closely linked lesions in a region of the x chromosome affect central and peripheral steps in...

Mol Gen Genet (1991) 226:265-276 002689259100101Z

© Springer-Verlag 1991

Closely linked lesions in a region of the X chromosome affect central and peripheral steps in gustatory processing in Drosophila Veronica Rodrigues, Swati Sathe, Ludwin Pinto, Rohini Balakrishnan and Obaid Siddiqi

Molecular Biology Unit, Tata Institute of Fundamental Research, Homi Bhabha Rd, Bombay 400005, India

Received August 9, 1990 / November 23, 1990

Summary. We have analyzed a set of closely linked mutations on the X chromosome of Drosophila that lead to defects in gustatory behavior. The mutations map to a small region of the X chromosome between 10E1-4. Two distinct complementation groups, gustB and gustD, map to the ends of this region. These groups show complex complementation patterns with the mutations gustC and GT-1, which also map to this region. We describe the behavioral and electrophysiological properties of the mutants. These mutations affect peripheral receptor properties as well as more central processing steps in the gustatory pathway.

Key words: Chemoreception- Taste mutants Gustatory behavior - Genetic interactions

Introduction

Complex behavior is a result of intricate neural processing of information. The taste system of Drosophila pro- vides a tool that allows the analysis of the role of genes in the development and function of a neuronal pathway. The taste sensilla are arranged in a stereotyped fashion on the proboscis, tarsi and wings (Falk et al. 1976; Nayak and Singh 1983; Hartenstein and Posakony 1989). The majority of taste sensilla located on the proboscis are innervated by five neurons - four chemosensory and one mechanosensory. Of the four chemosensory neurons one is responsive to sugars (S), two to salts (L1 and L2) and one to water (W) (Rodrigues and Siddiqi 1978; Fujishiro et al. 1984). As yet little is known about the circuits to which these receptors are connected (Nayak and Singh 1985), or how the discrimination of different stimuli is encoded in the brain.

Attempts to identify genes specifying the function and development of the taste pathway have involved the isolation of mutants defective in taste behavior (Isono and

Offprint requests to: V. Rodrigues

Kikuchi 1974; Falk and Atidia 1975; Rodrigues and Sid- diqi 1978; Tompkins et al. 1979; Tanimura et al. 1982; Morea 1985; Siddiqi et al. 1989). These mutations could, in principle, affect any step in the gustatory pathway from receptor specificity, transduction, coding and processing of information to the motor response.

The different chemospecificities of the taste neurons provide a system for the investigation of the mechanisms of specialization of closely related neuronal subtypes. One of the main molecular differences between the neurons is in the expression of acceptor sites for stimuli. The S neuron has distinct molecular acceptors for pyran- oses, furanoses and trehalose (Tanimura and Shimada 1981; Siddiqi and Rodrigues 1980; Tanimura et al. 1982). Biochemical and genetic evidence exists for the presence of two distinct acceptor sites for cations, on the L1 neuron (Siddiqi et al. 1989). One of these sites is specific for Na + while the other is more general, responding to both Na + and K + ions.

Mutations that perturb peripheral steps in the taste pathway could affect either the receptor sites per se or their specific expression among the subpopulations of neurons. The gene Tre specifies receptor sites for trehalose on the S neuron (Tanimura et al. 1982). The sucrose- insensitive mutation gustA specifically blocks the pyran- ose site of this cell (Rodrigues and Siddiqi 1981). Yet another gene, gustE, has recently been shown to alter the Na + specific response from the L1 neuron (Siddiqi et al. 1989). Mutations in the gustB gene lead to a rever- sal of response to sodium chloride. The neuronal corre- late of this behavioral change is an aberrant response to sodium chloride from the S cell. This locus has been proposed as a candidate for the regulation of the distribution of sites among the chemosensory neurons (Arora et al. 1987).

Mutations that alter the central processing of taste information are more difficult to analyze. Tompkins et al. (1979) have described a number of X-linked mutations that are associated with defective chemotactic responses. Some of the mutations show a conditional phenotype and two genes gusA and gusE appear to play

266

a role in development of the gustatory pathway (Tomp- kins 1979). The Lot-94 mutation alters the control of intake of taste substances (Falk 1979; Crnjar et al. 1983). Mosaic analysis has placed the focus of action of the mutation away from the proboscis and close to the pre- sumptive central nervous system (Falk and Atidia 1975). Siddiqi et al. (1989) have discussed a number of dominant autosomal mutations that result in pleiotropic taste defects. The step of the gustatory pathway that these mutations affect is not clear.

This paper analyzes a set of gustatory mutations that map in the 10EI-4 region of the salivary gland chromosome map. We used a variety of functional and genetic criteria to assign the mutations into two operational classess some of which show functional interactions in trans heterozygote combination.

Materials and methods

Stocks. The Canton Special strain (CS) was obtained from S. Benzer, Div. of Biology CalTech, Pasadena, USA. It was isogenized and used as the wild-type strain in all experiments. The source and genotypes of the deficiency and duplication strains are listed in Table 1. The balancer strains FM6 and FM7, the multiply marked strain y cv v f c a r and other markers are listed in Lindsley and Grell (1968). The gustatory mutations used in this study are listed in Table 2. gusF N5 was kindly provided by Laurie Tompkins, Temple University, USA. All cul- tures were reared in standard cornmeal-yeast medium at 22°-25 ° C.

