referred to as the bithorax-complex (BX-C) (LEWIS. 1978, 1981a, b). With one ... (MEl) caused by the X-linked recessive allele tuh-l* (h. = head defects) to cause ...
Copyright 0 1988 by the Genetics Society of America
Paternal Imprinting of Inversion Uub' Causes Homeotic Transformations in Drosophila David T. Kuhn and Gerhild Packert Department of Biological Sciences, University of Central Florida, Orlando, Florida 32816 Manuscript received July 30, 1987 Revised copy accepted September 30, 1987 ABSTRACT Paternal transmission of the bithorax-complex (BX-C) rearrangement, inversion Uab', causes a specific dominant gain of function phenotype in most abdominal segments. This representsa case of paternal imprinting since themutantphenotype will occur only if inversion Uab' is paternally transmitted. T h e transformations in males are toward genital arch tissue. For females the transformations are to tissue found on abdominal segment 7 (Ab7) and to structures normally restricted to the genital disc. Ninety-six percent of transformed areas appear on Ab5 andAb6 in both sexes and on Ab7 in females, coinciding with the Abd-B domain. Four percent of thetransformations occurred on Ab1 through Ab4, coinciding with the abd-A domain. T h e mutant phenotype can be dramatically enhanced by modifying genes such as the posterior BX-C mutant tuh-3. Expressivity is modulated by maternal effect alleles interacting with tuh-3. A region of function within inversion Uab' appears to be programmed during spermatogenesis to function in a legacy dependent manner during embryogenesis.
D
EVELOPMENT of thoracic and abdominal segments in Drosophila melanogaster is controlled by a series of homeotic genes clustered in 3R at 58.8, referred to as the bithorax-complex (BX-C)(LEWIS 1978, 1981a, b). With one exception the genes are aligned on the DNA inthe same order as the segments et al. 1983; KARCHet al. 1985). they control (BENDER The complex is divided into a left and right side. Mutants in the left side control function of the 3rd thoracic segment (T3) andanterior side of Abl, while mutants in the right side control functions from posterior Ab1 through Ab8. Mutants in the abdominal portion of the complex fallinto the two general functional domains, abdominal-A (abd-A) and Abdominal-B (Abd-B) (SANCHEZ-HERRERO et al. 1985; TIONG, BONE and WHITTLE1985; KARCHet al. 1985). The Ultraabdominal' (Uab') mutant actsas a cisdominant to transform Ab1 into Ab2 (KIGER 1976; DAVISand KIGER 1977). It is associated with a tiny inversion with one break point in bithoraxoid (bxd)and one in infraabdominal-8 (iab-8) (KARCHet al. 1985). Accompanying the gainof function phenotype for Uab' are two recessive loss of function phenotypes that are due to theinversion break points. Inversion Uab' hemizygotes showa strongbxd phenotype where Ab1 is transformed into the 3rd thoracic segment (T3), including a 4thpair of legs.The hemizygous condition at iab-8 results in a deficiency of all genital structures (D. T . KUHNand G. PACKERT, unpublished data).The Uab' gain of function phenotype and thetwo losses of function occur regardless of the inversion beingtransmitted paternally or maternally. Genetics 118: 103-107 (January, 1988)
The tumorous-head-? (tuh-3) mutant gene acts as a semidominant in the presence of maternal effect 1 (MEl) caused by the X-linked recessive alleletuh-l* (h = head defects) to cause eye and antenna transformations into tergites and genitalia (GARDNER and WOOLF1949; KUHN,ZUST and ILLMENSEE 1979) and tuh-3 acts as a recessive in the presence of maternal effect 2 (ME2), caused by the dominant allele tuh-Ig (g = genital defects) to produce genital disc defects (WOOLF1966, 1968). The tuh-3 mutant is the distal most gene mapped in the BX-C and is a lesion in the iab-8 region (KUHN,WOODSand ANDREW 1981; KARCHet al. 1985). The objectives of this paper are to: (1) show that paternal imprinting of inversionUab' causes homeotic areas on abdominal segments; (2)describe the phenotype; (3) demonstrate that tuh-3 is a potent modifier of inversionUab'; and (4) show that the tuh-3 maternal effect allelestuh-2"and tuh-lgmodulate the phenotype in inversion Uabi/tuh-? trans-heterozygotes. MATERIALSANDMETHODS
Drosophila stocks: All stocks were maintained at approximately 24' on a standard Drosophila medium consisting of cornmeal, agar, brewer's yeast, dextrose, sucrose, propionic and phosphoric acid. Tegosept was added to thesurface of the medium to inhibit fungal growth. T h e Drosophila stocks used were: (1) tuh-/E; Uab'/Xa; (2) C(1))", yz tuh-Ig/Y;tuh-3; ( 3 ) FM6, tuh-lg/tuh-l'; tuh-3;(4) C ( I ) R M , v tuh-I*/lY; tuh-3; (5) tuh-lh;tuh-3; (6) Oregon-R-C and (7) Canton-S. Explanations and symbols for mutants, chromosomes and wild-type stocks used are given in LINDSLEY and GRELL(1968), KICER (1976) and KUHN, WOODS and ANDREW(198 1).
