The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.

The adult cuticle of Drosophila is decorated with a large number of cuticular structures, such as hairs and bristles. These structures are polarized with respect to the plane of the epithelia and typically share a common polarity. For example, the wing is decorated with a large number of distally pointing hairs. Mutations in the tissue polarity genes disrupt the normal precise alignment of these structures (Gubb and Garcia- Bellido, 1982; Adler, 1992; Eaton, 1997). Most of the tissue polarity genes identified so far appear to be part of the frizzled (fz) signaling/signal transduction pathway, which controls the polarity of several types of cuticular structures (Adler, 1992; Gubb, 1993). Hair polarity is controlled by regulating the subcellular location for the initiation of the prehair – the cytoskeletal-mediated outgrowth that gives rise to the cuticular hair (Wong and Adler, 1993). In contrast, sensory bristle polarity is regulated by genes controlling the orientation of the cell divisions that give rise to the four cells of the bristle sensory organ (Reddy and Rodrigues, 1999; Gho et al., 1999). Ommatidia polarity is controlled by regulating the R3/R4 cell fate decision and the subsequent rotation of the developing ommatidia (Wherli and Tomlinson, 1998; Reifegerste and Moses, 1999). It is thought that there is a general tissue polarity intercellular signaling/signal transduction pathway that regulates specific downstream effector genes and proteins in the cells that give rise to the different cuticular structures (Adler, 1992).

The cadherins were originally identified as proteins that mediate Ca2+-dependent cell adhesion. These proteins typically contain four or five extracellular cadherin motifs, a single transmembrane domain and a cytoplasmic tail that contains a binding site for β-catenin (reviewed in Geiger and Ayalon, 1992). Multiple tissue type-specific cadherin proteins are found in all animals. Epithelial cells typically contain E- cadherin, which is localized to the adherens junction and is essential for the maintenance of epithelial tissue structure. The ability of classical cadherins to mediate cell-cell adhesion requires their being linked to the actin cytoskeleton via the catenins (Nagafuchi and Takeichi, 1988). Since β-catenin (armadillo in Drosophila) is also a key member of the wingless/Wnt signal transduction pathway, cadherins can, at least indirectly, affect Wnt signaling (Peifer et al., 1991). The realization that fz encodes Wnt receptors provides a clue for potential connections between cadherins and catenins in the context of tissue polarity formation (Bhanot et al., 1996). However, it does not appear that armadillo or shotgun (the Drosophila E-cadherin gene) plays important roles in the development of wing tissue polarity (Peifer et al., 1991; Uemura et al., 1996; Tepass et al., 1996).

In addition to the classical cadherins, there is a diverse group of proteins called protocadherins that have been found in Drosophila, C. elegans and mammals. These proteins are often substantially larger than classical cadherins. For example, the fat (ft) protocadherin contains more than 30 cadherin domains (Mahoney et al., 1991). Relatively little is known about the function of the protocadherins. Results of previous cell biological experiments suggest that protocadherins only weakly promote cell adhesion. The most interesting genetic data comes from the analysis of the dachsous (ds) and ft genes of Drosophila, which have recently been shown to encode protocadherins (Clark et al., 1995; Mahoney et al., 1991). Mutations in ds result in malformed flies that often die as pharate adults or as newly eclosed adults. The legs and wings are particularly abnormal in ds flies. Both are shorter and fatter than normal, and each also displays a number of specific defects including disrupted tissue polarity (Adler et al., 1998). Mutations in ft are pupal lethals. When clones of ft are generated in imaginal tissue, they result in tumorous outgrowths. Hence, ft appears to be a tumor suppressor gene in flies (Mahoney et al., 1991). Interestingly both ds and ft were among the genes identified in a large screen for new tissue polarity mutants. The underlying biochemical mechanisms by which mutations in these genes produce a tissue polarity phenotype are unknown. At the genetic level, it was shown that ds caused an altered anatomical direction of fz signaling and enhanced fz domineering nonautonomy (Adler et al., 1998).

The starry night (stan) gene was identified in the same mutant screen that led to the identification of ds and ft as tissue polarity genes. Mutations in stan produce a wing tissue polarity phenotype that is similar to that of fz and dishevelled (dsh; Krasnow et al., 1995), both in terms of the mutant wing polarity pattern and the small number of wing cells that produce more than the normal one hair. Genetic analyses in the present study have shown that stan likely plays a role in the fz signaling/signal transduction pathway that controls tissue polarity. Mutations that inactivate stan were found to spread across about 40 kb of genomic DNA. The stan gene produces a longer than 12 kb mRNA that encodes a protocadherin. This protein is homologous in general predicted structure to proteins identified in mammals (rMEGF2, mCelsr1) and C. elegans (CeCelsr). They contain a series of 8-9 cadherin motifs, 4-7 EGF-like motifs, two laminin G motifs, and a region of seven transmembrane domains that is reminiscent of trimeric G- coupled receptors (Hadjantonakis et al., 1997; Nakayama et al., 1998). The unusual structure of the Stan protein suggests it could function in both cell adhesion and cell signaling pathways.

Fly culture and strains

Flies were grown on standard media at 25°C, unless stated otherwise. Many mutants were obtained from the stock center at the Indiana University. A chromosome carrying Df(2R)17 was kindly provided by Dr R. Burgess (Stanford). Several different mutations that cytogenetically removed stan were used in our studies (Df(2R)E3363, Df(2R)stan1, Df(2R)stan2, Df(2R)stan9, Df(2R)stan14, Df(2R)stan38). For simplicity, they are all abbreviated as Df-stan. Alleles of stan were isolated independently in Charlottesville and Cambridge. In Charlottesville, the original stan alleles were identified in an F1 FLP/FRT screen for new wing tissue polarity mutations, while the original allele identified in Cambridge was found in an unrelated mutant screen. Additional stan alleles were isolated in both locations by F1 screens where we used a lack of complementation of viable alleles for screening. The alleles of stan were isolated by EMS, γ-ray, X-ray and P element mutagenesis. The gene was named starry night (stan) after the swirling brush strokes in the eponymous painting by Van Gogh.

