Molecular Structure offrizzled, a Drosophila Tissue ...have led to suggestions that tissue polarity...

Copyright 0 1990 hy the Genetics Society of America

Molecular Structure offrizzled, a Drosophila Tissue Polarity Gene

Paul N. Adler, Charles Vinson,' Woo Jin Park, Sharon Conover, and Lisa Klein2 Biology Department, Molecular Biology Institute and Cancer Center, University of Virginia, Charlottesville, Virginia 22901

Manuscript received January 20, 1990 Accepted for publication June 27, 1990

ABSTRACT The function of thefrizzled V;) locus is required to coordinate the cytoskeletons of pupal epidermal

cells so that a parallel array of cuticular hairs and bristles is produced. We report here the molecular cloning and characterization of the fz locus. The locus is very large. Mutations that inactivate the gene are spread over 100 kb of genomic DNA. The major mRNA product of the gene is a 4-kb RNA that is encoded by 5 exons spread over more than 90 kb of genomic DNA. Conceptual translation of this mRNA indicates that it encodes an integral membrane protein that is likely to contain both extracellular and cytoplasmic domains.

T HE adult cuticle of Drosophila melanogaster has a rich morphology, containing a large number of

polarized structures, principally bristles (4-cell sense organs) and hairs (cellular extensions of individual cells that later become sclerotinized). These polarized structures are evenly spaced, aligned in parallel, and typically point distally on appendages and posteriorly o n the thorax and abdomen, thus defining a tissue polarity.

The development of tissue polarity is expected to require intercellular signaling; however, very little is known about this process. Embryological experiments have led to suggestions that tissue polarity is a mani- festation of a gradient of cell adhesiveness (NARDI and KAFATOS 1976), a gradient of a diffusible morphogen (LAWRENCE 1966; STUMPF 1966), or direct cytoskel- eta1 and plasma membrane connections between cells (TUCKER 198 1). Examination of pupal wings indicates that the developing hairs contain large bundles of actin filaments, and that the developing hairs are aligned at the earliest stages of hair morphogenesis (IA. Wong and P. N. ADLER, unpublished results). Cell geometry appears to play a role in tissue polarity on the wing since the bundles of actin filaments that take part in forming the hair extend distally from the distal vertex of each hexagonally shaped wing cell.

In Drosophila a small number of genes have been identified on the basis of mutant phenotypes as being essential for the generation of tissue polarity (GUBB and GARC~A-BELLIDO 1982). These mutations, to a first approximation, do not alter the morphology of structures produced by individual cells (or developmental units). Rather they alter the orientation of these structures (e .g . , hairs) with respect to their neigh- bors and the organism as a whole. The best studied

' Cul-rent address: (hrnegie Institution of Washington, 1 1 5 West Univer-

' C:ur!-ent address: Crop Genetlcs International, Dorsey, Maryland 21076. b i t v Parkway, Baltin~ore, Maryland 21 210.

(;twetir\ 1 2 6 401-416 (Octoher. 1990)

member of this group of genes is the frizzled (fz) locus (ADLER, CHARLTON and VINSON 1987; VINSON and ADLER 1987). Most mutations in this gene disrupt normal tissue polarity in all body regions. We have primarily studied the effects of fz mutations in the wing (ADLER, CHARLTON and VINSON 1987; VINSON and ADLER 1987) because of the ease of examining this flat structure microscopically. In the most severely affected region of the wing, null mutations result in a loss of both proper polarity and the parallel alignment of neighboring hairs, giving rise to a relatively random pattern of hair polarity. In less severely affected regions of the wing, polarity is abnormal although neighboring hairs usually remain aligned, thus resulting in a distinctive swirling pattern. This swirling effect is the principal phenotype seen in weak mutations that only partially inactivate the fz locus. frizzled mutations also typically cause a rough eye. Here again the mutation does not grossly alter the differentiation of individual cells or ommatidia, rather it alters their spatial relationship to the rest of the eye (R. CARTHEW, personal communication; L. MARSH, personal communication). There are several exceptional fz alleles that do not fall into the hypomorphic to amorphic series defined by the majority of alleles. The exceptional alleles were originally identified as tissue specific alleles, because they do not result in rough eyes, as do all other moderate or strong fz alleles (ADLER, CHARL- TON and VINSON 1987).

Mitotic clones on the wing of several fz alleles (including a null allele) disrupt the polarity of wild type cells located distal (but not proximal) to the clone of mutant cells (VINSON and ADLER 1987). This non-cell autonomous behavior argues that the polarity information itself is altered in fz mutant tissue, and that the function of the fz locus is essential for the transmission of a polarity signal along the proximal-distal axis of the wing. When similar mitotic clones were

402 P. N. Adler et al.

generated for the two exceptional (tissue specific) alleles the polarity of the surrounding wild type cells was normal. This cell autonomous behavior suggests that these alleles do not alter the polarity signal, rather, they alter the ability of cells to respond to the signal. Thus thefz gene has two functions. It is required for the transmission of a polarity signal (non- cell autonomous function) as well as for the cellular response to (transduction of) the polarity signal (cell autonomous function).

The fz locus could encode either one bifunctional protein or two single function proteins. To try to distinguish between these possibilities and to help in our efforts to understand the molecular mechanism(s) underlying the development of tissue polarity we have undertaken the molecular cloning of thefz locus. We report here the cloning of thefz locus via the transposon tagging strategy (BINGHAM, LEVIS and RUBIN 1981; RUBIN, KIDWELL and BINGHAM 1982; SEARLES et al. 1982), and the mapping of the locations of 11 independent fz mutations. Somewhat unexpectedly, the fz gene is quite large-mutations were found to be located over approximately 100 kb of genomic DNA. A cDNA clone and Northern analyses indicate that the principal RNA product of the fz locus is a 4-kb RNA that is encoded by 5 exons that span over 90 kb of genomic DNA. Conceptual translation of this RNA indicates that thefz locus encodes an integral membrane protein that, if located in the plasma membrane, would be able to interact with both extracellular and cytoplasmic molecules, and thus potentially function in both the transmission of tissue polarity information between epidermal cells and the transduction of the polarity signal to the cytoskeleton. Evidence for a second substantially rarer@ transcript has also been obtained. Conceptual translation of the sequence of a cDNA clone of this RNA yields a truncated version of the protein predicted from the major transcript. Thefz gene is the first tissue polarity gene molecularly cloned.

METHODS AND MATERIALS

Drosophila strains and mutant isolation: The cytology, mode of induction, and phenotypic strength of fz alleles mentioned in the text are shown in Table 1. Marker mutations and balancer chromosomes are described in LINDSLEY and GRELL (1 968).

Hybrid dysgenesis was induced by crossing Harwich (P) males and Oregon-R (M) females. The dysgenic F, progeny of these flies were crossed tofz th st inlTM3 flies and the F2 progeny screened for newfz mutations. Flies carrying new mutations were then crossed to TM3 (st)#% th st flies and stocks carrying the new mutations established over TM3. Newfz mutations were isolated after y-ray (3500 R, from a I3'Cs source (Isomedix) or EMS (LEWIS and BACHER (1968)) mutagenesis by crossing marked mutagenized males to fz females and screening the F1 progeny for phenotypicallyfz flies. Stocks of each new mutation were established over a TM3 balancer chromosome.

Revertants and stronger variants were derived fromfzc'FKc by crossingfzCT8'/TM3 (P) males and TM3IDCXF (M) females. The dysgenic fz""" carrring female progeny from this cross were then mated tofzp th stlTM3 ( s t ) males. The f i c T 8 c ' I f p 2 1 progeny of this cross were then screened for flies with either a wild type or stronger mutant phenotype. Mutant or revertant stocks were then established using the TM3 balancer chromosome.

Cytogenetic analysis: Salivary gland squashes were done by standard technique except no stain was used. Chromo- somes were examined under phase optics. In situ hybridizations were done as described by BINCHAM, LEVIS and RUBIN (1981) using nick translated ('H-TTP) DNA as a probe.

Isolation of genomic clones: A genomic library was constructed in the Bam site of the EMBL4 vector (FRIS- CHAUF et al. 1983) from a partial MboI di est of DNA isolated from fzCTBc containing flies. The fi"'" containing chromosome had been outcrossed (with the fz""" stock as the female parent) to remove extraneous P elements. During this outcrossing the number of P elements apparently fell low enough to induce some mobilization of the P elements. The DNA was packaged in vitro, plated on the P2 lysogen (Q359), and this primary plating screened with a probe made by nick translating the P element containing plasmid pn25.1 (RUBIN and SPRADLING 1982; SPRADLING and RUBIN 1982). DNA from 31 P element containing recombinant phage was used to make probes for in situ hybridization to Oregon R salivary gland chromosomes.

Chromosome walking was done by probing filter plaque lifts of the Maniatis library (MANIATIS et al. 1978) with probes made by nick translation or random priming. On two occasions (to get across middle repetitive sequences) we isolated recombinant clones from a genomic library of the P2 strain constructed and kindly provided by L. SEARLES (University of North Carolina). We subsequently isolated bacteriophage from the Maniatis library that contained inserts that spanned the middle repetitive element.

Large scale preparation of bacteriophage DNA was car- ried out by the glycerol gradient technique (GARBER, Ku- ROIWA and GEHRINC 1983). Restriction mapping was car- ried out using single and double digests with six enzymes (WEINER, SCOTT and KAUFMAN 1984). The map was confirmed by genomic Southern analyses.

Isolation of cDNA clones: In our initial experiments three cDNA clones were isolated from a cDNA library constructed by POOLE, KAUVER and KORNBERG (1985) in the XgtlO vector using as a probe an 8-kb EcoRI fragment of genomic DNA from CV150. In subsequent experiments several additional clones were isolated using the insert from the cDNA clone ACVC22 (this clone was named CVC22 in a previous publication (VINSON, CONOVER and ADLER 1989)) as a probe. The procedures for experiments with this library and clones derived from it were the same as for the genomic libraries.

Many cDNA clones were recovered by screening the cDNA libraries constructed by BROWN and KAFATOS (1 988) using random primed DNA from previously isolated cDNA clones as probes. The cDNAs in these libraries were direc- tionally cloned into the pNB4O plasmid vector, thus facili- tating the determination of 3' and 5' ends. All of the clones have an oligo(A) stretch at their 3' end, although in some cases this is due to the oligo(dT) primer hybridizing to an internal oligo(A) sequence during reverse transcription. The library was screened by the colony hybridization procedure described by BROWN and KAFATOS (1988).

Isolation and analysis of genomic DNA: Drosophila DNA was isolated from adult flies as follows. Two hundred adult flies were anesthetized, and homogenized in a ground

Tissue Polarity Gene in Drosophila 403

TABLE 1

List offi mutations

Allele nanle Origin" Phenotypeb Het? Cytology Reference

CT8C GIIa

EA?

