Copyright 0 1995 by the Genetics Society of America

Hypomorphic Mutations in the hrual photokinesis A (&M) Gene Have Stage-Specific Effects on Visual System Function in Drosophila mehnogaster

Beth Gordesky-Gold, John M. Warrick, Andrea Bixler,' James E. Beasley and Laurie Tompkins

Department of Biology, Temple University, Philadelphia, Pennsylvania 191 22 Manuscript received August 29, 1994

Accepted for publication December 21, 1994

ABSTRACT Of the many genes that are expressed in the visual system of Drosophila melanogasteradults, some affect

larval vision. However, with the exception of one X-linked mutation, no genes that have larval-specific effects on visual system structure or function have previously been reported. We describe the isolation and characterization of two mutant alleles that define the lama1 photokinesis A ( Z p h A ) gene, one allele of which is associated with a P-element insertion at cytogenetic locus 8E1-10. Larvae that express lphA mutations are, like normal animals, negatively photokinetic, but they are less responsive to white light than ZphA+ controls. Larvae that are heterozygous in trans for a mutant lphA allele and a deficiency that uncovers the ZphA locus are blind, which indicates that the mutant allele is hypomorphic. lphA larvae respond normally to odorants and taste stimuli. Moreover, the lphA mutations do not affect adult flies' fast phototaxis or visually driven aspects of male sexual behavior, and electroretinograms recorded from the compound eyes of ZphA/deficiency heterozygotes and lphA '/ lphAz females are normal. These observations suggest that the lphA gene affects a larval-specific aspect of visual system function.

A fundamental question of biology concerns the na- ture of genes that determine the manner in which

systems develop and function. In holometabolous in- sects, in which many larval structures are replaced by their adult counterparts during metamorphosis, this question is of particular interest, since one can ask about the extent to which larval and adult homologues are regulated by the same genes. The visual systems of Drosophila melanogaster larvae and adult flies are ideal subjects for such analysis, since hundreds of mutations that affect the adult visual system have been identified ( e .g . , PAK 1979; HALL 1982), as have dozens of DNA sequences that are expressed there ( i . e . , SHIEH et al. 1989; HYDE et al. 1990). With regard to the question of whether these genes are also expressed in the larval visual system or affect its function, three mutations that affect various aspects of phototransduction in adult flies' eyes perturb larval responses to light, while five mutations that regulate other aspects of visual system development and function in adult flies have no detect- able effects on larval vision ( HOTTA and KENC 1984). Three of the four opsin genes that are expressed in the adult visual system are also expressed in the larval photoreceptor organ (POLLACK and BENZER 1988), as is the chaoptic gene, which codes for an adhesion pro- tein that is required for normal development of the rhabdomeres in the compound eye (POLLACK et al. 1990). Finally, mutations in the disconnected and imegu-

Corresponding author: Laurie Tompkins, Department of Biology

' Present address: Department of Biology, University of Tennessee, 015-00, Temple University, Philadelphia, PA 19122.

Knoxville, TN 3799600810.

Genetics 139: 1623-1629 (April, 1995)

lar chiasm C genes, isolated because they affect axon projections in the optic lobes of adults, exert their pri- mary effects on pioneering neurons in the larval visual system ( STELLER et al. 1987; BOSCHERT et al. 1990).

On the basis of these observations, genes that affect vision can be classified in three groups, distinguished by their developmental specificity: genes that are ex- pressed in the visual systems of larvae and adults; genes that are expressed in the larval visual system and, as an indirect consequence of their effects on larvae, also affect the adult visual system; and genes that are only expressed in the adult visual system. It would be reason- able to assume the existence of a fourth class of genes, those that exclusively affect the larval visual system. However, except for an unmapped X-linked mutation ( lnp) that affected larval responses to light without per- turbing adult flies' behavior in a fast phototaxis assay ( HOTTA and KENG 1984), which may no longer be extant (see below), there is no evidence for this class. Accordingly, we have begun to isolate and characterize mutations that affect larval vision. This paper describes the first fruits of our labor: two serendipitously recov- ered mutations that define the @hA ( lama1 photokinesis A ) gene, which appears to have a larval-specific effect on visual system function.

