Copyright 0 1987 by the Genetics Society of America

The Regulation of in Imaginal Wing Discs of Drosophila Hybrids: Evidence for cis- and trans-Acting Control Elements

Th. E. Sprey*" and David T. Kuhnt *Zoological Laboratory, University of Leiden, 2300 RA Leiden, The Netherlands, and TDepartment of Biological Sciences, University of Central Florida, Orlando, Florida 32816 Manuscript received March 21, 1986 Revised copy accepted October 23, 1986

ABSTRACT The aldehyde oxidase (Aldox) distribution pattern was determined for wing discs of partial hybrids between D. melanogaster and D. simulans. In these animals the regulation of Aldox activity is not uniform over the disc epithelium as both cis-dominant and trans-acting control were evident in different regions of the disc. The Aldox expression was shown to be regulated by loci on the X chromosome, 2L and 3R of D. melanogaster and 2R and 3R of D. simulans.

PATIOTEMPORAL patterns of specific gene RIGHT 1967; GLASSMANet al. 1968). In hybrids of me1 S products are basic features of multicellular orga- and sim the dimeric consists of a mixture of nisms. An example of such a pattern has been de- the homo-dimers of each parental species and the scribed for imaginal discs of Diptera (SPREY,1977; hetero-dimer (COURTRIGHT1967). A similar conclu- SPREY,ESKENS and KUHN, 1982). The pattern consists sion was reached by DICKINSON(1980a) for hybrids of a characteristic aldehyde oxidase (AO) distribution, between D. dijjferens and D.heteroneura. which is extensive in the wing discs of Drosophila Hybrids between me1 and sim show an intermediate melanogaster and Musca domestica. The A0 pattern wing disc pattern (SPREY,ESKENS and KUHN, 1982). provides an interesting model system for studies of A0 distribution suggested a cis- and trans-regulation genetic regulation in the imaginal disc cells during of pattern elements. To provide further evidence for larval development. these types of regulatory elements, we chose the ele- Several studies have revealed the regulation of this gant technique of "partial hybrids," devised by enzyme in different tissues of diverse Drosophila spe- MULLERand PONTECORVO(1940) and first used by cies. Like some other in D. melanogaster GRELL(1976) to locate the structural genes for aspar- (BEWLEY1981; CHOVNICKet al. 1976; THOMPSON, tate amino . GRELLproduced partial hy- ASHBURNERand WOODRUFF1977) a cis-acting regu- brids in which one chromosome or chromosome arm latory unit exists adjacent to the structural Aldox gene was homozygous for either me1 or sim genes in an (DICKINSON,1975, 1980a). Both cis- and trans-regu- otherwise hybrid background. We used this method latory control occur in different tissues of hybrids to systematically produce hybrids homozygous for me1 between closely related Hawaiian Drosophila species or sim chromosomal arms in order to survey the entire (DICKINSONand CARSON1979). genome for Aldox (pattern) regulating genes. Temporal regulation of enzymes is thought to be The objective of this study was to identify elements bipartite in that both cis-regulatory sites located close that regulate the A0pattern in the pouch of the wing to the structural gene and distant sites that exert a disc and to determine their mode of regulation. trans-regulation may be involved (PAIGEN1979). New insights into the regulation of patterns can be derived MATERIALS AND METHODS from studies of the A0 distribution in imaginal discs Drosophila stocks: The following stocks were kindly pro- of hybrids between D. melanogaster (mel) and D. si- vided by Dr. E. H. GRELL:D. melanogaster: (1) y; C(ZL),It; mulans (sim), since A0 pattern differences are found F(2R),bw; (2) y; F(2L), dp; C(2R) cn; (3) y; C(3L) ri; F(3R) for the wing disc (SPREY1977). In addition to the ca (modified from GRELL'Sstock); (4) y; F(3L), h2; C(3R)sr; species-specific differences, a second requirement for (5)y ac v/y2; bw/+; sim-4/y+ eyR (a me1 strain with one sim a model system is for enzyme coding for both species fourth chromosome) (6) y; Df(3R) ~bd"~/Xa,deficient for the Aldox structural gene and presumably regulatory ele- to be by the same structural gene. In me1 and sim the ment close to it (DICKINSON1975, 1978). The amount of A0 pattern depends on the Aldox structural gene A0 activity in tissues of deficiency heterozygous larvae is located in the right arm of chromosome 3 (COURT- about half that of wild-type levels. D. simulans: (1) T(Y;2)3/ net b py; (2) T(Y;3)1,Ubx"/cut ca'. Dr. WATANABEprovided ' To whom reprint requests should be addressed. the D. simulans strain bearing the marker Lhr (larval hybrid