Cytological procedures

To examine the process of hair morphogenesis, pupal wings were dissected in 4% paraformaldehyde/PBS, stained with a fluorescent- phalloidin which binds to F-actin (Wong and Adler, 1993) and examined by confocal microscopy (Molecular Dynamics).

Generation of genetic mosaics

Mosaic experiments were carried out using the FLP/FRT system to generate clones (Xu and Rubin, 1993). Larva of w, hs-flp; FRT42 pwn stan/FRT42 were heat shocked at 38°C for 30 minutes to induce the recombination at the FRT site. The stan clones could be recognized by the recessive mutation pawn (pwn) that resulted in cells forming thin wispy hairs and short deformed bristles. Clones homozygous for fz were marked with the autonomously acting trichome morphology marker, starburst (strb).

Scoring of mutant wings

Wings from relevant flies were mounted in Euparal (Asco labs) and examined under bright-field microscopy. The analysis of wing phenotypes is described in detail elsewhere (Wong and Adler, 1993; Krasnow and Adler, 1994; Adler et al., 1994). A distal to proximal gradient of fz expression was induced by the ‘waxing’ procedure (Adler et al., 1997).

Molecular biology

P1 clones were generously obtained from Dr G. Rubin (UC Berkeley) and Dr E. Nitasaka (Kyushu University). P1 DNA was prepared by the alkaline lysis method described previously (Hartl et al., 1994) with slight modifications. DNA and RNA isolation, Southern analysis, northern analysis, genomic DNA and cDNA library screening was performed by standard methods (Sambrook et al., 1989). Genomic walking with λ clones was performed as described previously (Park et al., 1996). DNA sequencing was primarily done in the Core Center of the University of Virginia.

Seven RT-PCR were performed to obtain the full stan transcript. Reverse transcription was carried out using SuperScript Preamplification System (GIBCO-BRL). The resulting cDNA was subject to polymerase chain reaction that consisted of 35 cycles of denaturation for 30 seconds at 94°C, annealing for 1 minute at 55°C, and extension for 2 minutes at 72°C. For RT1 (1962 bp), the primer sets were 5′-ACT TGC GAG TGT GAT TCC-3′ and 5′-ATT TAG GCG AAT GTG TGG-3′; for RT2 (1918 bp), 5′-AGC TCC TTG GCG ATT CTC-3′ and 5′-CGG CAG CAC AAC ACT TTC-3′; for RT3 (1897 bp), 5′-ACG TAA CGA TGC CCA CAT-3′ and 5′-CTC TTT CTG GCG TGT TCG-3′; for RT4 (1598 bp), 5′-GTG TAC CAC CCA AAT CGG C-3′ and 5′-GGC TTC GTT AAT TCC ATC CG-3′; for RT5 (1861 bp), 5′-ACG CCT CCT ATG CCA TTC-3′ and 5′-AGC GAC TGG GCA ATG GTA-3′; for RT6 (1891 bp), 5′-GGC CTG CTC CTC GTG CT-3′ and 5′-TCA CAT CAC CGG ACA TCG-3′; for RT7 (1194 bp), 5′-TAG TCG TCG TGG CGT AG-3′ and 5′-CCT GTG TCT CAT CTC ATT GTC G-3′.

In situ hybridization

The 4,513 bp stan cDNA was digested with ApaI and SacI, and the resulting 645 bp fragment was subcloned into pGEM-T-Easy vector (Promega). Sense and antisense RNA probes were prepared using the Dig RNA labelling kit according to the manufacturer’s instruction (Boehringer Mannheim). In situ hybridization was performed as described previously (Tautz and Pfeiffle, 1989) with minor modification.

Sequence analysis

Genomic DNA sequence was analyzed for the presence of a putative open reading frame or exons using computer programs such as WebGeneMarker and BCM gene finder (Smith et al., 1996). For RT- PCR, optimal primers were designed using Vector NTI (Informax) and Oligo 5.0 (National Biosciences). The PROSITE database was utilized for motif search of the Stan protein (Bairoch et al., 1997). The presence of putative signal peptide was determined using SignalP (Nielsen et al., 1997). Putative transmembrane domains were predicted using SOSUI (Hirokawa et al., 1998) and PHD (Rost et al., 1995) algorithms.

Isolation of starry night (stan) mutations

Mutations in the stan gene cause a tissue polarity phenotype over much of the epidermis. Epidermal hairs, sensory bristles and ommatidia are all affected by stan mutations, thus in this way stan resembles fz, dsh, Van Gogh(Vang)/strabismus (Taylor et al., 1998; Wolff and Rubin, 1998), and prickle (Gubb et al., 1999). We found the stan gene to be highly mutable. In one experiment, we simultaneously screened for mutations in Vang, kojak (koj) and stan after γ-ray mutagenesis. We isolated five times more stan (21 mutations) than Vang (4 mutations) mutations and seven times more stan than koj (3 mutations) mutations (P. N. Adler, unpublished). This suggested that stan was a large gene. The stan gene was mapped to position 62 on the second chromosome by meiotic mapping, and localized to 47B3-5 on the basis of several alleles that contained breakpoints in this interval and deficiencies that failed to complement stan point mutants (data not shown).

