In the Drosophila leg disc, wingless (wg) and decapentaplegic (dpp) are expressed in a ventral-anterior and dorsal-anterior stripe of cells, respectively. This pattern of expression is essential for proper limb development. While the Hedgehog (Hh) pathway regulates dpp and wg expression in the anterior-posterior (A/P) axis, mechanisms specifying their expression in the dorsal-ventral (D/V) axis are not well understood. We present evidence that slimb mutant clones in the disc deregulate wg and dpp expression in the D/V axis. This suggests for the first time that their expression in the D/V axis is actively regulated during imaginal disc development. Furthermore, slimb is unique in that it also deregulates wg and dpp in the A/P axis. The misexpression phenotypes of slimb clones indicate that the regulation of wg and dpp expression is coordinated in both axes, and that slimb plays an essential role in integrating A/P and D/V signals for proper patterning during development. Our genetic analysis further reveals that slimb intersects the A/P pathway upstream of smoothened (smo).

Cells in Drosophila imaginal discs proliferate and organize in larval and pupal stages to form adult structures with specific patterns. Several secreted factors are responsible for coordinating the precise patterning of imaginal tissues for proper limb development. Hh, expressed in posterior cells induces neighboring anterior compartment cells to express their own anterior determinants: the Drosophila TGFβ homolog decapentaplegic (dpp), and the Wnt family member wingless (wg) (Lee et al., 1992; Basler and Struhl, 1994; Capdevila et al., 1994; Tabata and Kornberg, 1994; Felsenfeld and Kennison, 1995). In the leg imaginal disc, expression of dpp in the dorsal-anterior stripe is required for specification of dorsal structures, while wg in the ventral-anterior stripe determines ventral structures (Ferguson and Anderson, 1992; Struhl and Basler, 1993; Wilder and Perrimon, 1995). The expression patterns of dpp and wg are defined in both the anterior-posterior (A/P) and dorsal-ventral (D/V) axes.

The restricted domains of dpp and wg expression are tightly regulated in the A/P axis by the Hh/Ptc and PKA signaling pathways (Phillips et al., 1990; Ingham et al., 1991; Basler and Struhl, 1994; Capdevila and Guerrero, 1994; Tabata and Kornberg, 1994; Felsenfeld and Kennison, 1995; Li et al., 1995). Inactivation of ptc and pka or ectopic expression of hh induces ectopic dpp expression in the dorsal-anterior and ectopic wg expression in the ventral-anterior of the leg disc (Phillips et al., 1990; Ingham et al., 1991; Basler and Struhl, 1994; Jiang and Struhl, 1995; Li et al., 1995; Pan and Rubin, 1995) (Capdevila et al., 1994; Lepage et al., 1995). However, mutations in components of the A/P signaling pathway do not alter wg and dpp expression patterns in the D/V axis. To date no evidence exists of a D/V signaling pathway. It is possible that the D/V axis defined during embryogenesis is retained in imaginal tissues. One mechanism that prevents misexpression of wg in the dorsal and dpp in the ventral is the antagonistic relationship between wg and dpp. Inactivation of Wg or Dpp signaling leads to ectopic expression of dpp or wg, respectively (Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffman, 1996; Theisen et al., 1996).

To identify recessive overproliferation mutations in genes which are lethal in homozygous mutant animals, we have performed genetic screens in mosaic flies containing homozygous mutant patches in otherwise wild-type backgrounds (Xu et al., 1995). Two classes of recessive overproliferation mutations have been identified. Mutations of the first group cause mutant cells to undergo extensive proliferation and form unpatterned, tumorous outgrowths in mosaic adults. Mutations of the second group induce both patterned and irregular outgrowths. Here we report a new gene of the second class, slimb, which affects developmental signals that regulate cell proliferation and pattern organization. We present evidence that slimb mutant cells induce outgrowths by misexpressing wg and dpp. slimb regulates wg and dpp in both the A/P and D/V axes, demonstrating for the first time that these signals are coordinated. Genetic epistasis experiments reveal that slimb intersects A/P signaling upstream of smo.

slimb fly strains

122 excision lines were generated from two P-alleles (slmb00295 and slmb05415) and about half of them reverted to wild type. More than 30 excision alleles behaved as a single complementation group. Strong slmb alleles, including the original P alleles and slmbe4-1 (Fig. 2A), caused embryonic lethality while weak alleles caused larval and pupal lethality.

