Molecular organisation and expression pattern of the segment polarity gene fused of Drosophila melanogaster

65

Mechanisms of Development, 44 (1993) 65 -80 © 1993 Elsevier Scientific Publishers Ireland, Ltd. 0925-4773/93/$06.00 MOD00194

Molecular organisation and expression pattern of the segment polarity gene fused of Drosophila melanogaster Pascal Therond l, Denise Busson 2, Elisabeth Guillemet, Bernadette Limbourg-Bouchon, Thomas Preat 3, R6gine Terracol 2, Herv6 Tricoire and Claudie Lamour-Isnard * Centre de Gdndtique Moldculaire du C.N.R.S., 91198 Gif sur Yvette, France. Received 3 March 1993; revision received 28 July 1993; accepted 5 August 1993)

The Drosophila segment-polarity gene fused (fu) is required for pattern formation within embryonic segments and imaginal discs. We previously reported that the 5' part of the fused gene is homologous to the catalytic domain of serine/threonine kinases. We present here the sequence of the complete transcription unit, which predicts a 805 amino acid long protein. The kinase domain actually corresponds to 268 amino acids in the N-terminal part, and no known function can be attributed to the rest of the putative FUSED protein. Transcripts from the fused gene have been characterized: a unique 3.2 kb fused transcript is produced in nurse cells, in low abundance, from stage 8 of oogenesis, and persistently through the rest of oogenesis. In embryos, this transcript is evenly distributed in all embryonic cells until the extended germ band stage, after which its amount strongly decreases. Ubiquitous expression is detected later in imaginal wing and leg discs. Possible roles of the FUSED protein in signal transduction pathways required for intercellular communication at different stages of development are discussed. Cell interaction; Drosophila; fused; Protein kinase; Segment polarity gene

Introduction

Establishment of metamerisation in the Drosophila embryo requires the activity of three classes of genes, called gap, pair rule and segment polarity genes (Jfirgens et al., 1984; Nusslein-Vohlard et al., 1984; Wieshaus et al., 1984). These segmentation genes were classified according to their mutant phenotype, which can affect several contiguous segments (gap genes),

* Corresponding author. Present address: Laboratoire de G~n~tique du D~veloppement et Evolution, Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05, France. Tel: 1-4427 5715. Fax: 1-4427 3660. i Present address: The George Hooper Research Foundation, University of San Francisco, San Francisco, CA 94743, USA. 2 Present address: Laboratoire de G6n~tique du D6veloppement et Evolution, Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05, France. 3 Present address: Theodor Boveri Institut fiir Biowissenschaften der Universitat Wiirzburg, D-8700 Wiirzburg, Germany. 4 Present address: lnstitut de physique nucl~aire, Universit~ Paris XI, B.P. no. 1-91406 Orsay Cedex, France.

alternate segments (pair rule genes) or every segment (segment polarity genes). The gap and pair rule genes act before cellularization through a regulation cascade which operates at the transcriptional level (for review, see Akam, 1987; Ingham, 1988). This cascade results in the establishment of positional information cues along the anteroposterior axis of the embryo. At the blastoderm stage, transcription of the segment-polarity gene engrailed (en) is initiated in 14 single-cell-wide stripes localized in the posterior part of each segment primordium, marking the anterior boundary of each parasegment (Martinez Arias and Lawrence, 1985; Ingham et al., 1985). At the same stage wingless (wg) expression appears in 14 stripes anterior and contiguous to the en expressing cells. After cellularization, pattern formation within segments is thought to be achieved through cell interactions mediated by segment polarity genes. Fourteen genes of this class have been identified (for review, see Ingham and Nakano, 1990; DiNardo and Heemskerk, 1990; Hooper and Scott, 1992). Mutations of these genes cause specific deletions in every segment; except in en mutants, these deletions are associated with duplications of some of

66 the remaining structures. The elements of pattern deleted are anterior (corresponding to denticle belts) in naked (nkd) (Jfirgens et al., 1984) and zeste white 3 (ZW3 sgg) (also called shaggy) (Bourouis et al., 1990; Siegfried et al., 1990), median in patched (ptc) (Niisslein Volhard et al., 1984) and costal-2 (cos-2) (Grau and Simpson, 1987), and posterior for all other known segment polarity genes (Ingham and Nakano, 1990). The occurrence of cell interactions mediated by these genes is suggested by different observations. For some of the segment polarity genes, the domains affected in mutant embryos are not coincident with the domain of expression of the wild type gene. This is true for wg (Baker, 1987), Cubitus interruptus Dominant (Ci D) (Orenic et al.,, 1990), and patched (ptc) (Nakano et al., 1989; Hooper and Scott, 1989). Furthermore, en expressing cells are dependent on the expression of wg in adjacent cells for the maintenance of en expression, and conversely (Di Nardo et al., 1988; Martinez Arias et al., 1988), implying some kind of cell-cell signaling between these neighbouring cells. Several segment polarity genes have been cloned and sequenced: engrailed (Fjose et al., 1985; Poole et al., 1985) and gooseberry (gsb) (Baumgartner et al., 1987; C6t6 et al., 1987) encode homeodomain containing proteins which arc probably transcription factors (Desplan et al., 1985); this could be also true for Cubitus interruptus Dominant (CiD), which encodes a zinc finger protein (Orenic et al., 1990). Other segment polarity genes correspond to proteins with possible functions in cell communication: wingless encodes a secreted protein, with significant sequence similarity to the mouse proto-oncogene Wnt-1 (Rijsewijk et al., 1987; Van den Heuvel et al., 1989), patched a putative transmembrane protein (Nakano et al., 1989; Hooper and Scott, 1989), armadillo (arm) a protein associated to the plasma membrane, closely related to proteins like human plakoglobin and Xenopus [3 catenin implied in cellular adhesive junctions (Riggleman et al., 1990; Peifer and Wieschaus, 1990; Mc Crea et al., 1991), and ZW3 sgg, a putative protein serine/threonine kinase, strongly homologous to the mammalian enzyme glycogen synthase kinase 3 (Bourouis et al., 1990; Siegfried et al., 1990). Here we describe the molecular organization and developmental expression pattern of the segmentpolarity gene fused (fu). This gene is maternally required for normal segmentation, as fu ~ mutant embryos born from homozygous f u - mothers present a characteristic segment polarity phenotype, with deletions of the posterior part of every segment (NfissleinVolhard and Wieschaus, 1980; Martinez Arias, 1985; Busson et al., 1988). This anomaly can be totally (for weak alleles) or partially (for strong alleles) rescued by a fu + allele paternally provided (Busson et al., 1988). Embryonic development of fu- mutants born from

heterozygous f i l + / f u females is normal, but they display adult cuticular anomalies, such as LV3-LV4 wing-vein fusion (see refs. in Lindsley and Grell ~, 1968; Fausto-Sterling, 1978) and ovarian tumors, whose incidence increases in fi~ homozygous females with temperature and aging (King, 1970). The fi~ product is thus also required during metamorphosis, and early oogenesis. We previously reported that the .fie genc contains a putative protein serine/threonine catalytic domain (Pr6at et al., 1990). In this study, we analyse the structure of the fi~ transcription unit, and its temporal and spatial expression, and discuss possible roles for the FUSED product.

Results

Identification of fit,e transcription units in the filsed region In a previous report, the ]u gene was found to lie within a 16.7 kb genomic region defined by the breakpoints of the Df(1)fu z4 and Df(1)fu m deficiencies and transitory phenotype rescue experiments (Mariol et al., 1987). The restriction map of the 16.7 kb fragment is shown (Fig. 1A). The transcription pattern of this region was investigated by Northern analysis using subclones of the 16.7 kb fragment as probes hybridized to poly (A) + RNAs extracted from various developmental stages. Five transcription units were identified within the range of the 16.7 kb interval (Fig. 1B). All these transcription units are developmentally regulated, with a strong maternal and embryonic expression, followed by lower expression in later stages (Fig. 1C). The fie gene was assigned to the C5 unit on the basis of molecular localization of small deficiencies associated with four viable fu mutants (Preat et al., 1990). This C5 unit encodes a 3.2 kb transcript, cDNA clones corresponding to these different transcripts were searched in ovarian, embryonic and pupal cDNA libraries (see Materials and Methods). An average of one positive clone was found among 10,000 clones hybridized with the 15.5 kb XbaI-KpnI probe (which corresponds to most of the 16.7 kb fragment). Alignment of these cDNAs with their genomic counterpart was performed in hybridizing them to genomic Southern blots: several clones were found for each of the C1 to C4 transcription units, but none corresponded to that of fused (C5). More extensive screening was undertaken using fused specific probes, leading to the isolation of only three fu cDNA clones out of 2.5 × 106 clones from different libraries (see Materials and Methods). The direction of transcription relative to the genomic map was determined, either directly by restriction mapping of directionally cloned cDNAs, or, in the case of the fused (C5) unit, by hybridizing strand

67 specific RNA probes to Northern blots (Fig. 1B). These results show that fused belongs to a genomic region which contains several developmentally regulated tran-

scription units, located very closed to each other. Furthermore, the patterns of expression of the RNAs encoded by the 5 units display obvious similarities, FU-L

