Wingless (wg), the Drosophila homologue of the mouse Wnt-1 proto-oncogene, is a segment polarity gene essential in each segment for normal Drosophila development. We here report the isolation of two novel Drosophila Wnt homologues, DWni-2 and DWnt-3, and thus the existence of a Wnt/wingless gene family in Drosophila. DWnt-2 and DWnt-3 map to chromosome 2 position 45E and chromosome X position 17A/B, respectively. DWnt-2 and DWni-3, like the other known Wnt genes, encode amino-terminal signal peptides suggesting that the gene products are secreted proteins. The putative translation product of DWnt-2 and the carboxy-terminal half of the deduced DiVzii-3 product are both rich in conserved cysteine residues. In comparison with other Wnt gene products, mostly about 40 × 103 relative molecular mass, the DWnt-3 protein has an extended amino terminus and a long internal insert, and its predicted relative molecular mass is 113 × 103. The expression patterns of these two Wnt/wg homologues are dynamic during Drosophila embryogenesis. The distribution of DWnt-2 transcripts is predominantly segmented, with the additional presence of transcripts in the presumptive gonads. Transcripts of both DWnt-2 and DWnt-3 appear to be associated with limb primordia in the embryo and may therefore specify limb development. DWnt-3 is also expressed in mesodermal and neurogenic regions. The distribution of D Wnt-3 transcripts in cells of the central nervous system (CNS) during Drosophila embryogenesis suggests that DWnt-3 could be involved in CNS development.

Wnt gene family members isolated from various organisms appear to be involved in aspects of body pattern organization during development. In the mouse, many of the Wnt genes are expressed in adult brain tissues and the expression patterns of the proto-oncogenes Wnt-1 and Wnt-3, and other mouse Wnt genes in specific regions of the brain and spinal cord during embryogenesis suggest a function for these genes in the development of the mouse central nervous system (CNS) (Nusse and Varmus, 1982; Jakobovits et al., 1986; Shackleford and Varmus, 1987; Wilkinson et al., 1987; McMahon and McMahon, 1989; Gavin et al., 1990; Roelink et al., 1990; Roelink and Nusse, 1991). Indeed, it has been demonstrated that Wnt-1 homozygous null mutants have severe defects in midbrain and hindbrain regions, indicating that Wnt-1, at least, is essential for the normal development of a substantial region of the mouse CNS (McMahon and Bradley, 1990; Thomas and Cappecchi, 1990).

wingless (wg), the Drosophila homologue of the mouse Wnt-1 gene (Rijsewijk et al., 1987; Cabrera et al., 1987), also has an essential role in development (Sharma, 1973; Babu, 1977). The segmental subdivisions of the Drosophila embryo are determined by the temporal expression of the maternal, gap and pair rule classes of genes. Genes of the segment polarity class, which includes wg, are required in each segment for correct pattern formation (for reviews, see Klingensmith and Perrimon, 1991; Hooper and Scott, 1992). Mutations in wg cause the posterior portion of each segment to be deleted and replaced by a mirror-image duplication of the anterior portion. During germ band extension, wg expression arises as a continuous stripe in the posteriormost portion of each of the fourteen parasegments (Baker, 1987; van den Heuvel, 1989), abutting the engrailed (en) expression domain which comprises the anterior fourth of each adjacent parasegment (DiNardo et al., 1985; Karr et al., 1989). en is a segment polarity gene encoding a homeodomain protein (Fjose et al., 1985; Poole et al., 1985) required to establish and maintain the compartment and segment borders (Morata and Lawrence, 1975; Kornberg, 1981). wg is required for formation of both the segment boundary and the parasegment groove, which forms at the junction of wg- and en-expressing cells (Perrimon and Mahowald, 1987). Although wg and en are not expressed in the same cells of the epidermis, wg function is necessary for maintenance of en expression and en function is required for the maintenance of wg expression (DiNardo et al., 1988; Martinez-Arias et al., 1988; Heemskerk et al., 1991). Analysis of the CNS of wg null mutant embryos has revealed only subtle defects suggesting a minor role for wg in Drosophila CNS development (Patel et al., 1989).

The predicted primary translation products of the Wnt genes are cysteine-rich proteins with amino-terminal signal peptides. There is biochemical evidence to suggest that processed, glycosylated mouse Wnt-1 protein is secreted (Papkoff et al., 1987) and may be associated with the cell surface (Papkoff and Schryver, 1990) and/or the extracellular matrix (Bradley and Brown, 1990). The protein product of wg has been demonstrated to reside both within and outside the cell (van den Heuvel et al., 1989), and there are genetic data to support the notion that wg can produce an effect in cells in which it is not expressed (Morata and Lawrence, 1977; Wieschaus and Riggleman, 1987; Baker, 1988a). Taken together, these findings suggest that the translation products of Wnt-1 and wg and, by analogy, the other Wnt genes are secreted proteins involved in cell-cell communication. The Wnt gene products may therefore function in positional signalling.

Despite extensive analysis of the function of wg as a segment polarity gene, its mechanism of action is largely unknown and the identity of the wg receptor has remained elusive. The phenotype of wg receptor mutants would be dependent upon possible interactions of the receptor with other ligands, perhaps related to wg. We here report the existence of a Drosophila Wnt gene family; two novel Drosophila genes, DWnt-2 and DWnt-3, which encode proteins with amino-terminal signal peptides, were isolated by virtue of their homology to the mouse Wnt-3 gene. Families of Wnt genes therefore exist in both flies and vertebrates, which may perform analogous functions. DWnt-2 and DWnt-3 are expressed during Drosophila embryogenesis in distinctive spatial patterns, as illustrated by in situ hybridization analysis on whole-mount embryos, and we discuss possible roles for these genes in Drosophila development.

Isolation and sequencing of DWnt-2 and DWnt-3 clones

A genomic library from the wild-type Oregon-S strain of Drosophila melanogaster was constructed in the lambda phage Charon 40 by subcloning of Sau3A fragments of selected lengths (15-20 kb) into the BamHI site of the phage polylinker. Hybridization under moderate stringency conditions (hybridization at 60°C in 5 × SSC; washes at 60°C, 1 × SSC), using a probe derived from Wnt-3 cDNA (Roelink et al., 1990) which contained most of the coding sequences but lacked the 3’ poly (A) tail, led to the isolation of genomic clones containing sequences from DWnt-2, DlWit-3 and wg. cDNA libraries prepared from Drosophila melanogaster pupae and 3 – 12 hour embryos (Poole et al., 1985) were hybridized with random primed 32P-labelled DWnt-2 and DWnt-3 genomic fragments, respectively (hybridization at 65°C, 5 × SSC; washes at 65°C, 0.1 × SSC). phage DNA was isolated according to Sambrook et al. (1989) and insert fragments were subcloned into pGEM3Zf(+) (Promega) or Bluescript (Stratagene). The nucleotide sequences of DWnt-2 and DWnt-3 cDNA and genomic DNAs were determined on both strands using specific oligonucleotide primers and directed subcloning. The intron-exon boundaries were sequenced from the genomic clones of DWnt-2. The dideoxy method of sequencing was employed using the Sequenase Kit (USB) according to the manufacturers instructions.