Cloning of slimb and H-slimb

Genomic DNA surrounding the P-insertion sites was obtained by plasmid rescue and used to isolate a genomic cosmid and cDNAs from an imaginal disc library. Comparison of genomic and cDNA sequences showed that slmb00295and slmb05415 inserted 150 nucleotides upstream of and within the coding region, respectively. Southern blot analysis of genomic DNA generated from the excision lines revealed that slmbe4-1 carries an approx. 3 kb deletion removing the 5′ end of the slmb transcript. The two P-alleles behave similarly to slmbe4-1 and are used interchangeably, while other excision alleles have weaker phenotypes. The 3.5 kb cDNA was sequenced to predict a protein product and cloned into the pCaSpeR-hs vector for germline transformation. Three of the transformant lines were able to fully rescue the lethality of the amorphic slmb alleles after 1 hour of heat shock every 24 hours during larval and pupal development. The human slmb homolog was identified by using the Drosophila cDNA as a probe to screen a human fetal brain library.

Generation and analysis of clones

Clones in adult flies and imaginal discs were generated by FLP-mediated mitotic recombination as previously described (Xu and Rubin, 1993; Xu and Harrison, 1994). Eggs from the appropriate crosses were collected for 24 hours and cultured at 25°C. Clones were induced in early second instar larvae by heat-shock induction of Flipase (38°C for 1 hour). Larvae from the following genotypes were used for clonal analysis: yw hsFLP1; P[FRT]82B P[πM]87E Sb63bP[y+]96E/P[FRT]82B slmbe4-1 or 00295 in a H1-1dpp-lacZ/+ background; and in a wg-lacZ/+ background. To detect hh-lacZ expression in slmb clones, hh-lacZ-P30 was recombined onto the slmb mutant chromosome and clones were induced in the following larvae: yw hsFLP1; P[FRT]82B P[πM]87E hh-lacZ-P30 P[πM]97E /P[FRT]82B slmb00295hh-lacZ-P30. Staining procedures followed standard protocols (Xu and Harrison, 1994).

Double mutant clones were induced in flies homozygous for the slmb null allele, but carried the hs-slmb31 rescue construct on the FRT40A chromosome arm. To ensure the full rescue of slmb− flies, eggs were collected every 24 hours and heat-shocked daily at 38°C for 60 minutes until hatched. Larvae of the following genotypes were generated and cultured at 25°C: yw hsFLP1; hs-slmb 31 P[FRT]40A/wgCX4ck P[FRT]40A; slmb00295/slmb00295, yw hsFLP1; hs-slmb 31 P[FRT]40A/dpp12ck P[FRT]40A; slmb00295/slmb00295, yw hsFLP1; P[FRT]82B P[πM]87E Sb63bP[y+]96E X/P[FRT]82B slmb00295hhrJ413, and yw hsFLP1; hs-slmb 31 P[FRT]40A/smoD16ck P[FRT]40A; slmb00295/slmb00295. slmb− clones were induced using the hs-slmb 31 P[FRT]40A chromosome at a frequency of 60% of discs. To verify that the smoD16ck P[FRT]40A chromosome that we used did not cause a cell-lethal phenotype, we examined clonal production by this chromosome and found it to produce smo− clones at a frequency of more than 25% of discs. In analyzing smoD16, slmb00295 double mutant clones, 40 mosaic leg discs were stained and found to have no ectopic wg or dpp expressed.

In a mosaic screen to identify recessive overproliferation mutations, we identified a new mutation, shiva, which causes outgrowths and disrupts pattern formation (Fig. 1) (Xu et al., 1995). In addition to two original P-insertion alleles, a deletion null allele (shivae4-1) was generated by excision of the P-elements and used for phenotypic analysis (Fig. 2A). Molecular and genetic characterization of shiva reveals that these mutations disrupt a single transcriptional unit and they can be rescued when the cDNA is expressed under control of the heat shock-inducible promoter (Materials and Methods). The transcript encodes a Cdc4-related protein containing F-box and WD-40 motifs. During preparation of this manuscript, Jiang and Struhl independently reported the identification of this gene as slimb (Jiang and Struhl, 1998). Thus, we are now renaming our gene slimb. Using a Drosophila slimb cDNA, we also isolated a human homolog (H-slimb) (Fig. 2B). The fly and human proteins share 78% amino acid identity throughout, suggesting that slimb is functionally conserved.