A IKb

t

J

FU-S I K E ABE

l Ill

H

CX CC

E

S

GC

HSp

KB

C GGG

I I

I

B

I BK

HSXBC

BE

K

Ill I IIIIII II I

4 CI

C2

C3

C4

C5 FUSED

3.3~

2.5~

2.35~ 1 .75

/ 3.2~

1.4~

Fig. 1. Transcriptional organization of the fu region. (A) Restriction map of a 16.7 kb wild type DNA fragment from Oregon-R strain corresponding to the fused region (A) XbaI; (B) BamHI; (C) Sacl; (E) EcoRI; (G) BglII; (H) HindIII; (K) Kpnl; (S)Sall; (Sp) Sphl; (X) Xrnal. Open bars represent the genomic fragments used for germ-line transformation assays: FU-S is a 5.1 kb BglII-Kpnl fragment, and FU-L a 7.4 kb SphI-KpnI fragment, which rescued fu- mutants in germ-line transformation experiments (see text). Dotted and stripped boxes represent the genomic probes used to detect the transcriptional units. (B) Schematic representation of the five transcription units. They were identified by Northern analysis and localized by restriction mapping of the corresponding cDNAs. Orientation of transcription relative to the genomic map (arrows) was determined as described in Materials and Methods. (C) Northern blots of poly(A) + RNA were hybridized with genomic (dotted and stipped boxes) or cDNA probes (see Materials and Methods) covering different regions. The sizes of the transcripts are indicated in kb. The developmental stages analyzed are as follows: female (9) and male (d) adults; (0-3), (3-6), (6-24) h after egg-laying in the embryonic period; (L1), first larval instar, 24-48 h after egg laying; (L2), second larval instar, 48-72 h; (L3) third larval instar, 72-120 h; (EP), early pupal period, 120 h-168 h; (LP), late pupal period, 168 h-216 h.

68 TABLE I Rescue of the pupal lethal and wing phenotypes of thc IU 'ntl(~3 allele by the 5.1 kb FU.S and 7.4 kb FU.L inserts Cross: F M 6 / f u ''H<~ × fumHa3/Y FU i n s e r t / + Genotypes

Expected phenotypes

Number observcd FU.S insert i

FU.L insert e

25

42

0

0

FM6/fu"U~'3 F M 6 / f u "tu,~

+/ + insert/+

B/2, fu

fu"H¢'~/lumNt'S

+ / +

lethal

fumlll'3/fu mlll~3

insert/+

B +, fu "

I0

20

FM6/Y FM6/Y

+/ + insert/+

B, fu

16

19

fun'U<~/Y

+/ +

lethal

0

(I

fumU~'~/Y

insert/+

B +. fu "

6

I0

I The FU.S insert is FU.S-5a, on the second chromosome. -' The FU.L insert is FU.L-29, on the second chromosome. If both the fused lethal pupal and wing phenotype are rescued by the insert.

especially a strong maternal and early embryonic cxpression.

Determination of the minimal genomic DNA sequence required for normal fused actit'fly We previously reported (Preat et al., 1990) that a 5.1 kb BgllI-KpnI genomic D N A fragment, named FU-S, for 'short fused fragment' (Fig. IA), which includes the entire C5 transcription unit, and the upstream part of the C4 unit, was able to rescue the wing and embryonic mutant phenotypes of ,fix~ and .fitA mutants, respec-

tively considered as a weak and a strong hypomorphic allele of fi,sed on the base of their wing phenotype (Busson et al., 1988)). In order to examinc whether the FU-S fragment actually carries the whole sequence required for a qualitatively and quantitatively normal fit exprcssion, we checked the ability of the FtI-S insert to rescue thc different ,ftlsed phenotypes, compared to the rescue obtained with a htrger insert, FU-L (for 'long fuscd fragment'), 7.4 kb long, which extends distally in the ('4 transcription unit (Fig. IA). We first examined (Table l) the rescue of the zygotically determined mutant phenotype of the ,fit '''ll~'~ allelc, considered as null or v e u strong (Busson cl al., 1988). Homozygous and hemizygous .f)tm~¢':~ flies die as pharatc adults, with a strong fused wing phenotype. Whcn F M 6 / t ' u '''~J''~ females were mated to fu / Y males bearing an autosoreal FU-S or FIJ-L insert, viable fu mile'3 :~f: F[J-N or FU-L males were obtained, which display a wild-type wing phenotype. These males when crossed I f [ : M ( ) / fu '''~'3 females gave rise to the expected number of ,filn'lI~"~: f'U-S or FU-L male and female progeny (compared to their FM6 sibling), disphiying a perfect wild type wing phenotype (Table 1). This observation shows that the zygotic ,/u 'm~''~ phcnotype is completely rescued by both FU fragtnents. Wc also checked oul the ability of different FU-S and FU-h insertions to rescue, when maternally provided, lhe maternal embryonic phenotype due to thc .tu ' \ a l l c l c (Tablc 2). Two FU-S insertions among lhe flmr tcsted (Sa and l()c) gave a complete maternal rescuc, since more than 9()C~ of the embryos hatched (Ogf in the control fh \,//u x femalcs mated to , / h \ / Y males; data larvac

not shown), and more

reached

the

adult

stage,

lhan

70%

of thcsc

half with a wild-type

TABLE II Maternal rescue of the fused mutant embryonic phcnotypc by the 5.1 kb KU-S and the 7.4 kb FU-h gcnomic fragments Cross 9

d

wfu A FU-S

wfu A

wfua

+

Y

wfu A F U - L

wfuA

+

fu mEl{~3

+

Number of eggs examincd

tlatched i lacvac

Adults

FU-S FU-S FU-S FU-S

247 570 260 327

220 (91 ~; ) 553 (97~) 1c)3 (75';) IC)5 (6{V,:)

1S 1 (811%) -: 403 (73e;) : 14~ (76G) ' 115 (59Gi -

FU-L 92 FU-L 5

70 43

,58 197~,: ) 4(I (93'~)

37 (54(~) " 39 (9gc~: } '

FU-S 5a

715

643 (9(/; }

5a 10c 19c 24c

wfu A Y

fu "tu~ FU-S

FU insertion

fu mu~'3 FU-S Y

+

241 (37<~)

t 100% expected for a complete maternal rescue. About half with wild-type and half with fused mutant wings. "~ 188 females and 53 males, all fu + as expected: fu tuna3 flies must possess at least one FU insert to bc viable: 75<~ flies of this genotypc arc expected in this cross, and only 58% (188/643 × I).5) are observed in the case of females, and 16c;;- (53/643 × 0.5) in the case of males. 2

69 wing phenotype (those having inherited the FU insert), half with a fused mutant wing phenotype. This rescue is similar to that obtained with both FU-L inserts tested in this experiment, FU-L92 and FU-L5. Rescue by the two other FU-S insertions was less efficient, as respectively 75% and 60% of hatched larvae were obtained. This lower rescue probably resulted from a position effect due to the insertion site. We also examined the rescue of the maternal embryonic lethality due to the fb/mH63 allele: the maternal rescue by the FU-S insert (FU-S 5a insertion) seems complete, as 90% of the embryos hatched. However, less adults than expected were obtained. As a matter of fact, in the breeding scheme used, 75% of the maternally rescued larvae possess one (50%) or two (25%) doses of the FU insert, and are thus expected, from the results shown on Table 1, to be viable and to possess wild type wings. Considering that 50% of the hatched larvae are likely to be females, and 50% males, we deduced from the results shown on Table 2 that 58% of the females larvae and 16% of the male larvae, instead of the 75% expected, reached the adult stage, giving a rescue of 78% for the females (which can be considered as satisfactory), but only of 22% for the males. This lower male frequency observed is unlikely to result from an incomplete zygotic rescue of the lethality of fH mH63 by the insert (see Table 1), but rather from an incomplete maternal rescue by this insert, which should be sufficient to allow hatching, but not development to adult stage, at least in most male larvae, more fragile than females. Such a weak maternal effect was observed with some hypomorphic fused alleles, such as fu DB5 and fu DB6 (Busson et al., 1988). Considering now the paternal rescuing effect of the FU-S and FU-L inserts (Table 3), in the breeding scheme used 75% of the embryos were expected to

T A B L E III Paternal rescue of the fused mutant embryonic phenotype by the 5.1 kb FU-S and the 7.4 kb FU-L genomic fragments Cross

F U inser- N u m b e r tion of exam-

ined eggs wfu A wfu A

wfu A wfu A

Pvj

Y

+

B

K

Pv

I

I

I

I

+ t 0' 0 0

+1

55 69 49 48

(63%) (67%) (66%) (56%)

38 41 26 23

0 4 1 2

FU-L29 FU-L 5 FU-L 92

82 (72%) 33 (48%) 27 (60%)

52 14 10

19 8 1

fu + F U - L y

+

112 69 45

I 75% expected f o r a complete paternal rescue: 100% of the f u A / f u + females and 50% of the males, those which are w f u A / y , F U insert/+ 2 All with wild-type wings

hatch (all of the females, and half of the males) if the rescue provided by the insert is complete, giving 1 / 3 of males and 2 / 3 of females in the progeny, whereas a maximum of 50% larvae hatch, all females, if no paternal rescue occurs. At least with FU-S 5a, 10c and 19c and FU-L 29 and 92, the percentage of hatched larvae actually observed could be considered to be satisfactory (respectively 63, 67 and 66% with the FU-S insertions, 60 and 82% with the FU-L ones). However,with the four FU-S insertions examined, very few males compared to females appeared in the adult progeny, whereas the expected number of males occurred with FU-L 29 and FU-L 5. Seven other FU-S insertions were examined in the same way. They all gave rise to very few males (data not shown). These results suggest that some additive sequences absent on FU-S, but

KINASE

I~++~S Sml B~...Sc,

O •2 9

+2000

-'-': i

C4 3 3~7

ATG C-DNA i 841

~: ~

GE32

Pv,

m.