In our collection of 49 stan alleles, there is a great deal of phenotypic variation (a detailed description of the stan alleles and their genetic interactions with other tissue polarity genes will be presented elsewhere). Most stan mutations are recessive lethals. In this study, we made extensive use of stan3, which is homozygous and hemizygous viable. Flies with this allele show a relatively strong polarity phenotype in the adult cuticle (Fig. 1B,D). The phenotype of stan3/stan3 wings is only slightly weaker than stan3/Df-stan wings (compare Fig. 1G and H), thus by this criteria stan3 is a strong hypomorphic allele for the wing tissue polarity phenotype. Since stan3 is healthy, the mutation is presumably functional for the stan vital functions. It is possible that stan3 is analogous to the dsh1 mutation, which is defective for tissue polarity, but functional for the essential functions of this gene (Krasnow et al., 1995).

Fig. 1.

Wing phenotype of stan. A and B are light micrographs of wings from wild-type and stan3/stan3 flies, respectively. C and D are confocal images of wild-type and stan pupal wings, respectively. Note that prehairs are initiating at central locations on the apical surface of the stan wing cells, whereas the prehairs form in the vicinity of the distal most vertex of the wild-type wing cells. Several cells in this field have formed two hairs. E, F, G and H are drawings of the polarity pattern on the dorsal surfaces of wild-type, fzK21/fzD21 (this represents the fz null phenotype on the wing), stan3/stan3, stan3/Df wings, respectively. Note the phenotype in H is only slightly stronger than that in G.

Fig. 1.

Wing phenotype of stan. A and B are light micrographs of wings from wild-type and stan3/stan3 flies, respectively. C and D are confocal images of wild-type and stan pupal wings, respectively. Note that prehairs are initiating at central locations on the apical surface of the stan wing cells, whereas the prehairs form in the vicinity of the distal most vertex of the wild-type wing cells. Several cells in this field have formed two hairs. E, F, G and H are drawings of the polarity pattern on the dorsal surfaces of wild-type, fzK21/fzD21 (this represents the fz null phenotype on the wing), stan3/stan3, stan3/Df wings, respectively. Note the phenotype in H is only slightly stronger than that in G.

The stan wing phenotype

We have used viable stan mutants (particularly stan3/stan3 and stan3/Df-stan) to examine the stan wing phenotype. As is the case for other tissue polarity mutants, stan mutants do not show a complete loss or randomization of hair polarity across the wing. Rather, they show a stereotypic abnormal polarity pattern (Fig. 1B,G,H). As noted previously, the polarity patterns that result from mutations in many tissue polarity genes are quite similar, albeit not identical. We call this pattern the fz/inturned (in) pattern after two of the best-studied genes in fz pathway (Fig. 1F). The stan mutant wings also have this general pattern. A second criterion that we have used to characterize tissue polarity mutants is the frequency of wing cells that form more than the normal one hair. Wings homozygous for stan3 have relatively few multiple hair cells (an average of 1.03 hairs/cell in our typical test region). This is similar to wings mutant for fz (1.02 hairs/cell), dsh (1.01 hairs/cell) and pk (1.02 hairs/cell). In contrast, it is much lower than that seen in wings mutant for inturned (in) (1.82 hairs/cell), fuzzy (fy) (1.92 hairs/cell) and multiple wing hair (mwh) (3.94 hairs/cell). We have also examined the process of hair morphogenesis in stan pupal wings. In wild-type wings, the prehairs that develop into the adult cuticular hairs are formed in the vicinity of the distal- most vertex of the cell and extend away from the cell in a distal direction (Fig. 1C). In stan mutants, many cells formed prehairs at a relatively central location on the apical cell surface (Fig. 1D). Other cells formed prehairs at abnormal locations along the cell periphery. This is similar to what we have found for mutations in the fz-like genes (Wong and Adler, 1993). Thus, based on several phenotypic criteria we place stan in the fz-like group of genes.

Cell autonomy of wing clones

Several recessive stan alleles were isolated due to a wing hair polarity phenotype seen in a wing in an FLP/FRT based F1 screen. Thus, it was clear that the presence of wild-type neighboring cells would not rescue all of the mutant cells in a clone. Several of the tissue polarity genes display domineering nonautonomy in wing clones – that is the presence of mutant cells in a clone alters the development of wild-type cells that are near the clone. To see if stan clones also displayed domineering nonautonomy, we generated mosaic wings where stan clones were marked with the hair marker pwn. Several different alleles were tested including the putative null allele stan24, the recessive lethal allele stan21 and the recessive viable allele stan3, and similar results were obtained. In all cases, the majority of clones behaved cell autonomously (53/60 for pwn stan21 clones, 72/83 pwn stan24 clones and 19/22 pwn stan3 clones) (Fig. 2A). Further, the extent of domineering nonautonomy for those clones scored as showing it was typically much weaker than we have seen with fz or Vang (Fig. 2B). We conclude from these experiments that stan principally functions cell autonomously.

Fig. 2.

Mitotic clone analysis. A and B show pwn stan24 somatic clones. The clone in A behaves cell autonomously as do most stan clones. The clone in B shows the weak domineering non-autonomy displayed by a minority of stan clones. The arrow points to several hairs with abnormal polarity that are not juxtaposed to the clone. The arrowhead indicates a cell that has formed two hairs. C and E show fzR52strb clones in stan3 wings. Equivalent regions of sibling wings where there are no clones are shown in D and F for comparison. The typical domineering non-autonomy of the fz clones in wild-type wing is shown in Fig. 1G. H shows the equivalent region of a wild-type wing.

Fig. 2.

