose response curve for light, the dark quadrants were prepared with high dye concentrations as described above, but the othertwo quadrants were prepared with agarose containing a varying, low concentration of the dye mixture. Adding dye to this pair of quadrants effectively reduced the difference between the light intensity passing through the two pairs of quadrants. After the plates had cooled to room temperature, 12 ml of 1% agarose maintained at 55" was distributed evenly over the surface of the plate. The plates were allowed to cool to room temperature, and larvae were prepared as for the contact chemosensory assay. Responsein the phototaxis assay was found to be a sensitive function of developmental stage; late third-instar larvae exclusively were used. After placing approximately 100 larvae in the center of the plate, the plate was placed, without its cover, on a light box (Film Illuminator 460A, S & S X-ray Products Inc.) in a dark room. The larvae were allowed to migrate for 5 min, and then a response index was calculated as described in the legend to Figure 6. Experimental errors refer to SEM. A control experiment inwhich the assaywas conducted in total darkness-with no transmitted light-suggested that the response may not be driven exclusively by visual input: the response to the greatest concentration differential, ie., on plates in which two quadrants contained no dye, was 0.20 f 0.04 ( N = 25), compared to a response of 0.78 k 0.05 ( N = 10) in the presence of transmitted light. This weak response in the absence of visual input may be due to the presence of an attractive contact chemosensory stimulus (e.g., MIYAKAWA 1980) in the dye. Surface temperatures of clear and dark quadrants after 5min on the light box differed by not more than 0.1 O .
295smellblind Olfaction and Drosophila Heat-sensitive lethality: For the experiment shown in Figure 8, flies in groups of approximately 60 were anaesthetizedlightly with C 0 2 and placed on apple juice plates sprinkled with dried yeast, underneatha 100-ml beaker (American SIP Tri-PourBeaker). After 224 hr at29”, fresh plates were introduced, and females were then allowed to lay eggs for an additional period of 2-3 hr. The plates were then removed, and small pieces of agarose containing approximately 10 eggs each were excised from the plates and transferred to the surface of 15 mm X 100 mm 1% agarose Petri plates. Following incubation at 29” for another 3034 hr, the small pieces of transferred agarose were scored for the number of empty eggcases and unhatched eggs to determine hatching frequency. The number ofeclosed adults was scored following >15 days of further incubation at 29”. Complementation analysis: Femaleshomozygous for o l f P y were crossed to sbl males, and the FI progeny were tested in the larval olfactory assay using a 10” dilution of propionic acidas stimulus. At the end of the 5-mintest period, larvae from each half of the plate were transferred separately to different culture vials. Following eclosion, the number of malesand females werecounted, and from these numbers response indices could be calculated independently for the males and for the heterozygous females. In order to determine if olfD“’ or oljD” complemented the olfactory phenotypes of sbl’ and o l f P Y , olfD’“lFM7 or olfDLZ/FM7 females were crossed to Canton-S-5, sbl’, or o l f P y males, and the FI larvae were tested and then scored as adults, as described above. Recombination mapping of sbl: For the initial meiotic recombination mapping of the sbl locus, o l f P y females were crossed to males carrying the multiply marked X chromosome yellow ( y , 0.0) crossveinless (cv, 13.7) vermilion (u, 33.0) forked (5 57.6) carnation (car, 62.5). FI progeny were allowed to mate with each other, and lines were established C ( I ) A y attached-X by crossingindividualF2maleswith females. Each line was examined for olfactory behavior and heat-sensitive lethality.Heat-sensitivity of these lines was assessed by allowing femalesof each line to lay eggs in vials at 29’, allowing progeny to develop at 29”, and then scoring for the absence of male progeny. Olfactory behavior was measured with the larval plate assay, using a 10” dilution of propionic acid as stimulus. At the end of the 5 min test period, larvae from each half of the plate were transferred separately to culture vials, and were then incubated at 25”. Following eclosion, the number of males and females were counted from each vial, and from these numbers, response indices werecalculated independently for the males carrying a recombinant chromosome and for females carrying the attached-X chromosome. Duplication and deficiency mapping of lethality: In order t o map the conditional lethality of sbl’ and olfDI’ and the unconditional lethality of three other sbl alleles, homozygous sbl’ and olfDxv females and olfD’“IFM7, olfDLz/FM7, and ~ b l ~ ( ” ” ~ / F Mheterozygous 7 females were crossed to males carrying deficiencies and/or duplications extending through parts of the 13F-16A interval on the X chromosome cytogenetic map (see Figure 9). Crosses with sbl’ and o l f F y were incubated at 29”; all other crosses were performed at 25’. RESULTS
The larval olfactory phenotype of orfo”’: Drosophila larvae exhibit a robust olfactory attraction to a and QUINN variety of volatile chemicals (ACEVES-PINA
FIGURE: 1 .“Larval olfactory plate assay. Larvae are placed on an agarose Petri plate containing two filter discs. An aliquot of 25 rl of odorant is placed on one disc; the other is a control. Larvae are allowed to migrate for 5 min, after which the number of animals on each half of the plate is counted. A response index (RI) is calculated by subtracting the number of animals on the control half of the plate ( C ) from the number on the stimulus half ( S ) and dividing by the total: RI = ( S - C)/(S C ) . R I thus varies from 1 (complete attraction) to 0 (no response). The RI in the assay shown here was 0.85. Often a small percentage of animals remained in the center of the plate; these were not counted as C or as S and therefore did not figure into the computation of the RI. A, f = 0 min; B, f = 5 min (taken from MONTE et al. 1989).