MATERIALS AND METHODS

Stocks: The wild-type Canton-S stock, which is M cytotype ( BINCHAM et al. 1982) , was derived from a male-female pair in 1976 and has since been maintained in mass culture (TOMPKINS et al. 1980). P cytotype Harwich ( JSIDWELL et al. 1977), attached-X ( C(l)DX, y f / F M 7 ( P ) y snx2 B ) ( ENGELS 1985), and FM7a balancer stocks were obtained from J. CARL-

1624 B. Gordesky-Gold et al.

SON. Two retinal degeneration A stocks (rdgA’ and rdgA3) were provided by W. STARK. cinnamon ( cin’) flies were collected from a stock obtained from M. WOLFNER. A stock designated as larval nonphototactic ( lnp) ( HOTTA and KENC 1984) was provided by Y. HOTTA. The Mid-American Droso hila Stock Center supplied three stocks: yellow ( y ) cut6 ( ct ) vermilion ( v ) forked ( f ) ; ocelliless ( o c ’ ) cut ( ct6)/FM6and M cytotype FM7a. A Of (1) C52/FM6 stock was obtained from the Dro- sophila Stock Center at Indiana University. Unless otherwise noted, all mutant alleles and special chromosomes are de- scribed in LINDSLEY and ZIMM (1992).

All stocks were maintained on a cornmeal-corn syrup me- dium at 22 or 25”.

Mutagenesis: The lphA mutations are the products of a hybrid dysgenesis cross, that is, mobilization of paternally de- rived Pelements in the germline of the progeny of P cytotype males and M cytotype females ( ENCELS 1983). To initiate the mutagenesis, Harwich males were mated to Cantons females. The F, male progeny of this cross, whose X chromosomes were inherited from their Canton-S mothers, were then mated to C(l)DX, y f / YP cytotype females to stabilize any Pelement insertions that had occurred and ensure that mutagenized X chromosomes would be inherited patroclinously. Since we were also interested in mutations that affected larval taste, the F2 larvae were screened in a taste assay ( GORDESKY-GOLD et al. 1988) to see whether they responded normally to 1 M NaCl, which repels wild-type larvae. Larvae that responded mistactically in two consecutive tests were transferred to vials to complete development and then sexed. Each male fly was individually mated to P cytotype FM7a females to establish, after two generations, a line in which all of the flies were homozygous or hemizygous for a mutagenized X chromo- some. Larvae from each line were then tested in the GORD- ESKY-GOLD et al. taste assay.

Since larvae from two of the lines responded abnormally to 1 M NaCl when the lines were first tested, those lines were retained for further analysis. In subsequent tests, larvae from the two lines behaved normally when they were tested with 1 M NaCl, but their responses to light were consistently abnor- mal in comparison with those of control larvae with unmuta- genized Canton-S chromosomes. Accordingly, the lines were retained for further analysis of their visual phenotypes.

Genetic analysis: Since the lphA mutations are X-linked (see below) , it was necessary to analyze the behavior of female larvae to assess the mutations’ dominance and their behavior in complementation tests. This analysis was facilitated by con- structing X chromosomes bearing the lphA mutations and a mutant cinnamon allele ( cin’) by meiotic recombination. For complementation tests and determination of dominance, fe- males homozygous for these chromosomes were crossed to cin+ males, generating cin’/ cin+ females and c i n ’ / Y males. Since homozygous or hemizygous cin’ progeny of cin’/ cin’ mothers die as embryos (BAKER 1973), this cross yields all- female populations of larvae that are heterozygous or homozy- gous for a l@hA mutation, depending on the genotype of the male parent with respect to the lphA gene.

To map the lphA gene by meiotic recombination, @ h A 2

females were crossed toy c t6 v fmales, and F2 males represent- ing single recombinant classes (e .g . , y+ ct6 v f ) were isolated. The males were mated to females from an FM7a stock ( P cytotype ) to generate stocks in which all of the flies were hemizygous or homozygous for the recombinant chromo- somes. Larvae from each stock were then tested in the photo- kinesis assay (see below) .

To correlate excision of a P element in the lphA ’ stock with reversion of the mutant phenotype, we isolated two &hA+ re- vertant lines. To do this, lphA’/Ymaleswere crossed to C(l)DX, y f / Y females from an M cytotype stock to initiate hybrid

B

dysgenesis ( ENCELS 1983). After eight generations, -20 indi- vidual males were crossed to females from a P cytotype FM7a stock to generate, in two generations, stocks in which all flies were homozygous or hemizygous for the mutagenized Xchro- mosome that was derived from the lphA’/Ymale progenitor. Larvae from each stock were then tested in the photokinesis assay. Two lines whose larvae responded normally to light were classified as revertant and retained for further analysis.

