a functional and structural analysis of the sex combs ... functional and structural analysis of the...

Copyright 0 1991 by the Genetics Society of America

A Functional and Structural Analysis of the Sex combs reduced Locus of Drosophila melanogaster

Angela M. Pattatucci, Deborah C. Otteson’ and Thomas C. Kaufman

Howard Hughes Medical Institute and the Program in Genetics, Indiana University, Bloomington, Indiana 47405 Manuscript received March 13, 199 1

Accepted for publication June 2 1 , 199 1

ABSTRACT We have undertaken a developmental genetic analysis of the homeotic gene Sex combs reduced (Scr)

of Drosophila melanogaster by examining embryonic and adult phenotypes of mutations affecting Scr gene function. Molecular mapping of Scr breakpoint lesions has defined a segment of >70 kb of DNA necessary for proper Scr gene function. This region is split by thefushi tarazu cftz) gene, with lesions affecting embryonic Scr function molecularly mapping to the region proximal (5’) toftz and those exhibiting polyphasic semilethality predominantly mapping distal (3’) to f t z . Gain-of-function mutations are associated with genomic rearrangements and map throughout the Scr locus. Our analysis has revealed that the Scr locus encompasses genetic elements that are responsible for functions in both the embryonic and larval to adult periods of development. From these studies, we conclude that Scr is a complex genetic locus with an extensive regulatory region that directs functions required for normal head and thoracic development in both the embryo and the adult and that the regulation of Scr during these two periods is distinct.

S TUDIES involving homeotic genes in Drosophila melanogaster have significantly advanced our un-

derstanding of the nature in which developmental processes are regulated. Mutations in homeotic genes cause a transformation of segmental identity characteristically recognized as the developmental replace- ment of one structure by another which normally occurs elsewhere (GARCIA-BELLIDO 1977; LEWIS 1978). These loci have been interpreted as ontoge- netic switches that govern the choice of developmental pathways (KAUFFMAN 1977). Specification of segmental identity is largely under the control of two major clusters of homeotic genes located on the right arm of the third chromosome, the Antennapedia complex (ANT-C) which directs developmental fates in the anterior thorax and head (KAUFMAN, LEWIS and WAK- IMOTO 1980), and the bithorax complex (BX-C) which regulates posterior thoracic and abdominal development (LEWIS 1978). The ANT-C is located in polytene chromosome interval 84A-84B 1,2 and contains five homeotic genes. This study presents a developmental genetic characterization of Scr, the gene which resides between Dfd and Antp in the ANT-C.

The Scr complementation group was originally identified by a spontaneously derived chromosomal inversion, S c e , which fails to complement Dx3R)Scr, a deletion of the entire ANT-C, and Dx3R)Antpl7, which has been molecularly determined to delete ANT-C sequences from the third intron of Dfd

’ Present address: Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403.

Genetics 149: 423-441 (October, 1991)

through Antp (KAUFMAN, LEWIS and WAKIMOTO 1980; our unpublished results). The S c e lesion be- haves as a recessive lethal, but as a heterozygote displays a dominant phenotype consisting of two com- ponents. First, ectopic sex comb teeth are produced on the first tarsal segment of mesothoracic and metathoracic legs of adult males. Second, there is a reduction in the normal number of eight to 12 sex comb teeth found on the prothoracic legs resulting in a sex comb with only two to five teeth. Df3R)Scr and Dx3R)Antpl7 heterozygotes were found to have an identical reduction in sex comb tooth number on their prothoracic legs, but did not exhibit the ectopic sex comb tooth phenotype on their mesothoracic and metathoracic legs. The Sc? lesion was shown to map proximal to the Antp locus, thus defining a second gene in the ANT-C required for proper specification of thoracic identity (KAUFMAN, LEWIS and WAKIMOTO 1980).

Molecular characterization of the Scr locus has revealed that the Scr transcription unit spans 25 kb of genomic DNA and is made up of three exons (SCOTT et al. 1983; KUROIWA et al. 1985; LEMOTTE et al. 1989). Proceeding from 5’ to 3’, these exons are 0.5, 1.0 and 2.5 kb in length. The two introns are 6 kb and 15 kb, respectively. A single 3.9-kb transcript has been detected early during embryogenesis (3-4 hr after oviposition) that continues to be expressed throughout the larval and pupal stages (KUROIWA et al. 1985). I n situ hybridization to wild-type embryos has revealed that Scr mRNA is first detected in early gastrulae and is localized to a band of cells immedi-

424 A. M. Pattatucci, D. C. Otteson and T. C. Kaufman

ateIy posterior to the cephalic furrow. Scr protein is not detected until later, during germ band elongation, when it is found in the region of the labial lobe. Subsequently, Scr mRNA and protein are detected during germ band retraction in the first thoracic segment, the subesophageal region of the CNS, in the labial ganglion, and in the mesodermal cells associated with the anterior midgut (MAHAFFEY and KAUFMAN 1987; MARTINEZ-ARIAS et al. 1987; RILEY, CARROLL and SCOTT 1987). The null state for Scr was described by DENELL et al. (1981). In the absence of Scr+ gene function, animals die as late embryos with associated abnormalities in the prothorax and pseudocephelon, consistent with the spatial pattern of mRNA and protein accumulation.

Although some initial molecular characterization has been completed and information regarding the wild-type expression pattern of Scr is available, an important question remaining to be answered is how Scr+ gene function contributes to the regulation of normal developmental processes in the animal. In this study, mutations affecting Scr gene expression were characterized and their effects on development examined. We demonstrate that Scr is unique among the ANT-C genes in that it is necessary for the proper development of both the head and the thorax in embryos and adults. Moreover, this locus appears to fall under a different regulatory paradigm in the embryo as compared to imaginal stages.

MATERIALS AND METHODS

Fly stocks: Flies were routinely maintained at 25" on standard Drosophila media supplemented with bakers' yeast, except where noted. Mutations used in this study are listed in Table 1 according to source, cytology, type of mutagen used in their recovery, and Scr mutant class designation accompanied by new and old nomenclature as referenced in LINDSLEY and GRELL (1968) and LINDSLEY and ZIMM

Isolation and classification of mutant alleles: Mutations affecting Scr gene function were isolated in several independent screens performed in our own as well as in other laboratories and sent to us for examination. In all experiments, wild-type third chromosomes were derived from the Oregon-R strain (represented as "+" in text and figures). The mutations under investigation were classified based upon their behavior in heterozygous combination with Dfl3R)Scr, a deletion of the ANT-C, and + third chromosomes. Lesions with >lo% adult survival over Dfl3R)Scr were considered viable and referred to as complementing; those exhibiting adult survival 510% over D,f(R)Scr were deemed semilethal or partially complementing lesions; and those showing no eclosing adults over Dfl3R)Scr were assigned as lethal and referred to as failing to complement. Hemizygous lethal lesions, displaying associated embryonic defects as described by DENELL et al. (1 98 1) for the null state of the Scr locus, and exhibiting a dominant reduction in sex comb tooth number in adult males were assigned as Scr null (Scr"""). Mutations exhibiting semilethality over Dfl3R)Scr and pro- ducing adult male survivors showing a moderate to strong reduction in sex comb tooth number were designated as Scr

(1991).

hypomorphs ( S C ~ ' ~ ~ ' ) . A majority of Scr mutations can be assigned to one of these two loss-of-function categories. However, a subset of the lesions also exhibit gain-of-function (neomorphic) qualities. Specifically, dominant mesonotal to pronotal as well as mesothoracic and metathoracic to prothoracic homeotic transformations are observed. In addition to their loss of function grouping, these lesions have also been classified as ScrCoF, based upon standard genetic criteria ( i e . , GOF/+ = GOF/+/+, where GOF = gain-of-func- t ion).

Molecular mapping of Scr breakpoint mutations: Break- point mapping was carried out according to the procedure of PULTZ et al. (1 988). Briefly, genomic DNA was prepared from balanced stocks of the mutant and Dfl3R)Scr chromosomes, as well as from stocks of the parent chromosomes. Each genotype was digested with four restriction enzymes and probed with radiolabeled lambda phage DNA contain-

~ c 7 r c x w ~ 5 ing genomic inserts from the Scr region. The location of

,ftr", S C ? ~ ~ ' , S c P W , and Dfl3R)Hu on the physical DNA map was reported by SCOTT et al. (1 983) and these have been incorporated into our map presented in Figure 1. The position of the Sc1"" breakpoint was determined by D. BARKER (personal communication).

Complementation analysis: Heterozygous males from each of the balanced mutant stocks were crossed to Dx3R)Scr/TM3,Sb females. The results of these crosses allowed the separation of the lesions into two groups, those which partially complemented the deficiency (semilethal) and those which failed to do so (lethal). The lesions were then crossed inter se according to the procedure described by LEWIS et al. (1980b) with the following modifications. Tests with cytologically normal alleles were performed at 18 O , 25 and 29 O . This facilitated the characterization of two temperature-sensitive alleles, Scr6 and Sc$. When present, Sb+ adult survivors were collected for all crosses and scored for phenotypic aberrations in the mouthparts and legs (see text and figures).