+
1979; MONTEet al. 1989). Figure 1 shows a simple assay for larval olfaction, inwhich animals migrate across aPetri dish toward asource of odorant. A response index reflects the fraction of animals on the half of the plate containing the odorant at the end of a five minute test period. Figure 2 shows that olfDXy larvae are defective in olfactory response to odorants representing three distinct chemical classes: propionic acid (an organic acid), ethyl acetate (an acetate ester), and acetone (a ketone). T h e mean response of o l f P y is lower than wild type at all concentrations shown of each chemical tested. We note that although olfactory responses are severely reduced in o l f P y , some response remains to all three chemicals. Formally, at least three explanations might account for this residual response: 1) theelf''
296
M . Lilly and J. Carlson
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FIGURE3.-Complementation analysis for olfDxyand sbl’ (abbreviated as sbl). oIf0”’ females were crossed to sbl’ males and the F, progeny were tested in the larval plate assay using a 10” dilution of propionic acid as the olfactory stimulus. At the end of the 5-min test period, larvae from each half of the platewere transferred separately to culture vials. A response index for the heterozygotes was calculated after counting the number of females in each vial after eclosion. Each value represents 5-15 assays. CS = Canton-S-
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FIGURE2.-Olfactory dose response curves for oljo”’ larvae with three chemicals. Each point represents 10-15 assays. Concentrations are expressed as logarithms of the dilution factors. A, propionic acid: B, ethyl acetate; C, acetone. At the highest concentration of ethyl acetate, wild-type response has declined from its peak value at adilution of Asimple interpretation of this decline is that at very high ethyl acetate doses, chemical concentrations over theentire platemay, duringthecourse of the assay, become sufficiently high as tosaturatethe wild-type animal’s olfactory response, thereby reducing the animal’s tendency to migrate up a concentrationgradient. T h e mutant’s olfactoryresponse, if less sensitive than wild type, might not be near saturation at this concentration, and a declining response would not then be expected.