RNA blot analysis

Total RNA from embryonic, larval, pupal or adult tissues was isolated by the guanidinium hypochloride procedure (Sambrook et al., 1989). 20 μg from each developmental stage was size-fractionated in a 1% formaldehyde agarose gel, then transferred to nitrocellulose filters, UV-cross-linked and hybridized under high-stringency conditions (65°C, 5 × SSC; washes at 65°C, 0.1 × SSC) to probes derived from DWnt-2 and DH’m-3 genomic clones labelled with [oPJdCTP by random-priming. Hybridizing transcripts were visualized by autoradiography.

Polytene chromosome mapping

In situ hybridization to salivary gland polytene chromosome spreads, prepared from wandering third instar larvae of Drosophila (CantonS-1), was carried out according to the protocol of Langer-Safer et al. (1982) with modifications by Pardue (1986). Probe DNA fragments were biotinylated by nick translation at room temperature (RT) for 1 hour using Bio-16-dU IP (ENZO Biochemicals). Labelled DNA fragments were separated from unincorporated nucleotides using a G50 medium Sephadex spin column. The hybridization mix (50% formamide, 5 × SSC, 5 ×Denhardt’s solution, 0.1% sodium dodecyl sulphate) plus biotinylated probe (∼0.2 μg) was boiled for 5 minutes, then pipetted onto the denatured, acetylated chromosomes. Hybridization proceeded overnight in a humid chamber at 25°C. The chromosomes were washed in 2 × SSC three times for 20 minutes at 53°C, in PBT (PBS plus 0.1% Tween 100) for 5 minutes at RT, and in PBS for 5 minutes at RT. Streptavidin-biotinylated peroxidase complex, made using reagents from the Vectastain ABC kit (Vector Laboratories) according to the manufacturers instructions, was incubated with the chromosomes for 2 hours at 37°C in a humid chamber. The slides were washed in PBS (2 ×5 minutes), PBT (5 minutes), then incubated for 20 minutes in a humid chamber at 37°C in a solution of 0.5 mg/ml diaminobenzidine in PBS plus 0.01% hydrogen peroxide. The chromosomes were washed five times in H2O, counterstained with Giemsa (1/100 dilution) in PBS for 30 seconds and washed in PBS, prior to examination under phase-contrast microscopy.

Whole-mount in situ hybridization

Embryos, from wild-type (CantonS-1) Drosophila flies, were pooled from 0 –6 and 0 –16 hour collections (25°C), dechorionated on the agar plates on which they were laid in a solution containing 5.25% sodium hypochlorite plus 0.1% Tween 20, then washed onto a filter with water. Fixation, pretreatment, hybridization and mounting procedures are essentially as described by Tautz and Pfeiffle (1989). Fixation was with 4% formaldehyde, 0.1M PIPES, pH 6.9, ImM EGTA, pH 8.0, and 2mM MgSO4 (PEM)-heptane. Proteinase K treatment was for 6 minutes. Prehybridization was at 48°C for 2 hours. Probe DNA fragments were labelled with digoxigenin according to the protocol supplied by Boehringer. The probe was precipitated, dissolved in Tris-HCl, pH 8.0 (10 mM); EDTA, pH 8.0 (1 mM), 0.2% sodium dodecyl sulphate and incubated at 37°C for 2 hours prior to use. Approximately 0.35 μg of labelled heat-denatured probe DNA was used per hybridization. The embryos were incubated in hybridization mix (Tautz and Pfeiffle, 1989) at 48°C overnight in a gently shaking waterbath, then washed, at 48°C for 20 minutes, once in hybridization mix, once in 1:1 hybridization mix.PBT, then five times in PBT. Antibody was preadsorbed to Drosophila embryos dechorionated, methanol treated, fixed, proteinase K treated and refixed as mentioned above. The hybridized embryos were incubated for 1 hour at room temperature with preadsorbed anti-digoxigenin antibody conjugated to alkaline phosphatase (Boehringer-Mannheim) at a final dilution of 1 in 1000. The hybridized embryos were washed four times for 20 minutes in PBT, rinsed twice in 0.1 M NaCl, 50 mM MgCl2, 0.1 M Tris-HCl, pH 9.5 and 0.1% Tween 20, and resuspended in 1 ml of the rinse solution plus 4.5 μl of nitrobluetetroleum and 3.5 μl of X-phosphate (Boehringer Mannheim DNA labelling and detection kit). The staining reaction was allowed to proceed for up to 3 hours in the dark, then rinsed with PBT. The embryos were dehydrated through an ethanol series, equilibrated in methyl-salicylate and mounted in Araldite plus hardener (Lawrence and Johnston, 1989). Embryos were photographed using Nomarski optics. The embryos were staged according to Campos-Ortega and Hartenstein (1985), and Wieschaus and N üsslein-Volhard (1986).

Double labelling

The following double-labelling procedure is essentially that of Cohen et al. (1991) with minor modifications. Embryos, from a P-element transformed strain carrying an en-lacZ. gene fusion (enβal/CyO-, N. Perrimon, unpublished), were pooled from 0-6 hour and 0-16 hour collections, dechorionated and fixed as described above except that fixation was for 20 minutes. Prior to devitellinization, the embryos were washed in PBX (PBS plus 0.3% Triton X-100), three times for 3 minutes, and stained in X-galactosidase staining solution (PBX with 100 mM K3(FeII(CN)6), 100 mM K4(FeIII(CN)6), 0.2% X-gal (from stock solution 8% X-gal in dimethylsulphoxide) for several minutes. The stained embryos were washed three times in PBX. The remainder of the double-labelling procedure is exactly as outlined above (see Whole-mount in situ hybridization), starting from the devitellinisation step. The double-labelled embryos were equilibrated and mounted in 70% glycerol/30% 0.1 M Tris-HCl pH 7.5 and photographed using Nomarski optics.

Cloning of the Drosophila Wnt homologues DWnt-2 and DWnt-3

By moderate stringency hybridization of a Drosophila genomic DNA library using a probe derived from the mouse Wnt-3 cDNA, we isolated several clones with different physical maps. The insert from one of these clones hybridized to wg at high stringency and had an identical restriction map. Two other clones, however, contained different sequences, homologous to the mouse Wnt genes and to wg at the amino acid level. We call these Drosophila Wnt homologues DWnt-2 and DWni-3. We isolated cDNA clones for these genes by screening Drosophila cDNA libraries.

The nucleotide sequences of DWnt-2 and DWnt-3 were determined from the cDNA clones and partly from the genomic clones. DWnt-2 contains five exons and the positions of the intron-exon boundaries, which were defined precisely by comparison of the nucleotide sequences from genomic and cDNA clones, correspond to those of wg-, the donor and acceptor splice sites correlate with the splice junction consensus for invertebrates (Shapiro and Senapathy, 1987). Restriction enzyme analysis and genomic sequence data have led us to conclude that the DWnt-3 gene contains no introns.

DWnt-2 and DWnt-3 cDNAs and putative protein sequences

The deduced amino acid sequences of DWnt-2 and DWnt-3 are shown in Fig. 1, together with the amino acid sequence of wg for comparison.

Fig. 1.