D. T. Kuhn and G . Packert
104
A ;.. .
TH'.:
,
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'12
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, ,,.;
"-7
-
n
FIGURE1.-Transformed areas on inversion Uab'/fuh-3 abdomens.(A) A male with transformed region on T r 6 and region lacking trichomes on S5. (B) Malewith transformed Tr6. GA, genital arch; Ab6, abdominal segment 6; Tr6, abdominal tergite 6; bars represent 0.05 mm.
Abdomen preparationand identification:All abdominal
tergites and sternites were analyzed in order to identify and categorize the transformed areas. No homeotic transformations occurred on thoracic segments or in genitalia. Abdomens were dissected free from the thorax in lactophenol mixture, cut along the dorsal midline, the internal soft tissue removed, cuticle flattened with dorsal side up in a drop of Hoyer's mixture, coverslipped and examined microscopically to identify the transformed tissue. The area and position
".
RESULTS
FIGURE2.-Transformations on fernale inversion Wab'/ruh-3abdomens. (A) Abdominal tergites 5 , 6 and 7 are reduced in size and two small transformed regions of Ab7 tissue are shown by arrows. (B) A trichomeless transformed region on Ab5 outlined by arrowheads. (C) Gainin function of genital disctissuein abdominal segment 7 where 8th tergite bristles appear on 7th tergite and vaginal teeth appearboth on 7th sternite and on vaginal plate. Ab7, abdominal segment 7; Tr8, abdominal tergite 8; VT, vaginal teeth. Bars represent 0.05 mm. Arrowheads are used to show the position and extent of the transformed tissue.
Examples of the Uub' caused transformations in male and female Uub'ltuh-3 are presented in Figures 1 and 2. Although not pictured, Uub'/+ flies show the same type of transformations. Heterozygosity for inversion Uub' and tuh-3 resulted in gains of function in males (Figure 1). Transformed areas on the tergites possessed genital arch-like tissue and the 6thabdominal segment (Ab6) was reduced in size (Figure 1, A and B) as compared to a wild-type Ab6. Pigmentation of transformed 6th abdominal tergite (Tr6) tissue was like that on the genital arch (Figure 1, A and B), with bristles darkly pigmentedand slender like genital arch bristles (Figure 1, A and B). Further, the transformed tissue was trichomeless. T h e suppression of Ab6 in males was interpreted to be a gain of function since Ab7, except7th spiracles, is suppressed in adult males. In extreme cases even the 7th spiracles disappeared. The homeotic transformations on Uub'ltuh-3 female abdomens (Figure 2, A and B) were morpholog-
ically like T r 7 and Tr8 tissue. The wild-type Tr7 and Tr8 are smaller than the more anterior segments. Thus, when distal abdomen/genital segments were expressed in more anterior positions, segments Ab5 through Ab7 became reduced in size (Figure 2A). Isolated regions showing transformations toward Ab7 tissue are identified by arrows in Figure 2A and encircled by arrowheads in Figure 2B. Tr7 was frequently transformed into Tr8 and vaginal thorn bristles appeared on sternite 7 (S7) (Figure 2C). These transformations suggest that T r 8 and thevaginal plate derived from the same primitive abdominal segment, Tr8 being dorsal and vaginal plate being ventral. The genital structures were never observed anteriorto Ab7. Data demonstrating that inversion Uub' must be transmitted paternally in order for the transformed spots to appear on the abdomen are presented in Table 1. The females usedin these crosses carried
of each transformed area were carefully traced onto a model abdomen to record the pattern and frequency of distribution. The criteria used to identify the transformed tissue were: (1) pigmentation of cuticle; (2) size, shape and texture of bristles; (3) presence, absence and distribution of trichomes and (4) pattern of bristles and trichomes on the cuticle.