To confirm the map osition of the lphA gene and charac- terize the mutant lphA allele, we analyzed the behavior of female larvae that were heterozygous in trans for the @ h A Z

allele and Of (1) C52. Since the deletion is embryonic lethal when homozygous, generation of lphAz/ Of (1) C52 larvae was facilitated by construction of a chromosome bearing the @hA’ allele and a mutant allele of shihre (shi‘”’) , which has heat- sensitive effects on larval and adult mobility ( POODRY et al. 1973), by meiotic recombination. To generate &Az/ Of (1) C52 larvae, shi‘”/ Y males were mated to Of ( 1 ) C52/ FM6 females, and the Df (1) C52/ shifs’ female progeny were crossed to lphAz shi”’/ Ymales. The progeny of this cross were reared at 22“, a temperature at which larvae that express the shi*’ mutation are mobile (POODRY et al. 1973). Since the response of wild-type larvae to light is temperature sensitive (J. M. WARRICK, D. P. KUTZLER and L. TOMPKINS, unpublished data) , the progeny of this cross were tested in the larval photo- kinesis assay at 22”, after which the assay plates were trans- ferred to 29” for 8- 10 min to paralyze the shi‘”’/ Y and shi‘”’/ lphA2 shi‘”’ larvae ( POODRY et al. 1973; L. TOMPKINS, unpub- lished data). Immediately after the 29” temperature shift, the mobile larvae, which were Df (1) C52/ lphA2 shi””, were observed to determine their distribution on the assay plate. To confirm that the deficiency-bearing chromosome did not have dominant effects on larval photokinesis, Df (1) C52/FM6 females were crossed to shi“’/Y males, and the Of (1) C52/ shit”’ female progeny were crossed to shi“”/Y males. Progeny of this cross were raised at 22”, tested in the larval photokinesis assay at 22” and then shifted to 29” as described above to paralyze the shifS1/ shi”’ and shi“”/ Y larvae. The Of (1) C52/ shi”’ larvae, which were not paralyzed, were immediately ob- served to determine their distribution on the assay plates.

Chromosome in situ hybridization: A pUCPT probe was biotinylated as specified in the protocol accompanying the nick translation kit (Enzo Biochemicals) , utilizing bio-16 dUTP as the biotinylated nucleotide. The biotinylated probe was hybridized to salivary gland polytene chromosomes, utiliz- ing a standard protocol (ASHBURNER 1989) with, in some cases, the following modifications: the chromosomes were pretreated with I X PBS rather than 2X SSC, they were not acetylated, and the predenaturing dehydration step was omit- ted. Peroxidase staining was performed according to the pro- tocol accompanying the Detek HRP kit (Enzo Biochemicals), utilizing a modified AEGsubstrate mixture ( 7 ml water, 1 ml of 8X reaction buffer, 160 pl of 0.5 M EDTA, and 80 p1 of AEC) . Chromosomes were counterstained with Giemsa.

Behavioral assays: The larval photokinesis assay has been described ( LILLY and CARLsON 1990). Briefly, two opposing quadrants of a partitioned X-Plate Petri dish (Falcon 1009) are filled with clear agarose, while the remaining quadrants are filled with an agarose solution that is dyed black with commercial food coloring. Approximately 100 larvae are transferred to the center of the dish, which is then placed on a light box to illuminate it from below. After 5 min, a stimulus index, which we define as the percent of larvae on the two quadrants that are filled with clear agarose, is calculated. In this assay, the larvae are responding to light, rather than a chemical stimulus associated with the food coloring in the black agarose, since wild-type (Canton$) larvae distribute themselves randomly on the black- and clear-agarose-filled

F

Mutations Affecting Larval Vision 1625

quadrants if they are tested on photokinesis assay plates in the dark (mean 5 SEM stimulus index = 50 5 2; n = 4 ) . Evidence that the response of wild-type larvae is a photokine- sis ( a change in behavior effected by light) rather than a negative phototaxis ( a directed movement away from light) is provided by the observation that individually tested Canton- S larvae, transferred to the center of a clear-agarose-filled quadrant, move quickly in the direction in which they were oriented when they were placed on the agarose; if they reach the edge of the Petri dish, they move rapidly along the periph- ery. Once they have crossed into a black-agarose-filled quad- rant, the larvae immediately begin to move more slowly, de- scribing randomly oriented circles. Thus, light changes the rate at which wild-type larvae move and their pattern of move- ment but does not elicit directed movement away from the stimulus.