K 2 1 GE26

GN6a

3 c2 I GL?a EAB2 GD46 KD4a

CT8CX2

KB4b

HD G

G

G G

G

X G G G G HD

HD

HD

W w

M

S S

S

S S M M M S

S

M

N Y

Y

N Y

Y

N Y N N N N

N

N

Normal, P element insertion Rearrangement,fr locus

juxtaposed to centromeric heterochromatin

2,3-Translocation, likely or- der 21-40/70D4,7-61;60- 41/70D4,7/100

In(3L)70D4-7;75A5-B12 Rearrangement,& locus

juxtaposed to centromeric heterochromatin

Complex rearrangement,fz locus juxtaposed to centromeric heterochromatin

In(3L)70D4-7;75D3-8 Tp(3,2;40;66B-D;70D4-7 Normal Normal Normal Normal, derived from CT8C

Normal, derived from CT8C

Normal, derived from CT8C

by hybrid dysgenesis



ADLER, CHARLTON and VINSON (1 987) VINSON (1 987)

ADLER, CHARLTON and VINSON

ADLER, CHARLTON and VINSON VINSON (1 987)

(1 987)

(1 987)

VINSON (1987)

VELISSARIOU andAsHBURNER (1 98 1) ADLER, CHARLTON and VINSON (1987) VINSON (1987) ADLER, CHARLTON and VINSON (1 987) VINSON (1987) KLEIN (1 987)

KLEIN (1 987)

KLEIN (1 987)

a HD, hybrid dysgenesis, G, 7-irradiation, X, x irradiation.

' Het? = heterochromatic breakpoint ? Y indicates that the mutation juxtaposesfr to centromeric heterochromatin. N indicates that it does W, weak; M, moderate; S, strong.

not.

glass homogenizer in 1.5 ml of buffer A [0.2 M NaCI, 0.2 M sucrose, 10 mM EDTA, 30 mM Tris-HCI (pH 8.0), 0.15 mM spermine, 0.15 mM spermidine, and 400 pg/ml ethidium bromide]. Then 1.5 ml of lysis buffer was added [0.25 M EDTA, 0.5 M Tris-HCI (pH 9.2), 2.5% sodium dodecyl sulfate (SDS)]. Proteinase K was added to 100 pg/ml and allowed to digest for 1 hr at 60". The DNA was then extracted twice with phenol, twice with phenol:chloroform:isoamyl alcohol (24:24: l), and once with ch1oroform:isoamyl alcohol (24:l). This was followed by a 1 hr RNase A digestion (20 pg/ml) at 37" and an ethanol precipitation. Whenever possible the DNA precipitate was spooled from the solution. For genomic Southern analysis, 5 or 10 pg of DNA was digested with an appropriate restriction enzyme, fractionated on a 0.8% agarose gel, blotted to Nytran (Schleicher and Schuell), and hybridized with appropriate nick translated or random primed probes. In the experiments where we were mapping the location of fz mutations, all genomic DNA samples were digested with 6 enzymes. All of the fz alleles listed in Table 1 were analyzed.

Isolation and analysis of RNA: RNA was isolated from various developmental stages by freezing the organisms in liquid nitrogen and then grinding them with a mortar and pestle on dry ice. The ground powder was added to a tube containing an equal volume of phenol:chloroform/isoamyl alcohol (24:24:1) and 2 X NETS buffer [200 mM NaCI, 2 mM EDTA, 20 mM Tris (pH 7.5), and 1% SDS], vortexed and centrifuged to separate phases. Both the aqueous and organic phases were reextracted and the aqueous phases pooled. The final aqueous phase was extracted with chloroform and precipitated with 0.25 M NaCl and 2.5 volumes

of 100% ethanol at -20" overnight. The pellet was dried, resuspended in sterile water and the RNA precipitated twice with LiCI. The final RNA pellet was resuspended in water. Poly(A)+ RNA was selected by oligo(dT)-cellulose chroma- tography, and 5-10 r g poly(A)+ RNA was fractionated by electrophoresis on a 1 % formaldehyde agarose gel (MANIA- TIS, FRITSCH and SAMBROOK 1982), and electroblotted to Nytran.

Several different procedures were used to probe North- ern blots. In some experiments the Northern blots were probed with '*P-labeled single-stranded RNA probes made using the T7 or T3 RNA polymerases from templates subcloned into Bluescript vectors (Stratagene). The hybridizations were at 65" for 16 hr in 50% formamide, 5 X SSC, 5 X Denhardt's solution, 50 mM sodium phosphate (pH 6.5), 0.16 mg/ml of salmon sperm DNA, 5 pg/ml each poly I , U , C, A and 10% dextran sulfate (MANIATIS, FRITSCH and SAMBROOK 1982). The blots were then washed twice with 1 X SSC, 0.1% SDS, followed by two washes in 0.1 X SSC, 0.1% SDS. All washes were at 65" for 0.25-1 hr. This procedure gave a reasonably strong signal, however a nonspecific band of hybridization (1 kb) was seen. This "hybridization" signal was specifically removed by including RNase in the next to last wash at a concentration of 0.5 pg/ml (DELEON et al. 1983) (in this case this wash was at 50" in 2 X SSC). This, of course, destroyed the blot for further probing.

As an alternative procedure Northern blots were hybridized at 42" using the procedure for Nytran supplied by Schleicher and Schuell and single-strand DNA probes made by doing an asymmetric polymerase chain reaction (SAIKI et al. 1988) in the presence of ["PIdCTP. The conditions for


making such probes (e.g., annealing and extension times) were optimized for particular fragments. This approach gave almost as strong of a signal as with the single stranded RNA probes but without the nonspecific hybridization. This approach also allows blots to be reprobed.

Northern blots were hybridized with probes for hsp83 (HOLMGREN et al. 198 1 ; MASON, HALL and GAUSZ 1984) or rp49 (O'CONNELL and ROSBASH 1984) to correct for RNA loading differences. The hsp83 probes were made by random priming of double-stranded hsp83 cDNA clone sequences. The rp49 probes were DNA probes made from an single-stranded M 13 subclone labeled with Klenow and the M 13 probe primer. Hybridization conditions for the hsp83 probes were as suggested by Schliecher and Schuell for Nytran, and for rp49 probes the procedure of BINCHAM and ZACHAR (1 985) was used. In cases where Northern blots were probed for fz with single-stranded DNA probes (or single-stranded RNA probes without RNase treatment) the blots were stripped by boiling for 5 min per side in 1 % SDS, 1 mM EDTA. The blots were then reprobed for hsp83 and/ or rp49. For blots where single-stranded RNA probes were used to detect fz RNA two other alternative procedures were followed. One consisted of probing the blot for rp49 prior to RNase treatment. This procedure did not yield very goodfz signals. The other consisted of taking an aliquot of the RNA samples in loading buffer and running those on a parallel gel, which was then blotted to Nytran and probed for either hsp83 or rp49. Although this procedure has theoretical weaknesses, in practice it gave very similar results to those experiments where the same blot was reprobed.

Radioactively labelled first strand cDNA was made for use as a hybridization probe by taking a mixture of poly(A)+ RNA isolated from wandering third instar larvae or early pupae, annealing random hexanucleotide primers (Phar- macla) and extending the DNA chains using cloned MuLV reverse transcriptase (Pharmacia) in the presence of ["'PI dCTP.

First strand cDNA for use in polymerase chain reactions was made using as primers oligonucleotides complementary to sequences found in the 3' exons of both classes of fz RNAs. The specific primer for the class I RNA was GCCCAGTAATCCCACGGCGGG (fzac 2172 (fz class I RNA, complement, starting at base number 2172)), and the primer for the class I1 RNA was GGACCAT- CAGGTCGAATTGGT (fzbc 2019 (fz class I1 RNA, complement, starting at base number 2019)). One microgram of poly(A)+ RNA was annealed with the two primers and cDNA synthesized by Mulv reverse transcriptase (Pharma- cia) using the conditions suggested by the supplier. The RNA was hydrolyzed with RNase, and the cDNA extracted with phenol/chloroform, chloroform, ethyl ether, and then passed through a spin column of Sephadex G-15 (Pharma- cia). The cDNA recovered from the spin column was diluted fivefold with H 2 0 (this resulted in a typical volume of about 500 PI). One to 5 PI aliquots of cDNA was then amplified via the polymerase chain reaction (SAIKI et al. 1988) using the ampliTaq kit (Perkin Elmer Cetus), in a thermal cycler (Perkin Elmer Cetus) using a 1-min denaturation at 94", a 3-min reannealing at 60 O , and a 3-min extension at 72 O . TO amplify class I specific sequences 30 cycles were run using GACCATTATGCCAAATC (fza 9 12-fz class I RNA, starting at base number 91 2) and fzac 2172 as amplimers. The product of this reaction was purified by a phenol/chloroform extraction and fractionation on a Sephadex G-15 spin column. The product was then diluted to 500 gl, and 1-5 wl of this DNA was amplified a second time for 30 cycles using CGGGTATGTGAAAAGTT (fza 1102) and CCA- ATTCGCAACATCAGGCG (fzac 2126) as primers. The

use of nested primers during a second round of amplification has proven in our hands to be very valuable in eliminating (reducing) the build up of artifactual amplification products. The class I1 cDNA was amplified using fza 912 and fzbc 2019 as the primers for the first round of amplification. The second round of amplification for the class I1 cDNA used fza 1 102 and GGTGTGGAACTGCCATAAGT (fzbc 1953) as the primers and was done for 40 cycles. Fifteen microliters of the 50-wl polymerase chain reaction (PCR) was then fractionated by electrophoresis on a 0.9% agarose gel. Both reconstruction control experiments and repeti- tions of the above experiments indicate that the amount of amplified cDNA is not strictly proportional to the amount of specific RNA present in the initial reverse transcription reaction. Large differences are however, likely to be a reliable indication of differences in the abundance of RNA molecules. Attempts to amplify both classes of cDNAs in the same tube were not, in general, successful-only the class I cDNA appeared to amplify. This may have been due to competition for the common 5' amplimers.

DNA sequencing: We have previously reported the sequence of the incomplete class I cDNA CVC22 (VINSON, CONOVER and ADLER 1989). This cDNA clone has been renamed ACVC22 for consistency with our new nomencla- ture-class I clone names begin with an A, class I1 names with a B. This sequence was obtained by first subcloning the insert into pEMBL 18+ and pEMBL 18- (DENTE, CESARINI and CORTESE 1983). Processive deletions were made using exonuclease 111 and S1 nuclease and a set of deleted clones obtained (HENIKOFF 1984). These were sequenced using the Sequenase reagents ( U S . Biochemicals). All clones were sequenced separately with both dGTP and dITP in the presence of single stranded DNA binding protein. The entire sequence was done in both directions. The full length class I cDNA AC2 was subcloned into the bluescript vector (Stratagene) in two pieces (1-2304, 2304-3809). The sequence of the 5' fragment was obtained by using a set of oligonucleotide primers that corresponded to the sequence of ACVC22 every 200 bp. The sequence of the 3' fragment was largely obtained from a set of nested deletions, although some sequence was obtained using specific oligonucleotide primers. Once again the Sequenase reagents were used. Much of the sequence of AC2 was only determined in one direction. The sequence of the 3' end of the class I cDNA clone AE6 was obtained using as a primer an oligonucleotide whose sequence corresponds to the T 7 promoter (a T 7 promoter sequence is found just 3' to the cDNA inserts in the libraries constructed by BROWN and KAFATOS (1988) and a single-stranded DNA template generated by an asymmetric PCR reaction using as a primer GGTCAAG- TACCTTTGCT (fza 2312). The sequence of the class 11 cDNA BE2 was determined by first subcloning the cDNA sequences into the bluescript vector, and then making a series of nested deletions which were sequenced as described for ACVC22. A few holes in the sequence were filled in using specific oligonucleotide primers. Part of the 3' untranslated region was only sequenced on one strand. The 3' end of the class I1 cDNA BC3 was determined using the T 7 promoter sequence oligonucleotide as a primer, and a single strand template generated by an asymmetric PCR reaction using as a primer GATCAATGCCTCTCTAA (fib 3681). The genomic sequence was obtained by subcloning exon containing restriction fragments into the bluescript vector. Single stranded templates were sequenced in one direction using specific oligonucleotide primers. Part of the genomic sequence that corresponds to the 3' untranslated regions of the mRNAs was not sequenced. The genomic sequence that corresponds to the 3' end of both classes of cDNAs was


sequenced and found to be in the expected genomic restriction fragments. Thus it is possible that there are additional small (<2 kb) introns that separate untranslated sequences present in exon 5 (and 5b) in Figure 8. The DNA sequence was entered using an IBI gel reader and the sequence assembled using KFMATCH (W. PEARSON) and the STADEN (1 980) programs. DNA and protein sequence analysis was done using a variety of software packages (IBI Pustell), DM (MOUNT and CONRAD 1986), FASTA, LFASTA and RDf2 (PEARSON and LIPMAN 1982), and the GCG package (Uni- versity of Wisconsin) on a variety of computers (IBM-AT, AT&T-3B15 and a VAXl1/70.

