An Analysis Using the hobo Genetic System Reveals That Combinatorial Signaling by the Dpp and Wg Pathways Regulates dpp Expression in Leading Edge Cells of the Dorsal Ectoderm in Drosophila melanogaster

Corresponding author: S. J. Newfeld, Arizona State University, Tempe, AZ 85287-1501., newfeld{at}asu.edu (E-mail)

Communicating editor: R. S. HAWLEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our laboratory has contributed to the development of a genetic system based upon the hobo transposable element in Drosophila melanogaster. We recently reported that hobo, like the better-known P element, is capable of local transposition. In that study, we mobilized a hobo enhancer trap vector and generated two unique alleles of decapentaplegic (dpp), a transforming growth factor-ß family member with numerous roles during development. Here we report a detailed study of one of those alleles (dpp^F11). To our knowledge, this is the first application of the hobo genetic system to understanding developmental processes. First, we demonstrate that lacZ expression from the dpp^F11 enhancer trap accurately reflects dpp mRNA accumulation in leading edge cells of the dorsal ectoderm. Then we show that combinatorial signaling by the Wingless (Wg) pathway, the Dpp pathway, and the transcriptional coactivator Nejire (CBP/p300) regulates dpp^F11 expression in these cells. Our analysis of dpp^F11 suggests a model for the integration of Wg and Dpp signals that may be applicable to other developmental systems. Our analysis also illustrates several new features of the hobo genetic system and highlights the value of hobo, as an alternative to P, in addressing developmental questions.

TRANSPOSABLE elements are invaluable tools for genetic analysis in many organisms. Experimental systems have been developed around P and hobo elements in Drosophila melanogaster. Structurally similar, the genetic systems of these elements share many characteristics. For example, both P and hobo systems are capable of efficient germline transformation (BLACKMAN et al. 1987 ), enhancer trapping mutagenesis (SMITH et al. 1993 ), and local transposition (NEWFELD and TAKAESU 1999 ). However, the hobo system is not as well developed as the P system. Here we report new features of the hobo system and describe the first use of this system as an analytical tool to address topical issues in developmental genetics.

Two techniques that we discuss have been reported once previously but not in the context of developmental genetic analyses: plasmid rescue of genomic sequences flanking hobo transgene insertions and the analysis of ß-galactosidase expression from hobo enhancer traps in embryos (SMITH et al. 1993 ). Two related techniques are described for the first time: hobo-specific primers for sequencing flanking genomic DNA and the analysis of ß-galactosidase expression from hobo enhancer traps in imaginal discs. Two larger issues related to the overall versatility of the hobo system are discussed: the stability of hobo transgenes in lab stocks and during crossing schemes and the feasibility of identifying enough suitable laboratory strains to conduct a thorough developmental genetics study.

As a point of departure we employed a unique allele of decapentaplegic (dpp^F11) generated in our local jumping study (NEWFELD and TAKAESU 1999 ). dpp is a well-characterized signaling molecule in the transforming growth factor-ß family (TGF-ß; NEWFELD et al. 1999 ). Dpp plays many roles in Drosophila development, including the specification of dorsal ectoderm during early stages of embryogenesis (RAY et al. 1991 ). The Dpp signal transduction pathway includes two cytoplasmic Smad proteins, Mothers against dpp (Mad) and Medea (Med). In response to a Dpp signal, a multimeric Mad/Med complex enters the nucleus and participates in the transcription of specific genes (WRANA 2000 ).

For some developmental decisions, Dpp signals are sufficient to specify the proper cell fate. However, Dpp alone can be insufficient to specify the appropriate cell type. In these cases, combinatorial signaling by several pathways appears to be required for correct cell fate specification. For example, the Dpp and Wingless (Wg) pathways are required to specify cell fates along the dorsal-ventral axis in the adult abdomen (KOPP et al. 1999 ) and along the proximal-distal axis in the leg (LECUIT and COHEN 1997 ).

wg is a well-characterized Wg/int-1 (Wnt) family member in Drosophila (SHULMAN et al. 1998 ). Wg plays many developmental roles, including the specification of segment polarity during early embryogenesis (BAKER 1987 ). The Wg signal transduction pathway includes a cytoplasmic protein complex made up of several proteins including Armadillo (Arm, homologous to vertebrate ß-catenin; PEIFER and WIESCHAUS 1990 ). In response to a Wg signal, Arm is released from this complex, enters the nucleus, and participates in the transcription of specific genes (POLAKIS 2000 ).