Chemicals. Sucrose, dextrose, fructose, quinine sulfate and quinine hydrochloride were obtained from Sigma, St. Louis, USA. Sodium chloride and potassium chloride were obtained from Sarabhai Chemicals, Baroda, India. Agar was obtained from Sisco, Bombay India and carmoisine red from Anand dyes, Bombay. Microtiter plates were obtained from Laxbro, Pune, India.

Behavioral tests

Proboscis extension test. This test has been widely used to assay gustatory responses in larger dipterans (Dethier 1976) and has been modified for use in Drosophila by Deak (1976). Two to four day old flies were starved in moist chambers for 15 h prior to the test. They were immobilized by cooling on ice and fixed ventral side up onto a microscope slide using myristic acid wax (m.p. = 58.5 ° C). The wax was applied with a tungsten wire loop whose temperature could be controlled by varying the current through the wire. The slides were placed in a humid chamber for a further 3 h to allow the flies to recover from handling.

A drop of deionized water from a glass capillary was applied to the labellum of the fly. Each fly was allowed to drink until satiated. The tarsus of the first leg was touched with a drop of the stimulus and the extension of the proboscis scored. Each fly was given five trials

Table 1. Breakpoints and sources of the deficiency and duplication strains used in the cytogenetic localization of the gustatory mutations

Strain Breakpoints Source of Def/Dup

Df(1)KA6/FM7 10E1;11A7

Df(1)m 259 - 4/FM7 10C2-3 ;10E1-2

C(1)DX,yf: 9F4;10E3-4 y+Yv + 4~3

Df(1)NI05/FM6 11A1 ;I1D1

G. Lefevre, Calif. State Univ. Northridge, Calif.

Dros. Stock Center, Bloomington, Ind.

Dros. Stock Center, Bloomington, Ind.

G. Lefevre, Calif. State Univ. Northridge, Calif.

For details refer to Lindsley and Zimm (1987)

Table 2. Phenotypes and tests of dominance of the gustatory mutants

Genotype Mean percentage acceptance SEM

a. Acceptance of 1 mM sucrose

CS 89.0 2.5 gustB × s 81.4 2.2 gustC 40.3 3.8 gglstD x 6 72.7 4.1 GT-I 13.6 1.4 gustC/+ 66.6 2.7 G T - 1 / + 64.3 1.3

b. Acceptance of 100 mM NaC1

CS 43.5 2.7 gustB × s 69.5 2.2 gustB × v 84.9 3.8 gustB gulf 70.7 2.9 gustC 80.8 2.6 GT-1 57.9 2.9 gustB × 5/+ 42.0 1.4 gustB × 7/+ 40.3 2.2 gustBgusF / + 39.0 1.5 gustC/+ 42.4 2.6 G T - 1 / + 50.1 1.7

c. Tolerance of 0.5 M NaC1

CS 7.9 1.3 gustB × 5 65.3 2.9 gustC 63.4 3.6 GT-1 69.7 1.1 gustB × s / + 11.7 3.1 gustC/+ 48.4 3.2 G T - 1 / + 33.9 3.3

d. Tolerance of 5 mM quinine hydrochloride

CS 4.0 1.2 gustC 43.3 4.1 gustD × 3 51.6 3.1 gustO x 6 38.9 2.5 GT-1 45.2 1.1 gustC/+ 19.1 2.1 gustD × 3/+ 18.3 3.3 gustD × 6 /+ 24.9 3.5 G T - I / + 17.1 2.5

The feeding preference test was the assay paradigm. The mean and standard error of the response was calculated from at least 10 readings in all cases

267

and taken as a responder if it extended its proboscis in at least three trials. To prevent adaptation, an interval of at least 5 min was allowed to elapse between stimula- tions. The fly was offered water between successive tests.

When the response of mutants was being evaluated, the wild type was tested in parallel under identical conditions. In each experiment, flies of both sexes were tested and the fraction of flies responding was noted.

Feeding preference test. The test was designed by Tani- mura et al. (1982). The wells of a microtiter plate were filled with 1% agar solution. Alternative wells also con- tained 2% of the food dye carmoisine red. When the response to an attractant was being assayed the stimulus was placed in the uncolored wells. For repellents, the substance was mixed into the wells with the food dye.

Flies for behavioral tests were grown in uncrowded bottles at 25 ° C and transferred onto fresh medium after emergence. They were aged for 2-4 days. Flies were placed in bottles containing moistened filter papers 18-h prior to the test. Approximately 100 flies were intro- duced into each test plate and left undisturbed in a dar- kened area for 1 h. They were immobilized by cooling and the color of their abdomens scored by inspection under a dissection microscope.

The percentage acceptance of a stimulus is measured by the fraction of uncolored flies in the population. The tolerance response to a repellent is given by the fraction of colored flies. Wild-type and mutant flies were tested in parallel under identical conditions. The mean and standard deviation of the response was calculated from a minimum of ten tests. The plates were run on at least three different days using independent batches of flies. Statistical difference between two means was calculated using Student's t-test.

Larval chemotaxis. The response of larvae to gustatory stimuli was measured as described by Miyakawa (1981) and Rodrigues and Siddiqi (1981). A 9 cm petri plate was filled with 1% agar solution. After the agar had solidified, half of it was cut away and replaced with agar containing the appropriate stimulus. Third instar larvae were washed off the culture medium and placed on a nylon mesh. They were washed extensively with deionized water and assayed immediately.

Approximately 100 larvae were placed in the center and the plate left undisturbed in the dark for 30 rain. Larvae on the stimulus and control halves of the plate were counted leaving out those that had not left the center. The Response Index was calculated from the difference between the numbers of larvae found on the stimulus and control halves of the plate divided by the total number of larvae participating in the test. An index of - 1 indicates total repulsion, + 1 indicates total attraction, and indices around 0 indicate a lack of response.