The LILLY and CARL~ON larval taste assay ( LILLY and CARL- SON 1990) is identical to the photokinesis assay except that 0.025 M or 1 M NaCl is substituted for the food coloring in one of the agarose solutions. For the taste assay, we define a stimulus index as the percent of larvae on the two quadrants that are filled with the agarose-NaCl solution.

In the GORDESKY-GOLD et al. assay for larval taste ( GORD ESKY-GOLD et al. 1988), -100 larvae are transferred to the interface between two 21.5 X 7 cm blocks of 0.4% agar, one containing 1 M NaCI. After 30 min, a stimulus index, which is the percent of larvae in the stimulus-containing agar block, is determined.

The larval olfaction assay is described in MONTE et al. (1989). A filter paper disc on one side of a Petri dish is saturated with 25 p1 of an odorant solution, diluted with dis tilled water, and a disc on the other side of the dish is satu- rated with 25 pl of distilled water. Approximately 100 larvae are transferred to the center of the dish. After 5 min, a stimu- lus index, which we define as the percent of larvae on the half of the dish with the odorant-saturated filter paper, is calculated.

Adult flies' fast walking phototactic behavior was assayed in a Y-maze ( QUINN et al. 1974), in which one arm is illuminated with white fluorescent light. Flies are shaken into a start tube and then agitated; after 30 sec, the flies in the illuminated arm, the dark arm, and the start tube and body of the appara- tus are counted.

Adult flies' sexual behavior was assayed by transferring a virgin male and female to a clear plastic cylindrical observa- tion chamber (volume ca. 0.2 cm') and then observing the flies under 7 X magnification for 10 min. At the end of the observation period, the male's courtship index (the percent of time spent by the male performing any courtship behav- iors) (TOMPKINS et al. 1980) is calculated. If the flies mate during the observation period, this is noted, and a courtship index is calculated for the time elapsing before copulation ensues. Flies that do not mate during the 10-min observation period are observed for an additional 20 min; if they mate during the second observation period, this is noted.

Electroretinograms: Electroretinograms were recorded from the compound eyes of adult flies as described in WOOD- ARD et al. (1989).

Statistical analysis: The significance of differences between means was determined by calculating t (Student's t test, two- tailed) after arcsin transformation of the data, if appropriate. The significance of differences in the distribution of individu- als into classes was determined by calculating contingency chi square with Yates correction factor. The significance of deviations from a 1:l ratio of the distribution of individuals into classes was determined by calculating chi square. Stat- graphics statistical software was used for all statistical analyses.

TABLE 1

Dominance and complementation of ZphA mutations' effects on larval photokinesis

Genotype Stimulus index n

Canton-S 14 5 2 25 lphA' 33 t 3" 10 lphA2 37 +- 3" 11 lphA'/ + 9 t 26 6 lphA2/ + 19 5 36 4 lphA'/lphA2 32 +- 3" 6 rdgA3 34 5 2" 8 rdgA' 47 5 3"s' 8 lphA2/rdgA' 8 5 2' 6

lphA2/Df(l)C52 52 5 2".' 5 Df(l)C52/+ 12 5 26 4

Larval photokinesis was assa ed as described in the text. For the Canton-S, lphA', lphA[ rdgA3 and rdgA' genotypes, male and female larvae were assayed (larvae from the 02 stock were not tested in the photokinesis assay, since the stock was maintained with a balancer chromosome because oc'/oc' fe- males are sterile). For the &A'/+, lphAz/+, lphAz/rdgA', and lphA2/oc' genotypes, only female larvae were assayed; the lphA chromosomes were n'n' lphA (the n'n mutation does not have a dominant effect on larval photokinesis; SI = 22 5 1 for larvae heterozygous in trans for n'n' and Canton-S chromo- somes, n = 3 ) . For the lphA2/Df(l)C52and Df(l)C52/+ geno- types, only female larvae were assayed; the l p h A 2 chromosome was @hA2 v shi'"' and the "+" chromosome was shi'"' (see text for details). Mean stimulus indices, +- SEM, are shown, as are the number of replicates for each genotype (n).