Fig. 1.

slimb clonal phenotypes in adult limbs. The yellow (y) and Stubble (Sb) cuticle markers were used to label slmbe4-1mutant cells in adult flies. Two types of outgrowths were observed in slmb mosaic legs: simple tissue outgrowths (A) and duplicated structures (B). (A) Scanning electron micrograph shows a leg tissue outgrowth composed of slmb+ cells (y+, Sb cells, y+ cannot be visualized on SEM). (B) Bifurcation of a third leg. The endogenous third leg (right) has signature posterior transverse and ventral bristles. The ectopic limb (asterisk) has slmb+ (y+, Sb) bristles, which are only of the dorsal type (close-up in C). As with the leg, wings from slmbe4-1 mosaic animals also displayed two types of outgrowths (D,F). (D) A slimb mosaic wing blade with multiple outgrowths. Outgrowths are found on either the dorsal or ventral surface of the blade, and in both anterior and posterior regions. (E) Magnification of an outgrowth indicated by the box in D. The outgrowth spans both sides of the third wing vein, and contains second row bristles at its apex (arrows). (F) A supernumerary wing (arrow) extends from the wing hinge-region and contains near-mirror images of anterior-most patterns and is composed of y+ cells (inset). (G) A slmbP00295 mosaic wing disc has extensive outgrowths. (H) High magnification of outgrowths in G. slimb− clones are located at the center of these outgrowths (H, arrows), inducing surrounding wild-type cells to proliferate.

Fig. 1.

slimb clonal phenotypes in adult limbs. The yellow (y) and Stubble (Sb) cuticle markers were used to label slmbe4-1mutant cells in adult flies. Two types of outgrowths were observed in slmb mosaic legs: simple tissue outgrowths (A) and duplicated structures (B). (A) Scanning electron micrograph shows a leg tissue outgrowth composed of slmb+ cells (y+, Sb cells, y+ cannot be visualized on SEM). (B) Bifurcation of a third leg. The endogenous third leg (right) has signature posterior transverse and ventral bristles. The ectopic limb (asterisk) has slmb+ (y+, Sb) bristles, which are only of the dorsal type (close-up in C). As with the leg, wings from slmbe4-1 mosaic animals also displayed two types of outgrowths (D,F). (D) A slimb mosaic wing blade with multiple outgrowths. Outgrowths are found on either the dorsal or ventral surface of the blade, and in both anterior and posterior regions. (E) Magnification of an outgrowth indicated by the box in D. The outgrowth spans both sides of the third wing vein, and contains second row bristles at its apex (arrows). (F) A supernumerary wing (arrow) extends from the wing hinge-region and contains near-mirror images of anterior-most patterns and is composed of y+ cells (inset). (G) A slmbP00295 mosaic wing disc has extensive outgrowths. (H) High magnification of outgrowths in G. slimb− clones are located at the center of these outgrowths (H, arrows), inducing surrounding wild-type cells to proliferate.

Fig. 2.

The slimb gene and its homologs. (A) The slimb transcript is illustrated on the genomic restriction map. An arrow indicates the initiation codon and direction of transcription. Hatched boxes indicate exon regions. The slmb P-alleles (slmb00295 and slmb05415) have P-elements in the first exon, and the excision line slmbe4-1 deletes the slmb promoter and transcript regions. Restrictions sites: G, BglII, H, HindIII; X, XbaI; R, EcoRI. (B) The Drosophila and human slmb transcripts predict protein products which share extensive homology throughout (boxes). Accession no. AF032878.

Fig. 2.