Genetics 115: 283-294 (February, 1987) 284 Th. E. Sprey and D. T. Kuhn rescue), a mutation that rescues the lethality of hybrid larvae PO (CYPHERet al. 1982) and was used to verify PO specific lacking the sim X chromosome (WATANABE,1979). A cv-c staining. Unless otherwise stated, the parental stocks had a Aldoxn’/sbd2 me1 strain obtained from the University of Cal- normal wild-type staining pattern. gary, Canada is mutant at the Aldox structural gene locus Photographs. Pictures of the wing discs were all printed in 3R (DICKINSONand WEISBROD1976). Other strains from with the anterior part of the disc at the left and prospective our own stock collection: (1) C( I)RM, w/Y; (2) C( l)RM,y U notum at the bottom. Magnification: Figures 2, 4, 7, 10 and bb/X.YB.B Y, y+; (3) w; (4) wild-type, Leiden mel; (5) wild- 1 1 at X260; Figures 1, 6 and 12 at X 190. type, Leiden sim. Descriptions of mutants and strains used are given in LINDSLEYand GRELL(1968) and in STURTE- RESULTS VANT (1929). All strains were raised on a standard Droso- phila medium containing 3% dried yeast, plus 0.3% yeast Pattern elements: The A0 staining in the pouch of hydrolysate, at room temperature. me1 wing discs was confined to four distinct staining A0patterns in parental stocks: The pattern of enzyme regions (Figure 1, a and d). (1) Region 1-an oval distribution as well as staining intensity were normal for the region in the center of the wing pouch within the parental me1 wild-type strain, the C(2L), C(2R), C(3L), C(3R) presumptive wing margin. (2) Region 2-an area sur- and for the me1 strain with a sim fourth chromosome. In general A0 patterns do not vary from one strain to the rounding the central spot extending as a stained mar- next. However, stain intensity does show some intraspecific gin band in an anterior-posterior direction and dor- variation. The Aldox null mutant, Aldox”’ showed no stain- soventrally as a band perpendicular to the first. (3) ing reaction. The parental sim wild-type homozygous Lhr Region 3-a darkly stained area that includes the strain and T(Y;2)3 strains possessed normal AO distribution presumptive wing surfaces and wing margin on the patterns and staining activity. T( Y;3)I showed normal distri- bution but reduced activity in males, while distribution and anterior side of the wing pouch and a stained band activity were normal in the females. The Aldox gene may along the posterior margin extending from the margin have been damaged during synthesis of the translocation band of region 2 to the posterior edge of the pouch. stock. The stained areas are equidistant from the central Crosses. Hybrids were produced by mating me1 females most region. (4) Region 4-A small extension of the to sim males. The number of larvae resulting from interspe- cific hybridization was low because of lowered rates of posterior margin band from the posterior most region mating and egg production and low embryonic viability. 