Recombinational mapping: Dfd', Dfd', Jz3, ftz", and AntpZ5 (see Table 1) are cytologically normal alleles that fully complement cytologically normal Scr""" and Scr"@" lesions for viability (WAKIMOTO 198 1 ; ABBOTT and KAUFMAN 1986; our unpublished results). S c P W is an Scr lesion that is associated with a 50-kb chromosomal inversion (SCOTT et al. 1983). Its proximal breakpoint is located between the 3' exons of theftr and Antp genes (Figure l), and its distal breakpoint is positioned just 5' of the Antp P2 promoter. Therefore, S~J""~lDfl3R)Scr animals are deficient in both Scr and Antp gene function and die as embryos. These above listed six mutations were used to determine the recombinational separability and proximal/distal order of the cytologically normal Scr4 null lesion vis-&vis the tester mutations. Due to the close proximit of these lesions (<0.2 map unit), females carrying C(I)Md,J (CRAYMER 1974) were employed to increase recombination frequency through the interchromosomal effect (LUCCHESI 1976). Females that were C(1)M4,yz; Ki Scr' pPlTM3,Sb pP were crossed to males carrying one of the tester mutations balanced over TM3,Sb @. Virgin female offspring of the genotype C(l)M4,f; Ki Scr' pltester, were collected and mated to Dfl3R)Scr/TM3,Sb @ males. The only viable Sb+ progeny are those derived from a recombination event between Scr' and the tester mutation. Additionally, all exchange events between Kinked (Ki) and pink peach ( p P ) were recovered over TM3,Sb f. Males were checked for the presence of Scr' by scoring for a reduction in number of sex comb teeth on the prothoracic legs. These males were then backcrossed to the testerlTM3,Sb pP stock to identify recombinant chromosomes carrying both Scr' and the tester mutation. Virgin females showing evidence

Scr Structure and Function 425

TABLE 1

Mutations and deletions of the Sex combs reduced locus used in this study

Allele" Synonym Cytology Mutagen Class Source or reference

Scr' SCP Scr' Scr' Scr' Scr6 Scr7 Scr' SCP Scr" Scr" Scrf2 Scrt3 Scr" Scr" Scrf6 Scrf7 Scr'* S C F S c P W ScrS'""

scrSc*T2

Sets""

step

SC2 p P 3%'' Dj73R)RC 7 Dj73R)AI I Dj73R)CPI Dfl;)R)Tpl9 Dj73R)Win I I Dj73R)Dfd I3 Dfl3R)MAP7 Dj73RPcr Dx3R)Hu

SC$"WW' ScrY"WW'

Dx3RPcx4 S C P W r n 3 ScrSCXW"6

Dfd'

Dfd5

3%' An&'

SCP8 S C P Scr'" Scr"" ScrW2' Scr f2&

Scrf7' S C P " scP68 SCf72 S C P SCr"O Scl/"O S C P ' S C P t 2 S C P ' ' SCrP'* scrwd3' S C F ' ScrW" ScrT'

ScrT2 ScrT3

SCPP'

Scrm"

RPl e W 2 0

A41 CPI 2 9 ~ 7 6

Dfd+&'' MAP7

H u f f i f

N ~ + R C ~

SCXW+"' scxw+R"l scxw+R"4 scxw+R'' scxw+R"6

DfCR"

DfCW2'

f t P Antpw'"

Normal Normal Normal Normal Normal Normal Normal Normal In(3LR)77D; 84B1,2 In(3LR)75B; 84B1,2 Normal Normal Normal Normal Normal Normal Normal In(3R)84B1,2; 95F In(3R)84B1,2; 84F1,2 111(3R)84B1,2~ Tp(3;3)84B1,2-

84D5,6; 80,81

In(2L)25D; 40+ T(2;3)25D; 84B1,2+ T(2;3)29B; 9 1 E

T(3;4)84B2; 102F+ In(3R)80-8 1 ; 84B1,2

T(2;3)40-41; 84B1,2

Tp(2;3)84A4,5-97CD; T(2;3)84A6-B1; 41 Normal Df(3R)84B1,2; 84D Df(3R)84B1,2-84D1,2 Df(3R)84A5; 84B1

Df(3R)83E1,2-84A4,5 Df(3R)83D4,5-84A4,5

Df(3R)83E3-84A4,5 Df(3R)83E3-84A4,5 Df(3R)84A1,2-84B1,2 Df(3R)84A6-B1;

8484D4,5 In(3R)84B1,2; 84F;86C7,8

T(2;3)84B1,2; 58F1,2 Normal Df(3R)84B3-84D1,2 In(3R)80; 84A4,5 T(2;3)22Dl; 63A1,2+

T(2;3)54A1,2; 80 Normal

Normal

Normal Normal

EMS X-ray EMS EMS EMS EMS EMS EMS X-ray

EMS X-ray

EMS EMS EMS EMS EMS X-ray X-ray Spont. EMS X-ray

X-ray X-ray

X-ray

DEB X-ray EMS EMS

Spont.' x-ray

x-ray x-ray X-ray X-ray X-ray X-ray

X-ray X-ray X-ray X-ray x-ray

EMS

EMS

EMS EMS

Null Null HYPO

HYPO Hypo Hypo HYPO

Null

Null Null Null Null Null HYPO HYPO Null Null Null Null/GOF Hypo/GOF Null/GOF

Null/GOF Hypo/GOF

Hypo/GOF

GOF Hypo/GOF Noned HYPO HYPO Null Null Null Null Null Null HYPO

HYPO

HYPO Null

Null Null

Noned

Noned

Noned Noned

LEWIS et al. (1 980a,b) LEWIS et al. (1980b) LEWIS et al. (1980a,b) LEWIS et al. (1980a,b) LEWIS et al. (1 980b) P. FORNILI and T. C . KAUFMAN P. FORNILI and T . C. KAUFMAN P. FORNILI and T. C. KAUFMAN ABBOTT (1984) ABBOTT (1 984) L. LAMBERT and T . C. KAUFMAN E. STEPHENSON K. A. MATTHEWS K. A. MATTHEWS K. A. MATTHEWS K. A. MATTHEWS M. A. PULTZ and T. C. KAUFMAN V. K. L. MERRILL and T. C. KAUFMAN LINDSLEY and GRELL (1968) LEWIS et al. (1 980b) S. Y . K. TIONC

S. Y . K. TIONC S. Y . K. TIONC

M. A. PULTZ and T. C . KAUFMAN

M. SEECER and T. C. KAUFMAN DUNCAN (1 986) LEWIS et al. (1980b) STRUHL (1 98 1) ABBOTT ( 1984) Unknown" DENELL and KEPPY (1 979) T. C. KAUFMAN HAZELRICC and KAUFMAN (1 983) M. A. PULTZ and T . C. KAUFMAN LEWIS et al. (1980a,b) HAZELRICC and KAUFMAN (1983)

HAZELRIGC and KAUFMAN (1983) HAZELRICC and KAUFMAN (1983) HAZELRICC and KAUFMAN (1983) HAZELRICC and KAUFMAN (1 983) HAZELRICC and KAUFMAN (1 983)

MERRrLL, TURNER and KAUFMAN

MERRILL, TURNER and KAUFMAN

L. CAIN and T. C . KAUFMAN ABBOTT and KAUFMAN 1986)

(1 987)

(1 987)

~~ ~~~~~~~~

Allele designations are as they will appear in The Genome of Drosophila melanogaster (LINDSLEY and ZIMM 1991). ' Cytologically this chromosome appears normal. However, molecular analysis has determined that this mutation is associated with an approximate 50 kilobase inversion with a proximal breakpoint between the 3' exon of Antennapedia (Antp) and 3' exon of fushi tararu ( f i r ) and its distal breakpoint just 5' of the Antp P2 promoter (SCOTT et al. 1983). ' This chromosome was found in our stock collection and its origin is not known.

These are not Scr alleles and were either used as controls or in recombination experiments.

426 A. M. Pattatucci, D. C . Otteson and T. C . Kaufman

PROXIMAL DISTAL

A82 A553 A439 A442 A1815

A78 A148 -A2015

"SCP - DfcIRIScx4

- S c P

- ftzm. -sc+xW. " M ( 3 R ) W n l l c. Df(3R)Tp19 -(3R)A41

-scrswn* -scrSsxn* "Scrsun.

"Scr- -scrSsrp* -Df(3f?)Hu

-Df(3R)CP1 C- Df(3R)Dfd13

FIGURE I.-Scr molecular map. The coordinates (in kb) are based on a zero point defined by SCOTT et al. (1983) in the Deformed (Dfd) locus. The known Scr transcription unit consists of three exons, solid bars depict coding exons and hatched bars represent noncoding exons or portions of exons. Thefir locus consists of two exons indicated by a solid bar in this figure, with flanking 5' and 3' regulatory sequences symbolized by vertical lines. The 3' most exon of Antennapedia (Antp) is shown below coordinate +loo. The central line is a restriction map of the region: blocks above the line represent EcoRl sites and blocks below the line are BamHl sites. Numbered solid lines below the restriction map are overlapping lambda clones spanning this region of DNA characterized in a chromosomal walk made by SCOTT et al. ( 1 983). Solid arrows denote DNA absent and the position of breakpoints for deficiency chromosomes. All other chromosomal rearrangements are indicated by solid lines and denote the resolution with which the breakpoint has been determined on the DNA. Scr gain-of-function alleles span this region and are identified by stars. The triangle adjacent tofir" indicates the position of the insertion element associated with this allele.

for exchange events between Ki and pP were first backcrossed to testerlTM3,Sb pP males to detect the recombinant chromosomes carrying the tester mutation. Male progeny of this mating were scored for a reduced sex comb phenotype, allowing the identification of recombinant chromosomes carrying both Scr4 and the tester mutation. The position of the Scr4 lesion relative to the tester was determined by linkage of the flanking markers Ki and pP in the recombinant classes. Recombination frequencies were cal- culated according to the method described by WAKIMOTO (1981).

Temperaturesensitive period: The temperature-sensitive period for the establishment of adult prothoracic identity was determined according to the procedure described by MERRILL, TURNER and KAUFMAN (1 987) with the following modifications. For broad determination of the temperature-critical period, ScrX/TM3,Sb virgin females were mated to Dx3R)Hu/TM3,Sb males. After eclosion, Sb' males (ScrR/ DfT3R)Hu) were collected and boiled in 10% KOH until the internal tissues dissolved. Internal debris was removed from the carcasses by the method of SZABAD (1 978). Prothoracic legs were dissected free and mounted on slides in Gurr's hydromount and assayed for the presence and number of sex comb teeth. T o more precisely define the temperature- critical period, experiments were performed which allowed a higher percentage of Sb' adult survivors, by mating Scr8/ TM3,Sb females to Dfl;R)A41/TM3,Sb males. Since the temperature critical period appeared to occur in the third larval instar and pupal periods, more precise staging of animals from late third larval instar (LIII) to eclosion was critical; therefore, the following criteria were used. Immobile third instar larvae and white prepupa (characterized by shorten-

ing of the body, eversion of the anterior spiracles, and hardening of the larval skin) that sank in distilled H20 were assigned to a 24-hr period that encompasses late LIII to the end of pupariation. Animals with pupal cases, but no apparent imaginal form which sank when placed in distilled H20, were considered to occupy a 12-hr developmental period subsequent to pupariation, ranging from apolysis to head eversion. Sinking pupae that had undergone head eversion but showed no evidence of eye pigment were assigned to the next 12-hr developmental period. The final develop mental period encompassed the pharate adult stage and ranged from the appearance of eye pigment in sinking pupae to eclosion. After eclosion, Sb' males were treated, mounted, and their prothoracic legs examined and sex comb teeth number counted according to the procedure described above.

Effective lethal phase determination and examination of terminal phenotypes: T o determine the lethal phase and to obtain animals for morphological analyses, OreR/ TM6B,Tb individuals were inbred to provide control values for lethality associated with the balancer chromosome. S C ~ " " ~ / T M ~ B , T ~ virgin females (where "MUT" represents one of the Scr mutations listed in Table 1) were then crossed to one or all of the following male tester genotypes, DA;fR)Scr/TM6B,Tb, Dx3R)Tp19/TM6B,Tb, Scr4/TM6B,Tb and DfT3R)Hu/Kgv. In most cases, reciprocal crosses were also performed. The flies were allowed to mate for three days and then egg collections were made according to the method of MERRILL, TURNER and KAUFMAN (1987). A minimum of 350 embryos were assayed for each mating.