mutation (or mutations) may not be amorphic, i.e., some residual gene functionis retained; 2) penetrance of the defect may not be complete,i.e., not all animals within the population express the defect; 3) a gene whoseactivity is abolished by the 01fDx9 mutation shares an overlapping function with a gene which is not affected by the mutation. In order to investigate furtherthe source of the residual activity-and in particularthe first possibility-we soughtadditional alleles of olfDxy. 01fDx9and smellblind are allelic: The mutant smellblind hasalso been reported as defective in larval
olfaction (ACEVES-PINA and QUINN 1982), although we are unaware of any published data documenting this phenotype. We emphasize here that the designation “smellblind” (ACEVES-PINA and QUINN 1979) was intended to indicate that the mutant was effectively “blind” to smell, and was not intended to imply any deficit in visual function. The mutant will be abbreviated here as sbl’ (in contradistinction to an earlier designation, smb), in accordance with the convention adopted by TOMPKINS et al. (1 982). Figure 3 demonstrates that sbl’ larvae, like 01fDx9, are defective in larval response to propionic acid. The figure also shows that both phenotypes are fully recessive, and that they fail to complement. With the approval of Drs. Rodrigues and Siddiqi, we therefore refer henceforth to 01fDx9 as sbl“‘fD,and we refer to these mutations as alleles of the sbl locus; we note that these designations receive further justification from the more extensive genetic analysis described below. The sbl defects extend to include contact chemosensory response: We sought to investigate the specificity of the sbl mutants with two purposes in mind. First, characterization of the extent of the sbl phenotypes might provide information useful in considering the function of the gene, and second, comparison of sbl’ and sbl“qD phenotypes might be useful in assessing the nature of the two alleles. Drosophila larvae exhibit a contact chemosensory
297
smellblind and Drosophila Olfaction
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~ k .l.--1..1r\.II . ( o 1 1 1 a I ( IICIIIO\CII\OI \ < I \ W \ . . I I I C ; I \ \ ; I V plate serlmwtl I'cl1.i phtc containing f h r quadrants. 'l'wo diametrically opposed q u x h n t s (the top and I>ottomquadrants) are filled w i t h 1 % agarosc containing a defined concentration of NaCI. Lar-
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,we I h c e t l i n the center of the plate and allowed to distribute for 5 rnin. A response index is calculated by subtracting the number of animals on the N;ICI-cont;lining quadrants (S) from the number on the control qtmdrants (C) and dividing by the total: RI = (C S ) / ( C S). The R I i n the assay shown here is 0.88; the stimulus \vas 1 .0 M NaCI. y.ICI
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response (TOMPKINS 1979).Figure 4 shows a paradigm used to measure this response. Larvae are allowed to partition among four quadrants of an agarose Petri plate, two of which contain defined concentrations of NaCI.The number of larvae on each quadrant are counted at the end of a 5-min test period, and from these numbers a response index is determined. High concentrations of NaCl repel the larvae, and Figure 5A showsthis contact chemosensory avoidance response to a range ofNaCl concentrations. The graph shows that sbl"'f' is defective in this response across a broad range of concentrations. Figure 5B shows that sbl' is also defective in this response. T h e response of sbl' at a concentration of 1 M NaCl is not significantly different from that of sbl"'f'' at the same concentration. sbl' and sblolm are normal in a test of larval visual response: Are the defects observed for the mutants in the two chemosensory behaviors simply a manifestation of a general defect in health or mobility? To address this question we developed a newassay to measure the larvae's response to a visual stimulus. The assay measures the partitioning of larvae between dark and light quadrants of a Petri plate (Figure 6). The paradigm was designed so as to require as many of the same skills as the chemosensory assays as possible, and it is structurally analogous to the contact chemosensory assav. Figure 7 shows that larvae exhibit a strong avoidance of light. Both sbl' and sbP'f'' exhibit a robust
Canton-S
sbl'v)
sbl
I
FIGURE5.-Cont;tct chemosensory response of sbl"'J" and sbl'. For each value, 10 5 N I34. A, NaCl dose response curve for sbl"". B. Response of sbl' to 1 M NaCI. Data for sb1"'J"are taken from panel A for comparison.
response, similar to thatof wild-typelarvae, across the entire response curve, i.e., underconditions which stimulateeither a strong or weak response. These results suggest that these two sbl mutations do not affect olfactory response by virtue of a generalized neural or motordefect, but rather they suggest a greater degree of specificity. sbl' and sbl"vDexhibit heat-sensitive lethality: Both sbl' and sbl"'1" are heat-sensitive lethal mutants. Figure 8 shows that when females of either mutant are allowed to lay eggs at 29", only a very small fraction (approximately 2%) of the eggs develop to adulthood. Most sbl"'f" eggs hatch, but then die as young larvae. In the case ofsbl', an appreciable fraction of eggs fails to hatch; most of those individuals which do hatch die as larvae. The lethality of the two mutants fails to complement (see below, Table 2), suggesting that the lethality is due to themutations at the sbl locus. If sbl is required for normal development to occur, stage? When is the genealso required during the adult raised at 25" and then transferred to 29", sbl' and sbt"") mate, lay eggs, and appear to survive normally (data not shown). These observations do not support the notion of a role for sbl after development to the adult stage, but neitherdo they exclude it. We do not
298
M. Lilly and J. Carlson
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FIGURE6.-Larval phototaxis assay. The assayis structurally identical to the NaCl assay. Larvae were placed at the centerof this Petri plate, which contains alternating dark and clear 1% agarose quadrants. The plate was placed on a light box in a dark room. Light passes through the clear agarose but is blocked by the dark agarose. Drosophila larvae are negatively phototactic and partition onto the dark quadrants, where the light is less intense. After 5 min a response index (RI) is calculated by subtracting the number of animals on the clear quadrants (C) from the number on the dark quadrants (D) and dividing by the total: RI = (D - C)/(D + C). The plate shown here was transferred from the light box onto a piece of gray paper for photography; the distribution of larvae shown here corresponds to an R1 of 0.85.