Bj HcE ,Zl IE

+3000

DOMAIN

818 904 1015 1086

C_DNA

c3

FU-S 5a 87 FU-S10c 103 FU-S 19c 74 FU-S 24a 86

Proximal

HC

c2

fu + FU-S

Distal

Bgl

Hatched t N u m b e r of larvae adults 2

2457

2516

3092

3159

3518

4271

TAG E92

C_DNA r 3 184

24~96 i'--T~ 2391

I

O l 14 + :,~

C-DNA

4271,

E 12 r::t

u 3840

Fig. 2. Structure of the fu gene. The restriction m a p is presented on the top line (abbreviations: B, BamHI; Bg, Bglll; E, EcoRl; H, HindllI; Hc, HinclI; K, Kpnl; S, Sail; Sc, SacI; Sm, Sinai). The coordinate + 1 on the map corresponds to the Bglll site limiting distally the 5.1 kb BgllI-Kpnl FU-S rescuing insert. Dark arrowheads indicate the locations of primers used in P C R experiments (see Materials and Techniques); 029: position 1712-1730; GE32: position 2570-2589. The kinase domain of the fu putative protein is indicated as a shaded box. The structure of the fu transcript is shown below: the coding sequence is represented as a black bar, and the untranslated region as a solid line. The three introns (open triangles) were identified by comparing c D N A nucleotide sequences to those of the genome and by S1 mapping. The arrow indicates the initiation site and direction of transcription. The fu and proximal part of the C4 c D N A s are represented as open boxes at the bottom of the figure.

70

-50 • 51 + 151

A'IV,C'IX~TCCAGGTCGTCAC C G ~ A G C G C aG~TAcn.~.:~.'I'CGaCGAATG'FIETA'a.T~'~'~ aGA~ ~ ~ ~ C CG~AC~AAT~GGTA~CAAC~TRECACCATGC C C G G A T C G A A C AI~ - ~ D G A G C R ~ A A G A C G G C C AGC & A ~ C G ~ C ~ G A ~ ~ CGATGGTACC~AG~'FI~AGacGGTGGGTGCGATGACGCAGCCC~GTGAAGC~AAG CACGGCGAGTCC GATAAGGG TGA AGACTAGCATcTC~AgGAAGCTGTACACGTA~TGC ATGI~[~ATC~TGCGI~VzGC G ~ A C C AC C ~ G ~ A ~ ~ C~ ~I~GA~GCCA~G~D~Aq cCC~CC~GC~ G G ~ J G C A C ~ T ~ C ~ C ~ G ~ A ~ C ~ ~ ~G'£'I'I~ A G T C ~ A ' I ~ . - L ~ . ~ T C ~ C A C ~ ' I ~ - - I ~ C G'~'I'I*sGCC A C T A C C t G G ~ C T G A GAT~C GA~'~CGGA~C C~ATCCTC CTC CTC AGTCGGCGTgcct t ggCC C C G T C G G T C ~ C G ~ % ~ F I ~ GCCCC~ C ~ G C ~ T C G C T ~ ~ ~ G T A ~ C C~TATCCTC~TAAAGTCGTCGC C ~ G G G C C ~ C ~ A C G C ~ C T G T g cg c ~ T ~ GTAG~GTAG~ ~ A ~ ' I " FI'I~A A T A C ~ T I ~ CG T ~ A C ~ T G T ~ A ~ G A ~ T C ~ C~ GATC~T~ CTC CAGC AE GTA~ GGC C~.-t~.~i%~D~C~C~TGAG~AC~~-~-Ft~GCCAC~GCCC~ATC~CAGCATC*ACCAcg C ~ A C A ~ TAEGG~TCATCGTC~CAC~ACAA~'I~ACCAC~GCGCGTACTGCGGA~'IV=CAGGCGGA~GAC~C~ C A " I ~ C T G ~ C CAGTC~AC ATAT~/fC ~4~ C ~ AE ACTGACC AC GCC AGC CCGA%~PC~Z~A~ ~ CGTC.G'L~JL " GaGCA~TGT~AGCATC~C~AAC~AACGAGAATAAGTCGCC G~fCAGCAGATCC~ GCGTG~ CCAG~ ~ C~ C ~ R ~ G ~ G C ~ % C ~ G C AC C A ~ T G T A A ~ C a ~ T A F ~ R ~ G A T ~ C ~ A G ~ ~ ~ C G~ GAC A ~ GT~CCAGCAGGTGCGAEAGC GaGTAGTCCAGCACGAAGGCG~T.ACCAG~CGTC~AGCGCG~g ~ C ~ GT CATCA~TC~TC~TC~TC~ATC~TAGAGAACTGCCAGGAGAGTAC~GCAG~ G G T C ~ G ~ G ~ A ~ ~ CAAAGATG~'fAC~AATGCAATAAAGCACACCTA~TAC CAT~AACATGCGAGATG~TGCCGCTGTCGG~TA~CGA~TAC A C AGATAAAACA TGAACGC~C~T'[CTC C C GCGCCAGTC~ CC~ATAGATC~CACGAAG~TGG4%AC ATC AGGTAC GAGATTACGCCGTATAT~CCACCC~T~GCT~AGC G~DCCG~ACAGGTRC*AGTACCAGC~CC ~ ~ A ~ C ~ G ~ CCA~ATAC4&ACT~CATCGG~"P~CACCATDG~ATTG~AGCGCACTACGTC4%AAGAAGTAC~-~T1~GA~C~AAAGT~CA~"~C A ~ A C ,Fi-ITI~ATCACTCL-Lt-I~C.CAG~CACTTACGCCGTC.ACCAGCT~GC~JEGTAC~%A~~A~~AT~T CCGTGACGTT~AX~DTC~GTAGC CGTG~ CAGAAGTCCGGCCL-I~I~2GGCCAGCT~TAGA~ATAGTACAGGGCATCC~ ~ C~ ~ +1 C GCCTCCCC~:TC C A C ~ A T T C . A G C T G ~ T G T I ~ T G C G C G T t ~ C . A R C . A T C T C GCC~IAC ATC.C ~ ~ G T A T ~ G ~ C ~ AC C ~ A T ~ C G G A % ' / ~ T G G T C A A % " G G C A G T ~ T C C C C A ~ " / ' ~ A C ~ C C ~ C ~ G ~ G T C ~ ' I ~ C ~ CGGA~ACCGAT~AGCGCG~k~CGACAC4~A~CACGTACAG~PC~GCAT~G~T~q~CGA~~G~~

+251 + 351 +451 +551 +651

AAATI~TCA%~fA~'ITATI~ AC AA~GA~fA~'DCC T G C G T C C C ~ A A T A A A A A C C AGCCAGC AA~-----~~ ~ G ~ T ~ T C~C CGTAE C C ~ A~/'I~T~'EGG~'I~ACT~'~'~TI"AGGTAC A~f AC GATATATAGGTAC A T ~ T ~ A ~A~FEATATTGCATATCA~'ITATTGCACTTATTAAGTAAAAAA~'I"GTAC A~TAAC~TATATACATAT AAATA%~ITATAATATATAATAATATATAAG~AAAG~PC G ~ AA~'ITI'EAT~AATAAATt~I'I"I~ ~ T ~ T ~ T A C ~ A ~ C AC AGATAAAAGCGCC4&ACTAGTC~TA%~DGTCT~%~fC~fATCATC*ATC~TCTATATAC*A'~'~'Frr~T~~ C A ~ G ~ A ~

• 751 +851

TA~CAGA~CCTGTATGAGCAA~AGTA'Fr t ' ~ A T A T A c t E ~ t ~ ~ C A C C T C ~ E ~ A C ~ C CACGCAC~AG~TAATCACA~CC CAATC CGCAGCAAAACAAAGAATAACC~CCGCTAC ~ T ~ ~ ~ A ~ M N R ¥ A V S S L V G ~ G S

-2250 - 2150 -2050 - 1950 -1850 -1750 -1650 -1550 - 1450 -1350 - 1250 - 1150 -1050 -950 -850 - 750 -650 -550 -450 -350 -250 - 150

• 951 +1051

C V Y K A T R K D D S K V V A I K c a t a c a a c t a g t t c a c a c c a t at t c a t g t t ct g c a g C G C G G A ~ I A G C C A C AGCATCCGCAC GTCA~GAGATGATCGAGTC P