The slimb gene and its homologs. (A) The slimb transcript is illustrated on the genomic restriction map. An arrow indicates the initiation codon and direction of transcription. Hatched boxes indicate exon regions. The slmb P-alleles (slmb00295 and slmb05415) have P-elements in the first exon, and the excision line slmbe4-1 deletes the slmb promoter and transcript regions. Restrictions sites: G, BglII, H, HindIII; X, XbaI; R, EcoRI. (B) The Drosophila and human slmb transcripts predict protein products which share extensive homology throughout (boxes). Accession no. AF032878.

slimb induces outgrowths in mosaic adults

Phenotypic analysis revealed that slmb clones induce tissue outgrowths and supernumerary limbs in mosaic adults (Fig. 1). To analyze the slmb mosaic phenotype, the yellow (y) and Stubble+ (Sb+) cuticular markers were used to label slmb cells (Xu and Rubin, 1993; Materials and Methods). In mosaic legs, outgrowths are composed of slmb+ cells (y+ and Sb) (Fig. 1A-C). In addition to irregular outgrowths, supernumerary legs derived from slmb+ cells are also observed in slmb mosaic animals (Fig. 1B,C). Outgrowths are also observed in the wing blade (Fig. 1D,F), where mutant clones for slmb frequently produced small outgrowths in the adult wing (Fig. 1D). Similar to the leg outgrowths, the wing outgrowths consist of slmb+ cells (y+ and Sb) (Fig. 1E). Moreover, these outgrowths project from both the ventral and dorsal surfaces of the blade and occur in both the anterior and posterior halves of the wing (Fig. 1D). Outgrowths were organized into wing blade-like structures with wing margin bristles normally seen at the corresponding wing margin (Fig. 1D,E). Rarely, supernumerary wings consisting of symmetric duplications of anterior-most structures develop at the wing hinge (Fig. 1F). Although y (slmb) cells are rarely observed in mosaic animals, examination of mosaic discs revealed overproliferation of wild-type cells surrounding slmb mutant clones (Fig. 1G,H). These observations lead us to conclude that the mutant cells did not survive to adult stage, and that the adult outgrowths they induced are vestiges of their presence.

slimb regulates wg and dpp expression in both the A/P and D/V axes of the leg disc

slmb-induced outgrowths are reminiscent of the phenotypes caused by misexpression of dpp and wg (Struhl and Basler, 1993; Basler and Struhl, 1994; Wilder and Perrimon, 1995). Thus, we examined dpp and wg expression in slmb mosaic leg discs using wg-lacZ and dpp-lacZ reporter genes (Blackman et al., 1991; Kassis et al., 1992). slmb clones ectopically express both wg and dpp in a cell-autonomous fashion (Fig. 3). In respect to the A/P regions, 58/72 A clones and 9/31 P clones ectopically expressed wg, and 43/81 A clones and 6/17 P clones ectopically expressed dpp. A composite view of wg expression in the 103 analyzed slmb clones are illustrated in five subregions (Fig. 3J). dpp expression of 98 slmb clones was analyzed and not found to fall into any distinct domains. slmb mutant clones deregulate wg and dpp in both D/V and A/P axes. Ectopic wg expression is observed in both ventral and dorsal regions (Fig. 3A-C,J). Similar results are also observed for dpp (Fig. 3D-I). In slmb mutant clones situated within or near the endogenous dpp expression zone, dpp was expressed in the mutant cells but down-regulated in adjacent wild-type cells (Fig. 3G-I). Previously it had been shown that Wg and Dpp signaling mutually antagonize each other’s expression, which prevents expression of the two molecules in the same cells (Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffman, 1996; Theisen et al., 1996). Ectopic expression of both wg and dpp in slmb clones in the dorsal-anterior of the leg disc indicates a disruption of this mutual antagonism.

Fig. 3.

slimb induces ectopic expression of wg and dpp in leg imaginal discs. In all panels, third instar leg discs are positioned with anterior to the left and ventral down. slmbe4-1 and slmbP00295 clones are marked by the absence of anti-Myc staining (green). wg-lacZ and dpp-lacZ and hhP30 expression patterns were visualized with anti-β-gal antibody (red). (A) Leg disc bearing slmbP00295 clones in the dorsal region associated with ectopic wg-lacZ expression. (B,C) Close-up images of the clone in A illustrating slmb− clone (lacking green; B) with ectopic wg expression (red; C). (D) slmbe4-1 clones also ectopically express dpp. (E,F) Close-up images of the clone in D illustrating a slmb− clone (lacking green; E) with ectopic dpp expression (red; F). (G) slmbe4-1 clones in the endogenous domain for dpp expression (arrow). (H,I) Close-up images of the clone indicated by an arrow in G, illustrating a slmb− clone (lacking green, H) in which dpp is expressed in the slmb− cells, but is suppressed in nearby wild-type cells (I, arrow). (J) Diagram illustrating wg expression based on 103 analyzed slmb− clones; five subregions are apparent. At the dorsal tip (I) and ventral-anterior (IV) regions, wg is ectopically expressed in all slmb− cells of a clone. Region III, however, which spans the D/V border is unique in that only a fraction of the mutant cells ectopically express wg. No ectopic wg expression has been observed in regions II and V. dpp expression in 98 slmb clones was analyzed and not found to fall into any distinct domains. (K) In contrast to their effects on wg and dpp expression, slmbP00295 clones do not alter hh expression. (L) Close-up images of the clone in K illustrating the anterior slmb− clone (top) does not express hh. (M,N) smoD16, slmbP00295 double mutant mosaic leg discs express no ectopic wg (M) or dpp (N).