"Values are significantly different from Canton-S controls ( t tests; P < 0.01 for all pairwise comparisons).

'Values are not significantly different from Canton-S con- trols ( t tests; P > 0.05 for all pairwise comparisons).

For each of the replicates, the distribution of larvae in the illuminated and dark quadrants was not significantly different from a 1:l ratio (chi square tests; P > 0.05).

l p h A 2 / O c ' 13 t 2b 5

RESULTS

Genetic characterization of the lphA gene: After screening ca. 1700 mutagenized X chromosomes, we recovered two lines (initially designated as 1 and 2 ) , each the product of a different mutagenesis cross, in which larvae responded abnormally to light. Like wild- type Canton-S controls, obtained from the stock from which the Xchromosomes in lines 1 and 2 were derived, the mutant larvae were negatively photokinetic, but their stimulus indices were significantly higher than controls (Table 1 ) . Observations of individual larvae from line 2 revealed that the larvae, unlike Canton-S controls, circled slowly on illuminated agarose, sug- gesting that the larvae did not respond normally to light.

Analysis of dominance revealed that female larvae heterozygous for a Canton-S X chromosome and an X chromosome from either line 1 or line 2 behaved like Canton-S larvae in the photokinesis assay (Table 1 ) , which indicated that the photokinetic mutations were recessive. Accordingly, we observed female larvae that

1626 B. Gordesky-Gold et al.

were heterozygous in trans for X chromosomes from lines 1 and 2. The trans heterozygotes responded to light like larvae from lines 1 and 2 (Table 1 ) . On the basis of these observations, we concluded that the muta- tions in lines 1 and 2 that affected larval responses to light were in the same gene.

To map the gene to an interval on the X chromo- some, we crossed y c t6 u f males to females from line 2, isolated recombinant F2 males, and generated homo- zygous stocks from the progeny of these males. We then assayed larvae from each recombinant class to see whether they behaved normally in the photokinesis assay in comparison to wild-type controls that expressed the same visible markers. Larvae from all eight of the recombinant stocks that were ct6 and u behaved nor- mally, regardless of their genotype with respect to the yellow and forked markers; conversely, larvae from all eight of the recombinant stocks that were ct+ and v+ behaved abnormally. On the basis of these observations, we concluded that the larval photokinesis gene mapped between the visible markers cut (map position 1-20.0; cytogenetic map position 7B3-4) and vermilion (map position 1-33.0, cytological location 1OA1-2) on the X chromosome.

The ocelliless and retinal degeneration A genes, which affect adult flies' vision (HARRIS and STARK 1977; LIND- SLEY and ZIMM 1992), had already been mapped to the cut-vermilion interval. Thus, it was of interest to deter- mine whether we had isolated new mutant alleles of either of those genes. As expected, since one rdgA allele affects larval responses to light in a different assay for visual behavior ( H O ~ A and KENG 1984), larvae that were homozygous or hemizygous for the extreme rdgA' allele or the less extreme rdgA3 allele responded abnor- mally to light in our assay, the extreme allele rendering the mutant animals atactic (Table 1 ) . In contrast, fe- male larvae that were heterozygous in trans for an X chromosome from line 2 and an Xchromosome from the rdgA' stock or the oc' stock responded normally to light (Table 1 ) , suggesting that the noncomplement- ing photokinetic mutations in lines 1 and 2 were not alleles of the retinal degeneration A or ocelliless genes. However, we were not able to ascertain whether the mutations in the two stocks complemented the X-linked lnp mutation isolated by HOTTA and KENC (1984), since the larvae from a stock designated as lnp were strongly repelled by light in our photokinesis assay (SI = 18 2 4; n = 4 ) .

Accordingly, we confirmed that the mutation in line 2 mapped within the cut-vermilion interval by analyzing the photokinetic behavior of larvae from 63 recombi- nant stocks, derived from F2 males in which the recom- bination event had occurred between cut and vermilion. As expected, the distribution of SIs was bimodal. Twelve of the 32 ct6 vOf stocks were classified as wild type since their mean stimulus indices were 525 (>95% of the stimulus indices for CS larvae are s 2 5 ) , while 20 were

classified as mutant because their mean stimulus indices were >25 (>95% of the stimulus indices for line 2 larvae were higher than 25) . Using the same criteria, 21 of the 31 ct' ZI stocks were classified as wild type, while 10 of the stocks were classified as mutant. The results of this analysis were consistent with the hypothe- sis that a single mutation at map position 29-30 is re- sponsible for the photokinetic defects exhibited by lar- vae from line 2.