1251 + 1351 + 1451 + 1551 + 1651

+ 1~ 1 • 1851 + 1951 +2 .' • 2151 +2251

+2351

+2451 +2551 • 2651 +2751 +2851 +2951 +3051 + 3151

H

V

• ]251

I

E

M

I

E

G

R

A

CTTC GAGTC C ~ S

F

E

S

K

V

I

S

K

K

E

L

K

C~'I'I"~CGTGG~ T

D

L

F

V

V

N

L

~

G

T

E

R

~ F

R

~ A

E

C

C

~ L

D

A M

I

C D

C C GGCq~A

Q

A

R

L

Q

S

D

S

D

A

A

S

V

R

L

A

G

C

~

L

A

L

M

S

C

V

59

C~ TACC~ L

S

R

Y

L

L

R

E

L

P

S

E

AAACGCGG~TAC~TI~TuT~'~'AATCCGCGGCTAAAC~-'~C~AC*AGCC~ C ~ ~ C G ~ N A E L V E R I V ~ N P R L N P V S L L Q S R H H L L R Q R S C Q Cq~M2~C ~ C C GC~/'fC A G C ~ ~ C AC~CGC A ~ " D G G A A ~ G AC4&Gt~I~C GA~I~/G~ "~i~AC~CAC C AL~fC G T L L R L L A R F $ L R G V Q R I W N G E L R F A L Q Q L S E H H S Y

+ 3451

ACCCGGCACTC~GTGGGGAGC'CCGC~AG~CCTCGACGAGATCAGTCAC"i~I~A~"F1TF~CGTCACCTA~C~.~TF~A~gC~G

• 3551 +3651

P A L R G E A A Q T L D E I S H F T F P V T ~I~'fA~ C ~TCC~V~%A~ "I'~GCCC~T~T~C~&GCA~IEG%~CGA~T~'DCCcGAAC~GG~~A~TAG~ C AG I ~ C ~ ' D C G T A C A ~ ] ~ C G 1~fTAC~I" D I ~ C ~ T * A C A C A ~ C ~ P G ~ C G G A ~ A G ~ C GCG'I~'~-~%~CCC2%GC~AC~ G

+ 3751 ÷ 3851 + 3951 +4051 +4151 +4251

K

C A G C G A g t a a g t g g c c a c t g cgg c c t ca t a g a a t t t ct t t t a c t a~act ctD~c~'cDc~Ii4tcaSatt~aacag~AATACTCCGCCGTG C C T C ~ CCGGGGTC~GAT S D N T P P C L L P G W D A G C q ~ M ~ G A T G A A T C C C # u % A G T C C G C C C A ~ G A G A A C GA~AG~V~C~CTA C ~ D ~ % C A G A ~ G ~ A ~ ~ ~ G ~ AT S C D E S Q S P P I E N D E W L A F L N R S V Q E L L P G E L D S L ~GAAACAC~_RE A A C C ~ G T ~ A G C A ~ A T ~ Z I ~ G ~ A C C G C ~ V ~ G C A A C ~ C A A F ~ C C A T ~ C C A C G G G T ~ G ~ C ~ ~ G ~ C K Q H N L V S I I V A P L R N S K A I P R V L K S V A Q L L S L P L~F~'~/DGC~TGGATCCTGTC~'I~Tq~ACCTC GAGCTCATCC GCAAC G%~TACGTC~GTAAAACT~ CC ~ A~TAC~ ~ ~G F V L V D P V L I V D L E L I R N V Y V D V K L V P N L M Y A C K C ~ C ~ A A E q ~ C ~ ~ CC~ CCC ~ C A E C~G~'PCGt~DC AG~CC4&ACG%~GCGTAC~CA%~DC c ~ ~ L L L S H K Q L S D S A A S A P L T T G S L S R T L R S I P E L T V TC G A C ~ C ~ AG'*C~Y~TKC G A A C T G G T C T G C C A C ~ T A C A C CTC~AGCAGCAG~'~CCT~C ~ ~ ~ C AT E E L E T A C S L Y E L V C H L V H L Q Q Q F L T Q F C D A V "A I ~IV~AAGCGATCTG~AACTT~ACC~CACC~ t & a g a t t a c c c t t a g a t g g t a t g a g c t g a c t cct g t a ~ g t ~gc~t t c~gt t L A A S D L F L N F L T H D ct c o c g t a g ~ R C - ~ # J % ~ C ~ A ~ "I~-*ACC~CCC~C~DC C,CC ~ - ~ A ~ C~ A ~ ~ ~ G ~ C C GA R

16 37

GAA~I~fGC G C A F ~ A ~ C G A C A ~ q ~ U A G G C

T

CTACAATC~GC CA~GAGGA~CC C~ACGTCGGGTC~CC GGGCATCTC~TGTCCGC~TACTAC CTGCATTCAAACC~A~ C~ CACC~T Y N G A M G E E P A R R V T G H L V S A L Y Y L H S N R I L H R D C T C A A A ~ C G C A A A A C G T C C T G C T C G A C A A G A A C A T G C AC G C G A ~ I L ~ G ~ CC G C A A C A T G A C C C T ~ C CAC G ~ C T L K P Q N V L L D K N M H A K L C D F G L A R N M T L G T H V L T S C GATCAAC~ GC C C C T C T A C A T G G C C C C G ~ GGAC GAGCCATAC GAC CATC A-~ GGAC ATGTGGTCAC%~C.C~ ~ ATA~ GTAC~ I K G T P L Y M A P E L L A D E P Y D H H A D M W S L G C I A Y. E AAGCATGGC CC~AGCCGCC C~TC~C AGCTCCATC CTGCATCTC~/.~ATGATCAAGCACGAC~G~G~C~AC~A~ S M A G Q P P F C A S S I L H L V K M I K H E D V K W P S T L T C GAGI~=C CGC~CC"I'~C CTACAGC~3CC TC~'~I'Ik~AGAAGGAC CC C G G ~ ~ = C G C A T A T C C T ~ 3 ~ A C C ~ C A C ~ ~ C C ~ G ~ A C ~ A ~ T E C R S F L ~ G L L E K D P G L R I S W T ~ L L C ~ P F V E G R I F c D N A E92 e n d ~ f A T ~ GCAG.%~J&C G C A C ~ C GC4%GGC GG~CC ~ u % G G A A ~ GC U~l'l'l~A C A A A ~ C C C ~ G C C A A G G ~ G ~ G ~ A G ~ C ~ C ~ A ~ T ~ I A E T Q A E A A K E S P F T N P E A K V K S S K Q S D P E V G D T C ~ C 4 ~ C C T ~ C C~'ITI~TI'I~=CGAGTC GC G A C A G C ~ C CACCTCCCGCC~%C~CAT~ ~ C ~ C CA~ ~ L D E A L A A L D F G E S R Q E N L T T S R D S I N A I A P S D V C~ A~AC~CGATGTGC~AC~ACAATA~C~-%C G T G T % % G ~ G ~ D C C C T I ~ GC G ~ A C ~ ~ C ~ ~ C G A D H L E T D V E D N M Q R V V V P F A D L S Y R D L S G V R A M P M TGGTAC ACCAGCCGGTC~TCAACTCGC~AC~GTCAGTGGC A A C ~ C A A T A T G A T C C T C A A C C A T A ~ ~ A ~ A ~ V H Q P V I N S H T C F V S G N S N M I L N H M N D N F D F Q A S C ~ G G ~ GGAG~C G~V~CGCTAAGCCAATAGTCGCTCCAACCGTACGC C A G T C G C G C A ~ ~ G T ~ ~ L R G G V V A A K P I V A P T V R Q S R S K D L E K R K L S Q N L ~ C~I~CAF-AC~ G ~ G A C C A-T~AGGC C ~ - ~ C G ~ - ~ C CACGGAC~%~'fC~'~TACAC~-~%A#J~C~tT~C C ~ ~ D N F S V R L G H S V D H E A Q R K A T E I A T Q E K H M Q E N K P cDMA El2 start - CTC C CC~GC~YFPC CTAC GCAAA~q~fC AGCCC CCGCAGC~CAGCCACAAC~AAACAC~AAq~CA~ CA C C ~ ~ P A E A I S Y A N S Q P P Q Q Q P Q Q L K H S M H S T N E E K L S

F

+ 3351

G

GT~CGTATACAAGGCGACACGCAAGGACGACAGCAAC~TGGTGGCCA~v-AAAGTGATCTC~AAG~tga~g~gc~ggcca~t~ataaa~caacaagtc R

+ 1151

G F

T A C ~ ~ ~ AG cDNA E12 end C~%G~T2C~TRC~%C~A~C A~C~2TC C C C C A A G % ~ A~AC-A#~%C A ~ C A ~ C ~ A A ~ A R C ~ C 4 E G T A ~ 2 & R E C A~fCAF~cT~ A A ~ A~TT~TAT~'I~C A R ~ A ~ C ~ ~C~L~TA~/C CTAGC ATR~%~IV~C~A~ C~*~ ~ T A T ~ CTAA'I~%~AATA%~A~ A ~ ~TA-~AT~ C~%A~"I~fAAATA'I"I"~"~ = A G C C ATA~'I" ~"I T I ~ A~ ~ ~ ~ A T ~ T G'~"~TI" ~~ & A ~ C A E A T A ~ P C A~&G~G~GG~'I'PG~C A A A A T A G A T % " G A T A C ~ C A ~ A T A & ' I T I ~ T A T A ~ C G A A A T A T A A C C ~ C A ~ A ~ T A G T ~ T TAAC~%TR~C C~&A~TT~DGTA~G~ C A T R F - K A T ~ C C A~~TAA%~TC C~CR~'TA~fAC T A ~ C 4 & A ~ ~ ~ ~ ~ T ~ ~ A AAATATA~'DCAA%~fAC A ~

92 125 159 192 225 259

292 325 359 392 425 459

492

505 539 572 605 639 672 686 716 749 789 805

71 present on FU-L, are required to get a FUSED activity similar to the one provided by an endogeneous fu + allele.