Comparison of the predicted amino acid sequences of DWnt-2, DWnt-3, wg. Asterisks indicate complete conservation of amino acids in the Drosophila Wnt genes. ^ indicates a cysteine residue present in wg and conserved in mouse Wnt-1, but absent from the other Drosophila Wnt genes; # indicates a cysteine conserved between DWnt-2, DWnt-3 and most mouse Wnt genes, but absent from wg and mouse Wnt-1; + indicates cysteine residues in the non-conserved amino-terminal portion of DWnt-3.

Fig. 1.

Comparison of the predicted amino acid sequences of DWnt-2, DWnt-3, wg. Asterisks indicate complete conservation of amino acids in the Drosophila Wnt genes. ^ indicates a cysteine residue present in wg and conserved in mouse Wnt-1, but absent from the other Drosophila Wnt genes; # indicates a cysteine conserved between DWnt-2, DWnt-3 and most mouse Wnt genes, but absent from wg and mouse Wnt-1; + indicates cysteine residues in the non-conserved amino-terminal portion of DWnt-3.

The DWnt-2 cDNA contains an open reading frame with the potential to encode a protein of 352 amino acids with a total relative molecular mass of 39950. The first ATG codon in the cDNA is followed by codons for a hydrophobic signal peptide sequence (Von Heijne, 1985).

The DWnt-3 cDNA contains an open reading frame which encodes a protein of 1010 amino acids with a relative molecular mass of 112881, almost three times larger than the proteins encoded by other Wnt gene family members. The ATG codon in the cDNA postulated to be the start of translation is preceded by stop codons in the same frame and the flanking sequences conform to the consensus for Drosophila translational initiation sites (Cavener, 1987). In the following sequence, a small cluster of positively charged residues followed by a hydrophobic domain are predicted to be part of a signal peptide sequence (Von Heijne, 1985). The amino-terminal region of DWnt-3 is considerably longer, some 500 amino acids, than the amino termini of the products of other Wnt genes characterized. It contains many charged residues, four cysteine residues, and two stretches of homopolymeric sequences containing either glycines or serines. There is, in addition, a 155 amino acid insert present in the carboxy-terminal half of DWnt-3. Some mouse members of the Wnt gene family encode short stretches of extra amino acids at the same relative location (Gavin et al., 1990) suggesting that the insert was present in ancestral Wnt genes prior to divergence of mouse and Drosophila species. Neither the extended amino terminus nor the 155 amino acid insert of DWnt-3 share significant homology with other known sequences. The wg gene encodes an 85 amino acid insert, in the fourth exon, which is absent from the mouse Wnt genes. Neither DWnt-2 nor DWnt-3 proteins contain an equivalent to the 85 amino acid insert of wg, although the DWnt-2 protein has an insert of 12 amino acids at the same relative location (Fig. 1).

DWnt-2 and DWnt-3 amino acid sequences are 40% identical to each other, and both sequences are around 40% identical to wg, not taking into account the nonconserved amino termini and the inserts of DWnt-2, DWnt-3 and wg. DWnt-2 and DWnt-3 gene products contain an abundance of cysteine residues, twenty-two of which are conserved between all known members of the Wnt gene family. An additional two cysteine residues (indicated in Fig. 1) which are absent from wg and mouse Wnt-1 gene products, are conserved between DWnt-2, DWnt-3 and all other mouse Wnt proteins known. There is also one cysteine residue unique to mouse Wnt-1 and wg gene products (Fig. 1).

The cytological map positions of DWnt-2 and DWnt-3

We localized the DWnt-2 and DWnt-3 genes on polytene chromosomes from the salivary glands of Drosophila (CantonS-1) larvae by in situ hybridization using biotinylated probes derived from the cDNAs of these genes. DWnt-2 maps to chromosome 2 position 45E, while DWnt-3 maps to chromosome X position 17A/B (data not shown). Although mutations that map to these loci may lie within DWnt-2 or DWnt-3, there is no immediately obvious correlation between any of the mutant phenotypes and the expression profiles of the respective genes.

Expression of DWnt-2 and DWnt-3 during Drosophila development

Genomic and cDNA fragments from DWnt-2 and DWnt-3 were used to probe Northern blots of RNA from different stages of Drosophila development (Fig. 2). The DWnt-2 probe detects a 2.5 kb transcript in embryos, larvae and pupae (Fig. 2, top panel). No DWnt-2 expression is seen in adult flies. DWnt-3 is expressed as a 3.8 kb transcript in embryos, larvae, pupae and adult flies (Fig. 2, middle panel). Both DWnt-2 and DWnt-3 transcripts are most abundant in late larval and pupal stages.

Fig. 2.

Northern blot analysis of DWnt-2 and DWnt-3. 20 μg of total RNA were loaded in each lane. Three separate blots containing the same samples were hybridized with a genomic probe for DWnt-2 (Top Panel), a cDNA probe specific for the amino-terminal half of the DWnt-3 gene (Middle Panel) and, as a control, a probe for the Drosophila Actin 5C gene (Bottom Panel). E, total embryos (0 –24 hours of development); LI, L2 and L3, first, second and third instar larvae, respectively; P, pupae; A, adult flies.

Fig. 2.

Northern blot analysis of DWnt-2 and DWnt-3. 20 μg of total RNA were loaded in each lane. Three separate blots containing the same samples were hybridized with a genomic probe for DWnt-2 (Top Panel), a cDNA probe specific for the amino-terminal half of the DWnt-3 gene (Middle Panel) and, as a control, a probe for the Drosophila Actin 5C gene (Bottom Panel). E, total embryos (0 –24 hours of development); LI, L2 and L3, first, second and third instar larvae, respectively; P, pupae; A, adult flies.

DWnt-2 expression during Drosophila embryogenesis We analyzed the spatial distributions of DWnt-2 and DWnt-3 transcripts during Drosophila embryogenesis on whole-mount embryos by in situ hybridization with digoxigenin-labelled cDNA fragments (Figs 3 and 5, respectively).

Fig. 3.

The distribution of DWnt-2 transcripts during embryogenesis. In situ hybridization using digoxygenin-labelled DWnt-2 cDNA fragments on wholemount embryos. Surface views, sagittal and parasagittal optical views as visualized by Nomarski optics. (A,B,C,D,E,H,N) Anterior is left, dorsal is up. (G,1,J,K,L,M) Dorsal views. (F) Viewed from ventral surface. Arrows indicate the third thoracic segment of the embryos. (A) Cellularizing blastoderm. Transcripts most concentrated between 15% and 70% egg length (0% is the posterior pole). (B) Early germ band extension, stage 7. mRNA localized in proctodeum (pr) (see also L). (C) Germ-band-extended embryo, early stage 10. Transcripts in discrete dorsolateral patches in the thoracic (Tl -T3) and abdominal (Al -A8) segments. (D) Stage 10. The dorsolateral patches in the thoracic and abdominal segments have broadened along anterior-posterior and dorsal-ventral axes. Transcripts are also detectable in the maxillary and labial segments (mx, li) and in the anterior region. (E,F,G) Early stage 11. Ventral spots appear in each of the thoracic and abdominal segments; the spot in abdominal 8 (A8) is usually faint or undetectable. (H) Stage 11. Transcripts have almost disappeared from the gnathal segments (mx, li). The dorso-lateral patches have retracted along both axes. RNA is visible in the labrum (Ir). (I) Germ-band-shortened embryo, stage 13. The segmented pattern of DWnt-2 transcripts has all but vanished. Transcripts are detectable in anterior (a) and posterior (p) regions. (J) Stage 14. DWnt-2 mRNA becomes apparent in the posterior portion of the presumptive gonads (g) (see also N). Posterior staining (p) is still detectable. (K) Stage 15/16. Transcripts disappear from the posterior region but persist in the presumptive gonads (g) and are visible at the anterior (a). (L) Detail, stage 8. DWnt-2 RNA in columnar cells of the anterior wall of the proctodeal primordium. (M) Detail (2 × magnification of I), stage 13. DWnt-2 RNA in posterior region. (N) Detail (2 × magnification of K), stage 15/16. DWnt-2 transcripts in posterior portion of presumptive gonads.