Imprinting Paternal
in D. melanogaster
105
TABLE 1
TABLE 4
Paternal influence by inversion Uab' and enhancementby tuh-3
Suppression andenhancement of transformed regions by ME1 and ME2
Offspring scored Males (X) Females
Group A tuh-3It~h-3(X) tuh-31tuh-3 Oregon-R-C (X) Oregon-R-C Cantons (X) Cantons Uab'lXa (X) tuh-3/tuh-3 Group B Uab'IXa (X) Uab'lXa Uab'lXa (X) Oregon-R-C Uab'/Xu (x) CantonS Uab'lXa (X) tuh-31tuh-3 Group C Oregon-R-C (X) Uab'fXa Cantons (X) Uab'lXa tuh-31tuh-3 (X) Uab'/Xa Group D tuh-3ltuh-3 (X) Uab'ltuh-3
Genotype
No.
No.
transform/ fly
Offspring sampled Maternal effect No.
Mothers
100 89 38 25
tuh-3/tuh-3 ORCIORC CSICS Xaltuh-3
45 53 73 44
0.00
Genotype
Transform per fly
ME2
37 Uab'ltuh-3
3.7
0.00 0.00
C(l)hU, y2 tuh-lr/lY; tuh-3lt~h-3 FM7, tuh-l'ltuh-1'; tuh-3/tuh-3
ME2
44 Uab'ltuh-3
3.9
Uab'lXa Uab'IORC Uab'fCS Uab'ltuh-3
0.75 0.75 0.62 4.09
C( I ) R M , u tuh-1 '/K t~h-31tuh-3 tuh-lh/tuh-Ik; kh-3/tuh-3
ME1
33 Uab'ltuh-3
1.8
ME1
40 Uab'ltuh-3
2.0
44 Uab'lORC 73 Uab'lCS 40 Uab'ltuh-3
0.00 0.00
TABLE 3
0.00
Uab'ltuh-3
0.00
Summary of several experiments mapping number and abdominal segment effected by transformed areas on female and male inversion Uab'Ituh-3 abdomens
23
0.00
All females mated with inversion Uab'lXa males.
All data collected in presence of tuh-1'.
tuh-lg (MEZ). Spots of transformed tissue never a p peared in wild-type flies (Oregon-R-C, Canton-S) or in the tuh-3 flies. When inversion Uub' is transmitted through themale, inversionUub'lXa, it caused a transformation frequency of 0.75 patches per abdomen. The transformations are definitelycaused by some factor associated with inversion Uab', since none of the Xu siblings show the phenotype. This dominant effect was similarly found for inversion Uub'/+ offspring when Oregon-R-C and Canton-S females were crossed with inversion Uab'/Xu males (Group B crosses). The tuh-3 mutant acted as a very potent modifier of Uub' since a greater than four fold enhancement of spot frequency occurred in the inversionUab'/tuh-3 heterozygotes. This was true only when inversion Uub' was paternally transmitted. When inversionUub' was maternally transmitted, 0.00 transformations occurred (group C crosses). In group D the hypothesis that tuh-3mediated a maternal effect which caused the transformed spots was tested and rejected because no transformations appeared in the Uub'ltuh-3 offspring from females carrying both mutants. However, the homeotic transformations did appear in trans-heterozygotes from the reciprocal cross, as seenin group B. The gain of function reported heremay actually be a consequence of the inversion break points. The inversion may have moved iub-8 next to genes controlling thorax development. Ab1 through Ab7 thus gain potential to produce genital structures, under this condition. The frequency of transformed spots on the abdomen of Uub'/tuh-3 flies could be significantly altered by the maternal effect alleles (Table 2). Expression was the greatest in the presence of ME2. The origin
FemalesA5A A4IA3A2
R Males
%
A6
A7
N
1 2 17 7 185 164 284 221 0.0 0.3 2.6 1 . 1 28.1 24.9 43.1 A IA4 A3 A2
0 0 0.0 0.0
2 4 1.9 3.8
A5
A6
49 46.2
51 48.1
No. transform/ fly
2.99
N
No. transform/ fly
110
0.96
of the tuh-P allele was unimportant. Females carrying an attached-X chromosome caused as many spots (3.7) asfemales heterozygous for a free X-chromosome carrying tuh-Ig (3.9). The tuh-/g allele was dominant since homo- or heterozygosity lead to high frequencies of transformed areas. Homozygosity for tuh-lhin the mother resulted in a significant suppression of spots (1.8 spotsper abdomen and 2.0 spots per abdomen). Data were pooledfrom many different experiments to map the transformed areas on theabdomens (Table 3). In females only 4% of the spots were found on Ab1 through Ab4,while 96% were in segments 5 through 7. Similar results were found for males, with 5.7%of the spots on Ab3 plus Ab4and 94.3%,equally distributed between Ab5 and Ab6. The sexual dimorphism in spot frequency may not be as great as it appears. Males have a suppressed Ab7, so the effect inversion Uub' might have on that segment could not be detected. DISCUSSION
The precise position within the BX-C responsible forthe gainof abdominal/genital function is unknown. The transformations could be caused by disruption of function anywhere between bxd at -20 kb on the DNA map (BENDERet ul. 1983) to the distal
106
T.