Mitotic clone analysis. A and B show pwn stan24 somatic clones. The clone in A behaves cell autonomously as do most stan clones. The clone in B shows the weak domineering non-autonomy displayed by a minority of stan clones. The arrow points to several hairs with abnormal polarity that are not juxtaposed to the clone. The arrowhead indicates a cell that has formed two hairs. C and E show fzR52strb clones in stan3 wings. Equivalent regions of sibling wings where there are no clones are shown in D and F for comparison. The typical domineering non-autonomy of the fz clones in wild-type wing is shown in Fig. 1G. H shows the equivalent region of a wild-type wing.

stan suppresses the domineering non-autonomy of fz clones

As an in vivo assay for fz pathway function, we used the domineering non-autonomy of fz clones. To do this, we induced fzR52strb clones in stan3 wings. In a wild-type wing, more than 80% of fz clones show distal domineering non-autonomy. That is, cells distal (and in part anterior/posterior) but not proximal to the clone show altered polarity that extends to cells that do not border the clone (Fig. 2G,H). We scored 54 fzR52strb clones in regions of stan3 wings, where the polarity was consistent enough for us to be able to score the clones for domineering non-autonomy. Forty two clones behaved cell autonomously (Fig. 2C,D) and only 12 clones showed evidence of domineering non-autonomy. Further, the extent of domineering non-autonomy in these 12 clones was modest (Fig. 2E,F). Thus, stan appears to be a suppressor of the domineering non- autonomy of fz. That there remains some fz domineering non- autonomy in stan3 wings may reflect stan3 not being a null allele. The ability of a stan mutation to suppress this fz phenotype argues that stan is downstream of fz and required for the cell non-autonomous function of the fz pathway.

stan is required for the ability of a gradient of fz expression to repolarize wing hairs

As a second in vivo assay for fz pathway function, we used the ability of a gradient of fz expression, with its highest point near the distal tip of the wing, to reverse the normal distal polarity of wing hairs (Adler et al., 1997). This result argues that cells can ‘sense’ the fz activity of neighboring cells and respond to this information. The production of a region of reversed polarity is likely to require both cell non-autonomous (e.g., a fz-dependent intercellular signal) and cell autonomous functions (e.g., transduction of the fz-dependent signal). We found that stan3 completely blocked the ability of a gradient of fz expression to reorganize wing hair polarity (Table 1). Hence we conclude that stan functions downstream of fz and is required either for the cells to be able to sense the fz activity of neighboring cells or to respond to this information.

Table 1.

starry night function is required for a gradient of frizzled expression to produce a region of reversed wing hair polarity

starry night function is required for a gradient of frizzled expression to produce a region of reversed wing hair polarity
starry night function is required for a gradient of frizzled expression to produce a region of reversed wing hair polarity

stan does not block the late fz gain of function

The overexpression of fz just prior to prehair initiation causes the formation of large numbers of multiple hair cells that are a phenocopy of the in-like mutations (Krasnow and Adler, 1994). We have previously used this fz gain-of-function phenotype as a test to identify genes that are downstream of and required for the transduction of the fz signal (Krasnow et al., 1995). It was shown that the function of the dsh gene, which is thought to function downstream of fz (Klingensmith et al., 1994; Theisen et al., 1994), was indeed required for this phenocopy (Krasnow et al., 1995). However, the function of several other tissue polarity genes, pk, ds and Vang, was not required (Krasnow et al., 1995; Adler et al., 1998; Taylor et al., 1998). To determine if stan was required for the transduction of the fz signal, we constructed stan; hs-fz flies and induced fz expression just prior to prehair initiation. We found that the stan3 did not block the ability of fz overexpression to induce cells to form multiple hairs (Table 2). Rather, it appeared to slightly enhance the ability of fz overexpression to induce multiple hair cells.

Table 2.

stan does not block the ability of fz overexpression to induce multiple hair cells

stan does not block the ability of fz overexpression to induce multiple hair cells
stan does not block the ability of fz overexpression to induce multiple hair cells

Mapping of the stan locus

We initially mapped stan to 47B2-4 based on the cytogenetic analysis of two independent stan inversions, stan10 and stan35 (data not shown). P1 clones covering this region were obtained and used to screen a Drosophila genomic λ phage library. The clones were assembled into a contig covering about 76 kb and a restriction map was constructed (Fig. 3). RFLPs associated with stan10 and stan35 were identified by genomic Southern blot analysis, and these were found to be separated by approximately 40 kb, consistent with the large genetic target size of stan. We also identified RFLPs associated with two cytologically normal alleles (stan15 and stan24) induced by treatment with ionizing radiation. Both mapped in the GC5 region relatively close to the RFLP associated with stan35.

Fig. 3.

Molecular cloning of stan. A represents two P1 clones used for initial molecular characterization of the stan locus. B shows a contig of cosmid and λ phage clones. C shows a restriction map of the stan locus made with EcoRI, the locations of stan and two unrelated transcripts, and the locations of the four stan mutations as determined from genomic Southern analysis. D shows the structure of the stan cDNA. Also shown are the regions of the stan cDNA that were confirmed by RT- PCR. E shows representatives of the genomic Southern analysis of stan mutations. 1 is an EcoRI digestion of wild-type and stan35 genomic DNA probed with a 3.5 kb EcoRI fragment of GC5. 2 is a NdeI digestion of wild- type and stan10 genomic DNA probed with a 4.4 kb EcoRI fragment of GC62. 3 is an EcoRI digestion of wild-type and stan15 genomic DNA probed with a 5.2 kb EcoRI fragment of GC5. 4 is an EcoRI digestion of wild- type and stan24 genomic DNA probed with a 5.2 kb EcoRI fragment of GC5.

Fig. 3.

Molecular cloning of stan. A represents two P1 clones used for initial molecular characterization of the stan locus. B shows a contig of cosmid and λ phage clones. C shows a restriction map of the stan locus made with EcoRI, the locations of stan and two unrelated transcripts, and the locations of the four stan mutations as determined from genomic Southern analysis. D shows the structure of the stan cDNA. Also shown are the regions of the stan cDNA that were confirmed by RT- PCR. E shows representatives of the genomic Southern analysis of stan mutations. 1 is an EcoRI digestion of wild-type and stan35 genomic DNA probed with a 3.5 kb EcoRI fragment of GC5. 2 is a NdeI digestion of wild- type and stan10 genomic DNA probed with a 4.4 kb EcoRI fragment of GC62. 3 is an EcoRI digestion of wild-type and stan15 genomic DNA probed with a 5.2 kb EcoRI fragment of GC5. 4 is an EcoRI digestion of wild- type and stan24 genomic DNA probed with a 5.2 kb EcoRI fragment of GC5.