T o characterize the terminal phenotypes, embryos that remained unhatched 36 hr after egg deposition were de-


chorinated, fixed and prepared for light microscopy by the method of VAN DER MEER (1 977). Cuticular structures and larval mouthparts were analyzed using phase-contrast microscopy and classified as normal or mutant according to the criteria of LOHS-SCHARDIN, CREMER and NUSSLEIN-VOL- HARD (1 979). Scanning electron microscope (SEM) analysis of embryos was performed according to the procedure described by WAKIMOTO, TURNER and KAUFMAN (1 984).

RESULTS

Classification of mutants: We have recovered and/ or characterized 39 mutations in the ANT-C that affect gene expression at the Scr locus. Although the phenotypes of these mutants suggest a graded series, in order to facilitate further discussion we have assigned the lesions to one of two categories based upon their ability to survive over a deficiency for the Scr locus. The source, cytology and classification of these mutations is listed in Table 1.

Lesions that are lethal as hemizygotes and exhibit the same array of abnormalities described by DENELL et al. (1981) for the null state of the Scr locus are designated as Scr null (Scr"""). These mutations also show a prothoracic to mesothoracic homeotic transformation in adults when in combination with a wild- type homologue (ScrnU"/+) that is diagnostically recognized as a reduced number of sex 'comb teeth present on the first tarsal segment of the prothoracic legs of males.

A second group of mutations exhibit adult survi- vorship as hemizygotes and have been designated as Scr hypomorphs (SC#~"). The number of eclosing adults is 110% depending on the lesion examined. These individuals show moderate or strong prothoracic to mesothoracic, pronotal to mesonotal, and labial to maxillary transformations. Within this class of mutations, are two temperature-conditional alleles, Scr6 and Scr'.

Although the criteria for this classification simplifies the discussion of a large group of mutations, the designations say little about the lesions at the molecular level. For example, the two temperature conditional alleles, Scr6 and Scr8, probably represent mis- sense mutations in the Scr transcription unit that destabilize the Scr protein. On the other hand, Dx3R)Hu has its proximal deficiency endpoint distal toftz, therefore not directly impinging upon the Scr transcription unit (see Figure l), but rather having its affect through an alteration in the Scr regulatory circuitry. Nevertheless, genetically both mutations confer similar phenotypes that place them in the Scr"ypo grouping, even though the molecular lesions apparently are quite distinct. Additionally, reductions in sex comb tooth number cannot necessarily be used as a reliable indicator of the type of lesion being discussed. For example, some Sc#fio/+ males exhibit sex comb tooth reductions just as extreme as certain

Scr"""/+ genotypes, but nevertheless allow survival to the adult stage as hemizygotes, thus still placing them in the Sc?.hYp' grouping. Although a majority of the lesions can be simply assigned to one of the two loss- of-function categories, a subset also exhibit gain-of- function (neomorphic) qualities as determined by standard genetic criteria ( i .e . , GOF/+ = GOF/+/+). These mutations are all associated with rearrangement breakpoints in or near the Scr locus (see Table 1 and Figure 1). In addition to displaying recessive loss-of-function phenotypes, these mutations also cause dominant homeotic phenotypes in both the dorsal and ventral aspects of the adult. The former is evidenced by the loss of pleural bristles and is interpreted as representing a mesonotal to pronotal transformation, while the latter is seen as a transformation from mesothoracic and metathoracic to prothoracic identity that is diagnostically recognized by the presence of ectopic sex comb teeth on the mesothoracic and metathoracic legs of adult males. One explanation for the dominant gain-of-function is that these mutations represent lesions which cause or allow ectopic expression of the Scr+ gene product and that Scr+ activity is necessary to promote a prothoracic identity (see PATTATUCCI and KAUFMAN 199 1). It is, however, necessary to rationalize the associated loss-of-function lethality with this gain-of-function ectopic expression.

Molecular mapping of Scr breakpoint mutations: Scr mutations have been previously localized on the physical DNA map both proximal (5 ' ) and distal (3') of the pair-rule segmentation gene fushi tarazu Pz] (SCOTT et al. 1983; KUROIWA et al. 1985), indicating that sequences in both of these areas are important for expression of the Scr gene. To better define the extent of the Scr locus, we have determined the position of 20 Scr breakpoint mutations on the physical DNA map by probing digested genomic DNA with radiolabeled lambda phage DNA containing genomic inserts from the Scr region (Figure 1). These breakpoint mutations span an interval of approximately 70 kb extending from the 3' end of the Antp gene to 30 kb proximal (5 ' ) of theftz gene. Sc#ypO breakpoints, associated with the semilethal adult phenotype, map to a region between $r and Antp. ScPU1l breakpoint mutations, on the other hand, map proximal to@.

Gain-of-function lesions (ScrCoF) are located in the Scr transcription unit itself (&eT1, ScTiMsc), as well as in the regulatory region upstream of the identified 5' start site (LEMOTTE et al. 1989) both proximal [5'] (SclscXTz) and distal [3'] ( S c P ' , Sc1scXT3, S c P w ) toftr, or mapping within the f tz transcription unit VtzRf"). An additional gain-of-function lesion not featured on the molecular map, S C ~ , is a transposition that moves a large portion of the right arm of the third chromosome to the distal end of the left arm of the second chromosome (Table 1). No molecular lesion has been


detected for this chromosome when probing with overlapping lambda phage encompassing the region from the 3' exon of Scr to the 3' exon of Antp (see Figure 1). Cytological analysis suggests that the breakpoint in the ANT-C DNA is likely to be proximal (3') of the Scr transcription unit (84A4, 5). Moreover, the S c 2 mutation complements all Scr lesions tested, but fails to complement Dx3R)Scr, supporting a location for the S c 2 breakpoint outside the Scr locus but within the ANT-C. Nevertheless, adult males heterozygous for this lesion and a wild-type chromosome have ectopic sex comb teeth on both their mesothoracic and metathoracic legs, indicative of a transformation of these segments toward a prothoracic identity. Fur- thermore, the establishment of a correlation between the observed phenotype and ectopic accumulation of Scr protein in mesothoracic and metathoracic leg imaginal discs of third instar larvae suggests that S C ~ exerts its effect by causing the misregulation of the Scr gene in these tissues (PATTATUCCI and KAUFMAN 1991). The fact that breakpoint mutations affecting Scr function cover such an extensive region of DNA, coupled with data indicating that these lesions fall into discrete phenotypic classes corresponding to particular regions of this DNA interval, leads us to conclude that Scr possesses a large and complex regulatory region upstream of the identified transcription unit.

Intra-allelic complementation between Scr mutations: Given the extensive segment of DNA over which Scr lesions are found, the possibility exists that separate regulatory regions may be responsible for discrete developmental functions. For example, AB- BOTT and KAUFMAN (1986) reported that certain lesions at the Antp locus complemented each other, a finding that presaged the identification of the Antp P1 and P2 promoters (LAUGHON et al. 1986).

To better understand the complexity of the locus, Scr mutations listed in Table 1 were crossed inter se. The results of these complementation tests are presented in Figure 2. Scr', Scr", S c P T 1 , Dx3R)CPl, Df3R)Tpl9, Dx3R)Winll , Dx3RWAP7, Sc7sc*wm5, and Dfl3R)Dfd 13 either disrupt or delete all or portions of the Scr transcription unit. These have been designated as Scrnu" mutations (see MATERIALS AND METHODS). Other mutants in the ScrnU" category are cytologically normal and fail to complement each other as well as the above breakpoint alleles. All inter se genotypic combinations of ScrnuL' lesions die as embryos, however, they tend to show a semilethal phenotype in combination with members of Scrhfio similar to that observed for Sc1-hYP0/Dx3R)Scr.

Inter se crosses between the various Sc1-hUPo cytologically normal mutations result in partial complementation (see MATERIALS AND METHODS). These same lesions when crossed to ScrnU" mutations exhibit semilethality and show varying degrees of adult prothor-

Df(3R)RC7

Df(3R)A41

Df(3R)Scx4

FIGURE 2.-Complementation map of the Scr mutations and deletions used in this study. The mutations in this representation have been subdivided into three groupings: lesions with breakpoints mapping proximal (ScrP'""'""') or distal (Scrd'""') toft2 but having no discernable affect onftz gene function, and those mapping within the limits of the definedftz locus and associated regulatory region that affect both Scr andftz gene function. The bars under the loci indicated at the top of the figure show the complementation behavior of deletions which approach Scr from its distal side [Dfl?R)RC7, DJT3R)A41, Dfl3RJScx4 and Dfl3R)HuJ and from its proximal side [Dff3R)CPI, Dfl3R)Tpl9, Dff3R)WinlI, Dff3R)Dfdl3 and Dfl3R)Map7]. A dark solid bar represents a failure to complement; a single line indicates complementation. The& breakpoints S c F T r andftzRP', fail to complement deficiencies in both the ScrProXima' and

groupings, whereas other non-rearranged ftz alleles fully complement these deficiencies. A star depicts an Scr gain-of-function mutation. Dfd = Deformed;ftz = fushi tarazu; Antp = Antenna- pedia.

acic to mesothoracic and labial to maxillary palp transformations.

Interestingly, Dx3R)CPl and DfT3R)Tpl9 delete ANT-C DNA with distal limits including the Scr transcription unit but that are proximal toftz, and fully complement mutations in that locus (Figure 1). How- ever, these same deficiencies either fail to complement or show only partial complementation with Scrhypo mutations, some of which have been molecularly mapped distal toftz. Similarly, Dfl3R)RC7, Dj(3R)A41, and Dx3R)Hu delete ANT-C DNA with proximal limits mapping 3' ofpz, fully complement mutations inftr, but fail to complement or exhibit partial complementation with ScrnU" lesions. This is particularly striking for Dx3R)RC7, which extends only a short

scrdistd


TABLE 2

Percent viability in combination with DJT3R)RC7

0 %" 5-1 5 % 20-30% 100%

scr"' sCr%xT2 Scr' s c r l Scr2 scr%rT3 Scr' Scr' Scr' ftP ftZ"

Scr'" Scr"

SC$"W

Dfl3R)Dfd 13 Dfl3R)Tpl9 Dfl3R)win 11

sc,,ScxTI

" Percentages represent a minimum total sample size of 130 animals for each genotype tested.

distance proximal to the Antp 3' exon and has been previously reported to only have effects on Antp gene expression (KUROIWA et al. 1985). Nevertheless, our inter se analysis indicates that DJT3R)RC7 has significant effects on Scr gene function as well (Table 2).