In an attempt to localize the mutations in lines 1 and 2 more precisely, we hybridized P-element DNA to polytene chromosomes from line 1 and line 2 larvae. As expected, since the two lines were the products of hybrid dysgenesis mutageneses, both of the lines had multiple P-element insertions. In line 1, in situ hybrid- ization revealed the presence of a Pelement in polytene chromosome bands 8E1- 10 (Figure 1 ) , although no hybridization to bands in the cut-vermilion interval could be detected in polytene chromosomes from line 2 lar- vae. Accordingly, we utilized hybrid dysgenesis to gener- ate two revertant lines from line 1 and then hybridized P-element DNA to polytene chromosomes from the two revertants. No P-element insertions were visible in the vicinity of polytene chromosome bands 8E1-10 in ei- ther revertant line. The location of the P-element inser- tion at polytene chromosome bands 8E1-10 in larvae from line 1 is consistent with the meiotic map position of the noncomplementing mutation in line 2 ( LINDSLEY and ZIMM 1992). Thus, the mutations in lines 1 and 2 define the larual photokinesis A ( 2phA) gene. We have designated the mutant alleles in lines 1 and 2 as lphA ' and IphA', respectively.

Finally, we assessed the nature of the @hA2 mutation by observing larvae that were heterozygous in trans for an X chromosome from the 2phA' stock and a defi- ciency-bearing Xchromosome. For this analysis, we uti- lized the Of ( I ) C52 deficiency, in which polytene chro- mosome bands 8E through 9C-D are deleted ( LINDSLEY and ZIMM 1992), since it is the only extant deficiency in which chromosomal material in the vicinity of the 2phA locus is deleted. As shown in Table 1, 2phA2/ Of ( I ) C52 larvae are blind in the photokinesis assay. Since the deficiency-bearing chromosome does not have a dominant effect on larval photokinesis (Table 1 ) , we interpret these results to indicate that 2phA' is a hypomorphic allele. This analysis also suggests that IphA' is a hypomorphic allele, since the phenotypes of @hA' larvae, @hA2 larvae, and those that are heterozy- gous in trans for the &hA' and &hA2 alleles are indistin- guishable.

Behavioral and physiological characterization of lphA mutations: Since the lphA mutations affected larval photokinesis, it was of interest to determine whether the mutations had any detectable effects on adult vision. Accordingly, we observed lphA' and lphA2 adults to see whether they responded normally to light in a photo- taxis assay. IphA' and lphA2 flies responded normally to

Mutations Affecting Larval Vision 1627

light in this assay (Table 2) , which implied that at least one aspect of adult visual system function was unaf- fected by the lphA mutations. In addition, we observed lphA ‘/ Y and lphA2/ Y males with Cantons females to assess the mutant flies’ courtship. Unlike blind males and those with impaired visual acuity (TOMPKINS 1984; TOMPKINS et al. 1982), the 1phA ‘ and lphA2 males per- formed vigorous courtship in response to wild-type fe- males and copulated quickly (Table 3 ) . Moreover, the mutant males routinely oriented their bodies to face the females’ abdomens; if the females circled the obser- vation chamber, the males pivoted to maintain this pos- ture. These behaviors, collectively referred to as orienta- tion (TOMPKINS 1984), are rarely performed by blind

TABLE 2

Effects of ZphA mutations on adult flies’ fast walking phototaxis

Percent leaving Percent of flies leaving Genotype start tube that responded to light

Cantons 69 t 2 99.7 2 0.2 IphA 66 2 5 99.8 2 0.1 lphA2 72 2 4 99.6 2 0.3

Adult flies’ phototaxis was assayed as described in the text. The percent of flies leaving the start tube is the percent of flies found in the dark and illuminated arms of the apparatus, rather than the start tube or the body of the apparatus, at the end of the experiment. The percent of flies leaving that responded to light is calculated by dividing the number of flies in the illuminated arm by the number of flies in the dark and illuminated arms, multiplied by 100. For each parameter, means for n = 10 replicates, 2 SE, are shown. Neither &/Af larvae nor lphA2 larvae were significantly different from Can- t o n s controls with regard to either parameter ( 1 tests; P > 0.05 for all comparisons).