Fig. 3.

The distribution of DWnt-2 transcripts during embryogenesis. In situ hybridization using digoxygenin-labelled DWnt-2 cDNA fragments on wholemount embryos. Surface views, sagittal and parasagittal optical views as visualized by Nomarski optics. (A,B,C,D,E,H,N) Anterior is left, dorsal is up. (G,1,J,K,L,M) Dorsal views. (F) Viewed from ventral surface. Arrows indicate the third thoracic segment of the embryos. (A) Cellularizing blastoderm. Transcripts most concentrated between 15% and 70% egg length (0% is the posterior pole). (B) Early germ band extension, stage 7. mRNA localized in proctodeum (pr) (see also L). (C) Germ-band-extended embryo, early stage 10. Transcripts in discrete dorsolateral patches in the thoracic (Tl -T3) and abdominal (Al -A8) segments. (D) Stage 10. The dorsolateral patches in the thoracic and abdominal segments have broadened along anterior-posterior and dorsal-ventral axes. Transcripts are also detectable in the maxillary and labial segments (mx, li) and in the anterior region. (E,F,G) Early stage 11. Ventral spots appear in each of the thoracic and abdominal segments; the spot in abdominal 8 (A8) is usually faint or undetectable. (H) Stage 11. Transcripts have almost disappeared from the gnathal segments (mx, li). The dorso-lateral patches have retracted along both axes. RNA is visible in the labrum (Ir). (I) Germ-band-shortened embryo, stage 13. The segmented pattern of DWnt-2 transcripts has all but vanished. Transcripts are detectable in anterior (a) and posterior (p) regions. (J) Stage 14. DWnt-2 mRNA becomes apparent in the posterior portion of the presumptive gonads (g) (see also N). Posterior staining (p) is still detectable. (K) Stage 15/16. Transcripts disappear from the posterior region but persist in the presumptive gonads (g) and are visible at the anterior (a). (L) Detail, stage 8. DWnt-2 RNA in columnar cells of the anterior wall of the proctodeal primordium. (M) Detail (2 × magnification of I), stage 13. DWnt-2 RNA in posterior region. (N) Detail (2 × magnification of K), stage 15/16. DWnt-2 transcripts in posterior portion of presumptive gonads.

Fig. 4.

The spatial distribution of DWnt-2/DWnt-3 transcripts in whole-mount embryos relative to en gene expression. Germ-band-extended embryos from a P-element transformant strain carrying an en-lacZ gene fusion (en β gal/CyO) double-labelled to visualize en by histochemical staining for β -galactosidase activity (turquoise) and transcripts from either DWnt-2 (A,B,C,D,E,F) or DWnt-3 (G,H) by in situ hybridization (purple). (A,B,C,D,G,H) Anterior is left, dorsal is up. (E) View of ventral surface. (F) View of dorsal surface. Note that the en expression domain lies in the posterior fourth of each segment, and, that wg-expressing cells lie directly anterior to the en domain. Abbreviations: AN, LI, LR, MX, Tl, T2, T3, A8; antennal, labial, labral, maxillary segments, thoracic segments 1,2 and 3, and abdominal segment 8, respectively. Arrows indicate thoracic segment 3. (A,B) Stage 10 embryo. The dorsolateral patches of DWnt-2 transcripts abutt the posterior edge of the en expression domain and extend in a posterior direction. It appears that DWnt-2 RNA is present in at least some wg-expressing cells. (C-F) Stage 11 embryo. The dorsolateral patches have retracted both toward the ventral midline and along the anterior-posterior axis away from the wg expression domain (C,D). In the thoracic segments, ventral spots of DWnt-2 transcripts abutt the anterior edge of the en expression domain and extend in an anterior direction over and beyond the wg domain (D,E). The ventral spots of DWnt-2 RNA in the abdominal segments also abutt the anterior edge of the en domain and extend over the wg expression domain (E), but cover a smaller area of the ectoderm than those of the thoracic segments. DWnt-2 transcripts are also visible in labral (LR) and anterior (a) regions (F). (G,H) Stage 10 embryo. The domains of DWnt-3 transcripts in the antennal (AN), maxillary (MX) and labial (LI) regions appear to overlap en expression domains and extend anteriorly over and beyond the wg expression domains.

Fig. 4.

The spatial distribution of DWnt-2/DWnt-3 transcripts in whole-mount embryos relative to en gene expression. Germ-band-extended embryos from a P-element transformant strain carrying an en-lacZ gene fusion (en β gal/CyO) double-labelled to visualize en by histochemical staining for β -galactosidase activity (turquoise) and transcripts from either DWnt-2 (A,B,C,D,E,F) or DWnt-3 (G,H) by in situ hybridization (purple). (A,B,C,D,G,H) Anterior is left, dorsal is up. (E) View of ventral surface. (F) View of dorsal surface. Note that the en expression domain lies in the posterior fourth of each segment, and, that wg-expressing cells lie directly anterior to the en domain. Abbreviations: AN, LI, LR, MX, Tl, T2, T3, A8; antennal, labial, labral, maxillary segments, thoracic segments 1,2 and 3, and abdominal segment 8, respectively. Arrows indicate thoracic segment 3. (A,B) Stage 10 embryo. The dorsolateral patches of DWnt-2 transcripts abutt the posterior edge of the en expression domain and extend in a posterior direction. It appears that DWnt-2 RNA is present in at least some wg-expressing cells. (C-F) Stage 11 embryo. The dorsolateral patches have retracted both toward the ventral midline and along the anterior-posterior axis away from the wg expression domain (C,D). In the thoracic segments, ventral spots of DWnt-2 transcripts abutt the anterior edge of the en expression domain and extend in an anterior direction over and beyond the wg domain (D,E). The ventral spots of DWnt-2 RNA in the abdominal segments also abutt the anterior edge of the en domain and extend over the wg expression domain (E), but cover a smaller area of the ectoderm than those of the thoracic segments. DWnt-2 transcripts are also visible in labral (LR) and anterior (a) regions (F). (G,H) Stage 10 embryo. The domains of DWnt-3 transcripts in the antennal (AN), maxillary (MX) and labial (LI) regions appear to overlap en expression domains and extend anteriorly over and beyond the wg expression domains.

Fig. 5.