3 to the edge of the disc. A0 activity in the wing Moreover, due to the special segregation of the different pouch of sim was found in region 3 only (Figure 1, b free, compound and translocated chromosome arms many and e). Presence or absence of staining within each combinations arise that are lethal during embryogenesis or region depended upon the genetic composition of the as young larvae. Thus, the number of larvae reaching the third instar was very low. partial hybrids. Each cross gave rise to a number of genetically different Full hybrids: Female hybrids from the cross be- classes. In order to reference these classes a representative tween me1 wild-type females and sim wild-type males chromosomal segregation scheme has been given with the had clear A0activity in region 1 and region 3 (Figure results. In most cases the mutant larval markers were such 1, c and f). Activity in region 1 was no more than half that the different classes of larvae could be distinguished. In some cases aneuploid larvae are known to reach the that seen in region 3, suggesting that the me1 Aldox pupal stage (FITZ-EARLand HOLM 1978). The me1 strains gene was active in 1, while both the me1 and sim genes with free-compound chromosomes were mated to wild-type were expressed in region 3. Female hybrids resulting sim so as to separate the effects of aneuploidy from the other from the reciprocal cross have an identical wing disc results. pattern. Several reasons prompted us to use predominantly female hybrids in our analysis. First, female hybrids have one X Partial hybrids (X chromosome): The influence of chromosome contributed by each species, whereas male the X chromosome on A0 distribution was examined hybrids have an X chromosome from one parent and a Y in larvae resulting from matings between C( l)RM,w/ chromosome from the other. Second, female hybrids are Y me1 females with Lhr/Lhr sim males. The Lhr muta- obtained in larger nunibers than male hybrids (STURTEVANT tion (located on chromosome 2) yields viable larvae 192 1). Third, the chance that a Y chromosome bears pattern regulation genes is rather small, since no pattern dimorph- without a sim X chromosome. Four different chro- ism exists between the males and females. mosome combinations occur. However, only partial Enzyme histochemistry: AO activity in imaginal discs was hybrids carrying two me1 X chromosomes were viable, demonstrated following the procedure of SPREY et al. being characterized by colorless malpighian tubes. (1 98 1). p-Dimethylaminobenzaldehyde, used as one of the The wing discs of these larvae showed the so called substrates for this study, also gives a reaction with pyridoxal oxidase (PO). PO is absent in wing discs of wild-type larvae “Minute”pattern, indicating the presence of both A0 but occurs in the wing pouch of some Minutes (FOGERTY, and PO (Figure 2a). Incubation of the discs with COURTRIGHTand KUMARAN1983). A combination of the heptanal revealed the A0pattern (Figure 2b). Many A0and PO patterns was demonstrated with para-p-dimeth- of these discs were not informative due to limited ylaminobenzaldehyde in mutant Minute wing discs and in activity. Those with sufficient staining showed uni- some wing discs showing “Minute” patterns in partial hy- brids. Therefore, it was necessary to eliminate the contri- form levels in regions 1, 2 and 3, with reduced activity bution of PO to the staining pattern by using the A0specific in the posterior of region 3. An XO partial hybrid with heptanal. Triniethoxibenzaldehyde is specific for a single sim X chromosome was produced by crossing Aldehyde Oxidase in Drosophila 285