This complex complementation pattern and molecular mapping are consistent with the view that DNA sequences associated with expression of the Scr locus can be found 40 kb upstream of the identified transcription start site for Scr (LEMOTTE et al. 1989). The S c P T 2 lesion is of particular interest in this light because its breakpoint has been located within theftz upstream regulatory region (see Figure 1). Hemizy- gous embryos that are ScFT2/DJT3R)Scr exhibit both a ftz- phenotype while simultaneously also displaying an ScP"" phenotype (see PATTATUCCI and KAUFMAN 1991). Since S c F T 2 (designated as Scr""") maps be- tweenftr and the 5' limit of the Scr transcription unit, but does not interrupt the defined transcription unit, we conclude that sequences on both sides offtz are necessary for normal Scr expression.

Recombinational mapping of Scr': The finding that Scr""" breakpoint mutations map proximal toftz, while Sc7hYpo breakpoints map distal, led us to question if cytologically normal mutations in these respective classes also map to discrete regions of DNA that are proximal or distal toftz. Using a set of 4-point crosses, we mapped by recombination Scr4, a representative of the Scr""" grouping. The results of this analysis are presented in Table 3.

Alleles used in mapping were: AntpZ5, Dfd', Dfd', f tz" and&'. These are all cytologically normal lesions that fully complement Scr mutations (see MATERIALS AND METHODS), and were used as markers for the two adjacent loci to Scr (Dfd and Antp) andftz, which maps within the upstream regulatory region of Scr. In addition, Scr4 was mapped relative to S c F W (Figure 1).

Recombinants were recovered for each of the four genotypes listed in Table 3 as well as for Dfd5/Scr4 andftz"/Scr4 (data not shown). The pattern of recovery of the flanking markers Kinked ( K i ) and pink-peach

(Pp) was consistent with the conclusion that Scr4 maps to the region of DNA that is proximal toftz and distal to Dfd ( i e . , the interval containing the Scr transcription unit). Additionally, the recombination frequencies for Dfd'-Scr4-ftz3 suggest that the distance between these lesions is approximately equal. This again is compatible with the placement of the Scr4 lesion some- where within the Scr transcription unit. This conclusion is consistent with Scr4 being a null allele that produces no detectable protein (RILEY, CARROLL and SCOTT 1987; A. M . PATTATUCCI and T. C. KAUFMAN, unpublished observations).

A much lower number of recombinants between Ki and Pp was recovered for the Scr4-ScpW mapping cross (a frequency of 2.1% compared to 4.1 % for all other crosses). However, suppression of recombination by the chromosomal inversion associated with the S c P W lesion could account for the reduced recombination frequency seen in this cross. Unfortunately, due to their semilethality, cytologically normal ScrhYPa mutations are not good candidates for this type of recombination mapping and their position relative to f tz remains conjectural.

Temperature-sensitive period: Two temperature- conditional Scr mutations have been recovered, Scr6 and Scr*. Both lesions behave essentially as a wild-type homolog over Dfl3R)Scr at the permissive temperature (25") exhibit slight decreases in viability at 29", but show polyphasic lethality at the restrictive temperature of 18" (see Table 4). The majority of the lethality occurs at the late embryo-first larval instar boundary, but a small percentage of individuals survive to adulthood. Depending on culture conditions (e.g., crowd- ing) the percentage survival of heterozygous combinations of these temperature-conditional mutations with Dfl3R)Scr ranged between 1 and 1 1 %. Surviving adults show oppositely oriented labial to maxillary and prothoracic to mesothoracic transformations (see Fig- ures 6, 7 and 8 for examples). For example, eclosing males are often completely devoid of sex comb teeth on their prothoracic legs. Because neither allele shows complete lethality at the restrictive temperature, we were unable to determine the temperature-critical period for viability at any stage.

The semilethality at the restrictive temperature allowed the determination of the temperature-sensitive period for the specification of adult prothoracic identity by assaying the number of sex comb teeth produced on the prothoracic legs of adult males. To broadly define this interval, a series of 24-hr. egg collections were taken from the cross of ScrB/TM3,Sb X Dfl3R)Hu/TM3,Sb at 18" or 29". These cultures were maintained at the collection temperature for varying durations of time and then shifted to the reciprocal temperature regime. The adults eclosing from this cross were scored for Sb+ and Sb, the former

430 A. M. Pattatucci, D. C. Otteson and T. C . Kaufman

TABLE 3

Results of selected recombination crosses between Scr mutations and adjacent loci

Total progeny scored

Heterozygous female genotype

No. of No. of Rec." Rec."

Ki X Pp 1 b x 1' order Left/right

Ki Scr' pp + ftz' + K i Dfd' roe p p

+ Scr4 + + K i Scr' + + Antpz5 p p

K i Scr4 + + ScrScxW p p

17.665

5,517

6,585

1 1,230

724

226

270

236

2

3

Scr-jtz

Dfd-Scr

Scr-Antp

Scr-Sex

Number of recombinant chromosomes recovered. ~ c r ' . Tester locus (ftz', Dfd', AntPZ5 or S C V ~ " " ~ ) .

identifying the mutant class. The results of this analysis are presented in Figure 3. Animals shifted from 29" to 18" during the first 4 days yielded adult males essentially devoid of sex combs. However, on day 5, an abrupt change in the mean number of sex comb teeth on the prothoracic legs was observed (0.5-3.5), corresponding to a developmental interval ranging from late L3 to the early pupal stage (immediately following apolysis). The mean number of 3.5 sex comb teeth remained constant for further temperature downshifts after the 5th day. Animals shifted from 18" to 29" yielded adult males with an average of four sex comb teeth on their prothoracic legs until approximately day 5, when a gradual decline in mean sex comb tooth number was observed. This trend continued until the 1 lth day, when an abrupt change

(from 3 teeth to 0) took place and this significant reduction in mean sex comb tooth number was maintained for subsequent temperature shifts.

To more precisely define the temperature-critical period within the L3 to pupal interval, a series of temperature pulse shifts was performed. For this set of experiments, we replaced the DJ3R)Hu chromosome with DJ3R)A41, which allowed for a higher percentage of the mutant class to reach adulthood. Larvae and pupae were collected and staged (see MATERIALS AND METHODS), and a series of 12-, 24-, and 48-hr pulses from 18 O to 29" was performed. The results of this analysis are diagrammatically illus- trated as a solid bar in Figure 3. These pulse experiments have defined the temperature-sensitive period for the specification of the adult prothoracic identity

1 2 3 4 5 6 7 8 -

I I I I I I I I

5.0 - - Shift Down

o..---"o Shift Up

4.0 - z t

\o/H \ v) c

\/'O'\/\

TSP - I-

! 0

- e \ 3.0

x

L

B - 2.0 a z E al I

1.0

'I\.*/ - /~"; I I I I I I I I I I I I I I I I I I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 -

Embryo Instar hstar 1st 2nd 3rd Instar 1 4 Pupa Adult -

FIGURE 3.-Temperature-sensitive period for the establishment of the adult prothoracic identity assayed by the production of sex comb teeth on the first tarsal segment of the prothoracic legs of adult males. Open circles connected by hatched lines represent temperature shifts from 18" to 29". and solid circles connected by solid lines depict temperature shifts from 29" to 18" for the genotype Sc$/DfljrR)Hu. The solid bar marked TSP represents the precise temperature-critical period for the establishment of the adult prothoracic identity determined using the genotype Scr8/Dx3R)A41. PP = prepupa.

Scr Structure and Function 43 1

TABLE 4

Summary of effective lethal phase studies on selected Scr mutations

Percentage lethalityb

Lethal genotype" Class Eggs Embryonic Larval Pupal Total Lethal phasec

OreRITM6B Control 400 15.8 18.7 10.3 44.8 Scr4/Dx3R)Scr Null 400 22.6 10.6 13.8 27.9 E ScTM"/Scr' Null/GOF 400 16.0 9.0 0 23.5 E Dff3R)Hu/Dj(3R)Tpl9 HYPO 500 0 24.2 0.6 24.6 Poly Dj73R)A4l/Djt3R)Tpl9 HYPO 400 0 19.4 3.3 22.2 Poly DfT3R)RC7/Dj(3R)Tp19 HYPO 600 0 15.4 3.0 17.8 Poly Scr5/Dx3R)Scr HYPO 440 14.7 4.9 4.3 22.3 Poly Scr6/Dx3R)Scr (1 8 o ) d HYPO(tS) 310 16.6 2.9 4.1 22.6 E-poly Scrs"'"/D~3R)Tp19 Hypo/GOF 400 11.3 4.2 1.6 16.2 Poly ftz'p'/scr' Hypo/GOF 360 30.6 1.9 0 31.9 E/L 1 ~ c r w 3 /Dx3R)Hu Hypo/GOF 480 1.9 27.1 0.2 28.7 Poly

a Tested chromosome is presented first; both third chromosomes were transmitted from parents heterozygous with TM6B. Dominant lethality associated with the TM6B chromosome is reported in ABBOTT (1984).

Data presented for experimental crosses have been corrected by subtracting the control percentage lethality from the percentage lethality ;~ctually observed. Totals represent the combined number of lethal animals from all stages minus control lethality divided by the total number of eggs counted.

' E = embryonic; L1 = first larval instar; poly = polyphasic. Genotypes were designated embryonic lethal if there was an absence of Tb+ larva and polyphasic if Tb+ larva were present.

Sere is a temperature-conditional cold-sensitive allele; restrictive temperature is 18'.

to an interval that spans late L3 (approximately 12 hr prior to pupariation) to the mid-pupal stage (approximately at head eversion).

At the present time, lesions that result in embryonic Scr gain-of-function phenotypes have not been recovered. Instead ScrCoF lesions show differential behavior, atting as Scr loss-of-function mutations in the embryo, while exhibiting gain-of-function phenotypes in adults. The fact that the temperature-sensitive period for the specification of adult prothoracic identity occurs during the late larval to mid-pupal stages may account for the apparent differential behavior of ScrCoF mutations in embryos as compared to adults.

Effective lethal phase and embryonic phenotype: Our complementation analysis has revealed that Scrnu" mutations are lethal as hemizygotes or homozygotes, while Sc#"f'' lesions show a semilethal phenotype. In order to determine when members of both groups die during development, effective lethal phase studies were performed for representative genotypes. The results of this study are presented in Table 4. Scrnu" hemizygotes die as embryos, the major lethal phase occurring late in embryogenesis, at approximately stage 16- 1 7 (CAMPOS-ORTEGA and HARTENSTEIN 1985). In contrast, Scr"Yp' lesions show polyphasic lethality. For these mutations, the majority of the lethality occurs either in late embryogenesis or through- o u t the larval stages.