The distribution of DWnt-3 transcripts during Drosophila embryogenesis as determined by in situ hybridization on whole-mount embryos using digoxygenin-labelled probes. Surface views, sagittal and parasagittal optical views as visualized by Nomarski optics. (A,B,C,D,F,H,K) Anterior is left, dorsal is up. (E,G,I,L) Viewed from ventral surface. (J,M) Dorsal views. (A) Cellular blastoderm embryo. Highest levels of transcripts between 20% and 80% EL (0% is the posterior pole). (B)Gastrulating embryo. Transcripts present in cells of presumptive cephalic and ventral furrows (cf, vf). (C)Germ-band-extended embryo, stage 8. DWnt-3 mRNA within cells of the mesoderm. (D,E) Stage 10 embryo. Transcripts in labral (lr), antennal (an), maxillary (mx) and labial (li) regions. (F,G) Stage 11 embryo. DWnt-3 RNA persists in mesoderm, and also, labral, antennal, maxillary and labia) locations. Transcripts appear in regions of the leg primordia in Tl-3 (arrows), and also, in the differentiating neurons of the CNS. (H,I,J) Germ-band-shortened embryo, stage 14. Transcripts in the ventral cord (vc) and suboesophageal and supraoesophageal ganglia of the CNS. (K,L,M) Stage 15/16. DWnt-3 mRNA in the CNS. Diagram. The locations of the limb primordia of an embryo at germ-band-extended stage; redrawn from Cohen (1990) with permission. Points indicate the primordia of sense organs of the vestigial larval appendages and stippled patches indicate the positions of the imagina! disc primordia in the antennal (an), labial (li), labral (Ir), maxillary (mx) and first thoracic (Tl) segments (Jürgens et al., 1986).

Fig. 5.

The distribution of DWnt-3 transcripts during Drosophila embryogenesis as determined by in situ hybridization on whole-mount embryos using digoxygenin-labelled probes. Surface views, sagittal and parasagittal optical views as visualized by Nomarski optics. (A,B,C,D,F,H,K) Anterior is left, dorsal is up. (E,G,I,L) Viewed from ventral surface. (J,M) Dorsal views. (A) Cellular blastoderm embryo. Highest levels of transcripts between 20% and 80% EL (0% is the posterior pole). (B)Gastrulating embryo. Transcripts present in cells of presumptive cephalic and ventral furrows (cf, vf). (C)Germ-band-extended embryo, stage 8. DWnt-3 mRNA within cells of the mesoderm. (D,E) Stage 10 embryo. Transcripts in labral (lr), antennal (an), maxillary (mx) and labial (li) regions. (F,G) Stage 11 embryo. DWnt-3 RNA persists in mesoderm, and also, labral, antennal, maxillary and labia) locations. Transcripts appear in regions of the leg primordia in Tl-3 (arrows), and also, in the differentiating neurons of the CNS. (H,I,J) Germ-band-shortened embryo, stage 14. Transcripts in the ventral cord (vc) and suboesophageal and supraoesophageal ganglia of the CNS. (K,L,M) Stage 15/16. DWnt-3 mRNA in the CNS. Diagram. The locations of the limb primordia of an embryo at germ-band-extended stage; redrawn from Cohen (1990) with permission. Points indicate the primordia of sense organs of the vestigial larval appendages and stippled patches indicate the positions of the imagina! disc primordia in the antennal (an), labial (li), labral (Ir), maxillary (mx) and first thoracic (Tl) segments (Jürgens et al., 1986).

As seen in Fig. 3, DWnt-2 transcripts are already present at the blastoderm stage of development. During cellularization, the highest levels of transcripts occur in the central region between approximately 15% and 70% egg length (EL) (0% is the posterior pole) (Fig. 3A). During the early stages of germ band extension (stage 7), DWnt-2 transcripts are visible within at least 12 columnar cells in the anterior wall of the proctodeal primordium (Fig. 3B,L).

DWnt-2 transcripts are detectable in the three thoracic and eight abdominal segments of stage 8 embryos, and by stage 9/10 the RNA is restricted to discrete ectodermal patches in the dorsolateral regions of each of these segments (Fig. 3C). The dorsolateral patches are half a segment in width, but during stage 10 the patches broaden toward the ventral midline and along the anterior-posterior axis (Fig. 3D). In order to delimit the DWnt-2 expression domains along the anterior-posterior axis and thus to identify genes with overlapping expression domains that might interact with DWnt-2, we performed double-labelling experiments using en expression as a positional marker. Fig. 4 shows the positions of the broadened patches of D Wnt-2 transcripts within the thoracic and abdominal segments with respect to en expression domains. The DWnt-2 transcripts occur across the anterior half of each segment and in the posterior portion of each segment, just anterior to the parasegment boundary, there is overlap with wg-expressing cells. There is, however, no overlap with the en expression domain in the posterior portion of the segments (Fig. 4A,B).

Appearance of DWnt-2 transcripts in two of the gnathal segments is more or less concomitant with the broadening of the dorsolateral patches (Fig. 3D). In the maxillary and labial segments, many of the cells in which DWnt-2 is transcribed lie anterior to the en expression domain, and there appears to be some overlap with the en domain (Fig. 4A,C,D). DWnt-2 transcripts also occur at low levels as spots in the primordial labrum and the dorsal area of the pro-cephalic lobe, and in the proctodeum (Fig. 3D). The positions of the DWnt-2 transcript domains in labral, maxillary and labial segments may correspond to the positions of primordia of the larval mouth part appendages (Jürgens et al., 1986).

At early stage 11, DWnt-2 transcripts arise in the ventral ectoderm, in a pattern of spots, two per each of the thoracic and abdominal segments, arrayed in two longitudinal rows along the entire length of the germ band. The ventral spots, which consist of clusters of cells, gradually enlarge during stage 11; the ventral spots in the thoracic segments become larger than those of abdominal segments 1-7, while those of abdominal segment 8 are often difficult to detect (Fig. 3E-H). The exact locations of the spots, between the intersegmental furrows, has been shown by double-labelling experiments to be directly anterior to the en expression domain, abutting the parasegmental boundary (Fig. 4C-F). The ventral spots therefore include cells from the wg expression domain, near the lateral tip of ventral wg stripes, as well as cells from the regions anterior and dorsal to this domain. DWnt-2 transcribing cells in the ventral regions of thoracic segments may therefore be within the anterior compartments of leg primordia (Bate and Martinez-Arias, 1991) as inferred from the pattern of Distal-less (Dll) expression in the thoracic segments (Cohen, 1990). It is also possible that the ventral spots in the abdominal segments are associated with precursors of the abdominal histoblasts, cell clusters that contribute to the larval epidermis and from which the adult epidermis of the abdominal segments is derived.

During germband shortening, DWnt-2 message disappears from the gnathal segments and the dorsolateral patches retract away from the ventral midline and shrink along the anterior-posterior axis such that D Wnt-2 transcripts are no longer detectable within cells of the wg expression domain (Figs 3H, 4C). By stage 14, the segmented pattern of DWnt-2 transcripts is absent. DWnt-2 RNA detectable in the posterior region of stage 13 and stage 14 embryos is apparently associated with the proctodeal opening (Fig. 31,J,M).

During stage 14, DWnt-2 message appears in the dorsolateral regions of the fifth abdominal segment, within the posterior portion of the presumptive gonads (Fig. 3J,N), associated with either germ-line precursor cells, recruited from the pole cells, or the mesodermal cells with which they intermingle to form the embryonic gonads. DWnt-2 transcripts are not detectable in pole cells prior to stage 14. From stages 15 onwards, DWnt-2 RNA is limited to clusters of cells in the anteriormost region of the embryo and to the posterior portion of the presumptive gonads (Fig. 3K).