Electrophysiological recordings for the labellar sensilla. The tip recording method of Hodgson et al. (1955) was modified for use in Drosophila (Rodrigues and Siddiqi 1978; Fujishiro et al. 1984; Arora et al. 1987). Two to

four day old flies were immobilized by cooling and intro- duced into a glass capillary. The capillary used to hold the fly was tapered using an electrode puller and cut to fit snugly around the fly such that the head and thorax could protrude from it. The fly was held in place with myristic acid wax. The proboscis was immobilized by a minute drop of wax applied at the haustellum of the proboscis. Care was taken to keep the labial palps free of wax and to avoid heating the labium.

A saline-filled glass capillary connected to a Ag-AgC1 junction served as the ground electrode. This was intro- duced into the thorax of the fly. The recording electrode was a tapered micro-capillary filled with a stimulus and connected to a preamplifier through a Ag AgC1 junction. Signals from the labellar hairs were passed through a low frequency filter (50 Hz) before being displayed on a storage oscilloscope (Tektronix 5113). Spike frequencies were measured within a 450 ms interval beginning 50 ms after the intitiation of the response. In a typical recording, at least eight hairs were sampled from each preparation. Each hair was stimulated twice with a 5 min gap between tests and the average spike frequency noted. The responses of sensilla from several preparations were pooled to give the mean, standard deviation and standard error of the mean. For compari- son of the means of two different samples Student's t-test was used.

Isolation of mutants. Mutagenesis with ethylmethanesul- fonate (EMS) was carried out according to the proce- dure of Lewis and Bacher (1968). Three independent experiments yielded the mutants that were analyzed in this study. 1. Isogenic X chromosome lines were set up with C(I)DX y f attached-X virgins. Male progeny from the lines were tested for their responses to sucrose, sodium chloride and quinine, gustB × s, gustC × 2, gustD × 3 and gustD × 6 were obtained in this screen. (We will drop the superscript notation of gustC X2 a s there is only one member of this class.) Details of this mutagenesis are given in Rodrigues and Siddiqi (1978). 2. The progeny of mutagenized males mated with C(1)DX y f virgins were screened in feeding preference tests using 10 mM glucose as the stimulus. At this concentration, 90% of CS flies eat from the wells containing the stimulus and appear uncolored. The red flies were selected and used to set up lines with C(1)DX yfvirgins. A screen of 5200 males yielded 2 pleiotropic mutations showing lowered responses to several stimuli. We will discuss only one of these mutations, GT-1, in this paper. The nomenclature designates a mutant and is not in- tended to indicate a locus. 3. In an attempt to obtain further alleles at the gustB locus, mutagenized males were crossed to females heterozygous for a deficiency of the gustB region (Df(1)KA6/FM7). The progeny were screened in the feeding preference test with 100 mM NaC1 as a stimulus in the uncolored wells. At this concentration, 40% of normal flies, but 70% of mutants eat NaC1. The flies with uncolored abdomens represent a population en- riched for mutants. These were selected and used to set

268

up lines with FM7 males, gustB × 7 was obtained in this experiment.

Meiotic mapping. A strain bearing the visible markers y cv v f ear, and showing normal gustatory responses was used in the recombinat ion mapping of the mutants. The gust mutants were crossed to a strain bearing the markers y cv v f car. The F1 progeny were selfed and at the F2, ten males bearing crossovers in each of the intervals were selected and used to set up lines with C( I )DX y f virgins. Male progeny f rom these lines were assayed for their performance in gustatory tests.

Cytological mapping. The gust mutants were crossed to a set of deficiency and duplication strains (Table 1). Progeny of the selected genotype were tested for their taste responses in the proboscis extension test and the feeding preference test.

of progeny of mutagenized flies for insensitivity to 10 m M glucose in the feeding preference test (W. Chia and S. Sathe, unpublished work).

Behavioral response of wild-type Drosophila

The responses of wild-type Drosophila adults and larvae are summarized in Figs. 1 and 2. Adults of the CS strain accept sugars and low concentrations of sodium chloride and are repelled by salts at high concentration and by quinine hydrochloride. The responses of the adults were assayed by the feeding preference test and the proboscis extension test. Despite the fact that these paradigms measure two different responses evoked by the stimulus, the dose response curves are similar. The respohses of the larvae were measured in a chemotactic assay that measures a positive taxis to favorable stimuli and an avoidance of repellents (Fig. 2).

Results

This paper deals with the phenotype and genetic charac- terization of seven EMS-induced mutat ions that result in defects in gustatory behavior, gustB ×s, gustC, gustD × 3 and gustD × 6 were isolated by testing progeny of mutagenized lines for their responses to sucrose, salts and quinine (Rodrigues and Siddiqi 1978). gusF is a salt- insensitive mutant isolated by Tompkins et al. (1979). Complementat ion analysis has revealed that gusF is allelic to gustB ×5 (Arora et al. 1987). We therefore re- designate this mutat ion as gustB ~"sv. gustB × 7 was isolated in an enrichment experiment (see Materials and methods) designed to select for alleles of gustB, gustD × 3 and gustD × 6 have previously been shown to be allelic. GT-1 is a new isolate obtained after selective enrichment

Behavioral responses of mutants

In an effort to assign the mutat ions to phenotypic groups, we examined their responses in four distinct behavioral paradigms - sucrose acceptance; acceptance of low concentrations of sodium chloride; tolerance to high concentrations of salts and quinine tolerance - and the electrophysiological responses of their labellar chemoreceptors to salts. Their phenotypes are summarized in Fig. 3 and in Table 2. We also tested each of these mutations in heterozygous combinat ion with a wild-type chromosome in order to ascertain whether the defect was dominant or recessive.