know the extent to which sbl function is disrupted by these manipulations, and it is possible that more severe disruptions of sbl function after eclosion might result in lethality. Recombinational mapping of sbl: As described above, sbl' and sbl"'f" have similar phenotypes with respect to larval olfaction, contact chemosensory behavior, and heat-sensitive lethality. Moreover, the olfactory phenotypes of the two mutants fail to complement, as do the lethality phenotypes. T h e simplest interpretation of these results is that in each mutant, all three phenotypes result from a single mutation, and that the two mutations affect the same genetic locus. If this interpretation is correct, then all phenotypesof each mutation should map to a common position. Both sbl' and sbl"'fD were isolated in screensdesigned to allow isolation of X-linked recessive mutations. As an initial step in the mapping of the sbl phenotypes, we attempted to determine whether the olfactory and conditional lethal phenotypes of sbloYD could be localized to aninterval of the X chromosome. For this purpose, we used the multiply marked X
A
LIGHT INTENSITY 4)
FIGURE7.-Dose-response curves for sbl' and sbP"/D phototaxis. Each point represents 10-20 assays. The indicated percentages of dye refer to the pair of quadrants containing variable, low dye concentrations; the other pair of quadrants contained a fixed, high concentration (see MATERIALS AND METHODS). Thus as the variable dye concentration increased, the light intensity differential between the two pairs of quadrants decreased.
T Canton4 sblolp
0 sbll
HATCHING
ECLOSION
FIGURE8,"Heat-sensitive lethality of sbPP and sbl'. Hatching percentages are based on examination of 500-625 eggs total ( N = 50-52 groups of -10 eggs each), which derived from three sets of flies for each genotype. Eclosion percentages are based on 200250 eggs for each genotype ( N = 10 groups of 20-30 eggs). Means are normalized to Canton-S values.
chromosome y cv v f car and established 98 attachedX lines, malesof each line carrying an identical X chromosome. Twenty-two of these lines carried parentalchromosomes and were included as internal controls. All of these control lines gave the expected results: all of 14 y cv v f c a r lines gave response indices considered to be normal (>0.5); all of 8 sbl"'f') chromosomes gave response indices considered to be abnormal (50.5). The other 76 lines carried recombinant chromosomes representing all 8 recombinant classes. T h e olfactory and lethality phenotypes of males from each line were tested, and both phenotypes
OlfactionDrosophila smellblind and
299
TABLE 2
TABLE 3
Complementation of heat-sensitive lethality
Complementation of olfactory phenotype
L1
sbl' sbloYD Canton-S
L2
20-137
Lethal Lethal Lethal Lethal Lethal Lethal Viable Viable Viable
023
sbl'
Lethal Lethal Lethal Lethal Viable Viable
Canton-S
Viable Viable Viable
clearly mapped to the u-f interval. (Among the 46 recombinants with breakpoints in intervals other than the u-f interval, all lines within a given recombinant class were scored uniformly as mutant or wild type for both phenotypes, with only one exception in the case of each phenotype.) Moreover, the two phenotypes cosegregated in 28 of the 30 recombinant lines with breakpoints inside the u-finterval. The two exceptional lines were not tested further to determine whether the failure to cosegregatesimply represented phenotypic variability; as noted above, two exceptions were also found among the 46 recombinants containing breakpoints outside the u-finterval. A second recombination analysis was conducted to mapthe heat-sensitive lethality of sbPyD at higher resolution. Females homozygous for sbPyD were crossed to males carrying they cu u f c a r chromosome. F1 heterozygous females were crossed to males carrying anFM7 balancer chromosome, in vials maintained continuously at29". Maleswhich developed and eclosed at 29" were scored for the y, cu, u , f , and car markers. This analysis confirmed our mapping of the s b P D lethality to the u-finterval: of 563 male progeny, all were marked with v and/or f. On the basis of 209 chromosomes recombinant in this interval, we tentasbl"yD tivelyassign amap position of 54.7tothe lethality. Four additional alleles of sbl: Four additional alleles of sbl have been identified. T w o EMS-induced alleles, sblL' and sblL2, were isolated by Dr. V. Rodrigues in a screen for mutations allelic to sbl"yD. We have identified two other alleles by testing lethal mutations previously mapped by others near the map position of sbl. One, 1(1)20-137, was identified from among a set of spontaneous lethal mutations isolated by SCHALET(1 986)in a screen for X-linked spontaneous lethals. The second, 1( 1)D23, was generated in a dysgenic cross by EEKENet al. (1985). Table 2 documents the allelism of these additional four mutations to sbl"yD and sbl' with respect to lethality at 29 " . This phenotype is recessive in all cases. As further evidence that the olfactory defect and heat-sensitive lethality both arise as a consequence of mutation at the same locus, Table 3 shows that both sblL' and sblL2 also fail to complement the olfactory defect of either sbl' or sbl"'f0. 20-137/sb11, 20-137/ sbl""", D23/sb11, and D23/sbl"yD heterozygotes were
sbl' sblo'fD Canton-S
Ll
L2
Canton-S
0.32 2 0.17 0.25 & 0.15 0.70 f 0.09
0.18 f 0.14 0.20 & 0.09 0.71 f 0.07
0.84 ? 0.08 0.73 ? 0.06
0.86 & 0.04
Response indices are for a 10" dilution of propionic acid. N = 10.