DWnt-3 expression during Drosophila embryogenesis DWnt-3 transcripts are present in blastoderm stage embryos with the highest concentration of RNA between 20% and 80% EL (Fig. 5A). During stage 5 of embryogenesis, a dorsoventral stripe of DWnt-3 RNA arises along the presumptive cephalic furrow. By stage 6, during gastrulation, transcripts are detectable in the mesoderm along the ventral furrow, and in cells of the dorsal folds (Fig. 5B). DWnt-3 continues to be transcribed in mesodermal cells throughout embryogenesis until at least stage 15 (Fig. 5C,F).

In embryos with an extended germ band at early stage 10, DWnt-3 message appears in clusters of ectodermal cells in the dorsolateral regions of the maxillary and labial segments, in the antennal region, and at the anterior tip within the primordium of the labrum (Fig. 5D,E). To determine the relative position of the DWnt-3 transcribing cells within the maxillary and labial segments, we performed double-labelling experiments using en expression as a positional marker. Cells containing DWnt-3 transcripts occur in a cluster in the region anterior to the en expression domain. The underlying mesodermal pattern of DWnt-3 transcripts makes it difficult to be certain whether DWnt-3 is, as appears, also transcribed in en-expressing cells (Fig. 4G,H). Nonetheless, DWnt-3 is apparently expressed in cells of the labral, antennal, maxillary and labial primordia of the vestigial larval appendages. In stage 11 embryos, DWnt-3 RNA is seen in small lateral clusters of cells in each of the thoracic segments in the region of the leg primordia (Fig. 5G); the precise location of these clusters could not be ascertained in double-labelling experiments due to the mesodermal staining.

DWnt-3 transcripts are first detectable in a metameric pattern about the ventral midline in cells of the developing CNS at stage 11 (Fig. 5F). By stage 14, after germ band retraction, head involution has commenced and the neuromeres corresponding to the gnathal segments have fused to form the suboesophageal ganglion. DWnt-3 transcripts are apparent in the suboesophageal and supraoesophageal ganglia, the antenno-maxillary complex, and the neuromeres of thoracic and abdominal segments which make up the ventral cord (Fig. 5H,J). Late in embryogenesis, at stages 15 and 16, DWnt-3 transcripts are still predominantly associated with cells of the CNS (Fig. 5K-M). The spatial distribution of DWnt-3 mRNA throughout the CNS appears to correspond to the pattern of differentiating neurons.

We have shown here that the Drosophila Wnt/wg gene family comprises at least three members. By moderate stringency hybridization of a Drosophila genomic library using the mouse Wnt-3 gene as a probe, the only Drosophila genomic clones that we isolated contained sequences from DWnt-2, DWnt-3 or wg. In an attempt to identify more members of the Drosophila Wnt gene family, we have used degenerate primers, similar to those used by Gavin et al. (1990), in polymerase chain reactions on Drosophila genomic DNA and succeeded in selectively amplifying only sequences from DWnt-2, DWnt-3 or wg.

The primary translation products of DWnt-2 and DWnt-3 share some of the characteristic features associated with products of the Wnt gene family, including putative glycosylation sites, amino-terminal signal peptide sequences and conserved cysteine residues. If the conservation of cysteine residues can be taken as a criterion for the relatedness of the Wnt genes, then Drosophila genes DWnt-2 and DWnt-3 are less related to wg than wg is related to mouse Wnt-1. Although we isolated DWnt-2 and DWnt-3 by hybridization to the mouse Wnt-3 gene, in terms of overall amino acid sequence homology (not taking into account inserts and non-conserved amino-terminal sequences) the best alignment of DWnt-2 is with mouse Wnt-7, while that of DWnt-3 is with mouse Wnt-5, (Gavin et al., 1990; A. Sidow, personal communication). The DWnt-3 gene differs significantly from other members of the Wnt family due to its potential to encode both an amino terminus considerably longer than the amino termini of the other Wnt proteins and an insert of some 155 amino acids.

The expression profiles of the Drosophila Wnt/wg genes during embryogenesis are quite distinct, despite some overlap, suggesting different developmental roles for these genes. The presence of DWnt-2 transcripts in the posterior region of the embryonic gonads may imply that DWnt-2 functions in gonadogenesis. Since the spatial distribution of DWnt-3 transcripts is closely associated with neurogenic regions of the embryo and transcripts are abundant throughout the CNS at later stages of embryogenesis, DWnt-3 could have a role in CNS development. In addition, roles for DWnt-2 and DWnt-3 in limb development are suggested by the apparent overlap between the expression domains of these genes and those of Dll, a homeodomain gene essential for limb development (Cohen and Jürgens, 1989; Cohen et al., 1989; Cohen, 1990) within regions which may correspond to the labral, antennal (except DWnt-2), maxillary and labial primordia of the vestigial larval appendages, and the anterior compartment of the leg primordia. There is also some overlap between DWnt-2, DWnt-3 and wg expression domains in these regions and there is evidence that wg expression may be related to specification of the imaginai disc primordia (Baker, 1988b). In view of the role of wg in anterior-posterior patterning during Drosophila development, perhaps combinations of Drosophila Wnt gene products provide the information required to position the developing limbs with respect to the anterior-posterior and dorsoventral body axes. Alternatively, the Drosophila Wnt genes may be involved in proximal-distal pattern formation in the developing limbs. In the absence of mutations within DWnt-2 and DWnt-3, however, the functions of these genes during Drosophila development remain uncertain. We speculate upon three possible complementations of wg function by the other Drosophila Wnt genes which may occur in embryonic domains in which wg is not expressed or not required, implying some level of functional redundancy between the genes.

Cohen (1990) has demonstrated that wg is required for Dll expression in the thoracic and labial limb primordia, but not in the labral, antennal or maxillary primordia. It is possible that DWnt-2 or DWnt-3 may be capable of inducing Dll expression in the latter regions in which wg function is not essential. Interestingly, there is an absolute requirement for wg expression in the activation of Dll only in those regions (labial and thoracic) that normally possess parasegment boundaries (Cohen, 1990). This suggests that in wg null mutants the absence of Dll expression in labial and thoracic regions could be finked to a defect in anterior-posterior pattern formation (Cohen, 1990) which apparently cannot be overcome by the presence of either DWnt-2 or DWnt-3.

A role for wg has been demonstrated in the localization of the protein product of the segment polarity gene armadillo (arm), the Drosophila homologue of the mammalian plakoglobin gene (Riggleman et al., 1990; Peifer and Weischaus, 1990). In the CNS, mesoderm and other regions of the embryo where wg is not expressed, arm protein is localized nonetheless, indicating that another factor is responsible for the process in these regions. It is conceivable that DWnt-3, expressed in the CNS and the mesoderm and highly related to wg, is involved in arm localization.