Because Scrnu" mutants die as late embryos, cuticle and SEM preparations of these animals were analyzed to determine if morphological abnormalities were present. Normal embryonic development is described in CAMPOS-ORTEGA and HARTENSTEIN (1985); for a

review of head segmentation, see DIEDERICH et al. (1 989). The following is a brief description of some of the salient features of anterior development affected by Scr""" mutants. During normal embryonic development, a small group of cells in the central region of the labial segment invaginate to form the salivary gland placodes. Head involution then begins at about stage 13 with the anterior movement of the gnathocephalic segments. The labial segments fuse at the ventral midline and move anteriorly, ultimately involuting into the stomodeum where they form part of the floor and sides of the oral cavity. Simultane- ously, the mandibular and maxillary lobes rotate dor- soanteriorly, eventually fusing with each other and the procephalic lobe. The dorsal fold then advances over the procephelon to form the dorsal pouch. Sub- sequently, the clypeolabrum and procephalic lobe involute (TURNER and MAHOWOLD 1976). The mandibular segment forms the sides of the oral cavity and ultimately gives rise to the dorsolateral papilla of the terminal sense organ. The maxillary segment remains outside the oral cavity and gives rise to the mouth hooks, the maxillary cirri, and a portion of the terminal sense organ (TURNER and MAHOWOLD 1976; FREDERICK and DENELL 1982; JURGENS et al. 1986).

Figure 4 presents SEM of the anterior end of Scr""" hemizygous embryos during early through late head involution. These embryos exhibit a general failure in that process. At first appearance, all three gnathocephalic segments of ScrnU" hemizygous embryos (mandibular, maxillary, and labial) are present (Figure 4A). There is, however, an apparent decrease in the size of the labial segment and no evidence of a salivary


FIGURE 4."Scanning electron micrographs of the anterior end of Scr'lDfl3R)Scr embryos during early (A) progressing through late (F) head involution. CI = clypeolabrum; Ma = mandibular segment; Mx = maxillary segment; Lb = labial segment.

invagination (Figure 4B). The labial segments do not migrate to the ventral midline and fuse. Instead the labial segments tend to coalesce with the maxillary segment. There is also a general failure in the dorsoanterior movement and fusion of the maxillary and mandibular segments with the procephalic lobe (Fig- ure 4, C, D and E). As a result, all three gnathocephalic segments fail to involute through the stomodeum and remain external throughout cuticle formation (Figure 4F). The reduced size of the labial segment and its inappropriate fusion with cells of the maxillary segment is consistent with the previous observation of WAKIMOTO and KAUFMAN (1981) that loss of Scr+ function results in a homeotic transformation of cells in the labial segment toward a maxillary identity. Interestingly, ScT"YPo hemizygous embryos appear to develop normally and do not show evidence of the morphological aberrations described above.

Figure 5 shows the embryonic cuticular phenotypes of representative Scrnu" and ScT"*' genotypes. Scr'*/ Dfl3R)TpZ9 (Figure 5D), Sc?lfttzRP' (Figure 5F) and

tions which display a cuticular phenotype representative of the null state for Scr. The anterior T1 denticle belt is reduced and resembles that of T2 (stemmed arrows in Figure 5, D and F) and the H piece of the mouth parts is absent (compare Figure 5, A with D). The goatee bristles are significantly reduced (open stars in Figure 5, D and F). Furthermore, the posterior portion of the mouth hooks is absent and a scleratized plate resembling the anterior portion is characteristically seen duplicated more posteriorly in the head. The SEM of an S~?~'/Dfl3R)Scr animal (Figure 5E) shows the duplication of a portion of the maxillary sense organ on the transformed labial lobe. However, the antennal sense organ is not duplicated. This labial to maxillary transformation is most clearly seen in the enlarged inset within Figure 5E, which is Scr"/ Dfl3R)Scr. Additionally, there is a duplication of the maxillary cirri on the labial lobe (large stemmed arrow in Figure 5E), providing further evidence for a labial to maxillary transformation in these animals. We con-

SC.,.%-xTI lDfl3R)Scr (Figure 5E) are mutant combina-

FIGURE 5.-Embryonic cuticle preparations of various genotypes demonstrating the loss of Scr function. These animals exhibit homeotic transformations of labial to maxillary and prothoracic to mesothoracic identities. Small arrows point to the leading edge of the prothoracic and mesothoracic segments; open stars identify the position and presence of goatee bristles on the prothoracic segment. mh = mouth hooks; mc = maxillary cirri; aso = antennal sense organ; mso = maxillary sense organ. A, Dfl3R)RC7/Dfl3R)Tpl9. B, Dfl3R)A4l/Dfl3R)Winl I . C , Dfl3R)Hu/Dfl3R)Tpl9. D, Sc+/jkRp'; E. ScrS'"''/Dfl3R)Scr. Inset: Scr"/Dfl3R)Scr. Star identifies an ectop icly produced maxillary sense organ on the labial segment. F, Scr''/ Df3R)Tpl9.

clude from these observations that Scr gene function is required during mid to late embryogenesis for normal head involution to occur and for the proper formation of embryonic prothoracic and head structures, particularly those derived from the labial segment.

T o determine if the above embryonic abnormalities are also associated with SCT"*~ mutations, we examined embryonic cuticle preparations of various genotypic combinations of Scrnu" and ScT"*O lesions. The results of this analysis are presented in Figure 5. Dfl3R)RC7/ Dfl3R)TpZ9 (Sc71?yp0/Scrnu") embryos are essentially wild-type in appearance (Figure 5A). The denticles at the anterior edge of the first thoracic segment (small arrow) and mouth hooks (large arrow) appear normal. Additionally, the proper position and presence of goatee bristles, which are diagnostic of the first thoracic segment, indicates that ostensibly normal prothoracic development is occurring in these animals. Comprehensive examination of Sc?*O/Scrnu" mutant combinations has revealed that the phenotypes of these animals is variable and that this variability is due to the Sc@" mutation.

Scr"*o/Scr"u" embryonic phenotypes range from wild-type in appearance to cuticular defects indicating that a weak or moderate prothoracic to mesothoracic and labial to maxillary transformation is occurring. Dfl3R)A41/Dfl3R)Winll (Figure 5B) and DfT3R)Hul Dfl3R)TpZ9 (Figure 5C) demonstrate the phenotypic variability observed for animals carrying deletions with breakpoints mapping distal to$%. The constella- tion of defects observed for both genotypes is variable; any given individual can exhibit all or none of them. For example, the anterior T1 denticle belt may be


FIGURE 6.-Scanning electron micrographs of adult heads. A, Head ofan OreRadult. B, Close-up view of OreR adult head showing the labial and maxillary palps. C , Head of an ScrX/ScrY adult grown at 18" (restrictive temperature). D, Close-up of an ScrR/ScrY adult grown at 18" (restrictive temperature) showing a homeotic transformation of the labial palps toward a maxillary identity.

reduced as in Figure 5C or not reduced as in Figure 5B. The goatee bristles may be reduced as in Figure 5B or not reduced as in Figure 5C. The posterior portion of the mouth hooks may be absent as in Figure 5B or present as in Figure 5C. Additionally, there may be a partial duplication of the maxillary sense organs in Sc?/?ypo/Scrnu" animals (data not shown).

Adult phenotypes associated with Scr losssf-function: Previous studies have suggested that the Scr+ gene product functions to promote normal develop ment of the prothorax and labial palps in adults (KAUF- MAN, LEWIS and WAKIMOTO 1980; LEWIS et al. 1980b; WAKIMOTO and KAUFMAN 198 1 ; STRUHL 1982; KAUF- MAN 1983; STRUHL 1983; SATO, HAYES and DENELL 1985). LEWIS et al. (1980a,b) reported an abnormal proboscis phenotype associated with adult survivors of the semilethal mutant alleles Scr) and Scr5 when heterozygous with Df3R)Scr. They found that the six pseudotracheal rows in a wild-type proboscis were reduced to three or less. Additionally, the lateral aspect of the labial palps exhibited an increase in the number and pattern of bristles to resemble a maxillary palp. We have extended the phenotypic analysis of adult hemizygotes carrying Scr"YPo lesions (Figures 6, 7 and 8). In the present study, we used the temperature-sensitive mutation Scr', which exhibits cold-sensitivity, although other Sc&" mutations were tested and gave similar results. Specifically, the lateral portions of the labial palps of Scr'/ScrY individuals, grown at 18", are transformed to resemble maxillary palps and the number of pseudotracheal rows is reduced relative to +/+ (compare Figure 6, C and D, with A and B). STRUHL (1 982) reported similar transformations for Scr- adult somatic clones.

We also examined the anterior and lateral aspects of the thoraces of Scr'/Scry mutant adults (Figures 7, B and D, and 8B) as compared with +/+ (Figures 7, A and C, and 8A). The anterior dorsal thorax of wild- type adults consists of the pronotum (solid triangles in Figure 7A), which is closely fused with the meson-

FIGURE 7.-Anterior view of adult dorsal and ventral thoraces. A, Anterior dorsal thorax of an OreR adult with black arrows pointing to the pronotum. B, Anterior thorax of Scr8/Scr9 grown at 18" (restrictive temperature). Stars indicate areas of ectopic mesonotal tissue; large arrows point to ectopic sternopleural bristles on the preepisternum: small arrow identifies an ectopically placed mesosternal bristle on the propleural plate. C , Ventral view of an OreR adult showing the region of the pleura. The star identifies the propleural plates; small arrows point to the mesosternal bristles located on the mesosternal plate; co = coxa. D, Ventral view of the region of the pleura of an ScrR/Scr' adult grown at 18" (restrictive temperature). Large arrows point to ectopically placed sternopleural bristles on the preepisternum; small arrow above the coxa (co) identifies and ectopic mesosternal bristle on the propleural plate; small arrows below the coxa point to normally positioned mesosternal bristles on the mesosternal plate.

otum. The anterior region of the mesonotum is called the prescutum. This consists of a narrow band along the mesonotum that swells out on each side to produce the humeral callus. The anterior third of the prescutum consists of smooth cuticle that is largely devoid of bristles. Moving posteriorly, this smooth cuticle is covered with approximately ten rows of moderately dense microchaetae, and is finally followed by dense microchaetae with interspersed macrochaetae including the notopleural and presutural bristles. Directly posterior to the prescutum is the scutum of the mesothorax, which occupies the largest portion of the mesonotum. This structure is densely populated with both microchaetae and macrochaetae. Proceeding ventrolaterally from the pronotum is the propleurum, which consists of the preepisternum laterally and the propleural plates ventrally. Together the pronotum and propleurum surround the cervical sclerites, which serve to link the head and the prothorax. This region is characteristically covered with microchaetae but contains no macrochaetae. Proceeding posteriorly, is the region of the mesopleurum, which is demarcated ventrolaterally by the dorsal coxal condyle, the pleural cleft, and the mesopleural suture. Within this region, are the two mesosternal bristles located ventrally (arrows in Figure 7C) and the characteristic pattern of 2 long and 4 to 5 shorter macrochaetae located on the lateral aspect of the mesopleurum (arrow in Figure 8A) known collectively as the sternopleural bristles (DEMEREC 1950; BRYANT 1978).