1234

FIGURE1 .-(a-c) A0distribution patterns in wing discs of a, mel; b, sim; and c, a full hybrid (female) between me1 and sim. (d-9 Schematic representation of A0patterns with subdivision of wing pouch into four regions that make up the patterns. A0activity in peripodial layer is not indicated.

*P

FIGURE2.-(a and b) A0distribution patterns in wing discs of partial hybrids homozygous for me1 X cliroinosome. a, "Minute" pattern (A0 and PO activity); b. A0only, showing restoration of activity in region 2 (arrow). (c) Wing disc of partial hybrid hemizygous for sim X chromosome. 286 Th. E. Sprey and D. T. Kuhn

C( l)RM,y v bb me1 females with wild-type sim males. folded and showed A0activity only in the peripodial Theoretically, four different classes occur, of which layer (Figure 4c). Similar results were found in larvae only the X,O class was viable and possessed imaginal trisomic for 2L that resulted from a cross between y; discs giving an A0 pattern. These discs showed an C( 2L)It; F( 2R)bw me1 females and wild-type sim males. A0pattern that was similar to the X, Y, male hybrids The effect of the right arm of chromosome 2 was (Figure 2c), although there was more activity in region assessed among offspring from a cross made between 2. y; F( 2L) dp; C( 2R) cn me1 females and T(Y,2)3/net b py Partial hybrids (second chromosome): To test the sim males. The different chromosomal combinations left arm of chromosome 2 y; C(2L)It; F(2R) bw me1 are given in Figure 5. Larvae that originated from females were crossed with T(Y,2)3/net b py sim males. this cross could be subdivided into two groups accord- Twelve possible chromosomal combinations arise (Fig- ing to the color of the malpighian tubes: (1) larvae ure 3). Several of these combinations were lethal in with pale yellow malpighian tubes, indicating homozy- an early embryonic stage. Larvae that are haploid for gosity for cinnabar (class 6); and (2) larvae with orange- half a chromosome are lethal (LINDSLEYet al. 1972), yellow malpighian tubes (classes 1, 2, 3 and 1 1). as are nullosomics and tetrasomics (SCRIBA1967, The wing disc pattern of class 6 larvae (homozygous 1969). Trisomic larvae for one arm of chromosome 2 for 2R mel) was like that of the full hybrid, although survive to the pupal stage (FITZ-EARLEand HOLM some activity was found in region 2 (Figure 6a). Lar- 1978). Euploid hybrids carrying C(2L) It from me1 vae with orange-yellow malpighian tubes showed ac- (class 4) have colorless malpighian tubes, due to homo- tivity in region 2 without clear-cut borders between 7ygosity for light. All other larvae had yellow malpigh- stained and unstained regions (Figure 6b). In most ian tubes. Homozygosity for the sim 2L was lethal (E. cases the posterior side of region 3 was broadened H. GRELL,personal communication). So all larvae with (Figure 6b). Other discs showed enhanced activity yellow malpighian tubes were trisomics (class 1, 2 and particularly on the posterior side of region 3 (Figure possibly 5). Other classes were lethal. 6c). The occurrence of distinct patterns within the Larvae with colorless malpighian tubes, homozy- group with yellow malpighian tubes pointed to the gous me1 2L, showed a “Minute” pattern in their wing presence of at least two types of larvae. To study the discs using p-dimethylaminobenzaldehyde as a sub- aneuploid pattern exclusively y; F(2L) dp; C(2R) cn strate (Figure 4a), while with heptanal the A0pattern me1 females were crossed with wild-type sim males. was hybrid with the addition of activity in region 4 Two possible chromosome combinations occur: (1) (Figure 4b). Region 4 staining is a species specific hybrids trisomic for 2R, with 2 me1 and 1 sim arms; component of mel. and (2) hybrids monosomic for the sim 2R. The tri- Larvae trisomic for 2L have yellow malpighian somic condition was viable whereas monosomy was tubes and appeared in the culture bottles later than lethal (FITZ-EARLEand HOLM1978). Offspring from their “Minute” sibs. Their wing discs were abnormally this cross also had orange-yellow malpighian tubes

FIGURE3.-Chromosonial combinations resulting from crosses between C(2L) It; F(2R) bw rnel females and T(Y,2)3/net b py sim males. Position of centromeres established by OHNISHIet al. (1982). Ychromosome in black. Lethal classes marked with +. Aldehyde Oxidase in Drosophila 287

c

a“ I FIGURE4.-(;1 ;itid 1)) A0patterns in wing discs of partial 1ivI)rids homozygous. ._ for mef 2L. a. “Slitiu *“ paitcrii (A0 and PO activity); b, A0only, showing restoration of activity in region 4 (arrow). (c) Abnormallv folded wing disc of an ;ineuploid hybrid, trisomic for 2L. with A0activity limited to peripodial layer.