FIGURE 1.-Hybridization of a bi- otinylated pUCPT probe to salivaly gland polytene chromosomes from 1phA’ larvae. The insert at 8E1-10 in the lphA ’ line, which is not pres- ent in either of the two revertant lines, is designated with an arrow.

males and mutant omh”” males ( TOMPKINS et al. 1982), which perform less optomotor turning behavior than males with normal vision ( HEISENBERG et al. 1978).

In addition to the various behavioral assays, we as- sessed the effect of the lphA mutations on the adult visual system by recording electroretinograms (ERGs) from the compound eyes of CS controls, lphA I / lphA I

females, lphA 2 / lphA2 females, lphA I / lphA females, and females that were heterozygous in trans for 1phA’ or lphA2 Xchromosomes and the Of (1) C52 deficiency. Three of the 14 lphA I / lphA I females from which ERGs were recorded had photoreceptor potentials whose am-

TABLE 3

Effects of ZphA mutations on male flies’ sexual behavior

Fraction Fraction Genotype copulating copulating of male CI (10 min) (30 min)

Canton-S 78 2 3 17/20 18/20

LphA’ 82 t 2 19/20 20/20 1phA ’ 78 2 4 12/20 17/20

Males were observed with Canton-S virgin females as de- scribed in the text. n = 20 pairs of flies for all genotypes. Mean courtship indices (CI) for the first 10-min observation period, 2 SE, are shown. The fraction copulating in 10 min includes males that began to mate during the initial 10-min observation period; the fraction copulating in 30 min includes males that began to mate during the initial observation period and those that began to mate during the second (20-min) observation period, if one was required. &hA‘ males nor IphA’ males were not significantly different from Canton-S controls with regard to their courtship indices ( t tests; P > 0.05 for both comparisons), fraction copulating in 10 min (contin- gency chi-square tests, P > 0.05 for both comparisons), or fraction copulating in 30 min (contingency chi-square tests; P > 0.05 for both comparisons).

1628 B. Gordesky-Gold PI 01.

FK;IXI;. ‘L.-~Elcctrorcrino~ra~~~s rrcortletl L’ro~n t h r com- pound eyes of C S ( A ) , / / h \ ’/ //)/!A ’ ( B) , / / 1 h A 2 / I / d r A ’ ( C ) , / p h A ’/ IphA’ (1)) , 1/1/r,\ ‘ / D f ( I ) (152 ( E ) , and //I/LA’/ l?f( I) (32 ( F ) females in response t o 500-mscc light pulses. EKGs were recortletl from at least six flies of each genotype; except for those recorded from / / h \ ’/ //drA ’ females (see text), EKGs from flies o f the same genotype were similar. An ERG from a //h\ ’/ 1/1/rA’ female whose photoreceptor potentials were o f normal amplitude, as was the case for the majority o f flies o f this genotype, is shown.

plitudes were lower than those of controls (in one fly, the ERG defect was intermittent). However, ERGS re- corded from the other 11 1pILA ’/ I/)hA ’ females and all of the 1/)hA2/ 1pAA2, I j , h A ’ / l f ~ k A ’ , l j h A ’ / D f ( I ) C52, and lj)hA’/Df (I) C52 females that were subjected to physiological analysis were similar to those of CS con- trols with regard to the amplitude and shape of the on-transients, the photoreceptor potential, and the off- transients (Figure 2 ) . Accordingly, we conclude that the l+hA mutations p r SF do not affect ERGS recorded from adult flies’ compound eyes.

Finally, to assess the specificity of the 1phA mutations’ effects on larval behavior, we observed IphA I and lplzA2 larvae to see whether they responded normally to nonvi- sual stimuli. In comparison to Cantons larvae, 1phA’ and 1,!)hA2 larvae responded normally to two olfactory stimuli, propionic acid and ethyl acetate; they also be- haved normally when they were tested with 0.025 M and 1 M NaCI in the LIL.I.Y-CARI.SON taste assay (Table 4 ) . In addition, as previously noted, 1pIzA’ and 1phA’ larvae responded normally to 1 M NaCl in the GORD- ESKY-GOI.1) taste assay (Table 2 ) .