Fig. 3.

slimb induces ectopic expression of wg and dpp in leg imaginal discs. In all panels, third instar leg discs are positioned with anterior to the left and ventral down. slmbe4-1 and slmbP00295 clones are marked by the absence of anti-Myc staining (green). wg-lacZ and dpp-lacZ and hhP30 expression patterns were visualized with anti-β-gal antibody (red). (A) Leg disc bearing slmbP00295 clones in the dorsal region associated with ectopic wg-lacZ expression. (B,C) Close-up images of the clone in A illustrating slmb− clone (lacking green; B) with ectopic wg expression (red; C). (D) slmbe4-1 clones also ectopically express dpp. (E,F) Close-up images of the clone in D illustrating a slmb− clone (lacking green; E) with ectopic dpp expression (red; F). (G) slmbe4-1 clones in the endogenous domain for dpp expression (arrow). (H,I) Close-up images of the clone indicated by an arrow in G, illustrating a slmb− clone (lacking green, H) in which dpp is expressed in the slmb− cells, but is suppressed in nearby wild-type cells (I, arrow). (J) Diagram illustrating wg expression based on 103 analyzed slmb− clones; five subregions are apparent. At the dorsal tip (I) and ventral-anterior (IV) regions, wg is ectopically expressed in all slmb− cells of a clone. Region III, however, which spans the D/V border is unique in that only a fraction of the mutant cells ectopically express wg. No ectopic wg expression has been observed in regions II and V. dpp expression in 98 slmb clones was analyzed and not found to fall into any distinct domains. (K) In contrast to their effects on wg and dpp expression, slmbP00295 clones do not alter hh expression. (L) Close-up images of the clone in K illustrating the anterior slmb− clone (top) does not express hh. (M,N) smoD16, slmbP00295 double mutant mosaic leg discs express no ectopic wg (M) or dpp (N).

Although lacZ reporter genes may not always reflect protein expression, these reporter genes have been previously shown to serve as faithful indicators for wg and dpp gene expression in the leg disc (Jiang and Struhl, 1995; Li et al., 1995; Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffman, 1996; Lecuit and Cohen, 1997). To test whether ectopic wg and dpp expression are responsible for the outgrowth phenotype in slmb mosaic animals, we generated flies carrying clones of cells mutant for both slmb and wg, or slmb and dpp. In comparison to slmb mutant clones, double mutant clones do not cause any significant outgrowths (Table 1). Therefore, Wg and Dpp are two primary effector molecules responsible for the induction of outgrowths in slmb mosaic animals. These results are consistent with previous observations that wg and dpp are both required for defining the proximodistal outgrowth center (Diaz-Benjumea et al., 1994; Campbell and Tomlinson, 1995; Lecuit and Cohen, 1997).

Table 1.