L ha -netb py

8a -netb py -ne1 b py

3 5 t

4 ._.ka .nelb py ne1 b py 4 -4 dp dP 9 1 11 FIGURE 5.-Chromosomal combinations resulting from crosses between F(2L)dp; C(2R)cn me1 females and T(Y,2)3/net 6 py sim males. and wing discs that had a similar enhanced activity in in male larvae. The latter were characterized by a the posterior part of region 3 (Figure 6d), as previ- general decrease in A0activity in all tissues. However, ously shown in Figure 6c. Therefore, we assume that after prolonged incubation a normal sim pattern be- larvae with orange-yellow malpighian tubes represent came visible (Figure 7b). Data suggest a correlation a variety of chromosomal combinations, including between reduction in Aldox activity and damage to euploids homozygous for the sim 2R (Figure 6b) as 3R during construction of the translocation strain. well as aneuploids trisomic for 2R (Figure 6c). Alternatively, it could be an effect caused by the Partial hybrids (third chromosome): The influence translocation itself, although the effect of the hetero- of the left arm of chromosome 3 was studied among chromatin of the Y chromosome probably does not the offspring of the cross between y; C(3L) ri; F(3R) extend to the Aldox gene. Whatever the explanation, ca me1 females and T(Y;3)1,Ubx”/cut ca2 sim males. lack of activity is a dominant condition. According to The A0 wing disc pattern of the parental sim strain Figure 8, two euploid classes occur, one homozygous was normal in female larvae (Figure 7a) but very weak for the me1 3L (class 4) and the other homczygous for 288 Th. E. Sprey and D. T. Kuhn enlarged haltere disc and reduced A0activity. Larvae with C(3R) me1 lacked a larval marker. A0 activity was expected to be normal in these wing discs. We found discs with normal activity and a me1 pattern (Figure 1Oa), and a second group showing only a trace of activity in region 1 (Figure lob). The haltere discs of this group were slightly larger. The combination of these two facts indicated that these larvae were homozygous sim 3R. After prolonged incubation the homozygous sim 3R discs showed staining in regions 2 and 3 (Figure 1Oc). Partial hybrids (fourth chromosome): In order to test the influence of chromosome 4 we crossed ey/ sim-4 me1 females with sim wild-type males. Abnor- mally shaped and sized eye-antennal discs gave an easy morphological marker for the ey'lsim-4 hybrid (Figure 1 la). Homozygous sim-4 hybrid larvae with normal eye-antennal discs (Figure 1 1 b) had the hybrid pattern (Figure 1 IC). The Aldox complex: structural gene and regula- tory region: To test the influence of the Aldox struc- tural gene and adjacent regulatory region on 3R we analyzed the offspring of three crosses. First cv-c Al- dox"' sbd me1 females were mated to Lhr sim males. Both male and female larvae had the entire sim Aldox complex on one 3R. while carrying the nonfunctional Aldox structural gene on the me1 3R. The wing discs FIGURE6.-(;I ;ind b) A0distribution pat~eriisin wing discs of of these hybrid larvae showed a distinct sim pattern partial hybrids homozygous for 2R: a. mcl; I). sim, with additional (i.e., without A0activity in region 1, Figure 12a). A0 activity in region-2 (arrow). (c and d) Wing discs of aneuploid activity was lower than that in wild-type discs, which hrbrids trisomic for 2R. is due to the transcription being limited to one dose of the structural gene. The second cross was made the sim 3L (Class 9). Two potentially viable trisomic between y; DJ3R) sbd'"'/Xu me1 females and wild-type classes (1 and 2) may be produced. Viability of the sim males. Hybrid larvae with the dominant Xu mu- aneuploid larvae was studied by crossing C(3L) me1 tation could be easily recognized by their deformed females to sim wild-type males and assessing egg hatch- wing pouches. Consequently, larvae with a normal ability. Since no eggs hatched, trisomy for 3L is lethal wing pouch lacked the me1 Aldox complex, but had during embryogenesis. Thus, only euploid larvae sur- the sim complex on the homologous chromosome. vive. Larvae homozygous for 3L me1 had enlarged Here too the discs showed a weak but distinct sim haltere discs due to Ubx" and had yellow malpighian pattern (Figure 12b). Third, wild-type me1 females tubes. Their wing discs showed reduced A0 activity were mated to T(Y,3)Ubxm/+;Lhr/+ sim males. For confined to region 1 and the anterior side of region the males we assumed a deficient Aldox complex 3 (Figure 7c). After a prolonged incubation, region 3 (either structural or regulatory), based on the strongly staining became heavy and light staining in region 2 reduced A0 activity caused by this translocation. appeared (Figure 7d). Larvae homozygous for the 3L So male hybrid larvae carried a functional me1 Aldox sim had colorless malpighian tubes and expressed a complex and a nonfunctional sim Aldox complex. full hybrid pattern (Figure 7e). Their wing discs showed a very weak A0 activity To test the effect of the right arm of chromosome (Figure 12c). However, after a very long incubation 3 y; F(3L), h2; C(3R) sr me1 females were mated to (70 min) a me1 pattern was visible, with activity in T(Y;3)1, Ubx"/cut cu2 sim males. The chromosome combinations for the cross are given in Figure 9. The regions 1, 2 and 3 (Figure 12d). Only the most prox- only viable larvae were hybrids with C(3R) me1 (class imal part of the posterior marginal band was absent. 4) and hybrids homozygous for the sim 3R (class 9). DISCUSSION all other combinations were embryonic lethals (LINDS- LEY et al. 1972). Class 9 larvae were identified by the Full species hybrids: A0 wing disc patterns of Ubx" bearing translocation, which resulted in a slightly hybrid larvae clearly show that presence of A0activity Aldehyde Oxidase in Drosophila 289 --