Sucrose acceptance. CS flies are strongly attracted to 1 m M sucrose in feeding preference tests. The three

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gustB alleles as well as both alleles of gustD do not exhibit a significant change in their sucrose phenotype (Fig. 3). Both gustC and GT-1 show a reduced response to sucrose. Stimulus response curves suggest that the defect is a change in the threshold of the response rather than an insensitivity to sugars. The defect in both mutations is semi-dominant (Table 2a). The response level of mu tan t /+ is lower than that of the wild-type strain (P<O.O1).

Sodium chloride attraction. The responses of the mutants to 100 mM sodium chloride are given in Table 2b. gustB × 5 gustB × 7, gustBgu~F, gustC and GT-1 all show an enhanced attraction to sodium chloride over the CS strain (P < 0.003). Both gustD × 3 and gustD × 6 show normal responses to sodium chloride (Fig. 3). The defect in the gustB alleles and in gustC is fully recessive. GT-1 shows a partially dominant phenotype.

Salt tolerance. Less than 15% of normal flies ingest 0.5 M NaC1. Several of the mutants are tolerant to this concentration of salt (Table 2c). In addition, gustC and GT-1 are more tolerant to 0.2 M KC1 (Fig. 3). Both gustD × 3 and gustD × 6 show normal responses to salts. The defect in the gustB alleles is fully recessive while gustC and GT-1 show partially dominant defects.

Quinine hydrochloride tolerance, gustB shows a normal avoidance responses to quinine. In gustD × 3 gustD × 6, gustC and GT-I , the threshold of avoidance of quinine is increased (Table 2d, Fig. 3). The defect in all these strains is partially dominant.

Third instar larvae of all the mutants show normal gustatory responses when assayed in the chemotaxis assay described in Materials and methods.

Electrophysiological recordings from the labellar sensillae of CS and mutant flies

Electrophysiological recordings from the labellar chemoreceptors were carried out from the medial and large sensilla on each half of the proboscis (Arora et al. 1987). In the wild type, the firing of the four neurons innervat- ing these hairs can be distinguished on the basis of their amplitudes under appropriate recording conditions. The amplitudes have however been shown to vary depending on the capacitance of the recording probe and the firing frequency of the neuron (Fujishiro et al. 1984). Spikes from the W and L2 cells are easy to distinguish. The amplitudes of the spikes from the S and L1 neurons are in the range 0.8 1.2 mV and 1.0 1.5 mV respectively.

In the analysis of the mutants, we have not attempted to distinguish S from L1 spikes but have rather, measured total spike frequencies elicited by a stimulus. The firing frequency was calculated in a 450 ms interval starting 50 ms after the initiation of the response. At least eight sensilla were sampled from each fly. Measurements from sensilla from different flies were pooled to calculate the average frequency.

gustB × 5, gustB × 7, gustBgusV and gustC all showed an elevated spike frequency in response to 100 mM NaC1. The firing frequency in gusF> gustB × 5 > gustB × 7 (Fig. 4). When ranked with respect to increased acceptance of NaC1, gustB × 7 > gustB × 5 =gustBgusv (Fig. 3). The spikes elicited from these mutants were heteroge- neous in amplitude varying from 0.7-1.8 inV. The spike frequency defects as well as the behavioral defects in all these strains are recessive in nature (Table 3). GT-1 and the gustD alleles do not show any alteration in the response of the peripheral neurons to salts. All seven mutants show normal electrophysiological responses to sucrose.

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-fi Fig. 3. Behavioral responses of the gustatory mutants to taste st im- uli. All responses were measured in the feeding preference test. To measure the acceptance responses of flies to 1 mM sucrose and 100 m M NaC1, the stimulus was placed in the uncolored wells. The percentage acceptance was calculated from the fraction of flies with uncolored adomens. Tolerance responses to 0.5 M NaC1, 0.2 M KC1, i m M and 5 mM quinine hydrochloride (QHC1) were measured by p lac ing the stimulus in the colored wells. The fraction of flies with colored abdomens gave the percentage tolerance• The bars represent the m e a n a n d s tandard d e v i a t i o n o f at least 20 readings

Genetics of gustatory mutations

Each of the mutations was tested for complementation in pairwise combinations of different mutant strains. This analysis allowed us to group some of the alleles into complementation groups. One member of each of the complementation groups was localized on the chromosome with respect to the visible markers, y cv v f

and car. Fine-scale mapping was carried out with deficiency and duplication strains.

Complementation analysis." the mutations define at least two complementation classes

We set up reciprocal crosses between all the mutants showing elevated acceptance of NaC1 and examined their

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Table 3. Electrophysiological responses of the labellar chemoreceptors of the gustatory mutants to 100 mM NaC1.