Double mutant clone analysis

Double mutant clone analysis
Double mutant clone analysis

slimb coordinates D/V and A/P signals to specify wg and dpp expression patterns

The slmb phenotype differs from those of all previously known genes, in that it is the first gene found to deregulate both wg and dpp expression in the D/V axis. Disrupting components of the Hh signaling pathway deregulates wg and dpp only along the A/P axis. For example, ectopic activation of hh or removal of ptc and pka results in misexpression of dpp and wg in anterior cells that normally do not express these genes. However, wg misexpression is always restricted to the ventral cells, while dpp misexpression is only in dorsal cells (Basler and Struhl, 1994; Jiang and Struhl, 1995; Li et al., 1995; Pan and Rubin, 1995). Thus, the control of wg and dpp expression in the D/V axis is not disrupted. The mechanism restricting wg and dpp in the D/V axis is not known. It is possible that the ability of dorsal cells to express dpp and of ventral cells to express wg is an inherent property of the D/V identity established during embryogenesis. The mutant phenotype of slmb clones in discs provides the first evidence that wg and dpp expression in the D/V axis is actively regulated during imaginal disc development, and is not solely defined during embryonic development. Since the Hh pathway regulates wg and dpp expression in the A/P axis, our results suggest that a pathway different from Hh may operate in imaginal discs to restrict their expression in the D/V axis (Fig. 4). This pathway cannot be either the Wg or Dpp signaling pathway since inactivation of Wg or Dpp signaling affects either dpp or wg expression, but not both (Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffman, 1996; Theisen et al., 1996). The slmb phenotypes described here were not observed in the previous study which used weak slmb alleles and revealed only A/P defects (Jiang and Struhl, 1998). The phenotypic differences probably reflect the fact that we have used a null allele instead of hypermorphic alleles.

Fig. 4.

A model for slmb function. slmb acts upstream of smo in A/P signaling which induces wg and dpp expression, and also participates in an unknown D/V signaling pathway (X) which restricts wg and dpp expression. Inactivation of slmb deregulates wg and dpp expression in both A/P and D/V axes.

Fig. 4.

A model for slmb function. slmb acts upstream of smo in A/P signaling which induces wg and dpp expression, and also participates in an unknown D/V signaling pathway (X) which restricts wg and dpp expression. Inactivation of slmb deregulates wg and dpp expression in both A/P and D/V axes.

In addition to D/V defects, slmb mutant clones also deregulate wg and dpp expression in the A/P axis. slmb is the first identified gene that regulates both wg and dpp expression in the A/P as well as D/V axes. The fact that mutations in slmb affect patterning in both axes suggests that the A/P and D/V signals are coordinated to specify wg and dpp expression patterns, and that slmb plays an essential role in integrating these signals (Fig. 4).

slimb intersects A/P signaling upstream of smo

To further explore how slmb regulation and function correlates with A/P signaling, we carried out double mutant analysis with slmb mutants and with mutants of hh and smo. No reduction of outgrowths was observed in slmb, hh double mutant clones (Table 1). Furthermore, slmb mutant clones have no effect on hh expression (Fig. 3K,L). This indicates that slmb acts downstream or independent of Hh signaling. In contrast, slmb, smo double mutant clones almost completely suppress slmb induced outgrowths (Table 1). Consistent with the adult phenotype, discs carrying slmb, smo clones fail to ectopically express either dpp or wg (Fig. 3M,N). These data suggest that slmb intersects the A/P signal upstream of smo (Fig. 4). The previous study suggested that slmb acts downstream of smo (Jiang and Struhl, 1998). This difference may be explained by the use of different alleles for smo and slmb. Many smo mutations are hypermorphic alleles which produce variable phenotypes (Alcedo et al., 1996; Heuvel and Ingham, 1996). smoD16 used in our analysis is caused by a DNA rearrangement which disrupts the smo transcript and produces the most severe embryonic phenotype (Alcedo et al., 1996; Heuvel and Ingham, 1996). The slmb product contains WD-40 repeats believed to act as a scaffold for the binding of multiple proteins (Neer et al., 1994; Sondek et al., 1996; Feldman et al., 1997; Skowyra et al., 1997). It is possible that this structure may allow for proteins such as Smo and components of a D/V pathway to converge. The Slmb-related protein Cdc4 from Saccharomyces cerevisiae along with Cdc53, and Cdc34 are part of the ubiquitin proteolysis machinery (Yochem and Byers, 1987; Goebl et al., 1988; Bai et al., 1996; Willems et al., 1996). Our data that Slmb acts upstream of Smo, together with its sequence homology with Cdc4, suggests that Slmb could be involved in the regulation of Smo protein degradation.

We thank members of our lab for helpful suggestions and discussion, U. Heberlein, D.J. Pan and the Berkeley Drosophila Genome Center for strains, S. Artavanis-Tsakonas for discussions, and J. Tamkun and A. Cowman for libraries. N. A. T., S. Z. and W. Y. W. were supported by NIH and Yale University predoctoral fellowships. This work was supported by grants from the Lucille P. Markey Charitable Trust and the NIH Cancer Institute to T. X.