Examination of the anterior thoraces of Scr*/Scry mutant adults reveals that the dorsal aspect of the


FIGURE 8.-Lateral v i w . 01' ; c d u l t tllolxes. A, Lateral view of the region of the pleura of an Oreli adult. Arrow is pointing to the nornlal tlistril)ution of sternopleural bristles on the mesopleura. B, I.ateral view of the region of the pleura of an Scr'lScr" adult grown at 18' (restrictive tenlperature). Large arrow points to the mesopleura with associated normally positioned sternopleural bristles: small arrow identifies an ectopically placed sternopleural bristle on the preepisternum. C, Lateral view of the region of the pleura of an Scr""/Scry'x'' adult (Sc~'~' '~/Scr'~' '~) . The star marks the area of the mesopleura. These animals show a complete absence of sternopleural bristles, indicating a homeotic transformation of mesopleura to propleura.

pronotum is absent and replaced by folds of cuticle covered with microchaetae (stars in Figure 7B) similar to those found on the dorsal mesothorax. A single long macrochaeta, and two or three shorter macrochaetae are also ectopically located in the preepister- nal region of the propleura just dorsal to the prothoracic coxa on both sides of the animal (large arrows in Figure 7, B and D). These are reminiscent of the sternopleural bristles found in the lateral region of the mesopleura (compare Figures 7B and 8 B with Figure 8A). The striking resemblance of these ectopic bristles on the preepisternum to sternopleural bristles is most graphically represented in Figure 8B by comparing the normal sternopleural bristles located on the lateral mesopleurum (large arrow) to the ectopic bristles found on the preepisternum (small arrow). In addition to these defects, an apparent ectopic mesosternal bristle appears on the propleural plate (small arrow in Figure 7B). The small arrows in Figure 7D identify both the ectopically placed mesosternal bristle on the propleural plate as well as the normal complement of mesosternal bristles on the ventral aspect of the mesopleurum. The first tarsal segment of the prothoracic legs is completely devoid of sex combs for males of this genotype (ScrR/Scr'" grown at 18 "). Fi- nally, there is a transformation in the shape of the prothoracic coxal leg segment and propleural plates

to one that more closely resembles the mesothoracic coxal leg segment and the mesosternum respectively (compare Figures 7, A and C, with B and D).

We interpret the group of defects associated with loss of Scr gene function in adults to represent oppositely oriented homeotic transformations moving from a labial to a maxillary identity in the head and a prothoracic to mesothoracic identity in the thorax. These transformations apparently encompass both dorsal and ventral tissues in the affected areas of the adult. An analogous pattern of homeotic transformations appears to occur with Scrnu" mutations in the embryo. However, the semilethal Scr/l-yPo mutants generally lack embryonic defects or transformations and appear to predominantly show evidence of Scr loss of function in the adult.

Analysis of the adult dominant gain-of-function phenotype: Our phenotypic studies have indicated that, as heterozygotes, certain Scr lesions exhibit a dominant ectopic sex comb tooth phenotype on mesothoracic and metathoracic legs of adult males. We have interpreted this to represent a mesothoracic and metathoracic to prothoracic homeotic transformation. To determine the extent of the transformation, we counted sex comb tooth number in their normal position on the prothoracic legs as well as assayed num- bers of ectopic sex comb teeth present on the mesothoracic and metathoracic legs of adult males. More- over, we examined the adult dorsal and ventral thoracic cuticle for further evidence that a transformation toward a prothoracic identity had occurred. Table 5 presents an analysis of sex comb tooth number on the legs of adult males carrying various mutations at the Scr locus. In addition to ectopic sex comb teeth on adult males, each of the dominant gain-of-function lesions in both males and females has associated with it a disruption or complete removal of the sternopleural bristles (star in Figure 8C), and mesosternal bristles are characteristically absent or disrupted. Moreover, the mesothoracic and metathoracic coxa exhibit transformations in shape that more closely resembles the coxa of the prothoracic leg (not shown).

Effects of Polycomb mutations on Scr gainsf-function alleles: We also examined the phenotypic expression of these alleles in a Polycomb (PC) mutant background, another haploinsufficient gene. Mutant alleles at the PC locus have been documented to result in the production of ectopic sex combs on the mesothoracic and metathoracic legs of adult males (DENELL 1978; LEWIS 1978; CAPDEVILA and GARCIA-BELLIDO 198 1; STRUHL and AKAM 1985; WEDEEN, HARDING and LEVINE 1986). We have shown that a direct correlation exists between the production of ectopic sex comb teeth resulting from mutations in PC and the misregulation of the Scr gene (PATTATUCCI and KAUF- MAN 1991). The present study was performed to


TABLE 5

Analysis of sex comb tooth number on the legs of males carrying selected Scr lesions

PC+/PC+ PCS/PC+ Number of sex comb teeth" Number of sex comb teeth"

Genotype Class T1 T2 T3 T1 T2 T3

+I+ ftz"/+ - b 10-12 0 0 10-12 0-2 0- 1

b - 10-12 0 0 8-12 0-2 0- 1

Dfl3R)Scr/+ - b 4-6 0 0 4-8 0-3 0

. b y / + Null 4-6 0 0 6-8 2-4 0 Scr'"/+ Null 4-6 0 0 4-6 0-2 0 S C F / + Null/GOF 4-6 0-3 0-2 6-8 2-6 0-6 Scr'/+ HYPO 6-8 0 0 6-8 0- 1 0 Scr7/+ HYPO 10-12 0 0 10-12 0 0 scrycxp/+ Hypo/GOF 7-10 0-3' 0-2 8-1 2 8-10d 4-8 Sed/+ GOF 8-10 0-4' 0-2 10-12 6- 10' 4-8 Scr""U/+ Hypo/GOF 6-10 0-6' 0-3 10-12 10-12d 3-10 ftZ"P'/+ Hypo/GOF 8-10 0-2 0 10-12 4-6 4-6 S c r W l /+ Null/GOF 6-8 0 0 4-6 2-6 0-6 Scr\"'Z /+ Null/GOF 8-10 0 0 10-12 4-6' 0-6 Scr\"73 /+ Hypo/GOF 6-8 0 0 4-6 2-4 0- 1

' Numerical values represent the range of sex comb tooth number found on a sample size of 40 legs. ' Control genotype. . ' Sternopleural bristles disrupted and/or reduced.

1

Sternopleural bristles missing.

determine if Pc mutations enhanced any of the dominant gain-of-function alleles or were capable of res- cuing the reduced sex comb phenotype associated with loss-of-function lesions. The results of this analysis are also presented in Table 5 . It would appear that the reduction in sex comb tooth number on the prothoracic legs of adult males associated with Scr loss-of- function cannot be rescued by the misregulation of Scr through a mutation in PC. This is most clearly seen when comparing Dfl3R)Scr/+ to DJT3R)ScrlPc. For both genotypes, the number of sex comb teeth on the prothoracic legs remains essentially the same. Never- theless, the presence of ectopic sex comb teeth on the mesothoracic legs of Dfl3R)ScrlPc individuals clearly indicates that misregulation of Scr is still occurring. On the other hand, the dominant gain-of-function lesions Sc?, S c P p , S c P W and Sex? are all significantly enhanced in a PC mutant background. These animals characteristically have a complete absence of sternopleural and mesosternal bristles with an accompanying change in shape as well as bristle pattern of the mesothoracic and metathoracic legs to closely resemble prothoracic legs. In addition, wings tend to be held out at approximately 90" relative to the thorax, and often there are a complete or nearly complete complement of sex comb teeth present on all six legs of adult males. Interestingly, S c P T 2 , f t z R P 1 , and S e e T 3 show little, if any, evidence of an Scr gain-of-function phenotype when heterozygous with a wild-type chromosome but do so in a PC mutant background. Each of these alleles, when present with a PC mutation, confers an ectopic sex comb tooth number that is significantly enhanced above that

normally seen with PC/+. It would appear that these mutations represent dominant gain-of-function at the Scr locus that is in some way "masked" unless they are in certain mutant backgrounds. Supporting gain-of- function for these lesions as opposed to merely en- hancing the Polycomb mutation in some manner is the fact that Scr' and Scr" exhibit ectopic sex comb tooth phenotypes when in combination with PC' that are not significantly enhanced above the control genotypes. However, the S c P T ' lesion is both similar in type and breakpoint position to Scr' and Scr" (Figure l), yet ScJcxT' displays ectopic sex teeth on the mesothoracic and metathoracic legs that are significantly enhanced in number above the control genotypes.

Phenotypic analysis of ScflcXw revertant alleles: A number of X-ray-induced lesions had previously been recovered that to some degree revert the dominant gain-of-function phenotype associated with S c P W . An extensive genetic analysis of these lesions is presented in HAZELRICC and KAUFMAN (1 983). These mutations appear to revert the S c P w gain-of-function phenotype by superimposing a second chromosomal rearrangement on the first. The position of the breakpoints of the S c F W revertant lesions relative to the original S c P w lesion on the DNA map, combined with an analysis of the degree to which each mutation reverts the gain-of-function phenotype should provide useful information regarding how the gain-of-function phenotype is conferred. The location of these lesions on the Scr molecular map is presented in Figure 1. Additionally, sex comb teeth were counted to ascertain the extent that each of these lesions reverts the gain-of-function phenotype (Table 6). Dfl3R)Scx4


TABLE 6

Analysis of sex comb tooth number for selected revertants of S c P w gain-of-function allele

Number of sex comb Position of rever- teeth" sion breakpoint

Genotype relative to& TI T 2 T3

scr\xmm' lTM3 Proximal 5-9 0-5 0-2 lTM3 Proximal 5-7 0-2 0-2

Proximal 4-8 0-3 0-1 Distal 6-10 0-1 0

DJ3R)Scx4/TM3 Distal 6-9 0 0

These selected data were originally presented in HAZELRICC and

Numerical values represent the range of sex comb tooth num-

Scr\"\V"'

&r\"'Vmo lTM3 Scr\"""f ITM3

KAUFMAN (1 983).

ber found on a sample size of 40 legs.

seems to be the only lesion that completely reverts the gain-of-function phenotype. We find this to be particularly notable because this deficiency removes Scr sequences located at the distal breakpoint of the S c F W chromosomal inversion situated just 5' of the Antp P2 promoter, while leaving sequences related to the proximal breakpoint found in the Scr locus intact. This suggests that both breakpoints associated with this chromosomal inversion contribute in some way to the gain-of-function phenotype. Generally, it would appear that reversion chromosomes with breakpoints distal t o j z more efficiently revert the gain-of-function phenotype conferred by S c F W than do those with breakpoints that are proximal toftz. In fact, Sc7scXww3,

are proximal tojtz, have reductions in sex comb tooth number on their prothoracic legs that parallel those previously observed for many of the S c P " alleles. Therefore, these lesions may not actually revert the S c F W gain-of-function, but instead cause a reduction in the number of ectopic sex comb teeth due to the introduction of a null lesion on the S C ? " ~ chromosome.