The mutual dependence of wg and en expression in the epidermis in the process of segmentation is well-established (DiNardo et al., 1988; Martinez-Arias et al., 1988). en is expressed in a segmentally repeated subset of neuronal cells of the embryonic CNS and may have an important role in CNS development (DiNardo et al., 1985; Patel et al., 1989). However, from studies of wg mutants, in which only subtle defects of the CNS were observed, it is clear that wg has only a minor role in CNS development (Patel et al., 1989) and is not essential for the regulation of en expression in the Drosophila CNS (Patel et al., 1989). Since DWnt-3 is expressed in the CNS, it is possible that it functions in the same pathway as en in neurogenesis. If so, it could be envisaged that expression of DWnt-3 and en are mutually dependent in certain regions of the developing CNS.

Wnt proteins possess secretory peptides and are predicted to interact with cell surface receptor molecules. There is some evidence to support the interaction of different Wnt-related proteins in the same receptor pathway, at least in situations involving Wnt expression at abnormal expression sites: MMTV-induced Wnt-1 and Wnt-3 expression in mouse mammary tumours and ectopically expressed Wnt-1 in Xenopus embryos, both produce effects in cells that do not normally respond to these gene products but which, conceivably, normally respond to other Wnt proteins. In Drosophila, recent evidence that ectopically expressed wg is able to function in the wg-signalling pathway suggests that a receptor responsive to the wg protein is present on cells that do not normally encounter this protein (J. Noordermeer, R. Nusse and P.A. Lawrence, personal communication). Perhaps such wg-responsive receptors normally interact with the translation products of DWnt-2, DWnt-3 or other members of the Drosophila Wnt gene family. Should such cross-reactivity occur between different Wnt gene products and their receptors, Wnt gene receptor mutants would not necessarily display the same phenotype as Wnt gene mutants.

We are especially grateful to Deborah Andrew, Matthew Scott, Laury Vonkalm, Joost Zomerdijk, and all members of the laboratory for advice and critical reading of this manuscript, as well as to, Nipam Patel, Corey Goodman and Stephen Cohen for their interest and useful comments, Deborah Andrew, Jasprien Noordermeer, Barbara Cohen and Kim Schuske for valuable advice concerning in situ hybridizations and Els Wagenaar for technical tips. J.R. was supported by an EMBO Long Term Fellowship. This research was supported financially by The Netherlands Cancer Institute and Howard Hughes Medical Institute.

The accession number for the sequences reported in this paper are: X64735 for DWnt-2-, and X64736 for DWnt-3.