RESULTS

Isolation of a P element insertion mutation in the fr locus: T o facilitate the molecular cloning of fz, six new fz mutations were recovered among the progeny of dysgenic parents (90,000 chromosomes screened). Using both genetic and cytogenetic criteria only one of these, fzcTBc, is due to a P element insertion. A high frequency (=1/50) of revertants is seen among the progeny of dysgenic fz""" flies, and in situ hybridization using a P element probe (pr25.1) (RUBIN and SPRADLING 1982; SPRADLING and RUBIN 1982) indicates the presence of P element sequences at 70D4-7 (the cytological location of fz) (VELISSARIOU and ASH- BURNER 198 1) in fz"TB". Revertants of fzCTBC show a loss of P element sequences at fz as assayed by in situ hybridization and genomic Southern analyses (see be-

Cloning of the fz locus: ThefiCTBC stock was outcrossed several times to M stocks to reduce the number of extraneous P elements. DNA was isolated from outcrossed flies, and a genomic library constructed in the EMBL4 vector (FRISHAUF et al. 1983). One P element containing recombinant phage (CV20) (out of 31 tested) hybridized to 70D4-7, the cytogenetic location off.. . Drosophila DNA from this recombinant phage was used as a probe to clone wild type fz DNA from the Maniatis Canton-S library (MANIATIS et al. 1978). A genomic Southern analysis of both revertants and more severe alleles derived from fzCTBC (described below) demonstrated that we had indeed cloned fz mutant DNA in CV20. A chromosome walk resulted in the cloning and restriction mapping of about 170 kb of fz region DNA (Fig. 1). Two middle repetitive elements were encountered during the walk, one was centered at map position -37 and the other at map position +42.

Mapping the location of fr mutations: T o delineate the extent of the fz locus we have mapped the location of all seven existing fz rearrangement mutations (Table 1) via genomic Southern analyses (see Figures 2 and 3). In addition we have identified (and located) DNA alterations associated with three cytologically normal, y-ray-induced mutations that appear to be due to insertions. Of the seven fz alleles associated with cytologically visible rearrangements, two stand

low).

out as not being strong alleles (ADLER, CHARLTON and VINSON 1987; VINSON 1987). One, f iG1 la , is a weak allele while the other, fzEA3, is a moderate allele. Both of these mutations are associated with heterochromatic breakpoints that juxtapose the fz locus to centromeric heterochromatin. We have localized both of these mutations to the interval between - 12 and -20 on our DNA map (Figure 3). These alleles are our two most distally mapping mutations (Figure 2). We suspect that these mutations break the chromosome outside of the fz gene and inactivate it via a position effect.

Four of the remaining five alleles associated with cytologically visible rearrangements appear to be null mutations (ADLER, CHARLTON and VINSON 1987; VIN- SON 1987). These alleles ( fzK2', fzGEZb, f zGN6a , and fz') have been mapped to a 46-kb region which must remain intact for fz gene function (Figures 2 and 3). The final mutation (fz"") associated with a cytologically visible rearrangement is a strong mutation, although some residual gene activity may remain (AD- LER, CHARLTON and VINSON 1987). We have localized this mutation to the interval between +66 + +70 on our DNA map (Figures 2 and 3). This is the most proximal location for a fz breakpoint.

We have used in situ hybridization to confirm the position of the breakpoints in the two euchromatic inversions ( fzKzI and fz'), and to orient the "walk" on the chromosome. The results are shown for fi"" (Figure 4). Salivary gland chromosomes from fi""/ TM3 larvae are not paired in the region affected by thefi"" inversion. Nuclei hybridized with nick translated bacteriophage CV535 DNA show a hybridization signal distal to the fz"" inversion (Figure 4A). When nick translated DNA from CV 1 12 is used as a probe, the hybridization signal is seen at the proximal end of the inversion (Figure 4C). These results orient the walk on the chromosome as shown in Figure 1. When DNA from CVl 1 1 is used as a probe, hybridization signals are seen at both ends of the inversion (Figure 4B), thus confirming that the breakpoint in

fzKZ1 is located within the Drosophila sequences in C V l l l .

Three of our four y-ray induced, cytologically nor- ma1,fi mutations ( f ~ ' ~ ' ~ , fzEABZ, and fzCD4') were found to be associated with gross DNA alterations. In all three cases, genomic Southern analyses suggest the alteration is due to the insertion of DNA sequences in the fz locus. The three apparent insertion mutations are located between map positions +21 + +22, +70 + +77, and +76 + +82, respectively (Figures 2 and 3). When these results are combined with the mapping of the P element insertion in fzCT8" [map position (-3.5)] we find insertion mutations mapping to over 80 kb of genomic DNA. When the rearrangement and insertion mutations are considered


CV602 cvlll CV1013 cwo1 (3'131 cv122 CV150

0'913 cv535 C m O -

cv112 Distal

-60 -50 -40 -30 -20 -10 0 10 20 30

EcoRl I I I 1 I I I 1 I I I I I I I I I I I I S a l I I I I I I I

Hindm 1 1 I I 1 I I I I I I I I 1 I l l

no1 I I I I I I I I I I

CV150 0'2105 (3'2303

cv220 1 CY2204 cv2402

30 40 50 60 70 80 90 100 2 Proximal 110 120

I I1 II I I I I I l l I 1 111 I I 11 I UEcoRl

I I I 1 II I I I I I 1 1 1 s a l J I I I I I I I I II 1 I I I I l l 1 I l l I 11 HindIII I I I I II I U II XhOI

FIGURE 1.-Cloned DNA from the$ region. The location of restriction sites in the approximately 170 kb of cloned) region DNA is shown, along with the regions contained within the inserts of individual recombinant phage. The orientation of the cloned region on the chromosome is given (distal toward the telomere, and proximal toward the centromere).

hll-4 b CTBCX2 -

KD4a FIGURE 2.-Molecular genetics of the& lo-

cus. The location of mutations that inactivate the$ locus is shown. The restriction fragments of cloned genomic DNA that hybridize to both the class I and class I 1 cDNA clones are shown

r? GI1 a

Cl3.Z

4" V d h xr;"., A EA3 cT8c K21 CEZb c2F2 A -20 0 20 40 60 80 100 below the map as exons of the presumed pri-

I I I I I I I I I I I I I I mary transcripts. Arrows indicate the direction dista

' % class u

together, we findfz mutations spanning about 100 kb of genomic DNA. No gross DNA alterations were detected for the two EMS induced tissue specific alleles (fz"' andfzF;").

Analysis of hybrid dysgenesis induced variants of fzCTBc: The P element insertion in fzcTEc results in a weakfz mutation that is unstable during hybrid dysgenesis. Both phenotypically wild-type revertants, and strongerfz mutations have been obtained among the progeny of dysgenic fz."TEc flies, and the associated structural changes have been analyzed by genomic Southerns (KLEIN 1987). Thirty-seven phenotypically wild-type revertants were isolated and analyzed. All showed gross DNA alterations in sequences detected by probing with the 0.5-kb EcoRI fagment into which the P element is inserted in fz"T8", and a majority appeared to be due to precise excision of the P element. Seventeen hybrid dysgenesis induced variants of fzcTEc that produced a stronger mutant phenotype

prorimaf of transcription.

were also obtained and analyzed. The most interesting consisted of eight variants where imprecise excision of the P element resulted in the deletion of flanking genomic sequences. Two of these secondary mutations are associated with deletions extending in both directions from the site of the P element insertion in fzcTEc. One of these mutations (fzKD4') results in a greater than 50-kb deletion (Figure 2). Four of the mutations are associated with small deletions that extend proximally from the P element in fzcTEc, resulting in the loss of the neighboring 0.3-kb EcoRI fragment, and the loss or reduction in size of the next (2.7 kb) EcoRI restriction fragment (for example,fiCTBcX2, Fig- ure 2). These mutations are strong mutations, thus sequences essential for fz function are located just proximal to the site where the P element is inserted infiCTEC. As is discussed later, the P element infiCTE" is inserted just upstream of the 5' most exon offz. In addition, one moderately strong allele,fiKB4b, was iso-

Tissue Pol;tl-ity Gene i n D1-osophil;t 407

F I ( ; I K I - . : ~ . - I . o ( . ~ I ~ ~ / ~ I I ~ ( I I I ol 1: n1utatiom vi.1 g:cmorrlic Soutll(m1 ;lnalv\cs. 111 c d 1 1);lnel DS;\ c s ~ ~ x ~ c t l l1.on1 wild-t\pc (or prcntal ) flies is con1pxrc(l 1 0 I)\:\ cstr;~c~ct l I'romj: nIu1a111 Ilk\ (either,/: Ilomo/vgou\ 0 1 . Iwmi/vgou\ 1lic.s). l)S:\ \\.;IS digested \v i l l i ;I rertric.- t i011 ( . t l / w I ( . . l ' ~ ~ ~ ~ ( . ~ i o ~ ~ ~ ~ ~ l ~ ( l 0 1 1 ;111 ;~g;~rosc gel. I)lottcd t o nirl.occ*llu- l o w . ; I I I ~ hvIwidi/ccl w i t l l ;I nick tr;~nsl;~tctl restriction Iklgmcwt liom the W I L . . \ ITO\\Y ildic;1tc novel rcstrictiorl fr-;qmcnts crc.;~tctl b v tlx. mtlt;llioll. 111 \ o 1 1 1 c 8 cligc\t\ 1 ) o 1 1 1 the prosinl;tl and distal ~ ~ ~ ~ ~ ~ r ~ ~ ~ ~ ~ ~ g e ~ ~ ~ c ~ l t l'r:lgmclus arc seen, while i l l others o n l v one of the t w o I I C . N I ' I X ~ I I I ~ I I I \ ~ I X . tlctcc~ctl. 1)igcstioll.c of genomic, DNA and l)o\ilious 1 1 1 ' I I I ( . 1)1d)c\ u s c d \vcrc:f:' I ' KroRI ( - 2 0 + -12): f:""". S h o l (-20+-12):,/:'f", f<coRl . ( - :K I - -3 ) : , f?J , S/rol. ( - 5 + +3): f:"';". k;roRl, ( + ? I - +~f""~""', k;foRl. ( + ? I + +3i);./:j.