lethal even at 25" andwere therefore nottested. With and EEKEN,we refer to 1( 1)20the assent of SCHALET 137 and I( 1 ) D 2 3 henceforth as sb120-137and sblDZ3. Cytogenetic mapping of sbl: In order to map the sbl locus at high resolution on the cytogenetic map, we have used duplications and deficiencies affecting the part of the X chromosome in which sbl was expected to reside onaccount of its recombinational map position. We have mapped the 29" lethality of both sbl' and sbPyD and the unconditional lethality of sblL1, sblL2, and each of these mutations was mapped using the entire set of seven duplications and deficiencies shown in Figure 9. The results of the five determinations agreed in all respects; moreover, the results obtained in each case were internally selfconsistent. Figure 9 shows the duplications and deficiencies used in this analysis, their cytogenetic boundaries, the results obtained with each, and the deduced map position of sbl: 14F6-15A2-3. The sbl locus, then, is expected to reside within the region spanned by four chromosomal bands on the cytogenetic map of the polytene chromosomes, 14F6, 15A1, 15A2, or 15A3. We note that this determination is consistent with the determinations derived by EEKENet al. (1985) for sblDZ3,and by SCHALET(1986) for sb120"37, who determined,at lower resolution,map positions of 14D1-2 to 15A4 for these mutations. Moreover, the cytogenetic position determined for sbl is very near that of rudimentary ( r ) , which has been determined 1979). This is to be between 14F6 and 15A2 ('JARRY consistent with the observation that the meiotic map position determined for sbl, 54.7, is near that determined for r, 54.5 (LINDSLEY and GRELL1968). We have also mapped the olfactory andcontact chemosensory phenotypes of bothsbl' and sbl"y" using the duplication T( l;2)r+75cand have obtained results consistent with the localization of the lethality (Figure 10). Both phenotypes of both mutants are covered by the duplication, indicating that the olfactory phenotype and the contact chemosensory phenotype reside between 14B13 and 15A9. We have not been able to attain higher resolution by using the deficiencies on account of the poor viability of sbllDe3ciency heterozygotes. DISCUSSION In this article we have defined and presented an initial genetic characterization of the sbl locus. We
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M. Lilly and J. Carlson 53.2
54.1
s4.5
56.7
FIGURE 9.-Cytogenetic localization of the sbl heat-sensitive lethality. The top line shows a meiotic recotnbination nlap of mutations in the vicinity of sbl. Under this line are the numbered and lettered intervals of the salivary gland polytene chromosonre cytogenetic map for the region. Empty rectangles indicate the extent of material missing in the deficiencies used i n this study; hatched rectangles indicate the extent of nraterial duplicated in the translocation stocks. Cytogenetic limits of eachduplication and deficiency are indicated adjacent to each rectangle. The dotted vertical lines indicate the inferred limits of the region to which the sbl locus has been localized. eas, easily shocked; para, paralytic; r , rudimentary; J forked. T h e "-"signs indicate that a nrutant heterozygous for either sbl' or s b P ' and the indicated chromosomal aberration is phenotypically indicates that the indimutant; cated heterozygote is phenotypically normal.