FIGURE7.-(a and b) 110 distribution patterns in wing discs ofsim T(Y,3)UbP/cuf col), incubation time 40 min. (c-e) Wing disc patterns of partial hybrids homozygous for SL ofc and d, md; e, sim. (c) Normal incubation (20 min). (d) Prolonged incubation (50 min). demonstrating diffuse activity in region-2 (arrow).

simulans

JLUbx -CUI ca melanogaster\

Ubx CUI w ri 5 &* rl ri ' ri 1 2

CPLUbx ._. CUI ca -ca e _cd & ce le _011 7 t 8 t 9 10 t 11 t 12 t FIGURE8.-Chromosomal combinations resulting from crosses between C(3L)R;F(3R) ca md females and T(Y,3)Ubx"/cut CO sim males. in region 1 is a dominant feature of mel, with recessive about half of that found in region 3 of the hybrid absence in sin. This is consistent with a &dominant pattern (Figure IC). This indicates activity of a single mode of regulation. The A0activity in region 1 is functional Aldox gene. Similar regulation of this gene clearly weaker than in the me1 wild-type pattern and has already been described by DICKINSON(1975, 290 Th. E. Sprey and D. T. Kuhn

simulans

._I cut ca melanogaster I I I I \ I I 1 I -curca

4

-cutce

4 4 4 4 i" -h 7 h h h h 7 i 8 ts 10 t 11 t 12 t

FIGURE9.-Chromosomal combinations resulting from crosses between F(3L)h; C(3R)sr me1 females and T(Y,3)Ubx"/cut ca sim males. ---- .. __ . - .

~ FIGURElO.--XO distribution patterns in wing discs of partial hybrids homozygous for 3R of a, mef; b and c, sim. (a) Restoration of me1 pattern in region 2 (arrow). (b) Normal incubation (20 min); (c) Prolonged incubation (50 min) demonstrating weak activity in regions 2 and 3. 1978). but for tissues other than imaginal discs. posterior part of the midgut of mel. Other data con- In region 2 of the hybrid discs there was an absence cerning different enzymes also imply that specific gene of A0 activity which is a species specific feature of expression is bipartite, including both proximal sites sim. Dominant sim suppression of A0activity in re- closely linked to the structural gene and distant sites gions normally expressing A0 in me1 suggests trans- (PAIGEN1979; BEWLEY198 1 ; KING and MCDONALD, regulation. DICKINSON(mob) described a similar cis- 1983). and trans-regulation for A0 activity in hybrids be- The difference in regulation between region 1 and tween D.formella and D. grimsham', where cis-control 2 is remarkable since it involves adjacent cell groups was found in one type of tissue, with trans in another. belonging to the same tissue type. The clonal restric- In the present study both types of regulation were tion found for the borderline of region 1 (KUHNet al. seen within a single tissue type. DOANEet al. (1983) 1983) now seems to make more sense, in that it may have found a comparable situation for the regulation prevent exchange of cells between territories with of the amylase structural gene in the anterior and different types of regulation. Aldehyde Oxidase in Drosophila 29 1 --

_- . i A J7

A-P FIGURE1 1 .-(a and b) A0distribution patterns in eye-antennal discs of a, a full hybrid (q"/sim-4)and b, a partial hybrid homozygous for the sim chromosome 4. Ey = eye disc (entirely reduced in a), A = antennal disc. (c) A0wing disc pattern of the partial hybrid (sim-l/sim-4).