+ 3 ) . (KO l l l ' l \ l)alld\ \\ere see11 for y " ' rcg"r'llcss 0 1 ' lv l lat

S h I . (+:{I -+.I;): ,/:'", f<coRI. (+.I; + +(<ti); f:f If'", IlintIIII, (+fi.I -+%I): ,/:'""" f k / m I I I , (+fi . f + +%*I): f:""". EcoRI. (+X +

I X . \ I I . ~ ~ I ~ ( I I I (,11/vlnc u c used. 1 1 1 a11 c ; w s ;I p r c n u l 1x11~1 did disap- ~ ) ( ' ; I I ' . (:onsid(,ring tIlis n w l I t anti the f ie1 I I I M ~ : " ~ " " is ;I Iwtcrochro- nwliu IwwLpoinI. n v wggcst ~ I I ; I I it just;~poscs f i t o a highlv 1 x p ' ; ~ t ( d wt(.llitc s(qtw1( c t l l ; l t t1oc.s n o t c.ontain ;I typical l i .eclwwy 01. wstriction \it<,\. 'l'hus t l~c l)rc.;lkl)oint I'ragnwnts ~ \ . o u l t l remain \ c ~ v I ; I I ~ . a n ( 1 I I O ~ I)( , dctc-c~cd b v the gcnon1ic Soutllcrn awlvsis.) 'I'hc I;IIK.~ U I I I I X ~ I I ~ I I ~ fi' I,'' and I I W I)hellotyl)ic.;lIIv \viltl-typc.f:' ""

I)\;\ dc111o~1\1ratc\ 1 1 w t i l l (:\'2? we II:ICI c lo~~ct l tl~c I' ~ I C I I I ~ I I ~ rcy)onsil)lc. f 0 1 . 111c 1:' '" n1ut;ttion. .l'llc insertion of the I' clcmen~ in /:' "' rc.sult(~l in 1 1 1 ~ o .>-k l ) k;coRI fragn~cnt from ( : \ ' I I I being qdit i l l t o ~ n ' o hrgcr l'r;IgmcI~ts. ~ I I w I' ckmcnt I I ~ S csc-iscd i n tlw ~ ~ v v c r t a n ~ I/' Ir". r-cworing I I W \viIcI-t\l)c restriction p t ~ c r n .

Iatetl t h a t deleted D N A sequences distally. This 2-3- k h tlrletion estentled into the EcoRI fragment distal to the 0..5-kh 1:'coRI fragment into which the P element Ilatl insel-ted. The analysis did not enable us to deter- mine if any sequences derived from the O..i-kb EcoRI remainetl, thus i t is possible that the deletion estended a short distance proximally as well. This allele suggests that sequences located 2-9 k b distal to the insertion

l ' l ( X ' K F 4.-fn .$/ /I / h\I)ridi/ation oricnls rl~cj: w a l k o n the chromosome. .llw orientation of t h nwlccul:tr \ \ x l k w i t h respect 1 0 rhc centromere and tclomerc W:I\ dc*tcrnlinctl b v in s i / ? / hyIwidi/ation using I~Ic.,/:"~ inversion. I n salivary gland chromosome sclu;dlcs o f

f~"~/lT.\fjr Iarv;lc t I ~ c * j: rc'gion is unlmircd. I)s~\ from I)actcr-io- pll;lgc.s (;\~.-):rl(p;llld ;\). ( : \ ' I 1 I (p;lllcI R). ;111tl ( : \ ' I 12 (pm1cI (:) were 1lic.k transl;ttrd and hv1)ridixd t o S I ~ I 1)rcp;lr;ltions. Tllr

arron tlclincs the l o w t i o n ol'thc end p i n t s of 111c inversion 011 the fPJ chron~osomc. (:\'3:%3 DS;\ hvbridix-s distal t o the inversion, while (:\ ' I I2 DS:\ hv1)ridi~cs prosinla1 t o 111c inversion thus ori- enting the w l k ;IS s h o w , i l l Figurt- I . ( ; \ ' I I 1 I)\:\ hvbridircs t o

I ) o ~ h sides of tl~c.,/:~'' inversion indicating that tIl~.f:"~ I)rc;tkpoint is cont;linctl within the insert i n ( ; \ ' I I I . thus ronlirming t h c results fro111 gcnowic Sourllcrn an:jlvsis. ~l'lle Iociltion of t w o chromosonw l:lndm~rks ((iXIK: ; 1 1 d 7 O h ) is s I 1 o w I .

of the P element infz','rx','are important forfz function, although since this allele retained some gene function these sequences are not absolutely essential for fz function.

Identification of transcribed regions: T o identify potential exons, a Southern blot of restriction digests of recombinant phage containing inserts spanning the interval from -45 to +38 on our DNA map was probed with racliolabeled cDNA made from a mixture of late larval and earlv pupal R N A , which are two developmental stages when we presume fz will be active. Four regions of hvbridization were seen (data not shown). Two strong hvbridization signals (-36 + -39, +36 + +38) correspond to restriction fragments that contain repeated sequences, and presum- ablv represent transposable elements. Two regions located at -2.7 + 0, and +26 + +35 gave weak hvbridization signals, and are within, or close to, the region that the breakpoint mapping argues must remain intact forfz function. The deletion of the more distal of these two regions (-2.7 + 0) results in a severe mutation (fz'~'7;u'~s-7-see Figure 2). Thus, these two regions appeared to containfz exons.

Isolation and mapping of fz cDNA clones: The 9- kb EcoRI fragment from CV 150 (+26 + + 3 5 ) , identified above as being likelv to contain fz exon sequences, was used to probe three cDNA libraries. Several clones were obtained from the librarv constructed bv L. KAUVAR (see POOLE, KAUVER and KORNRERG 1985) from R N A obtained from earlv pupae (5..5-7.5 davs of development). DNA sequences from the largest of these clones (ACVC22-2..i-kb insert) were used as probes to isolate additional cDNA


TABLE 2

cDNA clone analysis

Number isolated

Developmental stage“ Class I Class 11 Total

Early embryo 5 0 5 Mid embryo 10 1 11 Late embryo 4 1 5 Third instar imaginal disc 4 0 4 Early pupae 7 0 7

30 2 32

*Al l of the cDNA libraries screened were constructed by N. BROWN (BROWN and KAFATOS 1988), except for the early pupal library, which was constructed by L. KAUVAR (POOLE, KAUVAR and KORNBERG (1985).

clones (Table 2). Most of these clones were obtained from cDNA libraries constructed by BROWN and KA- FATOS (1988). Clones were initially classified by restriction mapping with both four- and six-cutter enzymes. This analysis suggested that there were two basic classes of clones that shared sequences over about two-thirds of their length, but which differed over the remainder of their sequence (at the 3’ end). The major class was found to be heterogeneous and appeared to contain three different 3’ endpoints. Subsequent blotting and sequencing experiments (see below) have confirmed these conclusions.

The location of the genomic sequences represented in both classes of cDNA clones was determined by hybridizing a Southern blot of restriction digests of the recombinant phage encompassing the genomic walk with radioactive cDNA sequences. For the major class (class I) hybridizations were seen (Figure 5) to the 2.7-kb EcoRJ fragment from CV111 and CV13 1 (-2.7 -+ 0), to the 5-kb EcoRI fragment of CV112 (+21 + +26), to the 9-kb EcoRI fragment of CV150 (+26 + +35) and the 4-kb EcoRI fragment of CV2303 (+87 -+ +91). In other experiments we have also seen hybridization to the 0.5-kb and 0.3-kb EcoRI fragments of CV 1 1 1. Thus the class I cDNAs represent an RNA encoded by exons that span about 90 kb of genomic DNA. Probes derived from class I1 cDNA clones hybridized to all but one of the restriction fragments that hybridized to the class I clones (the exception being the 4-kb EcoRI fragment from CV2303). In addition, hybridization was seen to two restriction fragments (found in CV2 105) that the class I clones did not hybridize to. Thus, the class I and class I1 clones differed by the use of an alternative 3’ exon. For the sake of clarity the names of all class I cDNAs begin with an A, and all class I1 cDNAs with a B.

Northern analyses of frizzled RNA: We have probed Northern blots of poly(A)+ RNA with single stranded riboprobes (MELTON et al. 1984) derived from class I cDNAs. A 4-kb band of hybridization is seen (Figure 6) in RNA isolated from embryos, larvae,

pupae and adult females. The longest class I cDNA clone (AC2) is about 3.8 kb, thus it is likely to be close to full length. A second band of hybridization (3.6 kb) is also seen in RNA isolated from adult females and embryos. No hybridization was seen to RNA isolated from adult males. This suggested that the RNA seen in females would be found in the ovary, as has been confirmed by in situ hybridization experiments (data not shown). The results with the single stranded probes indicate that thefz gene is transcribed from distal to proximal (-3 + +91) on the chromosome. Somewhat surprisingly, the developmental stages in whichfz mRNA is most abundant are early embryos and adult females. Substantially morefr RNA is detected in pupae (0-96 hr) than wandering third instar larvae. Both bands (4 kb and 3.6 kb) of hybridization to adult female RNA are seen when probes derived from either end of the cDNA clone are used (data not shown). Since the 3’ probe used in this experiment is specific for the class I cDNA clones, the two bands of hybridization to adult female and embryonic RNA do not represent the two classes of cDNA clones recovered. The relative intensity of hybridization to the 3.6-kb RNA, as compared to the 4-kb RNA, was reduced when a 3’ specific probe was used, suggesting that the 3.6-kb RNA lacks some sequences present near the 3‘ end of the 4-kb RNA. As was described above, our cDNA clone characterization provided evidence for three different 3’ ends on our class I cDNA clones. One cDNA clone (AE6) had a 3’ end 321 bp upstream of the 3’ end of our longest class I clone (Figure 8). Thus, it is possible that this clone represents the 3.6 kb RNA, and that this RNA arises from the use of an alternative poly(A) addition site.

When Northern blots were hybridized to probes specific for the class I1 cDNA clones, the only evidence of hybridization was a very faint signal (slightly longer than 4 kb) from embryonic RNA. This result, along with the low frequency of recovery of class I1 cDNA clones (frequency for all libraries screened 1/1 06, which is 1/10 to 1/20 the frequency for class I clones), suggests that the RNA represented by the class I1 cDNA clones is very rare.