"+"
have provided evidence that the olfactory phenotypes of two independently isolated mutants, sbl' and sbl"Y", arise as a result of mutations of this gene. The locus has been mapped to position 54.7 on the meiotic recombination map and to a small cytogenetic region, 14F6-l5A2,3, on the X chromosome. Six alleles have been identified. Two alleles, sbl' and sbPYD, are characterized here with respect to several phenotypes, and are found to be very similar. In addition to their common defect in larval olfactory response, both show similar behavior in a test of contact chemosensory response and in a new test of larval visual response. Both exhibit heatsensitive lethality early in development. Other similarities between the two mutantshad previously been found in studies of courtship behavior. A study of sbl"'fD by GAILEY,LACAILLADE and HALL(1986) showed that 1) sbl"YD females have a lower tendency than wild type to slow their locomotion in response to male courtship, and thus copulate later; 2) sbloYDmales are not induced to court to the same extent aswild type by extracts of courtshipstimulating flies; 3) sbl"" males show abnormal courtship behavior following exposure to mated females. All three of these courtship phenotypes have been documented for sbl' in separate studies (TOMPKINS et al. 1982, 1983; TOMPKINS, HALL and HALL 1980).
The map position which we have determined for the sbl locus does not agree with that of a previous report (SIDDIQI 1987), which placed sbl"'fD near singed-a locus in cytogenetic region 7. The basis for this discrepancy is not clear. We do not know the function of the sbl gene or its role in olfactory response. Althoughboth sbl' and sbl"Y" mutants are grossly abnormal in their response to acontact chemosensory stimulus, bothrespond strongly when driven by a visual stimulus in the same paradigm. These results argue against a generalized neural or motor defect for these two mutants; rather, they suggest a degree of specificity. Any speculation as to the function of the sbl gene, however, must be constrained by the fact that these two mutants exhibit heat-sensitive lethality atan early developmental stage, and that four additional alleles exhibit unconditional lethality. Thus if one were to postulate a role for sbl in chemosensory transduction, for instance, it would be necessary also to postulate either that some form of chemosensory function is required early in development or that sbl plays an additional role, such as in the transduction of another signalin an early developmental process. The genetic characterization of sbl which we have initiated here provides a foundation for further investigation intothedevelopmentalrole played by this
smellblind and Dl-0sophila Olfaction
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FIGURE10.-Duplication mapping of the olfactory and contact chemosensory phenotypes of sbF'fn and sbl'. Males bearing the duplication Tp(I;2)r+'" (and a second chromosome marked with C H I ) were crossedto homozygous sbl', sbF'fD,or Canton-S-5 females, and the F, progeny were tested. After the 5-min test period, larvae from the stimulusand control sectionsof the plates were transferred to separate culture vials.Following eclosion, adult flies were counted and scored for the presence or absence of the duplication. From these numbers, response indices were calculated independently for each genotype. A, olfactory assay using a 10" dilution of propionic acid as stimulus. N = 8 assays; B, contact chemosensory assay, using 1 M NaCl as stimulus; 8 5 N 5 10 assays.
gene, its role in olfactory response, and the relationship between these two roles. We are very grateful to VERONICA RODRIGUES,OBAIDSIDDIQI, MARTIN HEISENRERC, ARE SCHALET, JAN EEKEN,SHANKAR KULKARNI and JEFFREY HALLfor providing stocks, and to DONALD GAILEY, JEFFREY and HALL VERONICA RODRIGUES for communicating unpublished results. We would like especially to acknowledge the helpful advice of ABE SCHALET. This work was supported by National lnstitutes of Health grant GM 36862 to J.C. J.C. is an Alfred P. Sloan Research Fellow.
LITERATURE CITED ACEVES-PINA, E., and W. QUINN, 1979 Learning in normal and mutant Drosophila larvae. Science 206: 93-96. BARROWS,W.. 1907 The reactions of the pomace fly, Drosophila ampelophila lorn, to odorous substances. J. Exp. Zool. 4 515537. BENZER, S.. 1967 Behavioral mutants of Drosophila isolated by countercurrent distribution. Proc. Natl. Acad.Sci. USA 5 8 11 12-1119. BOLWIG, N., 1946 Senses and sense organs of the anterior end of the house fly larvae. Vidensk. Medd. Dan. Naturhist. Foren. 109 81-217. CRAYMER. L.. and E. ROY, 1980 Drosophila Inform. Serv. 55: 200-204.