Identification of the stan transcript

To identify and map gene products in the putative stan locus, northern blots were screened with the inserts of all the λ phage clones. Distinct RNA bands of 4.9 kb, >10 kb, and 3.8 kb in length were detected with the inserts from the clones GC71, GC5 and GC1 respectively. A late embryonic cDNA library (Brown and Kafatos, 1988) was then screened with probes from these clones. We isolated cDNA clones representing these three independent mRNAs and sequenced members of each cDNA class. One group (obtained after screening with GC71) was found to have 93% sequence identity to the Fw repetitive elements found in the white locus of wi flies. These sequences show an extensive sequence similarity to L1 sequences, a major family of repetitive DNA elements dispersed in the mammalian genome (Di Nocera and Casari, 1987). The second group was found to encode the Drosophila rab3 gene, which was previously mapped to 47B1-14 (Johnston et al., 1991).

The third group was represented by a 4.5 kb cDNA that hybridized to a larger than 10 kb mRNA on northern blots, suggesting it was an incomplete cDNA. Sequencing showed that it encoded part of a novel protein. A consensus poly(A) signal (AATAAA) was found and followed by a stretch of poly(A) tail, implying that the cDNA contains a true 3′ end. The cDNA mapped to restriction fragments altered by three of the stan RFLPs arguing that it was a product of the stan gene. To identify the remainder of the stan transcript, we sequenced genomic DNA encompassing about 50 kb and identified potential coding regions by computer analysis. We then used RT-PCR to test if the identified regions were indeed found in stan mRNA and they were present in the same RNA molecule as our original cDNA clone. The RT-PCR products were sequenced to confirm the predicted structure of stan. As summarized in the Fig. 3, the stan gene consists of at least four exons, and the stan mRNA is over 12 kb in length encoding a putative 3,579 amino acid protein.

stan expression

Northern blot analysis showed that the large stan mRNA was present in a number of developmental stages (Fig. 4A). It was most abundant in 6-9 hour embryos and more abundant in pupae than larvae. We used in situ hybridization to examine the expression of stan in pupal wings. We found stan mRNA to be present at relatively even levels in all regions of the pupal wing (Fig. 4B). This is consistent with the genetic experiments showing that stan mutations have a phenotype over most if not all of the wing.

Fig. 4.

Expression of stan. A shows a developmental northern blot. About 5 μg of poly(A) RNA from various developmental stages was fractionated and blotted to Nytran. The blot was probed with the original 4.5 kb stan cDNA. The lower panel is the same blot reprobed with rp49 as a loading control. Lane 1, 0-3 hour embryo; lane 2, 6-9 hour embryo; lane 3, 9-12 hour embryo; lane 4, 12-24 hour embryo; lane 5, first instar larva; lane 6, second instar larva; lane 7, third instar larva; lane 8, 1 day pupa; lane 9, adult. B shows an in situ hybridization of pupal wings with stan. The pupal wing was prepared and hybridized with an antisense stan RNA probe as described previously (Tautz and Pfeiffle, 1989).

Fig. 4.

Expression of stan. A shows a developmental northern blot. About 5 μg of poly(A) RNA from various developmental stages was fractionated and blotted to Nytran. The blot was probed with the original 4.5 kb stan cDNA. The lower panel is the same blot reprobed with rp49 as a loading control. Lane 1, 0-3 hour embryo; lane 2, 6-9 hour embryo; lane 3, 9-12 hour embryo; lane 4, 12-24 hour embryo; lane 5, first instar larva; lane 6, second instar larva; lane 7, third instar larva; lane 8, 1 day pupa; lane 9, adult. B shows an in situ hybridization of pupal wings with stan. The pupal wing was prepared and hybridized with an antisense stan RNA probe as described previously (Tautz and Pfeiffle, 1989).

The Stan protein is a protocadherin

The conceptual translation of the stan mRNA yielded a protein with several notable motifs (Figs 5, 6). When analyzed with SignalP algorithm (Nielsen et al., 1997), the amino-terminal peptide consisting of the first 29 amino acids was predicted to serve as a signal peptide. This and the presence of seven putative transmembrane domains in the carboxy terminus suggest that Stan is a type 1 membrane protein. In addition, the huge extracellular part of the protein contains nine cadherin motifs, two laminin G motifs, and four EGF motifs implying that Stan may communicate with many signaling molecules or receptors. Database searches showed that stan is closely related to mammalian genes, rMEGF2 (Nakayama et al., 1998) and mCelsr1 (Hadjantonakis et al., 1997) and a C. elegans gene, CeCelsr. Their function is largely unknown except that rMEGF2 is specifically expressed in cerebellum and olfactory bulb (Nakayama et al., 1998) and mCelsr1 is expressed in the developing central nervous system (Hadjantonakis et al., 1998). While carrying out the molecular analysis of stan, we learned that the same gene had been cloned by the Uemura group (Kyoto University) due to the cadherin domain homology (Usui et al., 1999). They have called the gene flamingo and obtained cell biological data showing that flamingo is downstream of fz.

Fig. 5.

The amino acid sequence of the Stan protein. The signal sequence is in a hatched box; nine cadherin motifs are boxed; four EGF-like motifs are shaded; two laminin G motifs are in bold; seven transmembrane domains are underlined. The nucleotide and amino acid sequence of stan have been deposited in GenBank under the accession number AF172329.

Fig. 5.