To identify developmental stages where the class I1 RNA was present we have used the polymerase chain reaction to amplify fz sequences from first strand cDNA. The amplimers chosen for these experiments would be expected to yield a 1024-bp fragment for the class I RNA and an 851-bp fragment for the class 11 RNA. As seen in Figure 7, this approach provided evidence for both types of RNAs in embryos, pupae and adult females. f, encodes a transmembrane protein: We have

previously published (VINSON, CONOVER and ADLER 1989) the 2466-bp sequence of the class I cDNA clone ACVC22. We report here the 3800-bp sequence of

A B C D E F G H I J K L M N O P p- . -...I- m " - 1

b

.̂

(AC2) our longest class I cDNA clone, the 5762-bp +4 (CTCAGTT) matches for nucleotides 2 "-* 7 the sequence of (BIC2) our longest class I1 cDNA clone, consensus start site for transcription in Drosophila and a s well the genomic sequences that encode and ATCAG/TTC/T (HUI.TMARK, KLEMENZ and GEHR- flank thefz exons. The composite sequence is shown ING 1986). We suggest that this region is the start site i n Figure 8. The longest cDNA clones of both classes for transcription of fi We note that if the cDNA (AC2 and RE2) start at the same nucleotide, which we clones are truly "complete" then the start of transcrip- have given the number 1 . The sequence from -5 + tion woulcl be at the A at position 4 of this sequence,

P. N. Adler et al. 410

I-, 11 -

b

A B C D E F G H I

J

which is the same position seen for the sevenless (sev) gene (BOWTHALL, SIMON and RURIN 1988). T h e first TATA sequence upstream of this site is at - 1 19. This is too far upstream to function as a typical T A T A box. A number of other Drosophila genes, such as ddc and s a , have been found to lack typical T A T A sequences (BOWTHALL, SIMON and RUBIN 1988; BRAY and HIRSCH 1986). A 15-bp sequence (-1 7 + -32) upstream of the proposed start site forfz transcription matches 14 bp of a 17-bp sequence (GGCTTGGTTA- AAAAGCC) in a similar position (-19 + -36) in the sev gene. There is not however, a remarkable match between the proposed “TATA” sequence for the ddc gene (BRAY and HIRSCH 1986) and any sequence in this region of thefz gene (the best match is 5/12).

Five exons were detected by comparing the class I cDNA and genomic sequences. T h e introns range in size from the 296-bp intron 3 to the approximately 60-kb intron 4. A consensus poly A addition site (AATAAA) is found 32 bp upstream of the 3’ end of the AC2 cDNA-thus this is likely to represent a true 3’ end. We thus consider AC2 to be a full length cDNA clone (the slightly larger estimated size of the fz mRNA on Northern blots would presumably be due in large part to the poly(A) tail which is not part

A B C D E F G H

FIGL’RE 7.-IY:R ; ln ; t l \& oI KS;\ ; t ~ ~ ~ ~ ~ ~ ~ ~ ~ l ~ ~ t i o ~ ~ . Poly(/\)’ RNA x x s reverse tr;tnscrihctl using prinlcrs complenlentary to the 3’ class I and 11 specific exons. Shown are the res111ts of amplifying these cDNAs. 1.ancs 13. I), and F show the results of amplifying c1;tss I ;tnd C, E. and G the results of ;tmplifying the class I I cDNAs. The cDNA ;tmplified in lanes B and C x’as made from 0-12-llr embryonic RNA. in lanes 1) and E from 0-24-hr pupal R S A . and in lanes F and G from ;tdult female RNA. The ;mplified fragments are of the expected sizes. ;Ind in other experiments the identity of thr bands from adult fenlxles h a s been confirmed by hlotting and sequencing. J4olecular weight markers ( s i x in kh) are shown in lanes A and H . Reconstruction experiments indicate that this procedure is not reliahle for obt;Iining quantitative data.

of the 3800-bp sequence shown in Figure 8). T h e sequences found at all of the intron-exon junctions (Figure 9) are typical of those seen in most eukaryotic genes (PADGETT et al. 1986). As we had speculated earlier (VINSON, CONOVER and ADLER 1989), ACVC22 had a “fake” 3’ end due to internal priming of cDNA synthesis at an internal oligo(A) stretch. Almost all of our class I cDNA clones shared this 3’ endpoint. T h e 3‘ end of another class I cDNA (AE6) was determined by DNA sequencing and found to be at nucleotide 3480. This site is 51 bp downstream of a near match for the consensus poly(A) addition site (AATAAG) that could serve as an alternative poly(A) addition site used to generate the 3.6-kbfz RNA seen in embryos and adult females. Since the sequence immediately downstream of the 3’ end of AE6 is A rich (8/10) it is possible that this cDNA clone arose from an internal priming event during reverse transcription. As we have reported elsewhere (VINSON, CONOVER and ADLER 1989), the sequence of the class Ifz cDNA clones contains a single, long open reading frame that contains an ATG initiation codon at nucleotide 708 and a T A G termination codon at nucleotide 2451. A few sequence polymorphisms were seen when the sequences of the two cDNA clones ACVC22 and AC2, and the Canton S genomic DNA were compared. T h e conceptually translated proteins from all three sequences were however, identical. This open reading frame shows typical Drosophila codon usage, and the CAAA sequence proceeding the ATG translation start sequence is a good match for the Drosoph- ila consensus sequence of C/AAAA/C (CAVENER 1987). There are termination codons in all three

Tissue Polarity Gene in Drosophila 41 1

reading frames upstream of this start site. The sequence of the class I1 cDNA clone BE2

(Figure 8B) is identical to the sequence of the class I clone AC2 for 1921 nucleotides (corresponding to exons 1 --.* 4), and it differs by the use of an alternative 3’ exon. The oligo(A) stretch found at the 3’ end of BE2 (and also in our other class I1 cDNA clone BC3) is encoded in genomic DNA, thus this cDNA is the result of an internal priming for reverse transcription. A consensus poly(A) addition sequence (AATAAA) is found 220 nucleotides downstream of the 3’ end of BE2. We hypothesize that the 3’ end of the class I I f i RNA will be just downstream of this site. This would yield a 4-kb (plus a poly(A) tail) RNA, consistent with our limited Northern data for this transcript. The sequence at the alternative splice junction that gives rise to the class I1 cDNA is different from that at otherfz splice junctions in that there is a 22-nucleotide stretch 30 bases upstream of the 3’ end of the intron and upstream of the consensus pyrimidine-rich region that lacks any T residues (Figure 9). This region is relatively T rich in the other) splice junctions.

As reported elsewhere (VINSON, CONOVER and AD- LER 1989) the conceptually translated class I Fz protein contains 581 amino acids, and an analysis of hydrophobicity (KYTE and DOOLITTLE 1982; ENGEL- MAN, STEITZ and GOODMAN 1986) reveals seven strongly hydrophobic stretches that are typical of transmembrane domains of integral membrane proteins. Individual exons do not appear to encode specific protein domains, as the second intron is located in between sequences that encode parts of the second putative transmembrane domain. Conceptual translation of the class I1 cDNA yields what is basically a truncated class 1 protein (415 amino acids), as the 12th codon in the class I1 specific exon is a stop codon (UAG). Four of the seven strongly hydrophobic stretches found in the class I Fz protein would be found in the class I1 Fz protein, thus, it is also likely to be an integral membrane protein.

DISCUSSION

Two fz proteins: The analysis of fi function in mitotic clones (VINSON and ADLER 1987) demonstrated two mutably separate functions.fz function is essential for the transmission of an intercellular polarity signal, which is presumably at least a partly extracellular process. fz function is also required for cells to be able to respond to the polarity signal, which is presumably an intracellular function. There are two alternative ways for how the fz locus could encode these two separate functions. One way would be to encode two distinct proteins. One protein (presumably located at least partly extracellularly) would function in the transmission of polarity information between cells, while the other protein (presumably located

intracellularly) would function in the transduction of the polarity information to the cytoskeleton. Alter- natively, thefz locus could encode a single multifunctional protein. Our finding a single major 4-kb fz mRNA on Northern blots of larval and pupal RNA that encodes a protein expected to contain both extracellular and cytoplasmic domains is consistent with the hypothesis thatfz encodes a single multifunctional protein (this argument assumes that the Fz protein is located in the plasma membrane, as opposed to other cellular membranes). We have however, obtained evidence for a secondfz mRNA species which encodes what is basically a truncated version of the protein encoded by the major fz mRNA. It is possible that each of the two Fz proteins has a single function in the generation of tissue polarity. The mapping of three mutations downstream of the class I1 transcription unit ( f i c z l , f i E A b Z , f i G D 4 b ) argues against the class I1 RNA (and protein) being of primary importance for tissue polarity. TheficZ1 mutation, which breaks the chromosome 15-20 kb downstream of the 3’ end of the class 11 transcription unit (but in the fourth intron of the class I transcript), is a strong mutation that almost completely inactivates the gene (ADLER, CHARLTON and VINSON 1987). The two apparent insertion mutations ( f i E A b Z and f iGD4’ ) , which are located 20-30 kb downstream of the 3’ end of the class I1 transcription unit (but in the fourth intron of the class I transcript), and 70-80 kb downstream of the promoter, result in a moderately strong phenotype, as does an insertion mutation in the first intron ( f z C L 3 7 . Other experimental approaches will however, be required to rigorously distinguish between the one and two protein models, and what is the function of the individual proteins. It also remains possible that there is more than 1 species of 4-kb fz RNA (e.g., differences due to the alternative use of microexons (HOGNESS et al. 1985; O’CONNOR et al. 1988), although we have not seen any evidence for this.

The structure of the 3.6-kbfz RNA seen in adult females and embryos is not certain. What data exists is consistent with this RNA being derived from the use of an alternative poly A addition site for a fraction of the class I transcripts in ovaries and early embryos. Such an RNA would encode the same protein as the 4-kb mRNA. This hypothesis is consistent with the relatively weaker hybridization on Northern blots for this RNA, as compared to the class I RNA, seen when 3’ specific probes are used. This hypothesis is also consistent with our failure to obtain any clear cut examples of cDNAs for this RNA, since almost all of our class I cDNA clones were derived from an internal priming event (e.g. , ACVC22) upstream of the presumed 3‘ endpoint of the 3.6-kb RNA. One candidate cDNA clone for a full length 3.6-kb RNA was obtained (AE6), but based on the sequence near the 3‘

412 P. N. Adler et al. A CAG CCC GGG GGA 1CC ACT AGT 1Cl AGA GCG GCC GCC AAC CGC GlG GM GCl CCA U T 11C

GCC ClA TAG 1GA GlC GTA 1AC GCG CGC 1CA ClG GCC GlC Gll T. . . . .............. ...

ltl Cll

AGA CAC 111 1TC

ClG GCl TAT AGC WG CAG

All

CAT ClG

ClC 1 W GAG

TAG 11G 1Cl GCl CCA M C

AlA AM CCG CCG

M C GAT

M l 111

M A AGA CCl ACG

GlT W l CCT C M CAC WG AGG CAT M C ClG TCA M l CCl ClC ACA GTC M A WG

GCC M G M G 11C

AGC CCl GTC 1CA AGG 1CG CCA 11C GAG CGG Cll GW

TlG 111 CCC

AAG C W

AGA GW CAC 11C CCA GlG M G AGC AGC CCA

W C GCT ClA GCA 1Cl

AGA ClC 1AC TAT 111

GlG CGA 111 GGC 1AC AM 1CA GCG CAC 11C

GCG TCl GCC

CTC 1Gl

-240 -180 -120 -60

1

-ACZ,BEZ AGT 11G 1lA GCG TAT GCC CTA CCA 1GC GGA 1GT GCl CGC GGA 11G AlT 1CG ACT CCA ACT 60

TCG GCl CCG 111 TCG K T 111 M C GlA U T CCC GlG CW AM AGl TW All CCA ClC CGl GlT 1Gl 1CA Cll All 111 11G CCC GGl 111 CCA AM AGT G M AW U T M G G M AlT CIA M A GCG GGG WG GCA T U A10 AlA TIC AGC 1 M CGC TIC 1TA TGT AlG TGC A T 1 CAC CIA W l AM U T GTC CCl GCl AW AM GlA M G CCC CGl ACT GTG CCA GAT AM AGC TAA CGC TIC T U GlC ClA M G ACC CCC AM U T 1 M AT1 11G ACA C M M A 1Al 111 GCC M C M C AM TAG CM AGl C W All C M ACG GlT AM CAI M T 1 M AGC ClG A M ITA ACA M A AAG