Genotype Mean spike frequency SEM N /450 ms

CS 46.2 0.2 350 gustB × s 60.0 0.5 90 gustB × 7 56.7 0.4 40 gustB gusv 65.4 1.1 48 gustC 59.7 3.1 80 GT-I 45.2 1.0 30 gustB × 5/+ 43.7 1.0 20 gustB × 7/+ 40.1 1.5 10 gustBgusF / q- 41.1 1.3 10 gustC/+ 48.4 1.0 25

The spike frequency was calculated from a 450 ms interval beginning 50 ms after the initiation of the response. N indicates the number of hairs sampled Unless otherwise indiacted, the flies tested were homozygous for the mutant allele

phenotypes in feeding preference assays and in electrophysiological recordings. The data f rom some of these combina t i ons are presented in T a b l e 4 a . gustB ×5 gustB × 7 and gusF behave as a single complemen ta t i on group. Both gustC and GT-1 fully complemen t gustB in behaviora l assays. In the electrophysiological tests, gustC fails to complemen t gustB ×5, while GT-1 fully complements gustB.

gustC, G T - I , gustD ×3 and gustD ×6 are par t ia l ly d o m i n a n t m u t a n t s and complemen ta t i on results were analyzed by compar ing the pheno type of the heterozygous m u t a n t c o m b i n a t i o n with tha t of each m u t a t i o n in t rans- to wild type. Heterozygotes of gus tC/GT-1 showed reduced responses to sucrose (Table 4b). The

Table 4. Complementation analysis of the taste mutants a. Mutants showing an elevated acceptance of 100 mM NaC1

271

Genotype Complementation with gustB × 5

Percent acceptance Spike frequency

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CS 42.0 1.4 43.7 1.0 20 gustB × 5 69.5 2.2 60.0 0.5 90 gustB × 7 83.9 1.3 69.1 2.4 12 gustB gusv 75.3 1.9 70.7 2.6 14 gustC 40.0 3.3 66.3 1.9 35 GT-I 35.5 1.9 38.1 2.0 21

All the mutations showing an elevated acceptance of NaC1 were tested for complementation with gustB × 5. Electrophysiological recordings were carried out against 100 mM NaC1 using the tip recording method. Spikes were counted in a 450 ms interval starting 50 ms after the initiation of the response. The values from different sensillae were pooled to calculate the mean and standard error of the mean. N indicates the number of hairs sampled. All behavioral measurements were made in feeding preference assays

b. Mutations showing pleiotropic gustatory defects

Genotype Complementation with gustC

Percentage Percentage Percentage acceptance tolerance tolerance (1 mM sucrose) (0.5 M NaC1) (5 mM Quinine)

CS 66.6__2.7 48.4__3.2 19.1 _+2.1 gustC 40.3 +_ 3.8 63.4 + 3.6 43.3 ± 4. l GT-1 20.1 ± 1.9 70.3 ± 4.1 45.5 ± 1.7

All the behavioral measurements were made in the feeding preference test. Mean + standard deviation was calculated from a minimum of 12 readings in all cases. Mutants were tested for complementation with gustC

c. Mutants showing increased tolerance to quinine

Genotype Complementation with gustD × 6 Percentage tolerance (5 mM quinine)

Mean SEM

CS 24.9 3.5 gustD × 6 38.9 2.5 gustD × ~ 40.1 1.5 gustC 20.3 2.9 GT-I 37.8 2.5

Mutants showing defects in quinine perception were tested for complementation with gustD ×6. The mean and standard error of the mean were calculated from at least 10 readings. All measurements were made in the feeding preference test

behaviora l phenotype of the heterozygote is significantly more severe than that of either of the homozygous mutants suggesting a func t iona l interact ion.

W h e n assayed for tolerance to quinine , gustD ×3, gustD ×6 and G T - I failed to complemen t one ano the r (Table 4c). gustC fully complemented the defect in the gustD alleles, bu t failed to complemen t the G T - I strain.

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Table 5. Meiotic mapping of the gustatory mutants

Genotype Recombinants

ycvv + + + + f

mutant normal mutant normal

gustC 3 17 20 4 gustD × 6 2 11 10 2 GT-] 2 21 30 7

gustC and GT-1 recombinants were assayed in the feeding preference test against 1 mM sucrose, gustD was measured in the feeding preference test against 5 mM quinine hydrochloride

Table 6. Cytological localization of the gustatory mutations using rearrangement chromosomes a) Testing of mutants showing a change in receptor physiology with deficiencies

Genotype Spike frequency/450 ms

Mean SEM N

Df(1)KA6/+ 38.5 1.7 10 gustB × 5/Df(1)KA6 62.5 1.5 20 gustBg~SF/Df(1)KA6 82.2 0.5 40 gustC/Df(1)KA6 67.1 2.1 20 Df(1)m259-4/+ 48.5 1.3 20 gustB × 5/Df(l)m259 -4 62.2 0.8 83 gustC/Df(1)mZ59-4 69.2 1.1 40

Electrophysiological measurements were carried out from the labellar sensillae using 100 mM NaC1 as the stimulus. N is the number of sensilla sampled. Phenotypes are compared with that of the Deficiency/+, mutation/+, and mutation/mutation values (see Ta- ble 2)

b. Location of the mutations with respect to Df(I)KA6

Genotype Percentage Percentage acceptance tolerance ( lmMsucrose) (5mMquinine)

+/Df(1)KA6 80.6 ± 2.9(13) 25.7 ± 4.3(9) gustC/Df(l)KA6 44.8 ± 3.3(10) 40.3 ± 3.0(9) gustD x 6/Dr( I)KA6 - 50.7 ± 5.4(13) GT- 1/Df(I)KA6 44.1 _+2.0(14) 30.1 ± 2.1 (11)

Values are mean ± standard error of the mean (number of tests). Phenotypes are compared with that of the deficiency/+, mutation/ + , and mutation/mutation values (see Table 2)

Our conclus ions f rom the c o m p l e m e n t a t i o n analysis s u p p o r t ou r c lass i f ica t ion based on p h e n o t y p i c cri ter ia . The lesions define two c o m p l e m e n t a t i o n g roups gustB and gustD, with three and two alleles respect ively, gustC and G T - I show complex c o m p l e m e n t a t i o n pa t te rns . The two m u t a t i o n s fail to c o m p l e m e n t one a n o t h e r in all the p a r a d i g m s tested. Howeve r they do no t behave as a single class, gustC fails to c o m p l e m e n t the e lec t rophys i - o logica l p h e n o t y p e o f gustB a l though it fully comple - ments the behav io r a l pheno type . GT-1 , on the o the r h a n d fails to c o m p l e m e n t the quinine to le rance pheno - type o f gustD while gustC does so.