Structural analysis of the fused gene The fused cDNAs As described above, after screening different libraries, only three fu cDNA clones were isolated. The Dil4 cDNA, 1.8 kb long, arose from an imaginal disk library (Brown and Kafatos, 1988). The E92 and El2 cDNAs were found in an embryonic (0-3 h) library (Poole et al., 1985), and their sizes are respectively 0.9 kb and 1.4 kb. None of these three cDNAs actually covers the entire fu transcription unit, which encodes a 3.2 kb transcript (Fig. 1C). Based on their restriction map, Dil4 and El2 are very similar, whereas E92 corresponds to a different sequence.

Nucleotide sequence The 7.4 kb FU-L genomic fragment which fully rescued the fu mutant phenotype and the Dil4 and E92 cDNAs were sequenced on both strands. The nucleotide sequence of the ends of El2 was also determined. The detailed structural organization of the fu gene deduced from these studies is shown in Fig. 2. The 7.4 kb genomic sequence, and the deduced aminoacid sequence are shown in Fig. 3. The coordinate +1 on the map corresponds to the BgllI site which limits distally the 5.1 kb FU-S rescuing insert. The FU-L fragment starts distally at position -2250. The genomic and cDNA sequences were compared. The E92 cDNA is likely to represent the 5' part of the fu transcription unit as it has an extra G at its 5'end, which probably corresponds to the mRNA capping nucleotide. The Dil4 cDNA represents the 3' part of the fu unit since it terminates at its 3' end by a polyadenylated tail of 68A. Between the 3' end of E92 and the 5' end of Dil4, there are 653 bp on the genomic sequence which are not present on any of the cDNA clones. The El2 cDNA lacks the last 419 nucleotides of the 3' region and is not polyadenylated; it is mostly included in Dil4, except a few nucleotides present in the 5' region (see below). Taken together, these observations suggest that the fu transcription

unit starts at +841 (which is the start of the E92 cDNA) and ends at +4271 (which is the Dil4 cDNA end), in good agreement with the size of the transcript (3.2 kb). In order to get further information on the fu transcription unit, primer extension analysis using a primer localized in the 5' part of the gene was performed (Fig. 4). Surprisingly, the actual 5' transcription site determined in this analysis was localized at position +818, 23 bp upstream the E92 cDNA 5' end. S1 mapping experiments are consistent with this result (data not shown). Other discrete bands were visible in the primer extension analysis, but none actually corresponds to the E92 cDNA 5' end. These observations suggest that other minor initiation sites might be used for fused RNA transcription. It must be noticed that none of the two putative 5' ends (+841 and +818) are preceded by the Drosophila consensus for transcription initiation ( A T C A G / T T C / T ) , and that no typical RNA polymerase II promoter sequence (TATA box) is found upstream. As expected, there is a AATATA sequence in the 3' untranslated region of the Dil4 cDNA, 12 bp from the poly (A) + stretch. This is known to be a minor variant of the polyadenylation signal consensus (Birnsteil et al., 1985). Three small introns interrupt the gene, respectively 72, 60 and 68 bp long. These three introns display donor and acceptor sites fitting the invertebrate splice junction consensus (Shapiro and Senapathy, 1987) and contain an internal sequence corresponding to a putative lariat branch point upstream to the 3' splice site (Intron 1: TTCAC at - 1 8 from AG, Intron 2: CTAAC at - 1 8 from AG; intron 3: CTGAG at - 3 4 from AG) (Keller and Noon, 1985). Although none of the cDNA clones found in the different cDNA libraries screened had the middle part of the gene (between positions + 1843 and + 2495), no other intronic sequence was suspected to occur in this region: the open reading frame is continuous between the first and the second intron, and S1 mapping confirmed the occurrence of only three introns in positions previously deduced from the cDNA sequences (data not shown). Finally, we used oligonucleotides 029 and GE32 (Fig. 2), respectively located at the 3' end of the cDNA E92, and at the 5' end of the cDNAs Dil4 and El2 as DNA primers for first strand cDNA synthesis by reverse transcriptase followed by amplification by

Fig. 3. Genomic nucleotide sequence of the fu gene and deduced translation product. The nucleotide sequence shown corresponds to most of the genomic sequence of the XS insert which totally rescued fu mutant phenotypes.Numbers on the left refer to the position of nucleotides; the + 1 coordinate marks the Bglll site limiting distally the FU-S insert (Fig. 1). The sequence starts at - 2 2 5 0 , at the SphI site which limits FU-L distally, and ends at +4269, at the G nucleotide which precedes the poly(A) tail on the Dil4 cDNA. The major transcription start site is indicated at position +818. The arrowheads indicate the limits of the c D N A s mentioned in the result section; direction of transcription is indicated by horizontal arrows. Intronic sequences, as deduced from genomic and c D N A sequence comparison, are written in small letters.The translation start consensus sequence, and the polyadenylation signal located 12 bp before the poly(A) tail are underlined. The A T G potential translation start site (+904), and the potential T A G translation arrest codon ( + 3 5 1 8 ) are indicated in bold letters. The translation product is shown in the single letter code u n d e r n e a t h the coding sequence. Numbers on the right refers to the amino acid residue positions. The predicted kinase catalytic domain of the fused protein is underlined.

72

....

~iii

:i!i !!iii if!

i!ii ii

iii~i~ iii

PCR (see Materials and Methods). Thc double stranded DNA obtained was directly sequenced and this sequence is coherent with the structural organization presented in Fig. 2. However, despite several attempts, this DNA fragment could not be cloned. One possible explanation is that this fragment is lethal t\~r bacteria. It must be noticed that the homologous gcnomic DNA region, which in addition contains the 6(i bp intron, was easily cloned in bacteria. The Dil4 eDNA displays a peculiar feature: its 5' end is located within the second intron, at position + 2396, which could be the spinning branch point, the surrounding sequence fitting with the consensus C / T T G / A A C / T . It might thus represent a maturation intermediate, as unspliced cDNAs are not uncommon in the library in which it was found (Brown and Kafatos, 1988).

The .fhsed putatil~e protein

!,i i

~i~I~:!ii!!i'i~~i

filial
7

~:

:if!

iiili

Fig. 4. Delelminaton of the 5' end of the .li~ gene by primer extension. The lane on the left contains the extended product from a S2p-end-labeled fit primer localized in the firsl exon (position: +928; +947). The four lanes labeled G, A. T and C show the dideoxy sequence reactions of the Fun44 fragment ( 1062: + 1424) primed with the same primer. Nucleotide sequence shown on lhe right hand margin is a portion of the Fun44 sequence. The extended produc! corresponds to the (7 in position 118; on the lit map, which is indicated by an arrow.

The methionine codon initiating the fit coding sequence is located at + 904. It was determined to he the first A T G on the E92 eDNA sequence opening a large open reading frame 2,415 bp long interrupted by three introns. The codon usage of this O R F is consistent with that known for Drosophila. The A T G located in +904 is preceded by a favourable Drosophila translation initiation consensus AACC (Consensus C / A A C / A C / A ATG, Cavener, 1987). Translation of the nucleotide sequence shows that .fit encodes a 805 aminoacid long protein (Fig. 3), with a predicted molecular weight of 90,300 Da. This prediction is in good agreement with the size of the ,/it protein recognized on Western blots by polyclonal antibodies (P. Th6rond, unpublished results). The putative catalytic kinase domain extends over 268 amino acids in the N-terminal part of the protein (Preat et al., 1990). Comparisons with protein sequence data bases show that higher homology scores were obtained with yeast s e r i n e / t h r e o n i n e kinases than with any known Drosophila kinase. The FUSED protein has 34% identity over 272 amino acids with the cAMP-dependent s e r i n e / t h r e o n i n e kinase encoded by the SNF1 genc of Saccharomyces cerecisiae (Celenza et al., 1986) and Cf 32/c identity over 233 amino acids with the cell division control products of the CDC28 gene of Saccharomyces cerecMae (Lorincz and Reed, 1984) and the CI)C2 gene of Schizosaccharomyces pornbe (Hindley and Phear, 1984), whereas it has only 25% identity over 279 amino acids with the Drosophila ZW3 ~gg protein (Bourouis et al. 1990).The remaining part of the putative FUSED protein, on the C-terminal side (537 residue long), does not display any significant homology with known proteins, and no putative function can be proposed for it. However, this part of the FUSED protein appeared to be essential for .[used activity a n d / o r regulation since some fused mutants have been shown to carry alterations in this sole region (Preat et

73 al., 1990; Therond, unpublished results). The regulatory domain of the FUSED kinase might reside in this C-terminal domain. It is known that many protein kinases are negatively regulated via an 'autoinhibitory' domain which interacts with the catalytic domain; it is supposed that the binding of an allosteric activator could disrupt this interaction, allowing phosphorylation of exogeneous substrates to occur (reviewed in T.R. Soderling, 1990). Although no phosphorylation consensus (RRXS/T) are present in the FUSED protein, RXXS/T sequences (serines in positions n°352, 422, 747, threonine in position n°445), which are known to be recognized by the catalytic domain of some serinethreonine kinases (Colbran et al., 1988), are present in the 3' domain. Hydropathy analysis reveals a particularly hydrophilic domain in the middle of the protein (from amino acid 410 to amino acid 530) which could correspond to a hinge region between catalytic and regulatory domains. Four potential N-glycosylation sites were encountered at positions 151,311,327 and 527 of the putative protein.