The amino acid sequence of the Stan protein. The signal sequence is in a hatched box; nine cadherin motifs are boxed; four EGF-like motifs are shaded; two laminin G motifs are in bold; seven transmembrane domains are underlined. The nucleotide and amino acid sequence of stan have been deposited in GenBank under the accession number AF172329.

Fig. 6.

Domain organization of the Stan and related proteins. MEGF2 has been identified by its multiple EGF- like domains. Northern analysis showed that it is highly expressed specifically in cerebellum and olfactory bulb (Nakayama et al., 1998). Celsr1 (Cadherin EGF LAG seven-pass G-type receptor) also appears to be implicated in the development of mammalian brain (Hadjantonakis et al., 1997). CeCelsr1 is a C. elegans homolog (F15B9.7). Motifs were assigned on the basis of the PROSITE analysis (Bairoch et al., 1997). The putative signal sequence and transmembrane domains are indicated by S and M, respectively.

Fig. 6.

Domain organization of the Stan and related proteins. MEGF2 has been identified by its multiple EGF- like domains. Northern analysis showed that it is highly expressed specifically in cerebellum and olfactory bulb (Nakayama et al., 1998). Celsr1 (Cadherin EGF LAG seven-pass G-type receptor) also appears to be implicated in the development of mammalian brain (Hadjantonakis et al., 1997). CeCelsr1 is a C. elegans homolog (F15B9.7). Motifs were assigned on the basis of the PROSITE analysis (Bairoch et al., 1997). The putative signal sequence and transmembrane domains are indicated by S and M, respectively.

stan interacts with fz

Several lines of evidence point to stan function being required for the function of the fz pathway in wing tissue polarity. Mutations in stan produce an abnormal polarity pattern that is typical of tissue polarity genes whose function is thought to be essential for the pathway, such as fz and dsh. We also found a number of strong genetic interactions between stan and several fz pathway genes, including an allele-specific interaction between stan and fz (data not shown). Further, the domineering non-autonomy of fz is strongly suppressed by the stan3 mutation (Fig. 2). Finally, the ability of a directed gradient of fz expression to redirect the polarity of cells on the wing is blocked by the stan3 mutation (Table 1). All of these observations argue that stan function is essential for the function of the fz pathway.

Since stan mutations do not block the ability of the late overexpression of fz to produce an in-like phenotype (Table 2), the interaction between fz and stan is complex. It appears that fz carries out multiple (both cell autonomous and non-autonomous functions) roles in tissue polarity. The requirement for stan function for some but not all fz functions provides further evidence that fz has multiple roles in the development of tissue polarity. The two functions of fz could represent fz functioning in two distinct pathways or fz functioning at two times/locations in a single pathway. In the former model stan could be downstream of fz in one of the two pathways, and in the latter model it could function between the two times/locations where fz functions and hence be downstream of some fz functions and upstream of others. The requirement for stan function for a gradient of fz expression to redirect wing hair polarity and for the domineering nonautonomy of fz clones, but not for the late overexpression of fz to induce an in-like phenotype is not unique to stan, but is also a property of Vang (Taylor et al., 1998).

The Stan protein has multiple domains

Conceptual translation of the stan open reading frame reveals that it encodes a member of a protocadherin family found in both mammals and C. elegans. The protein has a remarkable structure that includes nine cadherin motifs, four EGF motifs, two laminin G motifs and seven transmembrane domains. The protocadherins are not nearly as well studied as classical cadherins, and relatively little is known about their in vivo function. The classical cadherins were identified as proteins that mediated Ca2+-dependent cell adhesion (reviewed by Geiger and Ayalon, 1992). E-Cadherin is present at adherens junctions in epithelial cells and is essential for the maintenance of tissue structure. Cadherin-dependent cell adhesion requires the linkage of the cadherin to the actin cytoskeleton, which is mediated by the catenins (Nagafuchi and Takeichi, 1988). Some protocadherins, such as Ds and Ft have potential catenin binding sites although these are interrupted (Clark et al., 1995; Mahoney et al., 1991). Stan does not apparently have such catenin binding sites. Ds and Ft have been suggested to have an adhesion function based on a number of phenotypes. For example, large ft clones in the wing often end up evaginating inward to form cuticular nodules. This might be a consequence of altered adhesion. Smaller ft clones as well as ds clones exhibit a marked tendency to assume an oval shape, once again consistent with the hypothesis that the clone cells have altered adhesivity (P. N. Adler, unpublished data). In contrast, we saw no such phenotypes with stan. Hence, we think it unlikely that stan mutations have a major effect on the adhesion of wing cells. However, we note that the Uemura group has obtained evidence for stan being an adhesion protein in cultured cells (Usui et al., 1999).

The large size of the Stan protein provides it with many potential sites for binding to other proteins. The presence of both cadherin domains and a region whose topology is similar to a G-coupled receptor suggest that Stan could potentially function either in cell adhesion and/or in signal reception. The failure to see any evidence of altered wing cell adhesion in stan mutants suggests that at least in tissue polarity Stan functions primarily in signaling/signal transduction. If Stan does not function as an adhesion protein, what is the reason for the cadherin domains that are conserved in both vertebrates and invertebrates? One intriguing possibility is that the cadherin domains serve to localize the Stan protein to a plasma membrane where it functions in signal reception and/or perhaps to localize other tissue polarity proteins. Alternatively, the cadherin domains could be required for stan function in other tissues or for subtle but important effects on cell adhesion in the wing that we could not detect by obvious changes in clone shape.

During this work, W. J. P. was supported by a grant (G70800- GG01570) from KRF (Korea Research Foundation) and a grant (G01130) from KOSEF (Korea Science and Engineering Foundation), and P. N. A. was supported by a grant from the NIH (GM37136). S. C. and D. G. were supported by a programme grant from the British Medical research Council to M. Ashburner and D. Gubb. W.J.P conceived this work and supervised the molecular analysis of stan. J. C. and M. J. K. carried out most of the molecular experiments together. J. H. G took a part in the initial characterization of P1 and λ clones. S. C. and D. G. independently identified stan and isolated several key alleles (stan3 and stan10), J. C. and P. N. A. independently identified stan and carried out most of the genetic experiments. W. J. P. and P. N. A were co-authors of the paper.