TAl TlG ClT ATA AGG CCA AAC T U ATA 111 CCC GCC GGC A M TAT 111 A M GGl GlT M G GlC T U ACA AM 1 M AW GCA AM GCA M C GCA M l M G AW 111 CAG A M AAl AT1 1Al

TAT M G 1 W GlG CM GTA W l GAG CM CAT GTG 1Gl Gll 111 AAG 1Cl 111 M G AGA TCC 1CT 11C GlG CGA ClC M G 111 TlA M C AGG GM M l AM 1CA AAl M C TAT TCA 116 GTG

AW AM AGC CCC AM AGl 1Cl TlG ACA GCG ATA TCG AM U T 1CC AM ATC TCG CGl CM Met Trp Arg Gln

-ACVCZZ 120 180

300 240

360 420 480

600 540

M O 720 4

ATC ClC 111 All 11A CCC ACC ClG AlA CAG GGG CTC CAG CGC T I C GAT CAC AGC CCC CTC 780 I l e Leu Phe I l e Leu Pro lhr Leu I l e Gln Gly v.1 ~ l n Arg l y ~ ASP ~ l n ser pro L ~ U 24

GAT GCG ACT CCG 111 TAT CGC AGC GGC GGG GGA 111 ATG CCC AGl 1Cl GGC ACC CIA ClA 840 Asp Ala Ser Pro lyr lyr Arg Ser Gly Gly Gly Leu Met Ala Ser Ser Gly lhr Glu Leu 44

GAT GW ClG CCA CAT CAC AAl CGC 1Gl GAA CCC AlC ACC AlA TCG ATC 1GC AAG M T AlA 900 Asp Gly Leu Pro His His A m Arg CYS Glu Pro I l e lhr I l e Ser I l e Cys Lys 1%" I l e 65

CCA TAT M C AlG ACC All AlG CCA M T CTl AlT GGC CAl ACC M G CAG GAG GAG GCG GGl 960 Pro Tyr As" Met Thr I l e net Pro Asn Leu l l e Gly His lhr Lys Gin Glu GI0 Ala Gly 84

C1C GAG GlC CAT CAG 111 CCl CCC ClC GlG M C ATC GGC 1CC AGT GAT CAC ClC CAG TTG 1020 L e u Glu Val His Gln Phe A18 Pro Leu Vel Lys I l e Gly Cys Ser Asp Asp Leu Gln Leu 106

1TC ClC 1Gl 1CC ClG TIC Gll CCG GlC K C ACA All 11G GAG CGA CCC AlT CCG CCT TGT 1080 Phe Leu Cys Ser Leu lyr Vel Pro Val Cy$ lhr I l e Leu G I " Arg Pro I l e Pro Pro Cys 124

CW 1Cl ClG TGC G M TCG GCG CGG GTA TGl GAA M G TlA AlG M A ACC TIC AAC 111 LAC 1140 Arg Ser Leu CyS GlU Ser Ala Arg Val Cys G I " Lys Leu Met Lys Thr Tyr A m Phe A m 144

TGG CCG G M U T ClG GAG 1GC TCC AM 111 CCC GlC CAT GGA CGC GAG GAT 11G 1GC GlG 1200 Trp Pro G I " A m Leu Glu Cyr Ser Lyr Phe Pro Val His Gly Gly Clu Asp Leu Cys Val 16b

GCG GAG M T ACC ACA 1CA K G GCC 1CC ACG GCG GCC ACG CCC ACA AGG AGT GlG GCT M G 1260 Ala Glu Asn lhr Thr Scr Ser A l a Scr lhr A I S Ala lhr Pro lhr Arg Ser Val Ala Lys 184

Val lhr lhr ArP LyS H i s Gln Thr Gly Vel GIu Ser Pro His Arg A m I l e Cly Phe Val 2oI GlC ACT ACC COT AM CAC CAG ACG GGC GTA GM AGl CCG CAC CGA M C AT1 GGA 1TC GlG 1320

1GC CCC GlG C M ClG AM ACC CCG ClG GW AlG GGC TIC tu. ClA M A GTl GGC GGA AAG 1380 Cys Pro Val Gln Leu L y s lhr Pro Leu Gly Wet Gly lyr G I " Leu Lys Val Gly Gly Lyr 22b

ClA CGl 1Cl 111 ATG AM ATA M l 1CA ClA AM AGC GCC GM AM 1AA GCA 1CA TAl AM M l AWI GGG 11A TAG GCG CAT ACT GlA TIC CTl TlA M A CGT T U 111 1Cl All Gll A M

ACG AAG 111 CAC AM 1M All 11G GGG ACC T U CCA 1Cl Gll TAG CGA AlG 111 11A GCG AAC AAG TTC TAT M i CAT CCG TCG GGl GAG CAC AM CTT TlA ACC ATG 1CA AAA CTA A M

..._.__ 11C ACA U T TAC 1CA C M All ACT CAC 1GG CCT ClG 11C GTl GlT CCl TlG CAG

1

Cll ................................. Ivs-I (r26 kb).... .......................

I GAT ClC CAT GAC 1Cl GGA GCC CCA 1Gl CAT GCC AlG 11C TlC CCG WG AGA GAA AGG ACT 1440 ASP L e u His Asp CYS Gly Ala Pro CYS His Ala Wet Phe Phe Pro Glu Arp Glu Arg lhr 244

GlG CTl C W 1AC K G Gll GW 1CC 1GG GCA GCG GlC 161 GlA GCC AGC lbc TlG 111 ACG 1500 Val Leu Arg 1Yr 1rD Val G I Y Ser 1rD Ala A l a Val CY* Val Ala Ser CVS Leu Phe Thr 266

CTA ACT Cll TAT TIC 111 M l TGl ACT CAG M l TAT M G K G AM AGA 11A ACA ACT TlA Mf ACT 1Gl Cll AM CCC AM AM AM M l GGG 111 AM 111 M l AM TIC GAA AM U T AGl 11C Gll T U 1Gl TCT ClA %A GlC 1GC 1T)i AGl 111 11G 11C Gll All GGl 1CC CGC

1 M ..................................................... IVS.Z(~lkb) ............ 1AC CAT 111 1GC ITA CCC 11G ACT GTC 1 W GlG CTG CCA TAT 111 CTT TAG ACC 1 W M G

..__........... WG AlG GW W l CTC ClA ITA GlT ClC CTl T U 1Gl CCC CGC ACT W l AlA M C ACT M l GCA 111 111 AGG GM CAT CM AGC ATA 111 GGG Gll U T 1 M GGC GlA

1 GTC ClC ACC 11C 11G All W C 1CG 1CG CGl 111 CGC 1AC CCG WC AGG CCC All Glt 11C 1560 ACG T U 111 U T TCA M l 11A TAT 111 A11 1CC All ClC CCC 1Cl 1Gl W C 1CG 11G CAG

Val Leu lhr Phe Leu I l e Asp Ser Ser Arg Phe Arg Tyr Pro Glu Arg Ala I l e Val Phe 286

LN A h Val Cvs lyr Leu Vel Val Glv Cvr Ale lyr Val Ala GI" Leu Gly Ala Gly Asp YU 1% GCC Gll 1CC 1AC 11G GlA Gll GGA 1Cl GCC 1AC GlG GCG GGA ClG GGG CCG GCC GAC 1620

Ser Val Ser Cy0 Arg Glu Pro Phe Pro Pro Pro Val Lys Leu Gly Arg L e u Gln Met Met 324 IC1 GlG 1CG 1GC CGC GM CCA 111 CCG CCG CCC GlC AM CTC GGC CGC ClG CAG AlG ATG 1680

1CC ACC AlC ACC CAG GlC AW T W GCC M G AGA AGC AlC 1Cl GlA GCG ACT GGG A M

1

I

Ser lhr I l e lhr Gln

CCG G T t GlC M T TAT 111 T U TGC CAC CW M G TGC AM TlT TAT TTG ClG M G 11A M C C W M l T U w1 CC1 ACA W l ITA WG M l U T AM U T AM U T ATA AM AlG TlA ITA (IVS-3)

1 M AAC 1Gl GGC AGC 1CA M G (11 1 M CTT AAl TCl TI1 111 All GAC C W GlT TCl 111

1Cl 111 ClC ClA CAC GGC CAC CW C M ACC ACG TCC 1GC ACC GlT 111 11C AlG GCA ClC 1740

M AAl All TAT CCl Cll AGG All AlA 1 M All T U TAT ATA 11C T U GCA 1 W ClA CCl (2% bp)

1

Gly His Acg Gln lhr lhr Scr CYS lhr Val Leu Phe Met Ala Leu 3U M3

T I C 11C 1GT TGC AT& GCG GCC TTC GCG 1GG 1GG 1CG 1Gl ClG G U T l C GCC 1GG llt TIC 1800 TYI Phe CYB C m Met Ala Ala Phe Ala T ~ D TIT) Ser CYS Leu Ala Phe Ala l m Phe Leu 364

GCC GET GGC ClC AM 1GG WIC CAC WG GCG AlT GAG M C M G 1CG CAC 11A TlC CAC ClG 1E.O Ala Ala G I Y L e u Lys lrp Gly His GIu Ala [ l e Glu A m Lyr Ser His Leu Phe His Leu 384 W4

CfT CCC 1GG GCG GlG CCC GCC Cll CAC ACC AlC 1CC Gll ClG GCC ClG GCl AM Gll GM 1920 Val Ala l r ~ Ala Val Pro Ala Leu Gln lhr I l e Ser Val Lev Ala Leu Ala Lys Val Glu UY

I GGl M G 1TC 1Gl GW U T CCC GGC K G All All ATC WT AAG ClT W l ATC GM 1TC ClG G

ACT 11G GCC 11A CM CAT 1 W AAl AlG C M AGG AM AAG GAT 11G MA GGC CTA TAT M G .... IVS-4 (I 60 kb)...... ......................... GlC TlA A T 1 111 ATG U T AlC

1GA ClA All CAl All GAC 111 1 M Cll 1CA AlA All 111 M l ACA AlA CCl 1AA M l ACG AlA Cll ITA 111 AGA 1AC AlC AM 1GC TAT AGl AM 111 111 CGl 111 A l T C M Cl1 1CA

1 GCT GAC AlC CTT TCT GGC GlT TGT 11C GTG GGl CAG ClG GAT ACG CAC TCC CTG GGC CCG 1980

l y Asp I l e Leu Ser Gly Vel Cy* Phe Val Gly Gln Leu Asp lhr H I S Ser Leu Gly A18 &24

TlC CTG ATC Cll CCA CTC 1GC AT1 TAT CTC TCG AlC GGA GCA CTA 11C CTG ClG GCC G L A 2040 Phe Leu I l e Leu Pro Leu Cy% I l c l w Leu Ser I l e Gly Ala Leu Phe Leu Leu Ala G l Y u1

Phe I l e Ser Leu Phe Arg I l c Arg lhr Val Met Lyr lhr Alp Gly Lys Arg lhr Asp Lys e.4 111 All 1CG ClT TIC CGG ATC CGG ACA GlT AlG AM ACG GAC GW AAG AGG ACA GAC AAA 2100