Mapping of gustatory mutations to the IOE1-4 region o f the X chromosome

M a p p i n g crosses were i ndependen t l y car r ied ou t for gustB × 5, gustC, G T - I and gustD × 3. Each o f the mu ta - t ions was m a p p e d with respect to the visible ma rke r s yellow (y, 1-0.0), crossveinless (cv, 1-13.7) vermilion (v, 1-33.0), forked (f, 1-56.7) and carnation (car, 1-62.5). A n analys is o f pu re lines bea r ing single c rossovers allowed us to loca te the m u t a t i o n s in an in terva l be tween v and f . The m u t a t i o n s are closely l inked to v (Table 5), Tha t gustB × s and gustB gusv are loca ted in this in terval

c. Location of mutants with respect to Df(l)m 259-4

Genotype Percentage Percentage acceptance tolerance ( lmMsucrose) (5mMquinine)

+/Df(l)mZ59 4 87.1+3.3(15) 3.5+ 1.1(9) gustC/Df(1)m259 - 4 46.2 ± 3.5(1 I) 41.7 ±_ 3,2(10) gustD × 6 / D f ( 1 ) m 2 5 9 - 4 - 18.1 ± 1,9(9) gustD × 6/+ _ 24.9 ± 3,5(9) GT-I/Df(1)m zsg-'~ 87.0 ± 3.7(12) 4.1 _ 1.2(12) GT-1/+ 64.4 ± 3.9(16) 17.1 ± 2,1(10)

The values of mutation/+ are given in some cases fi'om compari- son. Values are mean+standard error of the mean (number of tests)

d. Location of gustD

Gcnotype Percentage tolerance (5 mM quinine)

Mean SEM N

+ ;y+Yv + ~3 2.3 1.0 10 gustD×6;y+Yv +~3 1.5 1.3 10 gustD × 6/Df(1)N105 23 2.3 12

has been shown previously (Tompkins et al. 1979; Arora et al. 1987).

Cytological localization of these mutations on the salivary gland chromosome map was carried out using strains bearing rearrangements in the region near vermilion (Table 1). The mutations were crossed individually to flies of genotype Deficiency/Balancer. The mutat ion/ Deficiency progeny were tested in behavioral assays and, in some cases, by electrophysiology. The responses of these flies were compared in each case to that of flies bearing the mutat ion in trans- to wild type. Where the deficiency heterozyotes gave mutant responses, the deficiency was inferred to span the region containing the mutation. The breakpoints of the deficiencies used are summarized in Table 1. It must be emphasized that the positions of the breakpoints listed are based on cytological examination. Preliminary molecular analysis of the Df(I )KA6 and Df(1)m 259-4 strains suggests that the two deficiencies do not overlap as indicated by their cytological positions, but the breakpoints map close to one another (W. Chia, R. Jackson, personal communication). This makes the mapping of these mutations within this region only tentative.

The electrophysiological responses of gustB alleles and gustC in trans with deficiencies are given in Tab- le 6 a. These mutations show defective electrophysiological patterns when in trans with either Df(1)KA6 or Df(1)m 259-4. These data map the receptor lesion to the 10El-2 region.

Df(1)KA6 (10El ;11A7) uncovers all the seven mutations discussed in this study (Table 6b). Df(l)m 259-4 (10C~3 ; 10El) also uncovers the "non-per iphera l" phenotypes of gustC. GT-1 and gustD are not included in Df(1)m 259-4 (Table 6c). gustD is covered by the duplication v + e3 (9F4;10E3~4) (Table 6d). GT-1 in combination with Dp(1 :Y)v ÷ e 3 is lethal; this will be discussed later. These data place gustD proximal to gustB and gustC within 10El ;IOE3-4.

The behavioral phenotypes of gustC in trans to deficiency are comparable in severity to that of the homozygous strain (Table 6b, c). These heterozygotes, however, show a more extreme sensitivity of the labellar chemoreceptors to NaC1 (Table 6a). GT-1/Deficiency flies show a less severe defect as compared with GT-1 homozygotes. This suggests that GT-I is an antimorph. The phenotype of gustD × 6/Df(1)KA6 to quinine hydrochloride is significantly more severe than that of the homozygous strain (P<0.01) . Df (1)KA6/+ females also show an increased tolerance to quinine (Table 6d). The response of gustD × 6; y + Yv + ~ 3 animals is comparable to that of +; y+Yv + # 3; this is unexpected since the gustD mutations produce a semi-dominant phenotype. The reason for this discrepancy is unclear.