,=

A

,,,

FUSED

3,2,.~

B ACTI N 50

wm~ C IFUSED/ACTIN5C (%)1

Northern blot analysis Genetic and cellular analysis have shown that the fu gene is required for normal oogenesis (R. King, 1970), embryogenesis (NiJsslein-Volhard and Wieschaus, 1980; Martinez Arias, 1985; Busson et al., 1988), and development of imaginal discs (Fausto Sterling, 1978; Wurst and Hanratty, 1979). The 3.2 kb fu transcript is accumulated at highest levels in adult female and early embryonic stages (Fig. 1C). An over exposure of the blot shows that the transcript is present in small amounts in adult male and in larval and pupal stages (Fig. 5A). The abundance of the fu transcript at different stages was estimated by hybridizing the blots with an actin 5C cDNA fragment (Fig. 5B). The detection of the signals in both hybridizations was done using the SOFI detector (Mastrippolito et al., 1991; Th6rond et al., 1992). Comparison of signal intensity provided by the two probes indicated that in adult females and in embryonic stages the fu transcript amounts are respectively 3.6 and 2.3% of the 2.0 kb actin 5C transcript ones (Fig. 5C).Thus they represent 0.001% of total poly (A) + RNA at the adult stage and 0.006% of total poly (A) + RNA in the early embryonic stage (Fyrberg et al., 1983; Anderson and Lengyel, 1984).

Spatial distribution of the fused transcript To determine whether fu RNAs are expressed in spatially or temporally restricted patterns, in situ hybridization using digoxygenin-labeled DNA probes (Tautz and Pfeifle, 1989) were performed on whole embryos, ovaries and imaginal discs (Fig. 6). In ovaries (Fig. 6A), fu transcripts are not detectable in early

1,OOE+00

~s"

~e

1,00E-01

e~e............_

1,00E-02 1,O0E-O3 J Female Male 0-3 H 3-6 H 6-24 H

L1

L2

L3

P1

P2

Fig. 5. Compared amounts of fu to actin transcripts during development. Northern blot of poly(A) + RNAs from different stages of development were successively hybridized with fu (A) and actin probes (B) and imaged with the SOFI detector as described in Materials and Methods. (C) Ratio between the amounts of fu and actin transcripts during development. The highest values for this ratio is in adult females and early embryonic stages: in those stages fu transcripts represent respectively 3.6% and 2.3% of the quantity of the actin 5C 2.0 kb. The ratio tends to zero in the latest stage (13; P1; P2).

stages, and become first visible in the nuclei of nurse cells at about stage 8 of oogenesis (King, 1970). During vitellogenesis, they are present in the cytoplasm of nurse cells and appear to flow into the oocyte as oogenesis proceeds. They persist in degenerating nurse cells till the end of oogenesis and are not detectable in the follicular epithelium. In embryos, fu transcripts are uniformly distributed but temporally restricted from early to mid-embryogenesis, in accordance with Northern blot data. At the preblastoderm stage, fu RNAs, most likely maternal in origin, appears evenly distributed (Fig. 6B). At the cellular blastoderm stage, fu RNAs are present in all cortical cells but not in yolk cells (Fig. 6C). During germ band extension, fu tran-

74 scripts a r e f o u n d in all g e r m layers at similar levels (Fig. 6 D ) b u t w h e n g e r m - b a n d retracts, they d e c r e a s e a n d are no l o n g e r d e t e c t e d above t h e b a c k g r o u n d (see Fig. 6E on which t h e staining o f two d i f f e r e n t embryos, o n e stage 13 e m b r y o a n d o n e y o u n g e r e m b r y o , p r o b a bly stage 10 can b e c o m p a r e d ) . In imaginal discs, f u t r a n s c r i p t s a r e p r e s e n t in all cells but are m o r e abund a n t in the wing t h a n in t h e leg disc (Fig. 6G).Thus, no localized e x p r e s s i o n o f fu t r a n s c r i p t s could be d e t e c t e d e i t h e r in e m b r y o s at any stage or in imaginal discs. In contrast, analysis with a c o n t r o l ftz p r o b e on e m b r y o s (Fig. 6F) a n d an en p r o b e on imaginal discs (Fig. 6 H ) e x h i b i t e d t h e k n o w n c h a r a c t e r i s t i c localized p a t t e r n of expression.

Discussion T h e fused g e n e is i n c l u d e d in an actively t r a n s c r i b e d region: five t r a n s c r i p t i o n units were c h a r a c t e r i z e d in a r a n g e of 16.7 kb o f D N A . T h e s e five units a r e d e v e l o p m e n t a l l y r e g u l a t e d , a n d t h e i r d e v e l o p m e n t a l p a t t e r n of e x p r e s s i o n exhibits striking similarities: they all show strong m a t e r n a l a n d early zygotic expression. Such g e n e c l u s t e r i n g is not u n c o m m o n in Drosophila. It might c o r r e s p o n d to e v o l u t i o n a r y or functional relat i o n s h i p s b e t w e e n the genes o f the cluster. In s o m e cases, the g e n e s a r e t h o u g h t to have o r i g i n a t e d from d u p l i c a t i o n s o f an a n c e s t o r gene. This hypothesis is unlikely in the case o f fused, as no cross h y b r i d i z a t i o n was seen b e t w e e n it a n d the o t h e r g e n e s in the region. F u r t h e r m o r e , the c o m p l e t e n u c l e o t i d e s e q u e n c e of the C3 a n d C4 g e n e s was e s t a b l i s h e d , r e v e a l i n g no h o m o l ogy with fused ( d a t a not shown). T h e s e c l u s t e r e d genes (or s o m e of t h e m ) m i g h t be u n d e r the c o n t r o l of c o m m o n cis-regulatory e l e m e n t s . This h y p o t h e s i s cannot be excluded, as only 480 b p lie b e t w e e n the transcription start sites of fused a n d C4, which are trans c r i b e d in o p p o s i t e direction. A l t e r n a t i v e l y , these genes could have n o n - o v e r l a p p i n g , very short cis-regulatory regions. T h e possibility of a c o m m o n cis-regulation for t h e s e two g e n e s will be e x a m i n e d in d e l i m i t i n g pre-

cisely the cis-acting e l e m e n t s necessary for correct expression of fused and C4. Recently, section 17 of the X c h r o m o s o m e was extensively s c r e e n e d for new lethal m u t a n t s ( E b e r l et aI., 1992). This analysis shows that in a d d i t i o n to fused, four c o m p l e m e n t a t i o n g r o u p s are u n c o v e r e d by Df(1)fuH4, the smallest )'h deficiency used in this study (Busson et al., 1988). It w o u l d be i n t e r e s t i n g to d e t e r m i n e w h e t h e r s o m e of t h e s e genes might c o r r e s p o n d to the t r a n s c r i p t i o n units C1 to C4. Germ-line transformation experiments demons t r a t e d that most g e n o m i c s e q u e n c e s r e q u i r e d for normal expression of the fu gene a r e c o n t a i n e d within thc 5.1 kb F U - S f r a g m e n t , which includes the entire fused t r a n s c r i p t i o n unit a n d 818 bp u p s t r e a m . This f r a g m e n t is able to rescue all fused zygotic p h e n o t y p e s , and, w h e n m a t e r n a l l y p r o v i d e d , the m a t e r n a l e m b r y o n i c lethality of the h y p o m o r p h i c fu A and a m o r p h i c fu ml~,-~ alleles. In this l a t t e r case, however, less adult males than e x p e c t e d arise, suggesting that the m a t e r n a l activity of the F U - S insert might be slightly insufficient. W h e n p a t e r n a l l y p r o v i d e d , both F U - S and F U - L fragm e n t s arc able to rescue the m a t e r n a l e m b r y o n i c lethality due to fi~,x but the e x p e c t e d n u m b e r of r e s c u e d a d u l t s is only o b t a i n e d with the larger fragm e n t F U - L . T h e s e results suggest that s o m e additive s e q u e n c e s c o n t a i n e d in the 2.3 kb g e n o m i c f r a g m e n t p r e s e n t on F U - L , but a b s e n t on F U - S , are necessary to get a c o m p l e t e l y n o r m a l fu + activity. Howcvcr, thc d i f f e r e n c e o b s e r v e d b e t w e e n the activity of these two f r a g m e n t s is m o r e likely q u a n t i t a t i v e than qualitative, since the small F U - S f r a g m e n t acts at the s a m e develo p m e n t a l stages than an e n d o g e n e o u s fi~ + allele. It is able to function maternally to rescue thc m a t e r n a l fused e m b r y o n i c p h e n o t y p e of all fused m u t a n t alleles, with a slightly w e a k e r efficiency than a r e s i d e n t .fi~ + allele in the case of the strong fu ~H63 m u t a n t . It is able to function zygotically to rescue the zygotic p h e n o t y p e s of all fused m u t a n t s , including Jilm~I6~; this zygotic activity is also able to partially rescue the m a t e r n a l e m b r y o n i c p h e n o t y p e . F r o m these observations, we p r o p o s e that the d i f f e r e n c e b e t w e e n F U - S and F U - L resides in the q u a n t i t y of F U S E D p r o d u c t synthetizcd.