D.
Kuhn and G . Packert
lesion in iab-8 at approximately +180 kb (KARCH et al. 1985). We currently feel that a position close to the iab-8 break point is the most probable, both because the transformations are to genital type tissue and because a similar transformation has been described as part of the iab-8 mutant Transabdominal (Tab) phenotype (CELNIKER and LEWIS1987). Both Uab' and Tab are associated with inversion break points in the iab-8 region.However, the similarity between the inversions ends there since the second breakpoint in inversion Uab' is proximal to iab-8, while the second break point with Tab is distal to iab8 (CELNIKER and LEWIS1987). Further, it does not appear as though parentallegacy playsa rolein transmission of the Tab spots (CELNIKER and LEWIS1987). Three lines of evidence indicate that the Uab' mutant does not produce the paternal imprinting. First, because Uab' is one of a series of Ultraabdominal mutants (LEWIS1978), two other mutants, Uab4 and Uab', were analyzed for transformations like those seen with inversion Uab'. Neither mutant caused the and D. T. abdominaltransformations (G. PACKERT KUHN, unpublished data). Second, a revertentof Uab' was tested that retained theability to cause the abdominal transformations, yet had lost much of the traditional Uab' phenotype (I. DUNCAN, personal communication). Third, transformation of Ab1 into Ab2 occurs in Uab' flies when passed through the female or the male, while the abdominal spots appear only when inversion Uab' is paternally transmitted. This is the first report of a paternally transmitted defect in the BX-C. Indeed cases of paternal effects are rare in animals. There are at least two general models that can adequately explain paternaltransmission effects: (1) inclusion of some sperm extragenic factor in the egg; or (2)a differential conditioning of the paternal and/or maternal genome during gametogenesis. Inclusion of some sperm extragenic substance is not a likely explanation because inversion Uab'/Xa males passon to one-half of the offspring the Xu balancer chromosome, yet no Xu animal has ever been found with abdominaltransformations. The transformed offspring must carry inversion Uab'. T h e more plausible explanation is a differential conditioning of the genomes during gametogenesis. We propose here that theresponsible factor in inversion Uab' is conditioned to function differently during embryogenesis by events occurring duringspermatogenesis. Inversion Uab' then acts postzygotically to cause the abdominaltransformations (G. PACKERT and D. T. KUHN, unpublished data). The type of genetic transmission seen with inversion Uab' is quite similar to gamete conditioning reported in mice where the paternal and maternal genomes appear to have different functions during early development (MCGRATH and SOLTER1984; SURANI, BAR-
and NORRIS1984). Very recently SWAIN,STEWand LEDER(1987) described a specific example of parental imprinting of an autosomal mouse transgene and provided the molecular explanation. The underlying mechanism involves differential DNA methylation during gametogenesis. Paternal transmission of the transgene results in an undermethylation pattern of cytosine nucleotides, leading to specific expression in the heart. Conversely, maternal transmission results in amethylatedpatternwhere the transgene is not expressed. Although this molecular mechanism for parental imprinting in mice accounts nicely for our results, it is generally held that D. melanogaster, unlike other eucaryotes, lacks methylated cytosines (RAZIN and RIGGS 1980). Thus, the underlying mechanism for imprintingseen with inversion Uab' may be quite different than that reported for mice. The tuh-3 mutant is not the only posterior BX-C mutant that enhances the paternal transmission effect. SGA62 (G. PACKERT and D. T. KUHN, unpublished data), an iabd mutant, is also a potent modifier of inversion Uab', producing the same array and frequency of transformations seen with tuh-3. There are two similarities between SGA62 and tuh-3. First, they are both Abd-B mutants. Second, both mutants cause a dominantgain of Abd-B function in the head (KUHN and PACKERT 1988). Therefore, theirability to act as modifiers could relate to either one or both of the similarities. ME1 causes a reduction in frequency of abdominal transformations in Uab'/tuh-3 trans-heterozygotes (Table 2). This highly significant suppression results in part because tuh-3 function approaches normal in the posterior embryo when the tuh-1" maternal effect is in force. No apparent loss of function is found in tuh-3 homozygotes in the presence of this maternal effect (KUHN and PACKERT 1988). So, Uab' acts as though it is in trans with a wild-type chromosome. Enhancement of the phenotype occurs in inversion Uab'ltuh-3 abdomens where ME2 (tuh-17 causes the tuh-3 lossof function (KUHN, WOODS and ANDREW 198 1). TON
ART
We thank W. BENDER,D. H. VICKERS and C. M. WOOLF for comments on an earlier version of this manuscript, and I. DUNCAN for sending his Uab' revertent strain. Project supported by National Science Foundation grant PCM 830124.