Alcedo
,
J.
,
Ayzenzon
,
M.
,
Ohlen
,
T. v.
,
Noll
,
M.
and
Hooper
,
J. E.
(
1996
).
The Drosophila snoothened gene encodes a seven-pass membrane protein, a putative receptor for the Hedgehog signal
.
Cell
86
,
221
232
.
Bai
,
C.
,
Sen
,
P.
,
Hofmann
,
K.
,
Ma
,
L.
,
Goebl
,
M.
,
Harper
,
J. W.
and
Elledge
,
S. J.
(
1996
).
SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box
.
Cell
86
,
263
274
.
Basler
,
K.
and
Struhl
,
G.
(
1994
).
Compartment boundaries and the controlof Drosophila limb pattern by hedgehog protein
.
Nature
368
,
208
214
.
Blackman
,
R.
,
Sanicola
,
M.
,
Raftery
,
L.
,
Gillevet
,
T.
and
Gelbart
,
W.
(
1991
).
An extensive 3′ cis-regulatory region directs the imaginal disk expression of decapentaplegic, a member of the TGF-beta family in Drosophila
.
Development
111
,
657
66
.
Brook
,
W. J.
and
Cohen
,
S. M.
(
1996
).
Antagonistic interactions between wingless and decapentaplegic responsible for dorsal-ventral pattern in the Drosophila leg
.
Science
273
,
1373
1377
.
Campbell
,
G.
and
Tomlinson
,
A.
(
1995
).
Initiation of the proximal axis in insect legs
.
Development
121
,
619
628
.
Capdevila
,
J.
,
Estrada
,
M.
,
Sanchez-Herrero
,
E.
and
Guerrero
,
I.
(
1994
).
The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development
.
EMBO J
.
13
,
71
82
.
Capdevila
,
J.
and
Guerrero
,
I.
(
1994
).
Targeted expression of the signaling molecule decapentaplegic induces pattern duplications and growth alterations in Drosophila wings
.
EMBO J
.
13
,
4459
68
.
Feldman
,
R. M. R.
,
Correll
,
C. C.
,
Kaplan
,
K. B.
and
Deshaies
,
R. J.
(
1997
).
A complex of Cdc4p, Skp1p, and Cdc53p/Cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p
.
Cell
91
,
221
230
.
Felsenfeld
,
A. L.
and
Kennison
,
J. A.
(
1995
).
Positional signaling by hedgehog in Drosophila imaginal disc development
.
Development
121
,
1
10
.
Ferguson
,
E.
and
Anderson
,
K.
(
1992
).
Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo
.
Development
114
,
583
97
.
Goebl
,
M. G.
,
Yochem
,
J.
,
Jentsch
,
S.
,
McGrath
,
J. P.
,
Varshavsky
,
A.
and
Byers
,
B.
(
1988
).
The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme
.
Science
241
,
1331
1335
.
Heuvel
,
M. v. d.
and
Ingham
,
P. W.
(
1996
).
smoothened encodes a receptor-like serpentine protein required for hedgehog signaling
.
Nature
382
,
547
551
.
Ingham
,
P.
,
Taylor
,
A.
and
Nakano
,
Y.
(
1991
).
Role of the Drosophila patched gene in positional signalling
.
Nature
353
,
184
187
.
Jiang
,
J.
and
Struhl
,
G.
(
1995
).
Protein kinase A and Hedgehog signaling in Drosophila limb development
.
Cell
80
,
563
572
.
Jiang
,
J.
and
Struhl
,
G.
(
1996
).
Complementary and mutually exclusive activities of decapentaplegic and wingless organize axial patterning during Drosophila leg development
.
Cell
80
,
563
572
.
Jiang
,
J.
and
Struhl
,
G.
(
1998
).
Regulation of the Hedgehog and Wingless signaling pathways by the F-box/WD40-repeat protein Slimb
.
Nature
391
,
493
496
.
Kassis
,
J.
,
Noll
,
E.
,
VanSickle
,
E.
,
Odenwald
,
W.
and
Perrimon
,
N.
(
1992
).
Altering the insertional specificity of a Drosophila transposable element
.