Adler
,
P. N.
(
1992
).
The genetic control of tissue polarity in Drosophila
.
BioEssays
14
,
735
741
.
Adler
,
P. N.
,
Charlton
,
J.
and
Liu
,
J.
(
1998
).
Mutations in the cadherin superfamily member gene dachsous cause a tissue polarity phenotype by altering frizzled signaling
.
Development
125
,
959
968
.
Adler
,
P. N.
,
Charlton
,
J.
and
Park
,
W. J.
(
1994
).
The Drosophila tissue polarity gene inturned function prior to wing hair morphogenesis in the regulation of hair polarity and number
.
Genetics
137
,
829
836
.
Adler
,
P. N.
,
Krasnow
,
R. E.
and
Liu
,
J.
(
1997
).
Tissue polarity points from cells that have higher Frizzled levels towards cells that have lower Frizzled levels
.
Curr. Biol
.
7
,
940
949
.
Bairoch
,
A.
,
Bucher
,
P.
and
Hofmann
,
K.
(
1997
).
The PROSITE database, its status in 1997
.
Nucl. Acids Res
.
25
,
217
221
.
Bhanot
,
P.
,
Brink
,
M.
,
Samos
,
C. H.
,
Hsieh
,
J. C.
,
Wang
,
Y.
,
Macke
,
J. P.
,
Andrew
,
D.
,
Nathans
,
J.
and
Nusse
,
R.
(
1996
).
A new member of the frizzled family from Drosophila functions as a Wingless receptor
.
Nature
382
,
225
230
.
Brown
,
N. H.
and
Kafatos
,
F. C.
(
1988
).
Functional cDNA libraries from Drosophila embryos
.
J. Mol. Biol
.
203
,
425
437
.
Clark
,
H. F.
,
Brentrup
,
D.
,
Schneitz
,
K.
,
Bieber
,
A.
,
Goodman
,
C.
and
Noll
,
M.
(
1995
).
Dachsous encodes a member of the cadherin superfamily that controls imaginal disc morphogenesis in Drosophila. Genes Dev
.
9
,
1530
1542
.
Di Nocera
,
P. P.
and
Casari
,
G.
(
1987
).
Related polypeptides are encoded by Drosophila F elements, I factors, and mammalian L1 sequences
.
Proc. Natl. Acad. Sci. USA
84
,
5843
5847
.
Eaton
,
S.
(
1997
).
Planar polarization of Drosophila and vertebrate epithelia
.
Curr. Opin. Cell Biol. 9, 860-866
.
Geiger
,
B.
and
Ayalon
,
O.
(
1992
).
Cadherins
.
Annu. Rev. Cell Biol
.
8
,
307
332
.
Gho
,
M.
,
Bellaiche
,
Y.
and
Schweisguth
,
F.
(
1999
).
Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell
.
Development
126
,
3573
3584
.
Gubb
,
D.
(
1993
).
Genes controlling cellular polarity in Drosophila
.
Development
1993
Supplement,
269
277
.
Gubb
,
D.
and
Garcia-Bellido
,
A.
(
1982
).
A genetic analysis of the determination of cuticular polarity during development in Drosophila melanogaster
.
J. Embryol. Exp. Morphol
.
68
,
37
57
.
Gubb
,
D.
,
Green
,
C.
,
Huen
,
D.
,
Coulson
,
D.
,
Johnson
,
G.
,
Tree
,
D.
,
Collier
,
S.
and
Roote
,
J.
(
1999
)
The balance between isoforms of the Prickle LIM domain protein is critical for planar polarity in Drosophila imaginal discs
.
Genes Dev
.
13
,
2315
2327
.
Hadjantonakis
,
A. K.
,
Formstone
,
C. J.
and
Little
,
P. F. R.
(
1998
).
mCelsr1 is an evolutionarily conserved seven-pass transmembrane receptor and is expressed during mouse embryonic development
.
Mech. Dev
.
78
,
91
95
.
Hadjantonakis
,
A. K.
,
Sheward
,
W. J.
,
Harmar
,
A. J.
,
de Galan
,
L.
,
Hoovers
,
J. M.
and
Little
,
P. F.
(
1997
).
Celsr1, a neural-specific gene encoding an unusual seven-pass transmembrane receptor, maps to mouse chromosome 15 and human chromosome 22qter
.
Genomics
45
,
97
104
.
Hartl
,
D. L.
,
Nurminsky
,
D. I.
,
Jones
,
R. W.
and
Lozovskaya
,
E. R.
(
1994
).
Genome structure and evolution in Drosophila: applications of the framework P1 map
.
Proc. Natl. Acad. Sci. USA
91
,
6824
6829
.
Hirokawa
,
T.
,
Boon-Chieng
,
S.
and
Mitagu
,
S.
(
1998
).
SOUSI: classification and secondary structure prediction system for membrane proteins
.
Bioinformatics
14
,
378
379
.
Johnston
,
P. A.
,
Archer
,
B. T.
,
Robinson
,
K.
,
Mignery
,
G. A.
,
Jahn
,
R.
and
Sudhof
,
T. C.
(
1991
).
rab3A attachment to the synaptic vesicle membrane mediated by a conserved polyisoprenylated carboxy-terminal sequence
.
Neuron
7
,
101
109
.
Klingensmith
,
J.
,
Nusse
,
R.
and
Perrimon
,
N.
(
1994
).
The Drosophila segment polarity gene dishevelled encodes a novel protein required for response to the wingless signal
.
Genes Dev
.
8
,
118
130
.
Krasnow
,
R. E.
and
Adler
,
P. N.
(
1994
).
A single frizzled protein has a dual function in tissue polarity
.
Development
120
,
1883
1893
.
Krasnow
,
R. E.
,
Wong
,
L. L.
and
Adler
,
P. N.
(
1995
).