Lethality of GT-1 in trans combination with duplication V + # 3

During our cytological mapping experiments, we observed that animals of genotype GT-1; y+Yv + 4e3 die (Table 7a). The lethal phase was in the second larval

273

Table 7. a. Genetic interaction of GT-1 with Dp(t ;Y)v + ~ 3

Genotype of Males Females M/F the male parent

CS 3550 4051 0.87 GT-I 106 2567 0.04 GT-l-revertant 1 402 603 0.66 GT-t-revertant 2 860 1000 0.86 GT-l-revertant 3 1011 1120 0.90

The numbers of male and female progeny from a cross with C( 1)DXyf;y + Yv ÷ ~ 3 females are given

b. Gustatory response of the GT-1 revertants

Genotype Percentage response to 1 mM sucrose

Mean SEM N

CS 89.0 t.9 30 GT-I 13.6 2.1 20 Revertant 1 87.5 3.1 10 Revertant 2 93.3 2.2 10 Revertant 3 95.2 3.2 10

instar. The lethality could, in principle arise because of a genetic interaction between the duplication and either the GT-1 mutation, or a second X-linked mutat ion in the strain. To choose between these two possibilities, we designed an experiment to test for segregation between the gustatory and the Dp(1 ;Y)v + ~ 3 induced lethality (GT-1 lethal) in the GT-1 strain.

GT-J males were crossed to females homozygous for the visible markers v and f . The F1 were allowed to mate among themselves and at the F2, 50 recombinants bearing single crossovers in the v-f interval were random- ly selected. These were crossed to C(1)DX y f females to generate pure lines. The male progeny were tested for their gustatory phenotype and also crossed to C(1)DXyf;y+Yv+ e3 females to test for lethality. All 32 lines showing gustatory defects also showed lethality. The 28 normal (gustation-positive) lines were also fully viable in combination with Dp(l ;Y)v + ~ 3

Further evidence that the lethality was, in fact, due to the GT-1 lesion comes from revertant analysis. In order to select suppressors of the lethal phenotype, we mutagenized GT-1 males with EMS. These were crossed to C(1)DXyf;y+Yv +~3 females. Viable males in the progeny were used to set up lines with C(l)DXyf;y+Yv + , 3 females. Three lines were obtained that reverted the lethal phenotype. These lines were ho- mozygosed for their X chromosome and tested for their gustatory phenotype in the feeding preference test. All these mutants showed a total reversion of their taste phenotype (Table 7b). Preliminary genetic analysis of these lines suggests that the revertants are extragenic and recessive (unpublished data).

Interactions between deficiency strains

Heterozygotes of Df(1) KA6 and Df(l) m 2 5 9 - 4 survive and produce an extreme miniature (m) phenotype. The

274

Table 8. Trans interactions between deficiencies Stimulus CS Df(1)KA6 Df(l)m 259-4 Df(1)KA6

+ + Df(1)m 259-'~

i mM sucrose 89.0_+1.9 80.6_+2.9 87.1_+3.3 56.9_+2.8 (30) (13) (15) (13)

100 mM NaC1 43.5_+2.7 41 .2_+2.7 44.4_+2.9 47.0_+2.2 (10) (12) (16) (10)

0.5 M NaC1 8.5_+2.1 10 .1 -+1 .1 11.1-+2.1 37.5-+2.3 (10) (10) (13) (i0)

Proboscis extension 75.3 90.9 83.5 35.7

Spikes/450 ms 46.2-+ 0.2 38.5 _+ 1.7 48.5 + 1.3 85.5 -+ 2.1 (350) (20) (20) (80)

The responses to 1 mM sucrose, 100 mM NaC1 and 0.5 M NaC1 were measured in the feeding preference tests. The means_+ standard error of the mean is given. The number of observations are given in parentheses. The proboscis extension response gives the mean number of flies that give a positive response to 1 mM sucrose of a sample of at least 50 flies. The spike frequency was measured in response to 100 mM NaC1. The total number of spikes were counted in a 450 ms interval beginning 50 ms after the initiation of the response. Mean_standard error of the mean is given. The number of sensilla sampled is given in parentheses

breakpoints of these deficiencies are in the 10EI-2 region. The sodium chloride-induced electrical activity f rom the labellar chemosensory neurons is greatly elevated (Table 8). However, these flies show a normal response to 100 m M NC1, but an altered response to sucrose and 0.5 M NaCL.

Discussion

We have described the phenotype, complementat ion behaviour and genetic localization of a set of seven X- linked gustatory mutations. All these mutat ions lie within a small region of the X chromosome between 10El 4 and affect different taste functions. A detailed analysis of the behavioral and physiological phenotypes of these mutants has led to the classification of the lesions into two operational classes - some affecting peripheral receptor properties and other involved in more central steps in the gustatory pathway. The observation that one of the mutat ions (GT-1) in combinat ion with a duplication of 10A1; 10E3-4 is lethal argues that some transcriptional unit 's within this region are involved in the development of the fly. Our results with trans-heterozygote combinations between mutations, and with mutations and deficiencies, suggest that these gene products interact to results in functional elements of the gustatory pathway. Our genetic analysis suggests the presence of two distinct complementat ion groups within this region: gustB and gustD.

Three independently isolated alleles at gustB all results in a similar phenotype. The increased acceptance of NaC1 is accompanied by an elevated firing frequency of the labellar chemoreceptors. Arora et al. (1987) have shown that the increase in spike frequency f rom gustB chemoreceptors is a result of activation of the sugar (S) neuron in addition to the salt (L1) neuron in response to salt. The mutant S cell must therefore possess acceptor

sites for Na +. The gene gustB presumably acts in the regulation of the distribution of Na + acceptor sites among the chemosensory neurons. The fact that mutations at this locus lead to an increase in the sensitivity of the neurons suggests that this regulation is negative. The idea is consistent with the recessive nature of the peripheral receptor phenotype, gustB gusv is the most extreme allele and shows a stronger phenotype when un- covered by a deficiency for the region.