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DOANE,W., 1967 Drosophila, pp. 219-244 Methods in Developmenial Biology, edited byF. WILT and N. WFSSELLS. Thomas F. Crowell. New York. EEKEN,J., R. SORELS, V. HYLAND, and A. SCHALET. 1985 Distribution of MR-induced sex-linkedrecessive lethal mutations in Drosophilamelanogasier Mutat. Res. 150 261275. GAILEY,D.. R. LACAILLADEand J. HALL, 1986 Chemosensory elements of courtship in normal and mutant,olfaction-deficient Drosophila melanogasier. Behav. Genet. 1 6 375-405. GANETZKY, B.. 1984 Genetic studies of membrane excitability in Drosophila: lethal interaction between two temperature-sensitive paralytic mutations. Genetics 108: 897-91 1. HELFAND, S., and J. CARLSON, 1989 Isolation and characterization of an olfactory mutant in Drosophila with a chemically specific defect. Proc. Natl. Acad. Sci. USA 86: 2908-2912. JALLON, M., C. ANTONYand T. IWATSURO, 1981 Elements of chemical communication between Drosophilids and their modulation, pp. 105-1 35 in Genetic Disseciion of Drosophila Behavior, edited by Y. HOTTA. University of Tokyo, Tokyo. JARRY. B., 1979 Genetical and cytological location of the structural parts coding for the first three steps of pyrimidine biosynthesis in Drosophila melanogaster. Mol. Gen. Genet. 172: 199-202. KULKARNI. S., A. STEINLAUF and J. HALL1988 The dissonance mutant of courtship song in Drosophila melanogasfer: isolation, behavior and cytogenetics. Genetics 1 1 8 267-285. LINDSLEY, D., and E. GRELL1968 Geneiic Variations of Drosophila melanogasier. Carnegie lnst. Wash. Publ. 627. MARKOW, T., 1987 Behavioral and sensory basisof courtship success in Drosophila melanogasier. Proc. Natl. Acad. Sci. USA 8 4 6200-6204. MCKENNA,M., P. MONTE, S. HELFAND, C. WOODARD and J. CARLSON, 1989 A simple chemosensory response in Drosophila and the isolation of acj mutants in which it is affected. Proc. Natl. Acad. Sci. USA 8 6 8 1 18-8 122. MIYAKAWA,Y., N. FUJISHIRO,H. KIJIMA,and H. MORITA, 1980 Differences in feeding response to sugars between adults and larvae in Drosophila melanogasier. J. Insect Physiol. 2 6 685-688. MONTE, P., C. WOODARD,R. AYER, M. LILLY,H. SUNand J. CARLSON, 1989 Characterization of the larval olfactory response in Drosophila and its genetic basis. Behav. Genet. 1 9 267-283. RODRIGUES, V., 1980 Olfactory behavior of Drosophilamelanogasier, pp. 36 1-37 1 in Developmeni and Neurobiology of Drosophila, edited by 0.SIDDIQI.P. BARU,L. HALLand J. HALL. Plenum, New, York. RODRIGUES.V.. and 0.SIDDIQI.1978 Genetic analysis of chemosensory pathway. Proc. Ind. Acad. Sci. 87B 147-160. SCHALET,A., 1986 The distribution of and complementation relationships between spontaneous X-linkedrecessive lethal mutations recovered from crossing long-term laboratory stocks of Drosophila melanogasier. Mut. Res. 163: 11 5-144. SIDDIQI,0.. 1987 Neurogenetics of olfaction in Drosophilamelanogasier. Trends Genet. 3: 137- 142. SINGH,R., and K. SINGH,1984 Fine Structure of the sensory organs of Drosophilamelanogaster Meigen larva. Int. J. Morphol. Embryol. 13: 255-273. TOMPKINS, L., 1979 Developmental analysis of two mutations affecting chemotactic behavior in Drosophila melanogaster.Dev. Biol. 73: 174-1 77. TOMPKINS, L., J. HALL andL. HALL,1980 Courtship-stimulating volatile compounds from normal and mutant Drosophila. J. lnsect Physiol. 2 6 689-697. TOMPKINS. L., A. GRO.SS, J. HALL,D. GAILEYand R. SIEGEL. 1982 The role of female movement in the sexual behavior of Drosophila melanogaster. Behav. Genet. 12: 295-307. TOMPKINS,L., R. SIEGEL, D. GAILEY and J. HALL,
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