ClG GAG CGC ClG AlG 1TG CGA AT1 GGl TlC TTC 1Cl GGA ClG TlC AT1 CTG CCC GCC GlG 2160 Leu Glu Arg Leu Met Leu Arg I l e Gly Phe Phe Ser Glv Leu Phe I l e Leu Pro Ala Val 466

GGA 11A ClG GGC 1GC 11C 11C TIC GAG TAC 1AC M C 111 GAC GAG 1GG AlG AlC C M 1GG 2220 G I Y Leu Leu Glv CYS Leu Phe lyr Clu lyr lyr Asn Phe Asp Glu lrp Met I l e Gln lrp 506

CAC AGC GAl AlC 1GC AAG CCC 11C TCA All CCG TGC CCG GCA GCC AGG GCG CCG GGA TCl 2280 H I S Arg Asp I l e Cyr Lyr Pro Phe Ser I l e Pro Cys Pro Ala Ala A'g A l a Pro Gly ser 52b

CCA G M GCC CGC CCC AlC 111 CAG AlC 111 ATG GlC M G T I C Cll 1GC 1CC AlG ClG GlG 2340 Pro Glu Ala Arg Pro I l e Phe Gln I l e Phe Met Val LYS Tyr L e u Cys Ser Met Leu Val 544

G ~ Y Val Thr Per Ser Val lrp Leu l ~ r Ser Ser Lyr Thr Wet Val Str lrp Arg ASD Phe 566 GGG GlC ACT 1CC AGC Glf TGG ClG TAT TCC ACC M G ACG AlG GlC AGC 1GG CGG M C 11C 2400

GlG GAG AGG 11G CAG GGC M G GAG CCC CGG ACC CGG GCG CAG GCG 1AC GlC TAG TAT GAG 2460

ACG GGl CCG GCG GGC GGG GCC M G 1CC ACG CCC ClT ACT CCC GAT CCG GAG GCG GCT MC 2520 Val Glu Arg L e u Gln Gly Lys Glu Pro Arg Thr Arg A18 Gln Ala Tyr Val 1ER

GAA Ab1 1AC 1TA GAG 111 AGC ACA 1AG ACG TlC CCl AlG CCC AM MG A M AM M A AlA 2580 ACA CGC ACT CGT A G l 1Cl 116 TGG CTl 1Gl A11 AAG 11C GTC ATC 1AC A11 1Gl CCC CGl 260

- ACVC22 AAA TCC ATA AM AGA wu MT CAA MG TTG ACC CTA GGC TAT TCT rrc AAT TU MG TAC 2700 All ACT 1GA TAC G M AGC M G CTl Gll C M CCA A M ClA 1GC GlA 1M Gll G A G ACT ClA 2760 ACT ClA AGl C G A GTl GTA 111 1Gl AGG All 1CA 11G CTA 1CG CGG TAT ACG M C ClG TAG 2820 CAC CIA TW TAT 1TC All 771 WC GCA ACT TTT M G TlA GCT TCC GAT 111 1GA M G Cll ZBBO

ACG 1CA GGC AlT CM AGG AlG 1GA CCC CCG CCl GCl ClG CAG TAG AlT ITA T U GCC M G 3000 1AA AlG 1GC CCA CIA 11A AlG AGA AlG TAG CAT AM TAG AGT AlT ACG 111 ACG ITA 1GA 2940

GCl T U U T 11C AT1 CAT 1AG 1CA C M M G AM 111 111 TCA ACC CAG ACA 1 M 1 M 11A 3060 1GC AlA TAG AGC ACA CAA 1CC GTA CAT AlA CCC AAG CAG Gll GGT G C l CAC AlC 1Al LC1 3120

AGl AM 1GT CCl CCA 1Gl GCl AM Cll ACT GCA U T ACG All All ATA AT1 1TA ACT AlA 3240 1CA TCT AlG ill GTA T U ACA M A M G G M ACC AlA AAC TAT ATA M l GTA 11A TGT AGC 3180

ACT GTA 111 GlA All 11A ACT GlA All GlA TAT TlG TlG AAC T U lTA All ClA M C GCG 3300 T U TGG AAl M l 111 T U 1TC AAT 111 1 M AlG ACG TlG TAG 1CA TAG CAC 11A AGC GM 3360 CAA ACA M C M A ACA CGC M A All 11G 11G CCA Cll TAT GGA M I 111 1CC Cll CAC CCC 3420

CM CCl A U A G 11G A M CTG CCA 111 AM TAT TlG TAG ACA CAT GAG AM 1CA CCG 3480 A M GA1 G M AGC AlG CGA 1GT TTG CCA ACT Gll GAG CAT 11G 111 TCT GCG CAT 11A All 3540

AGC All T M GCl GAT GGA MT GCA TAT T U TlA TAG CM 11A 1CG ATA GTC GGl GlA ACA 3M0 GAG 1CC TCA All 1AA AT1 GGC M l ACT U T ACG TW M T TAT GTC C M GCA W l 11G K T 3600

TAG CGA AlG CCC AGC CGC M G CGA ACA All AlA ACT GlA T U M C AM T U AM CAT GCA 3720 G M CAT CAT 1GA AGC AT1 C M TGG CAA ACA CAC AM CGG AM AlG C M M I A C 111 3780

- AE6

ACZ

GCA All GCl GGl GTl GAC C M CGC M C Cll 1TA GCG GTT 111 C M TlA Cll M G 11A TlT 3900 All 11A 11G 111 M C ACT CM AlA 1GA ACT Gll TTl M l 1GG ACT GGG AM All GCl TAT 3840

GAG M l 1 M GGA CCl 1CA AGA 111 M l 111 AT1 111 1CA TlT GlA AGC 111 CAT CGl AAG 3960 CGA CAT 1GC All CTl TCA TAT A11 AAl U T 1TG TAG AGC TlT 1

B.

AGT GGA ACT All ClT AT1 111 AGG GAT TAT U T TW TlC 111 AT1 1 M 1CT CAG 1TC AM .... IVS-4s (=20 kb)... .. TlT TAT TW AM ATA TTG TAG W l TTG TTA TlG 1CG AlG

ACT ClC AM CCA 11G CTC ACG TCG 1Gl CTl ACA C M M T AM CCG CCA All All M l GCA

CAC ACA CCA ACA CAC AGC GlA ClA AT1 1CC CAI 111 CCl CTl CTT TCA GGT ATG TAC 111 1932 1

l y Met 1yr Leu

TGG CAG 11C CAC ACC ATA M C TAG M l TAT GCA GAG 1Gl M G T U TW U T GTl CAT ACA 1992 Trp Gln Phe His Thr t l t A m TEU

ClG ATC ACC U T 1CC ACC 1 W TCC 1CC AT1 U T GAG 111 ACC CM ClA AAC T I C M G CCA 2052 111 ACA 1Gl AGC CAC AM GM CGC ClG GCA TAG AGC GCA AlC AM 1CG CW tu. T U ACC 2112 CAT TIC CGl ACT CAC AGC CCA TCG 1Cl ACC AAC CAC C W GW ClA ATC AlA AGC CCA 1TC 2172 ACA 1 M CM 1CA M G GCA All TCT GGA All CAG 11C GCA GGA 11A Cll ClA A11 All T U 2232 T U ACC CAT tu. M G 1CA AM AlA CW 1 M GM CGG TGG M C M C GGG Gll AlG GCl CAC 2-2 CCC CAC ACG GlC ClA 1 M AM TlC CCC ACC AM ACT TCl CAT T U AlA M C A11 CCC G M 2352 CCG M G ACA M G GCC All CCA TAT C W GlA CCC All CCG AM M C 1AG 1CC C M ACT M G 2412 GCC GCG T U TGT CGC TTG 111 GTA ClA ATG ATG GGC 1GG ATC CCA GTT CCl ClC C U TAT 2472 1Cl Cll GM TIC CCC CCC CCl GGG AM GCG CM AM T U All K T ACA 1Cl GCG 111 GM 2532 GCC GCG ITA TGG M G CCG 1GG CCA AW CCl CGG AAC CCC AAC M l GM AW CCC 11G CCC 2592 AT1 TCG CCl GCl 1Cl CAT AM 11G CCA 1GG AGC GCC M C M C AAG IT1 CCl 1CG ACT 1 W 2652 AGC CTT 11A GGC ClC 111 C M 111 ClA GGG ATG GTA CCG TW TCl 1TA GlA 1 M 1 M M l 2712 11C 11A U T ACT TAT CAA AM GTC 111 CCG ITA AGl All ClC TAT CTG TAT CAG 111 111 2772 111 All All TCA GM All GlC ClA Cll ClG AM ATA 111 AAC TAG TI1 ATA AM 111 A X 2832 TAT AlA Cll TGC U T 111 111 111 TlA AM ACT CTA U T U T TlA Cll TGC CGl ACC CCA 2W2

AM CCG ACT CGG CCA CAG TCC All G W TGC C W CCG 1CG CGl CCA GGC 1Gl AM U T T U 3012 ACC ClT 1TA CM GCC ATC TCl M C All 11G 1CA AW CW CCC CCA GGl CCG ACC GM ACA 2552

TAG CCA ACA TAA T U GCG GCA 111 TAT WC M G CGl GCA CCl CAC CAC LC1 1AC ACA CAG 3072 ACA CCC 1Cl CAC GW CAT GCG ACC ACG CCC ClA WC AGC GCA K T ACT W l 111 111 CGG 3132 CAC 11G GGC 111 1GG GCA M C AT1 TCl CAT AGG ClC ACA CGG C M MG TCA 11A GGl GAG 3192 GW 1CA CTl GCC CAC GCl TCl K G 1CG CAG 1CA CTl TW AM 111 CGC AM 11C AM Gll 3252 ACA 1CG 1TA 1Gl CCC CGG C M AlG GCG AM ATC AGG AGG AM ACG CCG CM ClC CCC ClC 3312 M C 1AG LC1 CCA ACC T U CCG ACA ACT TAT GGC CAT CGl All GGC GCT GCA ACA 11C CCC 3372 111 AlC 111 All ACA CGC AGC GlT WG All 1Gl 111 TAT ACC All TlG K C AGG AM All 3432

A W GGT W l ATG GM AGl 11A 111 111 1 M ACA AGC T U TGC All 11G 1GG CTl 11A AT1 3552 GM AM CTT 1TC GM GGC 1TC GGl Gll 1Gl GlG CCA GW AM 1AG AlG GTC W l TAT GlA 3492

GW TW WG ClA 1TC CCG CW TW 1 W AGl GCC CCA 1 M TGC 1GG 11A AGC 1 M AGT ACT 3612 T U CGG GM M C ITA TAT ClA AlG GGl ClC 11A TlT AAl ACT U T AGl U T AW TIC ACA 3672 111 1Cl 1TG AlC U T GCC TCl ClA U T ACC 1TG 1Gl CGT AM CAG M C ClG 1CA TAT AM 3732

- BEZ.BC3 AlC GTl AGl TlG M G ClA AM AM AM AM ACG TIC ClA ClC TIC TCA AAG ACT M l GCA 3792 T U M C 1Al M A W l ClC 1GC GGG C M T U 1TA A 6 1 GTC AM GCC C W CTA TlA ATG GlC 3852 CAC 111 1 W CCA M C GM ACC AAG C M AIG CAG All CW ACA CAT AM AAC T U GCA CM 3912 AGT M C C M GTl tu. 111 T U AlC G M 111 AW Cll AAC TTT 11A 11G TCl 111 1AC GGl 3972 TTG AGT CAC A

FIGURE X.-Sequence analysis of thefz gene. Shown in A is the sequence of thefz exons and flanking D N A for the class I cDNAs. In I"L1-t

13 t h c st.quence f o I the class I I specific exon is given. Horizontal arrows indicate the ends of the stated cDNA clones (e.g.. + AC'L). Downward artow indicate the locations of the intron-exon boundaries as deduced by cornparing cDNA and genomic clone sequences. T h e tr.;mshtion p~-otluct of the large open rt.;~ding frame is ;dso shown. Presumptive transnlenlbra1le domains ;1nd poly A addition sites are underlined.