Proc. Natn. Acad. Sci. USA
89
,
1919
23
.
Lecuit
,
T.
and
Cohen
,
S. M.
(
1997
).
Proximal-distal axis formation in the Drosophila leg
.
Nature
388
,
139
145
.
Lee
,
J. J.
,
Kessler
,
D. P. v.
,
Parks
,
S.
and
Beachy
,
P. A.
(
1992
).
Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog
.
Cell
71
,
33
50
.
Lepage
,
T.
,
Cohen
,
S.
,
Diaz-Benjumea
,
F.
and
Parkhurst
,
S.
(
1995
).
Signal transduction by cAMP-dependent protein kinase A in Drosophila limb patterning
.
Nature
373
,
711
715
.
Li
,
W.
,
Ohlmeyer
,
J. T.
,
Lane
,
M.
and
Kalderon
,
D.
(
1995
).
Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development
.
Cell
80
,
553
562
.
Neer
,
E. A.
,
Schmidt
,
C. J.
,
Nambudripad
,
R.
and
Smith
,
T. F.
(
1994
).
The ancient regulatory-protein family of WD-repeat proteins
.
Nature
371
,
297
300
.
Pan
,
D.
and
Rubin
,
G. M.
(
1995
).
CAMP-dependent protein kinase and hedgeog act antagonistically in regulating decapentaplegic transcription in Drosophila imaginal discs
.
Cell
80
,
543
552
.
Penton
,
A.
and
Hoffman
,
F. M.
(
1996
).
Decapentaplegic restricts the domain of wingless during Drosophila limb patterning
.
Nature
382
,
162
165
.
Phillips
,
R.
,
Roberts
,
I.
,
Ingham
,
P.
and
Whittle
,
J.
(
1990
).
The Drosophila segment polarity gene patched is involved in a position-signalling mechanism in imaginal discs
.
Development
110
,
105
114
.
Skowyra
,
D.
,
Craig
,
K. L.
,
Tyers
,
M.
,
Elledge
,
S. J.
and
Harper
,
J. W.
(
1997
).
F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex
.
Cell
91
,
209
219
.
Sondek
,
J.
,
Bohm
,
A.
,
Lambright
,
D. G.
,
Hamm
,
H. E.
and
Sigler
,
P. B.
(
1996
).
Crystal structure of a Ga protein β-gamma dimer at 2.1 Å resultion
.
Nature
379
,
369
374
.
Struhl
,
G.
and
Basler
,
K.
(
1993
).
Organizing activity of wingless protein in Drosophila
.
Cell
72
,
527
540
.
Tabata
,
T.
and
Kornberg
,
T. B.
(
1994
).
Hedgehog is a signaling protein with a key role in patterning Drosophila imaginal discs
.
Cell
76
,
89
102
.
Theisen
,
H.
,
Haerry
,
T. E.
,
O’Connor
,
M. B.
and
March
,
J. L.
(
1996
).
Developmental territories created by mutual antagonism between Wingless and Decapentaplegic
.
Development
122
,
3939
3948
.
Wilder
,
E. L.
and
Perrimon
,
N.
(
1995
).
Dual functions of wingless in the Drosophila leg imaginal disc
.
Development
121
,
477
488
.
Willems
,
A.
,
Lanke
,
S.
,
Patton
,
E.
,
Craig
,
K.
,
Nason
,
T.
,
Mathias
,
N.
,
Kobayashi
,
R.
,
Wittenberg
,
C.
and
Tyers
,
M.
(
1996
).
Cdc53 targets phosphorylated G1 cyclins for degradation by the ubiquitin proteolytic pathway
.
Cell
86
,
453
463
.
Xu
,
T.
and
Harrison
,
S.
(
1994
).
Mosaic analysis using FLP recombinase
.
Methods Cell Biol
44
,
655
682
.
Xu
,
T.
and
Rubin
,
G. M.
(
1993
).
Analysis of genetic mosaics in developing and adult Drosophila tissues
.
Development
117
,
1223
1237
.
Xu
,
T.
,
Wang
,
W. Y.
,
Stewart
,
R. A.
and
Yu
,
W.
(
1995
).
Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase
.
Development
121
,
1053
1063
.
Yochem
,
J.
and
Byers
,
B.
(
1987
).
Structural comparison of the yeast cell division cycle gene CDC4 and a related pseudogene
.
J. Mol. Biol
.
195
,
233
245
.