LITERATURECITED BENDER,W., M. AKAM,F. KARCH, P. A. BWCHY, M. PEIFER,P. SPIERER,E. B. LEWISand D. S. HOCNESS,1983 Molecular genetics of the bithorax complex in Drosophila melanogaster. Science 221: 23-29. CELNIKER, S . E., and E. B. LEWIS,1987 Transabdominal, a dominant mutant of the Bithorax Complex, produces a sexually dimorphic segmental transformation in Drosophila. Genes Dev. 1: 1 1 1-123.
Paternal Imprinting in D. melanogaster DAVIS,R. L., and J. A. KIGER, 1977 A clonal analysis of tergite development in Drosophila of Ultraabdominal and paradoxical genotypes. Dev. Biol. 5 8 114-123. GARDNER, E. J., and C. M. WOOLF,1949 Maternal effect involved in the inheritance of abnormal growths in the head region of Drosophila melanogaster. Genetics 34: 573-585. KARCH,F., B. WIFFENBACH, M. PEIFER,W. BENDER, I. DUNCAN, S. CELNIKER, M. CROSBY and E. B. LEWIS,1985 The abdominal region of the bithorax complex. Cell 43: 81-96. KICER,J. A., 1976 Cell determination in Drosophila with paradoxical genotypes. Dev. Biol. 50: 187-200. KUHN,D. T., and G . PACKERT, 1988 Tumorous-head type mutants of the distal bithorax~omplexcause dominant gain and recessive losses of function in D. melanogaster. Dev. Biol. 125: In press. KUHN,D. T., D.F. WOODSand D. J. ANDREW,1981 Deletion analysis of the tumorous-head (tuh-3)gene in Drosophila melanogaster. Genetics 9 9 99-107. KUHN,D. T., B. ZUST and K. ILLMENSEE, 1979 Autonomous differentiation of the tumorous-head phenotype in Drosophila melanogaster. Mol. Gen. Genet. 1 6 8 117-1 24. LEWIS,E. B., 1978 A gene complex controlling segmentation in Drosophila. Nature 276 565-570. LEWIS,E. B., 1981a Developmental genetics of the bithorax complex in Drosophila. pp. 269-289. In: Embryonic Development: Genes and Cells (Proceedings of the IX Congress of the International Society of Developmental Biology), Edited by M. BURGER. Alan R. Liss, New York. LEWIS,E. B., 1981b Developmental genetics of the bithorax com-
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plex in Drosophila. pp. 189-208. In: Developmental Biology Using Punjicd Genes (ICN-UCLA Symposia on Molecular and Cellular Biology), Edited by D. D. BROWN and C. F. Fox. Academic Press, New York. LINDSLEY, D. J., and E. L. GRELL,1968 Genetic variations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627. MCGRATH, J., and D. SOLTER,1984 Completion of mouse embryogenesis requires boththe maternal and paternal genomes. Cell 37: 179-183. RAZIN,A., and A. D. RIGGs, 1980 DNA methylation and gene function. Science 210 604-6 10. SANCHEZ-HERRERO, E., I. VERNOS,R. MARCO and G. MORATA, 1985 Genetic organization of Drosophila bithorax complex. Nature 3 1 3 108-1 13. SURANI,M. A. H., S. C. BARTON,and M. L.NORRIS, 1984 Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 308: 548-550. J. L., T . A. STEWART and P. LEDER, 1987 Parental legacy SWAIN, determines methylation and expression of an autosomal transgene: a molecular mechanism for parental imprinting. Cell 5 0 719-727. TIONG,S., L. M. BONE and R. S. WHITTLE, 1985 Recessive lethal mutations within the bithorax-complex in Drosophila. Mol. Gen. Genet. 200: 335-342. WOOLF, C. M., 1966 Maternal effect influencing male genital disc development in Drosophila melanogaster. Genetics 53: 295-302. WOOLF,C. M., 1968 Male genital disc defect in Drosophila melanogaster. Genetics 6 0 1 11- 121. Communicating editor: V. G. FINNERTY