(OHNO1969). At first glance these results seem com- patible with those of COURTRIGHT(1967) who found that in larval hybrids between me1 and sim the paternal genome was not transcribed before the end of the larval period. DICKINSON(1 97 1), however, concluded that the paternal genome was active in the first instar. The fact that female hybrids show the central spot irrespective of which species contributes the paternal genome, support the idea of an early activation. Partial hybrids homozygous for part of the melan- ogaster genome. Wing discs of larvae homozygous for the me1 X chromosome showed A0activity in regions 1, 2 and the anterior part of 3 (Figure 2b). Since the level of activity was normal in other tissues, the sim Aldox gene interacted normally with the X-linked me1 maroon-like+ and cinnamon+ gene products to produce functional protein (for review of interactions see COURTRIGHT1976). A0activity in region 2 of hybrids with me1 X chromosomes suggests presence of a locus or loci that influence the me1 pattern. Homozygosity for me1 2L gave a hybrid pattern with the addition of region 4 (Figure 4b). This clearly shows the existence of a recessive trans-acting control element on this chromosome arm. The me1 2R arm is not involved in regulation of the A0 activity since hybrids with me1 2R had the hybrid pattern (Figure 6a). FIGUREI2.-AO distribution patterns in hybrid wing discs. a. For 3L the situation is not straight forward because Aldox"'/sim*; b. Df(3R) sbd'"/sim+; c and d, mel+/T(Y,3) Ubx"; Lhr; c, normal incubation (20 min): d, prolonged incubation (70 min). of the damaged Aldox complex in the sim (Y;3)trans- location. Discs of larvae homozygous for me1 3L had The fact that the A0activity regulation shows cis- a weak activity in regions 1, 2 and 3 (Figure 7d). and trans-regulation in the same tissue excludes the However, the fact that these larvae bear the sim trans- possibility that only one genome is expressed through- location, makes it plausible that the appearance of out the tissue in any general way. Therefore, no activity in region 2 occurred because of the defective preferential activation of the maternal genome occurs Aldox complex rather than by homozygosity for con- as described for interspecific vertebrate hybrids trol elements on the me1 3L. 292 Th. E. Sprey and D. T. Kuhn The appearance of the me1 A0 pattern in discs of larvae homozygous for me1 3R (Figure loa) shows that important recessive control element(s) reside some- where in 3R. The same conclusion was reached by KUHNand SPREY(1987) with regard to control of the malic enzyme distribution pattern in eye-antennal discs. If A0 is regulated in the same way as demon- strated for enzymes that showed expression in trans- formed enzyme null strains (SPRADLINGand RUBIN 1983; GOLDBERG,POSAKONY and MANIATIS1983; SCHOLNICK,MORGAN and HIRSH 1983), then the in- structions for temporal and spatial distribution of the enzyme will be found next to the structural gene. Partial hybrids homozygous for part of the simu- lans genome: A0wing disc patterns of partial hybrids with a single sim X chromosome (X#) did not differ from those of the hybrids (Figure 2c), nor did those of hybrids homozygous for sim 3L and 4. No pattern was found without activity in region 1, although it is possible that this type of a pattern corresponded to the lethal group homozygous for sim 2L. Yet, in two situations a different pattern was observed. First, a pattern extension occurred in region 2 in discs of larvae homozygous for simulans 2R (Figure 6b) and is regarded as a change toward the me1 pattern. At present no suitable explanation can be given for this unexpected result. Interpretation of the pattern change found in hy- brids homozygous for sim 3R (Figure 1Oc) is compli- FIGURE 13.--Schematic overview of the patterns found for the cated by the effect of the translocated arm on the A0 different partial hybrids. a, Full hybrid; b through 0, partial hybrids. activity. Just as males of the parental strain, hybrids The homozygous or hemizygous chromosomal condition has been with this translocation had a strongly reduced A0 indicated for each disc. m, mel; s, szm. activity. The marker Ubx as such does not influence synthesis of another sim T(Y,3) strain with a normal the A0 wing disc pattern as shown by KUHN and A0 activity in males is a prerequisite to resolving this CUNNINCHAM(1977). The decrease seemed to be question. A further analysis of the present T(Y,?) stronger