Fig. 6. Expression of fu in ovaries, embryos and imaginal discs. Two different fu genomic DNA probes, the 1.4 kb BamHI-SalI fragment corresponding only to the kinase catalytic domain and the 3.6 kb KpnI-KpnI fragment covering both catalytic domain and 3' part of the gene, were used as probes for in situ hybridization experiments. Similar results were obtained with both probes. In situ hybridization with the f?z cDNA probe on embryos and en probe on imaginal discs were used as controls for localised expression. (A) Wild-type ovarioles..,,'il transcripts first appear in nuclei of nurse cells (n.c.) at about stage 8 (s8) of oogenesis (King, 1970). At stages 9 (sg) and 10 (sl0), fu expression is clearly detectable in the cytoplasm of nurse cells. Follicular cells (f.c.) are not stained. (B-F) wild-type embryos hybridized with fu (B-E) and t?z (F) probes. The fu transcript is uniformly distributed and found within all germ layers up to late extended germ band stage, but from the beginning of germ band retraction, fu expression decreases and can no longer be detected by this technique. (B) A preblastoderm stage embryo exhibiting a uniform staining in the cortical cytoplasmic region. (C) A blastoderm stage embryo with staining in the cytoplasm of peripheral cells, the yolk is not stained. (D) An extended germ band stage embryo showing uniform staining in all embryonic cells. (E) Unstained retracting germ band stage embryo (stage 13) (compare with the staining observed in a younger embryo on the same picture). (F) ftz localised expression is shown as seven stripes in an early gastrulating embryo and as a repeated pattern in the nervous central system of an extended germ band embryo. (G,H) Wing and leg imaginal discs stained with fu (G) and en (H) probes: the fu transcript is uniformly distributed but at a much higher level in wing disc than in the leg disk. This can be compared with the expression of en which is restricted to the posterior compartment in wing and leg discs.

-..J rji

76 Some cis-acting enhancer sequences necessary to get the maximum level of fused expression might be present on the 2.3 kb Sphl-BglII fragment present on FU-L and absent on FU-S; the lack of these hypothetical enhancers would only be visible when a high level of FUSED product is required, for example for paternal rescue. As a matter of fact, it was already known that, even with a resident fit + allele, the paternal rescue of very stong alleles, a s flt mtl~'3, was incomplete (Busson et al., 1988). Fused is likely to encode one single transcript: on Northern blots, at any developmental stage, fu probes hybridized to a 3.2 kb polyadenylated RNA, and no alternative splicing was found in S1 mapping (data not shown). However, in primer extension experiments (performed on maternal poly (A) ~ RNAs), several other discrete bands were found apart from the major band which corresponded to a start in the +818 nucleotide. Furthermore, the unique cDNA clone isolated including the 5' part of the gene (which possesses in 5' an extra G probably corresponding to the mRNA capping site) starts at position +843. It was found in a maternal eDNA library. These different results suggest that fused can be transcribed, at least during oogenesis, from several close transcriptional start sites. The .fit transcript is present in the germinal cells of ovaries, in embryos until the end of germ band extension at mid embryogenesis, and in imaginal disks. When present, the fu transcript is evenly distributed. At other stages, fu expression is low, or undetectable. This pattern is consistent with the .fit mutant phenotype, which reveals that the FUSED product is required during different developmental stages. Mutant females display ovarian tumors (King, 197(I); this tumoral phenotype, which supposes extra cystocyte divi~ sions in early stages of oogenesis, is not consistent with the fact that the fu transcript could not be detected before stage 8 of oogenesis. It is possible that )'~t transcripts actually present in the vitellarium could not be detected by in situ hybridization with the technique that wc used. If not rescued by a paternal Ju ~ allele, oocytes formed by fu non tumoral egg chambers, give embryos with segmental defects corresponding to the replacement of posterior parts of each segment by mirror image duplications of remaining anterior parts. These anomalies fit well with the presence of the .f. transcript in ovarian germ cells during vitellogenesis, and in the 0-3 h embryos as a maternal transcript. The occurrence of paternal rescue is in accordance with an early zygotic transcription of the Ju gene, probably just after cellularization. Maternal Ju activity alone is sufficient to allow normal embryonic development, but its lack can be compensated, at least partially, by zygotic expression: it is thus likely that the maternal and zygotic fit products display identical roles in embryos. How-

ever, although the lack of ./itsed product leads to pattern deletions in specific regions of every segment, the fu RNA is uniformly distributed within the embryonic segments. The same uniform transcription pattern is observed for the zw3 ~ gene, which is maternally expressed and encodes a putative serine-threonine protein kinase like fused (Bourouis et al., 1990; Siegfried et al., 1990). Several mechanisms are involved in the regulation of the activity of segment polarity genes. Some of these genes, such as engrailed (Fjose c t a l . , 1985; Poole et al., 1985; Kornberg et al., 1985), wingless (Baker, 1987; Rijsewick et al., 1987; Ingham ct al., 1988) and gooseberry (Baumgartner et al., 1987; C6td et al., 1987) display a spatially restricted transcription pattern, mediated by pair-rule genes, from the blastoderm stage. Others, like patched (Nakano et al., 1989: Hooper and Scott, 19891 and Cubitus interruptus (Orenic et al., 1990; Eaton and Kornberg, 1990) arc uniformly transcribed at first, before beeing spatially restricted via a negative regulation mediated by etzgrailed. A third mechanism, observed in the case of armadillo, is a spatial modulation of expression occurring during translation, as the ARM protein appears to accumulate preferentially in a segmental pattern coincident with wg expression (Riggleman et al.. 19901. Such modulation in protein expression might also occur in the case of Jit or zw3 ~ . Other mechanisms might also account for the regionalized pattern observed in fit mutants, such as local activation of the native protein, or spatial restriction of FUSED product targets. Finally, zygotic loss of fi4 activity leads to normal embryonic development through the action of the maternal product, but mutant adults have defects in the adult cuticular structures, in accordance with the presence of fit transcripts in imaginal disks. All fi~ alleles give wing, ovarian and embryonic defects, and fully complement in trans heterozygous combinations. Furthermore, all .fit defects are suppressed by loss of function alleles of the gene Suppressor of fused (Preat, 1992). From these observations, we previously inferred that the gene probably encodes a protein with a single function implicated in different developmental processes (Busson et a[., 1988). The putative protein s e r i n e / t h r e o n i n e kinase function attributed to fused is consistent with this hypothesis. After cellularization, the genes of the segment polarity class are known to mediate cell-signaling events that specify individual cell states within the parasegment (Martinez-Arias et al., 1988; Ingham and Nakano, 1990). The FUSED serine-threonine kinase could therefore be involved in transduction of intercellular signals at this stage. It was previously shown (Limbourg-Bouchon et al.. 19911 that in ,lit mutants, the expression of engrailed and wingless respectively disappears partially and totally at germ band retraction

77 (stage 10 of embryogenesis). One possibility could be that the ENGRAILED protein, of which several serine and threonine residues are phosphorylated (Gay et al., 1988), might be a target for the FUSED kinase, although it has been shown that at least some of the residues phosphorylated in vivo on the ENGRAILED protein are phosphorylated in vitro by CASEIN KINASE II (Bourbon and Kornberg, personal communication). Another possibility is that fused could be involved in the signal transduction pathway controlling wg transcription, in accordance with complete extinction of wg expression in fu mutants. In wild type ovaries, four cystocyte divisions occur, leading to an egg chamber containing 16 cells, which differentiate further into 15 nurse cells and one oocyte. In fu mutants, and in mutants for some other genes such as fs(1)1621 (Gollin and King, 1981) or otu (King and Storto, 1988), cystocytes undergo supernumerary divisions and fail to differentiate into nurse cells and oocytes, leading to ovarian tumors. At the present time egg chamber development is poorly understood. It was recently suggested that loci producing tumorous ovarian defects might interfere with the process of germ-line sex determination (N6thiger et al., 1989; Oliver et al., 1990). Fused kinase might therefore be involved in signal transduction leading to specification of germ cells during cystocyte divisions: if specification were affected, mitosis would not be arrested, and ovarian tumors would be formed. However, as serine/ threonine kinases are known to be implicated in cell cycle control in various organisms, an alternative hypothesis accounting for the ovarian fu- phenotype is that fused could be directly involved in specific control of cell division during oogenesis.