AClGTMCT AClC

AGIGTAGGT IVS-1 TTCACMATTACTUCTGGCCTCTGTTCGTTGTTCCTTTGCAGl~ CGIGTMGT IVS-2 MATTCMATTTATATTTMTATCCATTCTCCCCTCTTGT~CTCGTTGCAGIGT AGLGTGAGA IVS-3 TTMCTTMTTGTTATTTTATTWCCGAGTTTCTTTTTCTTTTCTCCTACAGIGG A G I G T M G T IVS-46 GCAWWCACCMCAGACAGCGTACTMTTTCC~TTTCCTCTTCTTTCAGlGT A G I G T M G T IVS-4 TTTAGATACATCMATGCTATAGTMATTTTTTCGTTTTATTCMCTTTCAGIGT

FIGURE 9,”Nucleotide sequences surrounding the splice sites used in the formation of the mature fz mRNA. The consensus sequences for splice sites in animal cells are shown above in bold.

end of this clone it seems possible that AE6 could have been derived from an internal priming event during reverse transcription. In any case, this RNA is not detected in larvae or pupae, hence it is unlikely to be important for the generation of tissue polarity in the adult epidermis.

A membrane protein functions in tissue polarity: The conceptual translation of the class I cDNA sequence yields a protein that is likely to be an integral membrane protein. If this protein is located in the plasma membrane it would be expected to contain both extracellular and cytoplasmic domains. The predicted structure of the Fz protein suggests it could function in the transmission of polarity information by transporting across cell membranes a morphogen present in a concentration gradient along the proximal-distal axis of the wing (LAWRENCE 1966). If so, we might expect a buildup of morphogen upstream of a clone of fz cells in an otherwise wild type wing ( i e . , the morphogen exiting the wild-type cells proximal to the clone could not be transported into the mutant cells at the proximal edge of the clone, and hence the concentration of the morphogen might build up locally and “back up” the system), and a deficiency of morphogen downstream of such a fz clone ( i e . , there would be no morphogen exiting the cells at the distal edge of the mutant clone and enter- ing the wild-type cells just distal to the clone). If polarity is determined by the vector of the concentration gradient (LAWRENCE 1966), then the polarity of cells located both proximal and distal to mitotic clones should be altered. Since only cells located distal to fz mitotic clones show altered polarity (VINSON and AD- LER 1987), we do not favor such models. Alterna- tively, the Fz protein could function in the transmission of polarity information via direct cell-cell inter- actions (NARDI and KAFATOS 1976; TUCKER 198 1). For example, there could be a gradient of “j2 activity” in the plasma membrane of a cell (e.g., more activity in the membrane located on the distal as opposed to proximal side of the cell). A high level of ‘‘fz activity” in the plasma membrane in a region of one cell could inhibit “$z activity” in juxtaposed regions of neighboring cells. Such a system could transmit polarity information from one cell to another. An intracellular gradient of “ji activity” might reflect an intracellular gradient of Fz protein abundance in the plasma membrane (e.g., more Fz protein in the plasma membrane on the proximal as compared to the distal side of the

cell), which may be detected when Fz antibodies are available.

The function of thefz locus is also required for cells to be able to respond to polarity information. As we have reported elsewhere (VINSON, CONOVER and AD- LER 1989), an analysis of hydropathy suggests that the Fz protein contains 7 transmembrane domains. This is interesting as seven transmembrane domains are found in the large family of G protein-coupled membrane protein receptors (GILMAN 1987; KOBILKA et al. 1988; DOHLMAN, CARON and LEFKOWITZ 1987). These proteins presumably function by ligand binding extracellularly, resulting in a conformational change that either activates or inhibits G protein activity. It seems plausible that fi functions via a G protein in transducing polarity information to the cytoskeleton during hair and bristle formation. The analysis also shows, however, that fz does not show substantial sequence similarity to any members of this large gene family, thus until there is direct evidence of involvement of the G protein signal transduction pathway one must remain skeptical of such involvement.

Both of the fz mRNAs are rare: Based on the frequency of recovery of fz cDNA clones from a number of different cDNA libraries (average 1/ 50,000) it seems clear that the fz mRNA is reasonably rare. Consistent with this conclusion, the strength of the signals obtained when Northern blots are probed with fz sequences is much lower (at least 50-1 00-fold) than that seen for control mRNAs (e.g., hsp83). Based on the frequency of recovery of cDNA clones we estimate its abundance in pupae as 0.002%. This is consistent with thefi protein functioning as a polarity morphogen receptor, but not with it being a major structural component of epidermal cells. Preliminary in situ hybridization experiments (W. J. PARK and P. N. ADLER, unpublished results) indicate that fz is not expressed in all cells in the pupae so that the above abundance estimate is likely to be an underestimate for the pupal epidermis (one of the tissues where fz is expressed, and the tissue where hair and bristle morphogenesis occurs). It is unlikely, however, that the abundance in the pupal epidermis would be greater than 0.0 1 % (based on the fraction of pupal cells that contain fz RNA and on the relative in situ hybridization signals in different tissues). The class I1 RNA is substantially (we estimate at least 10-fold) rarer than the class 1 RNA. Whether this RNA is functional or a product of a splicing “mistake” is not clear.

DNA alterations associated withfr mutations do not provide evidence for functional subdivisions: We have found no evidence for qualitative functional subdivisions in the locus. None of the rearrangement mutations shows any phenotypic tissue specificity. The two most distally located breakpoints (fzEA3, fzC’la)

partially inactivate the gene throughout the adult


epidermis. We suspect that the inactivation of thefz gene in these mutations is due to a position effect. Both mutations result in the juxtaposition of the fz gene to centromeric heterochromatin. The phenotype offzG”’ is variable, and could in fact, be due to position effect variegation (TARTOF et al. 1989). It remains possible, however, that the breakpoints associated with these mutations remove regulatory sequences that quantitatively enhance the expression of the fz gene in all parts of the epidermis. The other five breakpoint mutations are all strong mutations. Four of these (fzX2’,fzCRZb,fzGNhU, andfz’) appear to be null mutations. Thus, continuity of the chromosome from +2 to +48 appears to be absolutely essential for fz function. Considering this conclusion, it is not surprising that our major classfz cDNA clones represent an mRNA derived from a primary transcript that spans approximately -3 to +91 on our DNA map. The most proximal fz breakpoint ( fzC2’) results in a strong mutation, but one that may retain some low level of function (ADLER, CHARLTON and VINSON 1987). This breakpoint is in the same intron (intron 4-as defined by the class I cDNAs) as the breakpoint associated with the fz’ inversion, which appears to completely inactivate the gene. It is worth noting that the class I1 RNA could be transcribed from the fzCz’ chromosome but not the fz’ chromosome. One rea- sonable interpretation of this data is that the product of the class I1 RNA has a minor function (in a quantitative way) for tissue polarity that is not qualitatively different phenotypically from the function of the product of the class I RNA.

The P element insertion mutation ( fzcTBc) that we used for cloning fz gene sequences is a very weak mutation (ADLER, CHARLTON and VINSON 1987). Mu- tations due to the insertion of P elements that only partially inactivate genes are not rare, and in a number of cases the P element appears to have inserted just upstream of the transcription unit (SEARLES et al . 1982; TSUBOTA and SCHEDL 1986). This is the case for fzcTBC. Using this allele as a starting point we have isolated a series of hybrid-dysgenesis induced strong fz alleles. A number of these, which are strong mutations, are due to small (1-3 kb) deletions extending proximally from the P element insertion site offzcTBC into the 5’ most exon offz. These results argue that the cDNA clones we have identified are likely to represent functionalfz mRNA, and furthermore, suggest that these cDNA clones will share sequences with all mRNAs that contribute to fz function during the development of the adult epidermis.

The large size of thefz locus is somewhat surprising as the genetics of the locus are not particularly complex, as is seen for a number of other large loci [e.g., cut (JACK 1985), Ultrabithorax (BENDER et al . 1983), Notch (ARTAVANIS-TSAKONAS, MUSKAVITCH and YED-

VODNICK 1983; KIDD, LOCKETT and YOUNG 1983), or Antp (SCOTT et al. 1983)] in Drosophila. In a number of these genes (e.g., Ultrabithorax and Notch), mutations due to the insertion of transposable elements into introns result in tissue specific phenotypes (BENDER et al . 1983; ARTAVANIS-TSAKONAS, MUSKAV- ITCH and YEDBROVNICK 1983; KIDD, LOCKETT and YOUNG 1983). It is not clear, however, that this will be a general result. Three of our cytologically normal, y-ray-induced mutations ( f~‘~’~, fzEAE2, fzCD4’) appear to be due to the insertion of DNA sequences into fz introns. It is worth noting that these three alleles do not show any tissue specificity. The two EMS-induced tissue specificfz alleles (ADLER, CHARLTON and VIN- SON 1987) are not associated with gross DNA alterations (VINSON 1987).

The large size of the fz locus in Drosophila melanogaster appears to be conserved in Drosophila virilis. We have recently cloned genomic sequences from D. virilis that show strong cross hybridization to individual exons of D. melanogaster (K. JONES and P. N. ADLER, personal communication). D. virilis sequences that hybridize to exons 2 , 3 and 4 from D. melanogaster are present together in the inserts of individual recombinant bacteriophages. There is however, no overlap between the inserts present in recombinant bacteriophage that contain either presumptive exon 1, exons 2, 3 and 4, or exon 5 from D. virilis. While these data do not require that the presumptive D. virilisfz gene span about 100 kb of genomic DNA, as does the D. melanogaster fz gene, it does suggest that the general structure (i.e., exons 2, 3 and 4 clustered close together and far from exons 1 and 5) and large size of the D. melanogaster fz gene is conserved, and hence of some functional importance. What the functional importance is remains to be established

This work was supported by grants from the National lnstitutes of Health (NIH) (ROI-GM37136) and the National Science Foun- dation (PCM-8402519) to P.N.A. C.V. was supported by an NIH predoctoral traineeship in developmental biology (HDO-7 192). PNA was supported by an NIH Career Development Award (KHDO-0361) during the initial stages of the work. We thank J. CHARLTON for maintaining stocks. We thank B. P. BRUNK, A. L. BEYER, C. EMERSON, J. HENRY, B. GEORGE, B. BUCHER, P. BENDER, J. JOHNSON, T. CHARLESBOIS and E. SHIMAKAWA for help in various aspects of the work and/or for helpful comments on the manuscript.

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Molecular Structure offrizzled, a Drosophila Tissue ...have led to suggestions that tissue polarity...

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