than that caused by a single dose of Aldox"' strain, with regard to the decrease of A0 activity in in mel, indicating that the Aldox-complex on the ho- males, also deserves attention. mologous 3R arm might also be influenced. In these Influence of the Aldox complex: Full hybrids lack- partial hybrids, in which the Aldox gene and its cis- ing the me1 Aldox gene or complex show a weak sim acting control element were homozygous sim, a sim wing disc pattern. This represents further evidence of pattern was expected. The weak activity in region 1 a cis-acting control element, which determines the contradicted the conclusion obtained from discs of expression of the Aldox gene in region 1 of mel. The deficiency heterozygotes for the Aldox complex, absence of A0 activity in region 1 of hybrids lacking which indicated that it is normally the me1 complex the me1 Aldox gene shows that this gene is normally being expressed there (see Figure 12, a and b). More- expressed in region 1 of full hybrids. The reduced over, region 2 showed a weak activity as well. To staining in the regions common to both species shows explain the paradox we have to assume that in this that both genomes contribute to the final hybrid situation me1 regulating loci act, in trans, on the sim pattern. This further implicates the Aldox complex in Aldox gene, although with a very low efficiency. 3R as a major controlling region for activity in region If we also assume an inhibitory trans-acting sim 1. Yet, it is not proof of a cis-acting control. A regu- control element, which in full hybrids suppresses Al- lation by means of a diffusible substance, produced dox expression in region 2, it fails to act in the according to a species-specific program and recog- situation of 3R homozygous for sim. At present we nized only by the conspecific structural gene would can only speculate about a possible role of the defec- give similar results. tive sim Aldox complex in this inhibitory effect. The Overview (Figure 13): The results show that A0 Aldehyde Oxidase in Drosophila 293 activity in the hybrid wing disc is regulated in different amylase expression in Drosophila melanogaster. pp. 63-90. In: ways. In region 1 a cis-dominant type of regulation Isozymes: Gene Expression and Development (Current Topics in Biological Medical Research Series, Vol. 9), Edited by M. C. occurs (Figure 13a), while in region 2 a trans-acting RATTAZZIet al. Alan R. Liss, New York. control takes place (Figure 13a). Data are presented FITZ-EARLE,M. and D. G. HOLM,1978 Exploring the potential that show existence of Aldox expression regulating of compound free-arm combinathns of chromosome 2 in Dro- loci on the meE X chromosome (Figure 13b), 2L (Fig- sophila melanogaster for insect control and the survival to pupae ure 13d) (possibly 3L, Figure 13h), and 3R (Figure of whole-arm trisomies. Genetics 89: 499-5 10. FOGERTY, S. C., J. B. COURTRIGHTand A. K. KUMARAN, 13k) and the sim 2R (Figure 13g) and 3R (Figure 131). 1983 Expression of pyridoxal oxidase in the imaginal discs of The most pronounced effect was associated with the Drosophila. Genetics 104 s25. mel 3R (Figure 13k), presumably due to a factor close GLASSMAN,E., T. SHINODA, E. J. DUKE and J. F. COLLINS, to the structural gene. 1968 Multiple molecular forms of xanthine dehydrogenase and related enzymes. Ann. N.Y. Acad. Sci. 151: 263-273. The authors thank Dr. E. H. GRELLfor many of the stocks used GOLDBERG, D. A., J. W. POSAKONYand T. MANIATIS, during this investigation and his continued encouragement during 1983 Correct developmental expression of a cloned alcohol the course of the investigation. Dr. JOE DICKINSONis thanked for dehydrogenase gene transduced into the Drosophila germ line. his thoughtful comments on the manuscript. We additionally thank Cell 34: 59-73. SIMONPLOEGER for preparing the schemes and taking photographs GRELL,E. H., 1976 Genetic analysis of aspartate aminotransferase and CAROLINAJOST for her stimulating discussions and technical isozymes from hybrids between Drosophila melanogaster and assistance. D.T.K. was supported by National Science Foundation Drosophila simulans and mutagen-induced isozyme variants. research grant PCM-8302 104. Genetics 83: 753-764. KING,J. J. and J. F. MCDONALD,1983 Genetic localization and LITERATURE CITED biochemical characterization of a trans-acting regulatory effect in Drosophila. Genetics 105: 55-69. BEWLEY,G. 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