Both the gustD × 3 and gustD × 6 alleles increase the tolerance of the fly to quinine hydrochloride. This gene maps in 10E1-4 and shows a partially dominant phenotype. The two pleiotropic mutat ions gustC and GT-1 are difficult to classify as members of either gustB or gustD. These lesions fail to complement one another in behavioral paradigms, but there are striking differences in the phenotypes and genetics of the two strains. In addition to the defects in behavioral responses to sugars, salts and quinine, gustC also shows an elevated sensitivity of the labellar chemoreceptors to NaC1. The various properties ofgustC do not appear to be due to a unitary lesion. The elevated salt acceptance and the chemoreceptor phenotypes are fully recessive in nature. In this respect gustC is similar to gustB alleles. All other behavioral phenotypes are partially dominant and in this respect comparable to GT- I . The different phenotypes of gustC could result f rom multiple genetic defects all mapping in the 10E1-2 region. On the other hand, it is possible that the product mutated in gustC plays a role in several elements in the gustatory pathway and is required in different stochiometric amounts at different steps. Fur- ther evidence for this hypothesis comes f rom the fact that gustC fails to complement the elevated spike response in gustB, but fully complements its behavioral response to low salt concentrations.

Mutations that lead to dominant or semi-dominant effects are likely to affect genes that specify complex mult i -component systems. Ribosomes, spliceosomes, re-

275

ceptors and channels are example of structures com- posed of several identical or non-identical subunits. Other types of mult i -component systems include large macro-molecular assemblies, e.g. cytoskeletal elements (Stearns and Bostein 1988; Beall et al. 1989). In both these types of systems, the stoichiometry of the participating molecules is critical and would be expected to result in dose-related phenotypes in mutants (Last et al. 1987; H o m y k and Emerson 1988). There is some evidence for functional interaction between different do- mains of the genetic region in 10E1 4 that specifies taste function. The phenotypes of GT-1/gustC and GT-1/ gustD are more extreme than those of either homozygous gustC or gustD. In combinat ion with Df(1)KA6, GT-1 shows a phenotype that is significantly less severe than that of GT-1 homozygotes, but more severe than that of G T I / + . Duplication of the 10AI ;10E3-4 region in a GT-1 background results in lethality at the second larval instar. All these results suggest that GT- I makes an ant imorphic product. The wild-type product presumably associates with the gene products of linked genes to result in gustatory function and in the development of the organism.

Heterozygotes of Df(1)KA6 and Df( l )m a59-4 show a greatly enhanced firing frequency of the the labellar chemoreceptors in response to NaC1. The behavioral response to salts is, however, unaltered. In addition, these flies show a reduced response to sugars. In our working hypothesis of gustatory coding, we suggest that input f rom the peripheral neurons is "weigh ted" to result in a behavioral response (Siddiqi et al. 1989; Balakrishnan and Rodrigues, in press). Hence in gustB an aberrant response f rom the S neuron to NaC1 results in an increased behavioral acceptance (Arora et al. 1987). The increased tolerance observed in these strains to higher concentrations of salts could be a result of this enhanced acceptance, Flies of genotype gustB/gustC and Df( I )KA6/Df(1)m 259-4 show an elevated firing frequency f rom the chemoreceptors, but no concomitant change in the preference for salts. In our analysis, we have not a t tempted to distinguish the spikes f rom the S and L1 neuron and the firing could, in principle, arise f rom either of these cells. However the lack of an appre- ciable defect in salt perception despite the peripheral alteration, can be explained by assuming that the lesions in these genotypes affect multiple steps in gustatory function. An alternative explanation that behavior is insensitive to changes in spike rate is unlikely since in gustB an increase in firing frequency does result in an altered behavioral response.

In addition to the extreme hypersensitivity of the chemoreceptors to salt, deficiency heterozygotes also show a miniature (m) phenotype. None of the gustatory mutations nor these mutat ions in trans with deficiencies show an m phenotype. The relationship of the gust genes to the miniature-dusky complex is not clear. Various m alleles are known and these map in the 10EI-4 region (Dorn and Burdick 1962). We have examined the gustatory phenotypes of a number of viable alleles of m and found them to show normal gustatory responses (unpublished data). The circadian rhythm mutan t Andante

(And) also maps to this region (R. Jackson, personal communication). Neither And nor And/gustD animals show defects in their gustatory response. Furthermore, gustD has been found to possess normal eclosion rhythms (R. Jackson, unpublished).

We have shown that at least two loci that lie in the 1 0 E 1 ~ interval specify elements of the gustatory pathway in adult Drosophila. The genetic properties of the mutat ions suggest the presence of a complex locus specifying elements of the gustatory pathway. Our data are based on a small number of alleles and clearly require a much more detailed genetic and molecular analysis. The strategies employed for the genetic dissection of other gene complexes are likely to be fruitful in this regard (Garcia-Bellido 1979; Ruiz-Gomez and Modolell 1987).

Acknowledgements. The authors are grateful to Dr. K. VijayRagha- van for his many useful suggestions during the course of this work and his critical comments on the manuscript. We are indebted to Rob Jackson for sharing his unpublished data with us, for his comments on the manuscript as well as for helping us to obtain several of the strains. We also thank Bill Chia for his help in the mutant screens and for informing us of his unpublished data.

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C o m m u n i c a t e d by J.A. Campos-Or tega

closely linked lesions in a region of the x chromosome affect central and peripheral steps in...

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