Analysis of Scr gain-of-functionlscr loss-of-function heterozygotes: We examined various Scr heteroallelic combinations to determine if the dominant gain-of-function phenotype associated with ScrCoF mutations could be suppressed by ScrnU" or Scrhypo lesions. The results of this analysis are presented in Table 7. Heteroallelic combinations were made both with the Scrrcxw gain-of-function chromosome and the Dx3R)Scx4 revertant chromosome. This enabled us to determine if Dx3R)Scx4 actually reverts the gain-of- function phenotype or if it is in some way "masked" similar to the situation seen with the S c p T ' , S c p T 2 ,

ftz'p', and S c F T 3 lesions. Combinations of representative Scr lesions with Dx3Rpcx4, reveal that this mutation truly reverts the S ~ V ~ ~ ~ ~ gain-of-function phenotype. Ectopic sex combs were observed only for

ftzRp'/Dx3R)Scx4. However, this phenotype could be easily due to theftz'p' lesion itself (see Table 5). For

sc+scxww5 and Sc7scXwN6, which all have breakpoints that

each of these genotypes, there is also a reversion of the defects in the sternopleural bristles, providing further evidence for reversion of the gain-of-function phenotype. We find it notable that heteroallelic combinations between PC' and S c F W significantly enhance the presence and number of ectopic sex comb teeth, whereas combinations between Pc3 and Df3R)Scx4 show reductions consistent with the phenotype characteristic for PC"/+ (compare Table 5 , line 1 to Table 7, line 2). This strongly suggests that the Dx3R)Scx4 chromosomal rearrangement reverts the gain-of-function phenotype associated with S c F W and is not acting like one of the "masked" gain-of-function mutations. Furthermore, the apparent additive effects of the PC" and S c F W mutations, taken with the observation that Pc3 apparently still misregulates Scr in the presence of Dx3R)Scx4, implies that they may be affecting different aspects of Scr gene function.

Heteroallelic combinations of representative lesions with S c F W yielded complicated results. At first glance, it appears that the gain-of-function phenotype is completely suppressed for combinations involving Scr4 and Sc?, as males characteristically lack sex combs entirely. However, upon closer examination, Sc? /ScFW animals exhibit a very strong mesothoracic and metathoracic transformation to prothorax in all other aspects of the gain-of-function phenotype (e.g., the absence of sternopleural bristles) except the production of sex combs, suggesting that S c F actually enhances rather than suppresses the gain-of- function conferred by S c F W . Conversely, Scr4/Sc?scxw animals do not exhibit these striking aspects of mesothoracic and metathoracic to prothoracic and mesopleural to propleural transformation. Instead the gain- of-function phenotype appears to be suppressed for this genotypic combination (although a small percentage of adults do have disrupted sternopleural bristles). Other ScrnU" point lesions behave like Scr4, and in combination with S c F W produce phenotypes that are consistent with a suppression of the gain-of-function phenotype. However, Scrnu" breakpoint lesions in combination with Stew display phenotypes that we interpret to be an enhancement of the gain-of-function phenotype and therefore behave in a similar manner to Sc?. Evidence for this enhancement is difficult to ascertain by simply counting sex comb teeth because the loss of Scr gene function due to the Scrnu" lesion apparently reduces the number of ectopic sex combs found on the mesothoracic and metathoracic legs of males. However, the complete loss of sternopleural bristles, as well as a general increase in severity of the defects associated with the gain-of- function phenotype to resemble that observed with P c 3 / S c P W is consistent with the interpretation that heteroallelic combinations involving S c p W with rearranged Scr lesions enhance the gain-of-function phe-


TABLE 7

Analysis of sex comb tooth number 00 the legs of males carrying various heterozygous combinations of Scr alleles with S c P w and Dfl;R)Scx4

S C P W DA-7R)scx4 Number of sex comb teeth" Number of sex comb teeth"

Genotype Class T1 T2 T3 T1 T2 T3

p%"/- - b 8-10 0-6' 0 8-1 0 0 0 P 2 / - - b 10-12 10-126 3-10 10 0-2' 0- 1

scr"'/- Null/GOF 0 Od 0 0 0 0

ftz"P'/- HYPO 8-1 2 6-8' 6-8 4-6 0-3 0 1- Null/GOF 2-4 2-4' 2-4 0 0 0 l- Null/GOF 6-8 4-6' 4-6 4-6 0 0 1- Hypo/GOF 4-6 2-4' 0- 1 0 0 0

Scr'l- Null 0- 1 0' 0 0 0 0

Scr3/- HYPO 6 0-3' 0- 1 4-6 0 0

ScrY"" S c r S < X T 2

ScrY""

Numerical values represent the range of sex comb tooth number found on a sample size of 40 legs. * Control genotype. ' Sternopleural bristles reduced and/or disrupted on some individuals.

Sternopleural bristles absent.

notype; while presumed point mutations at the locus suppress. A detailed study addressing the seemingly inconsistent phenotypic results obtained with various ScrnU'l lesions when in combination with S c F W is presented in the accompanying analysis (see PATTATUCCI and KAUFMAN 1991).

Heteroallelic combinations of S c F W with Sc#YPo lesions do not show the variability in phenotype seen with ScrnU'l mutations. Scr/?ypo lesions tend to reduce the number of ectopic sex comb teeth associated with the gain-of-function phenotype but do not suppress their presence, and apparently fail to suppress the sternopleural phenotype. Results with Sc@'O mutations suggest that reductions in the expressivity of the Scr gain-of-function phenotype are possible through heteroallelic combinations of gain-of-function and loss-of-function Scr alleles, but that the penetrance of the gain-of-function remains essentially unaffected.

DISCUSSION

Sex combs reduced is a complex genetic locus: The objective of this investigation was to determine the effects of various Scr mutations on normal Drosophila development. The results of these studies have allowed the classification of Scr lesions into two broad categories. Mutations designated ScrnU" were shown to represent the null state of the locus based upon their recessive embryonic lethality and phenotypic abnormalities paralleling those previously described by DENELL et al. (1981). Sc#upo lesions have been classified as hypomorphs based upon their exhibiting recessive semilethality that is polyphasic. Adult survivors in this group display oppositely oriented labial to maxillary and prothoracic to mesothoracic homeotic transformations. However, these same genotypes show few or no embryonic defects. Mutations designated ScrCoF exhibit recessive behavior similar to what

has been described for ScrnU1l or Scrhupo, but also show dominant gain-of-function (neomorphic) phenotypes consisting of mesothoracic and metathoracic to prothoracic as well as mesopleural to propleural homeotic transformations. A subset of the ScrCoF lesions tend to show evidence of a gain-of-function component only when in heteroallelic combination with selected other ScrcoF lesions or a mutation at the Polycomb (PC) locus.

Molecular mapping of Scr breakpoint lesions has defined a region of >70 kb of DNA necessary for Scr gene function. Previous analyses have identified the Scr transcription unit as encompassing 25 kb of DNA (SCOTT et ai. 1983; KUROIWA et al. 1985; LEMOTTE et al. 1989). The remaining apparent regulatory sequences are split by the segmentation genefushi tarazu (ftz). Interestingly, Scrnu1' breakpoint lesions molecularly map proximal (5') offtz; whereas, Sc7hrPo breakpoint lesions map distal (3'). A cytologically normal Strnu" allele, Scr4, was also determined to map proxi- mally toftz. Although dominant gain-of-function mutations map throughout the Scr locus seemingly without respect to the position offtz (see Figure l), their recessive behavior also places them either in the Scrnul' or Sc#*O groupings. When viewed in this context, the positioning of the associated breakpoints relative to f t z shows strict adherence to the trend previously described for Scrnu" and Sc#*O mutations above.

Complementation studies involving the various Scr mutations have demonstrated that Scrnul1 lesions fail to complement each other for viability, but in most instances complement those lesions in the Scrh*' group. The exceptions are the deficiencies, DfTjrR)Hu, Dfl3R)A41, and Dfl3R)RC7. These lesions fully complement f t z mutations, but fail to complement most Scrnu1l lesions for viability and Sc#@O mutations for adult phenotype. This is most clearly seen in heterozygous combination of the above three deletions with


Dj(3R)CPl. The proximal end points of the former all fail to physically overlap the distal limit of the latter (see Figure 1). In contrast, Sc?scxp, S c F T 3 and Sc?scxww', also Scr"Jp0 lesions, complement Dj(3R)CPI for viability. Since these three lesions are all broken proximal to the largest of the three deficiencies discussed above and therefore displace more of the presumed 5' regulatory region of Scr, it is difficult to explain the complementation pattern on the basis of the removal of successively larger blocks of regulatory sequence collectively required for Scr expression. The one distinguishing feature between these two subgroups of rearrangements is that the deficiencies remove the Antp locus, while the translo- cation mutations leave it intact. This raises the possibility that Antp function is required in some fashion for the proper "regulation" of Scr. The validity of this speculation awaits experimental testing. In any event, we can safely conclude from the available data that Scr possesses a large, complex 5' regulatory region that extends minimally to the 3' exon of the Antp gene, with important elements located both proximal ( 5 ' ) and distal (3') relative to&.