dishevelled is a component of the frizzled signaling pathway in Drosophila
.
Development
121
,
4095
4102
.
Mahoney
,
P. A.
,
Weber
,
U.
,
Onofrechuk
,
P.
,
Biessmann
,
H.
,
Bryant
,
P. J.
and
Goodman
,
C. S.
(
1991
).
The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily
.
Cell
67
,
853
868
.
Nagafuchi
,
A.
and
Takeichi
,
M.
(
1988
).
Cell binding function of E-cadherin is regulated by the cytoplamic domain
.
EMBO J
.
7
,
3679
3684
.
Nakayama
,
M.
,
Nakajima
,
D.
,
Nagase
,
T.
,
Nomura
,
N.
,
Seki
,
N.
and
Ohara
,
O.
(
1998
).
Identification of high-molecular-weight proteins with multiple EGF-like motifs by motif-trap screening
.
Genomics
51
,
27
34
.
Nielsen
,
H.
,
Engelbrecht
,
J.
,
Brunak
,
S.
and
von Heijne
,
G.
(
1997
).
A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites
.
Int. J. Neural Syst
.
8
,
581
599
.
Park
,
W. J.
,
Liu
,
J.
,
Sharp
,
E. J.
and
Adler
,
P. N.
(
1996
).
The Drosophila tissue polarity gene inturned acts cell autonomously and encodes a novel protein
.
Development
122
,
961
969
.
Peifer
,
M.
,
Rauskolb
,
C.
,
Williams
,
M.
,
Riggleman
,
B.
and
Wieschaus
,
E.
(
1991
).
The segment polarity gene armadillo interacts with the wingless signaling pathway in both embryonic and adult pattern formation
.
Development
111
,
1029
1043
.
Reddy
,
G. V.
and
Rodrigues
,
V.
(
1999
).
Sibling cell fate in the Drosophila adult external sense organ lineage is specified by prospero function, which is regulated by Numb and Notch
.
Development
126
,
2083
2092
.
Reifegerste
,
R.
and
Moses
,
K.
(
1999
).
Genetics of epithelial polarity and pattern in the Drosophila retina
.
BioEssays
21
,
275
285
.
Rost
,
B.
,
Casadio
,
R.
,
Fariselli
,
P.
and
Sander
,
C.
(
1995
).
Prediction of helical transmembrane segments at 95% accuracy
.
Protein Science
4
,
521
533
.
Sambrook
,
J.
,
Fritsh
,
E. F.
and
Maniatis
,
T.
(
1989
).
Molecular Cloning: A Laboratory Manual
.
Cold Spring Harbor, NY
:
Cold Spring Harbor Laboratory Press
.
Smith
,
R. F.
,
Wiese
,
B. A.
,
Wojzynski
,
M. K.
,
Davison
,
D. B.
and
Worley
,
K. C.
(
1996
).
BCM search launcher – an integrated interface to molecular biology data base search and analysis services available on the world wide web
.
Genome Res
.
6
,
454
462
.
Tautz
,
D.
and
Pfeiffle
,
C.
(
1989
).
A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals a translational control of the segmentation gene hunchback
.
Chromosoma
98
,
81
85
.
Taylor
,
J.
,
Abramova
,
N.
,
Charlton
,
J.
and
Adler
,
P. N.
(
1998
).
Van Gogh: a new Drosophila tissue polarity gene
.
Genetics
150
,
199
210
.
Tepass
,
U.
,
Gruszynski-Defeo
,
E.
,
Haag
,
T. A.
,
Omatyar
,
L.
,
Torok
,
T.
and
Hartenstein
,
V.
(
1996
).
shotgun encodes Drosophila E-Cadherin and is preferentially required during cell rearrangement in the neurectoderm and other morphogenetically active epithelia
.
Genes Dev
.
10
,
672
685
.
Theisen
,
H.
,
Purcell
,
J.
,
Bennett
,
M.
,
Kansagara
,
D.
,
Sved
,
A.
and
Marsh
,
J. L.
(
1994
).
dishevelled is required during wingless signaling to establish both cell polarity and cell identity
.
Development
120
,
347
360
.
Uemura
,
T.
,
Oda
,
H.
,
Kraut
,
R.
,
Hayashi
,
S.
,
Kotaoka
,
Y.
and
Takeichi
,
M.
(
1996
).
Zygotic Drosophila E-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo
.
Genes Dev
.
10
,
659
671
.
Usui
,
T.
,
Shima
,
Y.
,
Shimada
,
Y.
,
Hirono
,
S.
,
Burgess
,
R. W.
,
Schwarz
,
T. L.
,
Takeichi
,
M.
and
Uemura
,
T.
(
1999
).
tFlamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled
.
Cell
98
,
585
595
.
Wherli
,
M.
and
Tomlinson
,
A.
(
1998
).
Independent regulation of anterior/posterior and equatorial/polar polarity in the Drosophila eye
.
Development
125
,
1421
1432
.
Wolff
,
T.
and
Rubin
,
G. M.
(
1998
).
strabismus, a novel gene that regulates tissue polarity and cell fate decisions in Drosophila
.
Development
125
,
1149
1159
.
Wong
,
L. L.
, and
Adler
,
P. N.
(
1993
).
Tissue polarity genes of Drosophila regulate the subcellular location for prehair initiation in pupal wing cells
.
J. Cell Biol
.
123
,
209
221
.
Xu
,
T.
and
Rubin
,
G. M.
(
1993
).
Analysis of genetic mosaics in developing and adult Drosophila tissues
.
Development
117
,
1223
1237
.