Babu
,
P.
(
1977
).
Early developmental subdivisions of the wing disk in Drosophila
.
Mol. Gen. Genet
.
151
,
289
294
.
Baker
,
N. E.
(
1987
).
Molecular cloning of sequences from wingless, a segment polarity gene in Drosophila-. The spacial distribution of a transcript in embryos
.
EMBO J
.
6
,
1765
1773
.
Baker
,
N. E.
(
1988a
).
Embryonic and imaginai requirements for wingless, a segment-polarity gene in Drosophila
.
Dev Biol
.
125
,
96
108
.
Baker
,
N.
(
1988b
).
Localization of transcripts from the wingless gene in whole Drosophila embryos’
.
Development
103
,
289
298
.
Bate
,
M.
and
Martinez-Arias
,
A.
(
1991
).
The embryonic origin of imaginai discs in Drosophila
.
Development
112
,
755
761
.
Bradley
,
R. S.
and
Brown
,
A. M.
(
1990
).
The proto-oncogene int-1 encodes a secreted protein associated with the extracellular matrix
.
EMBO J
.
9
,
1569
75
.
Cabrera
,
C. V.
,
Alonso
,
M. C.
,
Johnston
,
P.
,
Phillips
,
R. G.
and
Lawrence
,
P. A.
(
1987
).
Phenocopies induced with antisense RNA identify the wingless gene
.
Cell
50
,
659
63
.
Campos-Ortega
,
J. A.
and
Hartensteln
,
V.
(
1985
).
The Embryonic Development o/Drosophila melanogaster
.
Berlin
:
Springer-Verlag
.
Cavener
,
D. R.
(
1987
).
Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates
.
Nucl. Acids Res
.
15
,
1353
1361
.
Cohen
,
B.
,
Wimmer
,
E. A.
and
Cohen
,
S. M.
(
1991
).
Early development of leg and wing primordia in the Drosophila embryo
.
Meeh. Dev
.
33
,
229
240
.
Cohen
,
S.
and
Jürgens
,
G.
(
1989
).
Proximal-distal pattern formation in Drosophila: cell automous requiremnet for Distal-less gene activity in limb development
.
EMBO J
.
8
,
2045
2055
.
Cohen
,
S. M.
(
1990
).
Specification of limb development in the Drosophila embryo by positional cues from segmentation genes
.
Nature
343
,
173
7
.
Cohen
,
S. M.
,
Bronner
,
G.
,
Kuttner
,
F.
,
Jürgens
,
G.
and
JSckle
,
H.
(
1989
).
Distal-less encodes a homoeodomain protein required for limb development in Drosophila
.
Nature
.
338
,
432
4
.
DiNardo
,
S.
,
Kuner
,
J. M.
,
Theis
,
J.
and
O’Farrell
,
P. H.
(
1985
).
Development of embryonic pattern in Drosophila melanogaster as revealed by accumulation of the nuclear engrailed protein
.
Cell
43
,
59
69
.
DiNardo
,
S.
,
Sher
,
E.
,
Heemskerk-Jorgens
,
J.
,
Kassis
,
J.
and
O’Farrell
,
P.
(
1988
).
Two-tiered regulation of spatially patterned engrailed gene expression during Drosophila embryogenesis
.
Nature
332
,
604
609
.
FJose
,
A.
,
McGinnis
,
W. J.
and
Gehring
,
W. J.
(
1985
).
Isolation of a homeobox-containing gene from the engrailed region of Drosophila and the spatial distribution of its transcript
.
Nature
313
,
284
289
.
Gavin
,
B. J.
,
McMahon
,
J. A.
and
McMahon
,
A. P.
(
1990
).
Expression of multiple novel Wnt-l/int-l-related genes during fetal and adult mouse development
.
Genes Dev
.
4
,
2319
2332
.
Heemskerk
,
J.
,
DiNardo
,
S.
,
Kostriken
,
R.
and
O’Farrell
,
P. H.
(
1991
).
Multiple modes of engrailed regulation in the progression towards cell fate determination
.
Nature
352
,
404
410
.
Hooper
,
J. E.
and
Scott
,
M. P.
(
1992
).
The Molecular Genetic Basis of Positional Information in Insect Segments
.
In Results and Problems in Cell Differentiation
, (ed.
W.
Hennig
).
Heidelberg
:
SpringerVerlag
.
Jakobovits
,
A.
,
Shackleford
,
G. M.
,
Varmus
,
H. E.
and
Martin
,
G. R.
(
1986
).
Two proto-oncogenes implicated in mammary carcinogenesis, int-1 and int-2, are independently regulated during mouse development
.
Proc. Natl. Acad. Sci. U. S. A
.
83
,
7806
10
.
Jürgens
,
G.
,
Lehmann
,
R.
,
Schardin
,
M.
and
Nüsslein-Volhard
,
C.
(
1986
).
Segmental organisation of the head in the embryo of Drosophila melanogaster
.
Roux’s Arch. Dev. Biol
.
195
,
359
377
.
Karr
,
T.
,
Wier
,
M.
,
AU
,
Z.
and
Kornberg
,
T.
(
1989
).
Patterns of engrailed protein in early Drosophial embryos
.
Development
105
,
605
612
.
Klingensmith
,
J.
and
Perrinton
,
N.
(
1991
).
Segment polarity genes and intercellular communication in Drosophila
.
In Cell Activation: Genetic Approaches
, (ed.
J.
Mond
,
J.
Cambier
and
A.
Weiss
), pp.
251
274
.
New York
:
Raven Press
.
Kornberg
,
T.
(
1981
).
engrailed: a gene controlling compartment and segment formation in Drosophila
.
Proc. Natl. Acad. Sci. U.S.A. IS, 1095–1099
.
Langer-Safer
,
P. R.
,
Levine
,
M.
and
Ward
,
D. C.
(
1982
).
Immunological method for mapping genes on Drosophila polytene chromosomes
.
Proc. Natl. Acad. Sci. U.S.A
.
79
,
4381
4385
.
Lawrence
,
P. A.
and
Johnston
,
P.
(
1989
).
Pattern formation in the Drosophila embryo: allocation of cells to parasegments by evenskipped and fushi tarazu
.
Development
105
,
761
767
.
Martinez-Arias
,
A.
,
Baker
,
N. E.
and
Ingham
,
P. W.
(
1988
).
Role of segment polarity genes in the definition and maintenance of cell states in the Drosophila embryo
.
Development
103
,
157
170
.
McMahon
,
A. P.
and
Bradley
,
A.
(
1990
).
The Wnt-1 (int-1) protooncogene is required for development of a large region of the mouse brain
.
Cell
62
,
1073
1085
.
McMahon
,
J. A.
and
McMahon
,
A. P.
(
1989
).
Nucleotide sequence, chromosomal localization and developmental expression of the mouse int-l-related gene
.
Development
107
,
643
50
.
Morata
,
G.
and
Lawrence
,
P. A.
(
1975
).
Control of compartment development by the engraded gene of Drosophila
.
Nature
255
,
614
617
.
Morata
,
G.
and
Lawrence
,
P. A.
(
1977
).
The development of wingless, a homeotic mutation of Drosophila
.
Dev. Biol
.
56
,
227
240
.
Nusse
,
R.
and
Varmus
,
H. E.
(
1982
).
Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome
.
Cell
31
,
99
109
.
Papkoft
,
J.
,
Brown
,
A. M.
and
Varmus
,
H. E.
(
1987
).
The int-1 protooncogene products are glycoproteins that appear to enter the secretory pathway
.
Mol. Cell. Biol
.
7
,
3978
84
.
Papkoff
,
J.
and
Schryver
,
B.
(
1990
).
Secreted int-1 protein is associated with the cell surface
.
Mol. Cell. Biol
.
10
,
2723
30
.
Pardue
,
M. L.
(
1986
).
In situ hybridisation to DNA of chromosomes and nuclei
.
In Drosophila: A Practical Approach
, (ed.
D. B.
Roberts
), pp.
111
137
.
Oxford
:
IRL Press
.
Patel
,
N. H.
,
Schafer
,
B.
,
Goodman
,
C. S.
and
Holmgren
,
R.
(
1989
).
The role of segment polarity genes during Drosophila neurogenesis
.
Genes Dev
.
3
,
890
904
.
Peifer
,
M.
and
Wieschaus
,
E.
(
1990
).
The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin
.
Cell
63
,
1167
1178
.
Perrimon
,
N.
and
Mahowald
,
A. P.
(
1987
).
Mulitple functions of segment polarity genes in Drosophila
.
Dev. Biol
.
119
,
587
600
.
Poole
,
S. J.
,
Kauvar
,
L. M.
,
Drees
,
B.
and
Kornberg
,
T.
(
1985
).
The engrailed locus of Drosophila: structural analysis of an embryonic transcript
.
Cell
40
,
37
43
.
Riggleman
,
B.
,
Schedl
,
P.
and
Wieschaus
,
E.
(
1990
).
Spatial expression of the Drosophda segment polarity gene armadillo is post-transcriptionally regulated by wingless
.
Cell
63
,
549
560
.
Rijsewijk
,
F.
,
Schuermann
,
M.
,
Wagenaar
,
E.
,
Parren
,
P.
,
Weigel
,
D.
and
Nusse
,
R.
(
1987
).
The Drosophila homolog of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless
.
Cell
50
,
649
57
.
Roelink
,
H.
and
Nusse
,
R.
(
1991
).
Expression of two members of the Wnt gene family during mouse development; restricted temporal and spatial patterns in the developing neural tube
.
Genes Dev
.
5
,
381
388
.
Roelink
,
H.
,
Wagenaar
,
E.
,
Lopes da Silva
,
S.
and
Nusse
,
R.
(
1990
).
Wnt-3, a gene activated by proviral insertion in mouse mammary tumors, is homologous to int-l/Wnt-1 and normally expressed in mouse embryos and adult brain
.
Proc. Nad. Acad. Sci. U.S.A
.
51
,
4519
4523
.
Sambrook
,
J.
,
Fritsch
,
E. F.
and
Manlatis
,
T.
(
1989
).
Molecular Cloning: a Laboratory Manual
. Second ed.
Cold Spring Harbor Press
.
Shackleford
,
G. M.
and
Varmus
,
H. E.
(
1987
).
Expression of the proto-oncogene int-1 is restricted to postmeiotic male germ cells and the neural tube of mid-gestational embryos
.
Cell
50
,
89
95
.
Shapiro
,
M.
and
Senapathy
,
P.
(
1987
).
RNA splice junctions of different classes of eukaryotes: sequence statistics and functional impheations in gene expression
.
Nucl. Acids Res
.
15
,
7155
7174
.
Sharma
,
R. P.
(
1973
).
wingless, a new mutant in Drosophila melanogaster
.
Drosophila Inform. Serv
.
50
,
134
.
Tautz
,
D.
and
Pfeiffle
,
C.
(
1989
).
A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback
.
Chromosoma
98
,
81
85
.
Thomas
,
K. R.
and
Capecchi
,
M. R.
(
1990
).
Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development
.
Nature
346
,
847
50
.
Van den Heuvel
,
M.
,
Nusse
,
R.
,
Johnston
,
P.
and
Lawrence
,
P. A.
(
1989
).
Distribution of the wingless gene product in Drosophila embryos: a protein involved in cell-cell communication
.
Cell
59
,
739
749
.
Von Heijne
,
G.
(
1985
).
Signal sequences: the limits of variation
.
J. Mol. Biol
.
184
,
99
105
.
Wieschaus
,
E.
and
Nüsslein-Volhard
,
C.
(
1986
).
Looking at embryos
.
In Drosophila: A Practical Approach
, (ed.
D. B.
Roberts
), pp.
199
227
.
Oxford
:
IRL Press
.
Wieschaus
,
E.
and
Riggleman
,
R.
(
1987
).
Autonomous requirements for the segment polarity gene armadillo during Drosophila embryogenesis
.
Cell
49
,
177
84
.
Wilkinson
,
D. G.
,
Bailes
,
J. A.
and
McMahon
,
A. P.
(
1987
).
Expression of the proto-oncogene int-1 is restricted to specific neural cells in the developing mouse embryo
.
Cell
50
,
79
88
.