Scr is responsible for distinct developmental functions: Scanning electron micrographs of embryos hemizygous for Scr""" mutations show homeotic transformation of the labial lobes toward a maxillary identity and a general failure in head involution. The embryonic lethality associated with these genotypes can probably be directly accounted for by the labial to maxillary transformation, which sets up a cascade of aberrant morphogenic events that result in a failure of head involution. Rather than migrating to the ventral midline, the cells of the labial lobes are incorporated into the maxillary lobes, again suggesting that these cells have been transformed to a maxillary identity. Dorsoanterior migration and subsequent fusion of the maxillary and mandibular lobes with the procephalic lobe also occurs abnormally. One explanation for this is that the maxillary lobes are now significantly increased in size and cell number, thus mechanically preventing normal migration and fusion. Alterna- tively, transformation to a maxillary identity may not be complete for the labial lobe cells "recruited" into the maxillary lobe and they could still retain some labial identity. In this way, failure of migration and fusion might be due to "recruited" cells within the maxillary lobe attempting to move in two different directions. At present, we are unable to distinguish between these two possibilities. Embryonic cuticle preparations of Scr""" hemizygotes show that a homeotic transformation of prothorax toward mesothorax is also occurring in these genotypes.

ScrhYPo hemizygotes are semilethal. This lethality is polyphasic, the major lethal phase occurring during the larval stages. Adult S C . " ~ ~ " / D ~ survivors display

cuticle defects and bristle patterns indicative of labial to maxillary, prothoracic to mesothoracic and propleural to mesopleural homeotic transformations analogous to those previously described for Scr""" hemizygotes in the embryo. Nevertheless, Scd'YPO hemizygous embryos show little, if any, evidence of a homeotic transformation. Because Scrnu" and ScrhJpo breakpoint mutations molecularly map to distinct regions within the Scr locus that are 5' and 3' offtz respectively, it is possible that Scr"Jf" lesions still contain DNA sequences required for Scr embryonic function, but lack or disrupt sequences necessary for an adult specific function. Unfortunately, neither of the temperature-sensitive alleles display complete lethality at the restrictive temperature. Therefore, it has not yet been possible to determine two discrete temperature-critical periods for viability. It is also possible that the differential affects on adults us. embryos in the ScrnuL' and Scrh*' lesions results from a quantitative defect on the Scr gene product. That is, the adult or imaginal requirements for Scr are more stringent than those of the embryo. The fact that the adult responds phenotypically to a heterozygous deletion of the locus while the embryo does not is consistent with this possibility. The actual explanation however, awaits further genetic and molecular testing.

Based upon the results of the investigations described above, we conclude that the most easily discernable role of the Scr gene product is in the establishment of prothoracic identity. Nevertheless, Scr is unique among the ANT-C genes in that its function is also required for normal head development. Loss of Scr gene function results in oppositely oriented head and thoracic transformations. Thus, Scr can be thought of as a "border" gene in that the leading edge of the prothoracic segment and the trailing edge of the labial lobes, mark the embryonic "border" between the head and trunk. Interestingly, the product of the proboscipedia ( p b ) gene of the ANT-C is also expressed in the labial and anteriorly into the maxillary lobes of the developing embryo (PULTZ et al. 1988). Therefore, it too can be thought of as acting at the border between head and thorax. Recent evidence has demonstrated that the p b gene product directs maxillary development in the adult (D. L. CRIBBS, F. M. RANDAZZO and T. C. KAUFMAN, manuscript in preparation). We find it noteworthy that loss of Scr gene function in both the embryo and adult results in a homeotic transformation of labial toward maxillary identity. This result suggests that Scr and p b function independently to direct prothoracic and maxillary identity respectively, but in some manner may function together to establish labial identity. However, in the embryo at least one other gene product must be involved in the specification process because loss of p b function at this stage of development


does not result in the labial lobe being transformed toward a prothoracic or any other identity (PULTZ et al. 1988). At the present time, the best candidate would appear to be the product of the spalt (sal) gene. Loss of sal function has been demonstrated to result in a partial embryonic labial to prothoracic homeotic transformation (JURCENS 1988). Therefore, one function of Scr may be to act collectively with sal and pb to establish labial identity and effect the final refine- ment of the cephalic and thoracic boundary in the developing embryo.

Scr gene function is similarly required in subsequent stages of development for proper differentiation of the adult prothorax and labial palps. The latter again in some manner being dependent upon the proper functioning of the proboscipediu gene (D. L. CRIBBS, F. M. RANDAZZO and T. C. KAUFMAN, manuscript in preparation), but the former apparently independent of pb. Adult males that are Scrnu”/+ show reductions in sex comb tooth number and bristle patterns consistent with prothoracic to mesothoracic and propleural to mesopleural homeotic transformations. As noted above, this phenotype is apparently the result of Scr gene function falling below a certain threshold level. However, these same animals have labial palps that are wild-type in appearance. Adult labial to maxillary transformations are observed in Scrnu”/Scrnu” somatic clones or Sc@*O hemizygotes, perhaps due to Scr gene function now falling below a second threshold level. Although we cannot rule out the existence of specific promotor elements to affect these two functions, we also can find no positive evidence for their reality. Thus, we at present favor the quantitative interpretation.

Comparisons with Antp: The results of these investigations point to similarities, albeit superficial, between the Scr and Antp genes. Scr is a large locus with an apparent extensive regulatory region. It exhibits genetic complexity in that lesions affecting embryonic and adult aspects of Scr gene function apparently map within definable regions of the locus. Additionally, all gain-of-function mutations are associated with chromosomal rearrangements, the breakpoints of which map throughout the locus. However, each of these lesions also has a specific recessive component that correlates directly with the region of Scr DNA affected.

ABBOTT and KAUFMAN (1 986) reported a somewhat similar genetic complexity at the Antp locus and interpreted their results as reflecting two independent origins of expression for the locus. This was subsequently confirmed molecularly through the identification of the P1 and P2 promoters by LAUGHON et al. (1986). The parallels between the distribution and behavior of genetic lesions at the Antp and Scr loci might suggest that a second, yet unidentified adult-

specific promoter may also be found for Scr, specifically in the large region betweenftz and the 3’ ter- minus of Antp. However, preliminary experiments performed in this laboratory specifically addressing the identification of an additional promoter for Scr have failed thus far to produce evidence of its existence. Another dissimilarity between Antp and Scr is that temporal and spatial transcript heterogeneity appears to be an important component for function of the former, whereas transcript homogeneity seems to be the mode of expression of the latter. Unlike Antp, only a single transcript has been detected for Scr and the differing aspects of the Scr phenotype are apparently the result of precise quantitative regulation of this single transcript rather than qualitative expression of several transcripts as may be the case for Antp [see BERMINCHAM et al. (1990) and references therein]. This is supported by our genetic analysis which points to a dosage component for Scr suggestive of the following series in order of decreasing sensitivity for Scr gene function:

Adult prothorax > adult labial z embryonic-

prothorax > embryonic labial.

Similar to Scr, Antp gain-of-function mutations are all associated with chromosomal rearrangements. AB- BOTT and KAUFMAN (1986) proposed that gain-of- function at Antp is the consequence of the “juxtaposition of permissive non-Antp DNA sequences which result in the improper expression of the [Antp] leg- specific function in the eye-antennal disc.” This hypothesis was confirmed at the molecular level and demonstrated to be the result of fusion of Antp protein coding sequences with those of a second gene normally expressed in the eye-antennal disc or the introduction of enhancer elements capable of directing expression of Antp in the eye-antennal disc (FRISCHER, HACEN and GARBER 1986; SCHNEUWLY, KUROIWA and GEHRINC 1987; JORCENSEN and GARBER 1987). The fact that the S C ~ lesion exhibits a strong dominant ectopic sex comb tooth phenotype without disrupting any of the identified Scr locus suggests that gain-of-function at Scr may also be the result of a similar juxtaposition of permissive sequences. These permissive non-Scr sequences could contain enhancer elements capable of improperly directing Scr gene function in posterior regions of the thorax. Alterna- tively, these sequences could in some way override or interfere with normal Scr negative regulation in these regions of the thorax. The structure of the ScrscXw chromosomal inversion, which has its distal breakpoint located approximately 5 kb upstream of the Antp P2 promoter, suggests that Antp regulatory sequences involved in directing Antp expression in the posterior thorax may now be juxtaposed at Scr and are responsible for its misregulation.


Although juxtaposition of permissive sequences appears to sufficient for conferring gain-of-function at Antp, simple coupling of novel sequences to Scr regulatory DNA is not enough to result in Scr gain-of- function. This difference is most clearly seen by the number of chromosomal rearrangements that do not result in an Scr gain-of-function phenotype (see Figure 1). Additionally, any interpretation of gain-of-function at Scr must account for the apparent paradoxical behavior of the Sc? lesion, which exhibits character- istics of Scr null and gain-of-function mutations simultaneously. One explanation for the Sc? paradox is that the lesion somehow disrupts sequences involved in the activation of the cis-coupled structural gene while at the same time causing misregulation of the structural gene in trans. The S c P W lesion also appears to exert its gain-of-function effects on the Scr structural gene in trans. This is supported by the phenotypic suppression of the gain-of-function produced by S c F W when an Scrnu'* mutation is placed in trans (e.g., S c P W / S c r 4 ) . Indeed, it would appear that Sc? and S c P w effect their gain-of-function phenotypes solely through misregulation of the Scr structural gene in trans. This hypothesis is tested and confirmed in the accompanying analysis (PATTATUCCI and KAUFMAN 1991). A comparable lesion has yet to be found at the Antp locus. Instead, the fact that the gain-of-function phenotype is unaffected when Antp null alleles are placed in trans configuration to Antp gain-of-function alleles, along with the large number of revertants that have been obtained by disrupting the cis-coupled Ant# transcription unit on the gain-of-function chromosome, suggest that gain-of-function at Antp occurs through a different mechanism than gain-of-function at Scr.

The results of this developmental genetic analysis have allowed us to conclude that the Scr locus possesses distinct developmental functions which are necessary for proper head and thoracic development. Information regarding the molecular organization of Scr has facilitated the demonstration that the initiation of embryonic and adult functions is quantitative and dependent on information contained within unique portions of this locus. Therefore, the comprehensive role in development of the Scr locus would appear to be specified by the precise temporal and spatial activation of the Scr transcription unit at precise levels throughout the course of development.

The authors wish to thank F. R. TURNER for taking the SEMs used in this analysis. We are grateful to A. M. KAPOUN, F. M. RANDAZZO and C. KOPCZYNSKI for their critical reading of the manuscript. The authors would like to especially recognize the contribution of an anonymous reviewer, whose detailed comments significantly enhanced the quality of this presentation. This work was supported by a National Institutes of Health (NIH) Predoctoral Fellowship (GM07757) to A. M. P. and an NIH grant (GM24299) to T. C. K., who is an Investigator of the Howard Hughes Medical institute.

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a functional and structural analysis of the sex combs ... functional and structural analysis of the...

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