Materials and Methods

Drosophila stocks Flies were kept at 20°C on standard medium as described in Gans et al. (1975). The strains f/,/1, fu A and fL/mh63 used in this study were described previously (Busson et al., 1988); fL/1 and fitA are hypomorphic alleles; homozygous and hemizygous fitl and fitA mutant flies born from heterozygous mothers are viable as adults, displaying the fused wing phenotype, and, in females, ovarian tumors whose incidence increases with temperature and aging; mutant embryos born from homozygous mothers are lethal and display the characteristic segment polarity phenotype but heterozygous female progeny possessing a fit+ paternal allele are viable, fL/mh63 is considered as amorphic: homozygous and hemyzygous flies are lethal as late pupae; the maternal embryonic phenotype observed in germinal

clones is strongest that the one obtained with fit l and fitA. It is as strong as the one observed in germinal clones homozygous for the l(1)fit z4 deletion; the paternal rescue by a paternal fu + allele is incomplete: heterozygous females can hatch but die before adult stage.

Enzymes and isotopes Enzymes were purchased from Amersham International, Boehringer Mannheim, New England Biolabs and Pharmacia, and were used as recommended by the manufacturers. [a-32p]dATP (400 Ci/mmol), [735S]dATP (3000 Ci/mmol), [T-32p] dATP (3000 C i / mmol) were from Amersham International.

RNA isolation and Northern blot analysis Embryos, larvae, pupae and adults used for RNA preparation were raised according to standard methods (Roberts, 1986). Total RNA was extracted by the hot phenol/SDS extraction procedure, as described by Palmiter (1974). After ethanol precipitation, RNA was dissolved in water, and poly A + RNA was prepared by oligo (dT) cellulose chromatography (Sambrook et al., 1989). 15-20/zg of poly A + RNA from various stages were fractionated on 1.3% glyoxal-agarose gels and blotted onto nylon membranes (Amersham N). Hybridization was performed in 50% formamide, 5 x SSPE, 5 x Denhardt's solution, 0.5% SDS containing 100 m g / m l of denatured herring sperm DNA at 42°C overnight or for 36 h. The blots were hybridized with 32p-labeled DNA probes from either a 0.85-kb KpnI-HindlII fit genomic fragment (Fig. 1 and 5),or a 1.35-kb EcoRI-SmaI fragment of the DI14 fit cDNA, and a 453-bp HaelII-HindlII fragment from the 3' region of the actine 5C gene (Fyrberg et al., 1983) and from different subclones of the KpnI-KpnI 16.7 kb fu region (Fig. 1). In order to estimate the amount of fu RNA, the same filter was successively hybridized with fu and actin probes The membrane was imaged in both cases 48 h with the SOFI detector (Mastripollito et al., 1991) which allowed a direct quantification of the amount of radioactivity (N) found in the RNA band of each lane, after background substraction. The ratio between the amounts of fused and actin transcripts were calculated as:

gfused R

~

CPMactin x CPMfused

CPM denotes the total rate of the counts of the probe used, which includes both probe specific activity and probe concentration.

78

Germ line transformation

-4.300 to +5.100; they were designated as fun 114 (a

The 7.4-kb SphI-KpnI FU-L fragment and the 5.1-kb BglII-KpnI FU-S fragment were respectively cloned

BglII fragment, -4300; -664), fun 44 (a SacI-Kpnl fragment, -1062; + 1424), fun 64 (a KpnI-Sacl fragment, +1424; +2788), and fun 4 (a SacI-Kpnl frag-

into the pW8 and pW6 vectors (Klemenz et al., 1987), which contain the white + gene as a transformation marker, and injected into the w 1118; p(a 2-3); Sb/TM6 UbX host line (Robertson et al., 1988) under standard conditions (Spradling and Rubin, 1982). The surviving adults were mated to w 1~8 flies, and their progeny examined for white + activity. Stocks were established from independant transformant lines, and the insertions transferred by appropriate crosses into the fu 1, w fu A, o r f//mh63 strains.

Isolation of cDNA clones cDNAs corresponding to fu flanking genes (C1 to C4) were isolated from the 4-8 and 8-12 h cDNA librairies kindly provided by N. Brown (Brown and Kafatos, 1988). They were screened with a 15.5 kb XbaI-KpnI genomic DNA fragment (Fig. 1) according to the protocol described by Sambrook et al. (1989). One particular cDNA was recovered with a frequency of about one out of 5,000 to 10,000 colonies tested. As the cDNAs are cloned in a single orientation in the pNB40 vector, their restriction maps indicate the direction of transcription relative to the genomic fragments. The DI14 fu cDNA was recovered once from the imaginal disc library of Brown and Kafatos (1988) out of 2.6 105 colonies hybridized with a 3.6-kb KpnI-KpnI ( + 1432; + 4500) genomic DNA fragment. The E92 fu cDNA was recovered from the 0-3 h cDNA library constructed by Poole et al. (1985) over 6.5 105 colonies tested with a 1.4 kb BamHI-SalI (+940; +2281) genomic DNA fragment; the El2 cDNA was recovered in the same screen with a PvuII-BamHl ( + 1716, +2766) probe. No other fu cDNAs could be obtained in spite of extensive screening of more than 1.5 106 clones from different cDNA libraries (embryonic cDNA libraries of Brown and Kafatos (1988), ovarian cDNA library of M. Champ (personal gift), embryonic and pupal cDNA libraries of Clermont-Goldshmidt (personal gift). Using E92 and El2 fragments subcloned into the bluescript KS + vector as template, stand-specific RNA probes were synthesized (Sambrook et al., 1989). They were used to probe Northern blots, in order to determine the direction of transcription of the fused gene.

DNA sequencing and computer analysis To determine the sequence of genomic DNA, restriction fragments of the Cos C4' cosmid which contains the fu locus from the Oregon R stock (Mariol et al., 1987) were subcloned; they gave rise to four DNA fragments covering the fu region from coordinate

ment, +2788; +5100). These fragments were cloned into M13mpl8 and M13mpl9, and unidirectional deletions were generated by T4 polymerase. Overlapping clones were sequenced on both strands by the dideoxy chain termination method (Sanger et al., 1977) using the sequenase system (U.S. Biochemical Corporation). cDNA fragments were subcloned into the bluescript KS + vector (Stratagene), partially deleted by exonuclease III, and sequenced using the same method. DNA sequences were compiled using the UWGCG: University of Wisconsin Genetic Corporation Group software (Devereux et al., 1984). The deduced amino acid sequence was compared with the HBRF data base using the program FASTA (Pearson and Lipman, 1984).

Primer extension analysis The 5' end of the fit transcripts was mapped by primer extension, Using 20-bp antisense synthetic primer that is specific for the first exon (5'-AAGGATCCTTGCCCCACCAG-3'), localised between 928 and 947 (see Fig. 3). 10/zg aliquots of poly (A) + RNA, purified from females of the Oregon R stock, were hybridized overnight at 65°C with 60 ng of the primer labeled at the 5' end with [y-32P]dATP and extended by the method described in Berger and Kimmel (1987). Extended single-stranded fragments were separated on a 6% denaturing polyacrylamide gel along with dideoxy sequence reactions of fun 44, primed with the same primer, as a marker (fun 44 is a Sac I-Kpnl fragment cloned in M13mp18 and containing the most upstream region of the fused gene).

PCR amplification of cDNA A sample of a cDNA mixture obtained by reverse transcription of poly(A) + RNA issued from 0-3 h embryos was used in a PCR amplification. The reaction was performed in a 10 mM Tris pH 8.4, KC1,50 mM, MgC12 2 mM, 100 g / ml gelatin, 0.1% Triton X-100 buffer with Cetus Amplitaq polymerase. Primers were 029 (5'-GGGTGACACAACAGCTGCG-3' corresponding to positions + 1730, + 1712 on the genomic sequence) and GE32 (5'-ATCGTTCTCGATGGGCGGAC-3' corresponding to positions +2589, +2570 on the genomic sequence). See Figs. 2 and 3.

Whole-mount in situ hybridizations Whole-mount in situ hydrizations were performed according to Tautz and Pfeifle (1989), with some modifications provided by U. Waldorff (Biozentrum, Basel).

79 Embryos were fixed for 25 min at room temperature in a two phase mixture consisting of heptane and 10% formaldehyde, phosphate buffered saline (PBS), 50 mM EGTA. After devitellinization in methanol, embryos were stocked at -70°C in 100% ethanol. They were fixed a second time before use in 5% formaldehyde, PBT (PBS plus 0.1% Tween 20), for 20 min at room temperature. Hybridization and washes were performed at 50°C. Ovaries were dissected in PBS and fixed twice: firstly, in 4% paraformaldehyde-PBS, on ice, and secondly in the same medium plus 0.5% Triton X-100, at room temperature. Hybridizations were at 45°C and washes at 48°C. The cDNA restriction fragment probes were prepared using the digoxigenin DNA labeling kit from Boehringer Mannheim, following the manufacturer's instructions.

Acknowledgements The authors wish to thank Ren6e Charef, Eliane Foug~re and Janine Le Guennec for excellent technical assistance. This work was supported by grants n ° 6770 from ARC (Association de Recherche contre le Cancer) and n ° 502936 from INSERM (Institut National pour la Sant6 et la Recherche M6dicale)

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