DISCUSSION

We have isolated mutant alleles of the @A gene, one of which has been shown to be hypomorphic while the

TABLE 4

Effects of ZphA gene mutations on larval responses to olfactory and gustatory stimuli

Genotype

Stimulus Cantons / jhA’ //I/LA’

Ethyl acetate

Ethyl acetate

Propionic acid

0.025 M NaCl

1 M NaCl

1 M NaCl

( 1 :10 dilution) 7 6 2 2 7 7 2 1 8 3 2 1

(1:IO” dilution) 9 3 2 1 9 1 2 1 8 5 2 1

(1:10 dilution) 9.5 2 1 97 2 1 98 2 1

(LII.I.Y-CARISON assay) 5 5 2 2 5 8 t 4 5 9 5 2

(LII.I.Y-CARISON assay) 7 2 1 9 2 2 8 2 1

(GORIXSKY-GCXD assay) I3 2 2 21 2 3 23 2 4

Larval olfaction and taste were assayed as described in the text. Mean stimulus indices, 2 SE, are shown. n = 8-24 repli- cates for each genotype and stimulus. Neither ZphA’ larvae nor 1/1hA’ larvae were significantly different from Canton-S controls in response to any of the stimuli ( t tests; 1’ > 0.03 for all comparisons).

other, by virtue of its identical phenotype, is likely to be. The 1phA mutations appear to have larval-specific effects on visual system function. As noted above, elec- troretinograms recorded from the compound eves of lphA 2 / D f (I) C52 females, which are blind as larvae, are normal, as are electroretinograms recorded from the eyes of lphA2/ lphA2, lpltA’/ 1phA2, and lphA’/ Df ( I ) C52 females. Moreover, the ability of 1phA I and l/dzA2 adults to run toward light is not perturbed by the mutations. Finally, males that are hemizygous for @hA I

or lplzA2 alleles, like l{)hA+ males, perform vigorous courtship, orient toward females, and copulate quickly.

We have also shown that the 1phA‘ and IphA2 muta- tions do not affect larval responses to olfactory or taste stimuli. These observations suggest that the 1phA muta- tions affect the larval visual system specifically rather than having generalized effects on the larval nervous system o r musculature that would impair the animals’ ability to move quickly in response to light.

Since larvae from the stock designated as lnp were strongly repelled by light in the photokinesis assay that we employed, we were unable to do complementation tests to determine whether the 1phA mutations define a new gene with larval-specific effects on visual system function or are new alleles of the lnp gene, which was defined by an allele that may no longer be extant. With regard to the question of whether genes with larval- specific effects on vision are rare or common in the Drosophila genome, we recovered only two 1phA alleles in our mutant screen, which might be interpreted to suggest that mutations with larval-specific effects on vi- sion are relatively rare. However, it should be noted that we obtained the 1pIzA alleles by screening fewer

Mutations Affecting Larval Vision 1629

than 2000 mutagenized male larvae; moreover, our screening protocol was designed to detect only X-linked mutations that affected larval behavior. More impor- tantly, the mutagenized larvae were initially screened for gustatory abnormalities rather than visual system defects; hence, any mutagenized chromosome that af- fected larval vision without having at least a transitory effect on the animals' responses to 1 M NaCl in the GORDESKY-GOLD taste assay would have been discarded. Accordingly, we are currently attempting to identify genes with larval-specific effects on vision by screening directly for X-linked and autosomal mutations that af- fect larval photokinesis.

We are grateful to JOHN CARISON for encouragement, providing Drosophila stocks, teaching us how to do many of the larval assays, and providing facilities and equipment for recording ERGS. We also thank JUAN Rr~sc; t rEsc:ov~~ for recording some of the ERGS and assisting us with the other recordings; BILL STARK, MARIANA WOLFNER, and YOSHIKI HOTTA for stocks; KAREN PALTER for the pUCPT probe; LARRY YAGER for photography; and DAN LINDSLEY for comments on an earlier draft of the manuscript and confirmation of our suspicions that Df ( 1 ) C52 was the only deficiency that uncovered the lphA locus. This work was supported by a grant from the National Science Foun- dation (BNS865554) , awarded to L.T. A.B. was supported by a Na- tional Science Foundation Undergraduate Research Program grant, awarded to Temple University.

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Communicating editor: T. ScHiTmActI