Two studies have examined the mechanism of combinatorial signaling by TGF-ß and Wnt pathways. Both studies focus on Smad proteins and Arm/ß-catenin in Xenopus. In one study, coinjection of Smad2 and ß-catenin activated the transcription of siamois, a common target gene, significantly above the levels of Smad2 or ß-catenin alone (CREASE et al. 1998 ). In the second study, complexes containing Smad4 and ß-catenin synergistically affect the transcription of twin, a Wnt target gene (NISHITA et al. 2000 ), suggesting that Smad4 participates in Wnt signaling. However, the authors are careful to say that no evidence exists for Med (homologous to vertebrate Smad4; WISOTZKEY et al. 1998 ) activity in Wg signaling in Drosophila. How Wg and Dpp signals are integrated in Drosophila is currently unknown.

Here we address this question through a developmental genetic analysis of dpp^F11. We report that lacZ expression from dpp^F11 accurately reflects dpp mRNA expression in leading edge cells of the dorsal ectoderm. Our analysis of dpp^F11 suggests that combinatorial signaling by the Wg and Dpp pathways occurs via transcription factor complexes. Further, this study illustrates the value of the hobo genetic system for analyzing developmental mechanisms.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Molecular biology:
Plasmid rescue of genomic DNA flanking the H[Lw2] transgene in the hobo enhancer trap strain H[Lw2] dpp^F11 Dp (2;2) DTD48 dpp^d-ho/CyO was conducted as follows: 5' flanking DNA was recovered by digestion with BamHI (BamHI cuts at nucleotide 10514 in the dpp sequence; GenBank accession no. U63857) and sequenced with primer pH 5 (5'-AATTGTAGGGTGTGAGTCGAGTG-3'); 3' flanking DNA was recovered with HindIII (HindIII cuts at nucleotide 17688 in GenBank accession no. U63857) and sequenced with primer pH 6 (5'-ATCGGGTGGACGTAGAGTGCGAG-3'). Genomic Southerns to detect endogenous hobo elements were conducted as described (BLACKMAN et al. 1987 ). A list of 78 strains analyzed for the presence of endogenous hobo elements is presented in WALDRIP et al. 2001 .

Fly stocks:
The dpp^F11 hobo enhancer trap strain is as described by NEWFELD and TAKAESU 1999 . Two armadillo (arm) alleles: arm² (arm^XM19, moderate hypomorph) and arm⁴ (arm^YD35, genetic null) are as described by PEIFER and WIESCHAUS 1990 . Two nejire (nej) alleles: nej¹ (strong hypomorph) and nej³ (protein null) are as described by AKIMARU et al. 1997 . The arm² nej³ and arm² FRT 101 strains are as described by WALTZER and BIENZ 1998 . The nej¹ FRT 101 strain is as described by WALTZER and BIENZ 1998 . The Med¹ (genetic null) strain is as described by DAS et al. 1998 . The kayak (kay) allele kay¹ (genetic null) is as described by RIESGO-ESCOVAR and HAFEN 1997 . The blue balancer strains are as described: FM7c P[eve-lacZ] (WALTZER and BIENZ 1998 ), CyO P[wg-lacZ] (KASSIS et al. 1992 ), and TM3 P[Scr-lacZ] (GINDHART et al. 1995 ).

Genetics:
All experimental chromosomes were maintained over blue balancers. In matings with dpp^F11, the arm and nej mutant strains (both genes are on the X chromosome) did not need to be hobo-free since mobilization of the transgene in the germline of experimental embryos was inconsequential. For tests of dpp^F11 expression in arm and nej zygotic mutants, males carrying dpp^F11 were crossed to females heterozygous for an arm allele (arm² or arm⁴) or a nej allele (nej¹ or nej³). For tests of dpp^F11 expression in Med zygotic mutants, a double balanced stock was generated that carries dpp^F11 and Med¹. A hobo-free Med¹ strain was used to construct this stock. No hobo-free Mad strains have been identified to date. For tests of dpp^F11 expression in kay zygotic mutants, a double balanced stock was generated that carries dpp^F11 and kay¹. A hobo-free kay¹ strain was used to construct this stock. For tests of dpp^F11 expression in arm nej zygotic double mutants, males carrying dpp^F11 were crossed to females heterozygous for an arm² nej³ chromosome. For tests of dpp^F11 expression in germline clone (GLC) mutant embryos (embryos lacking maternal and zygotic gene activity), females bearing GLC of arm² or nej¹ were mated to males carrying an X chromosome blue balancer and dpp^F11. The hypomorphic alleles arm² and nej¹ were used to make GLC because the null alleles arm⁴ and nej³ do not come through the germline (PEIFER and WIESCHAUS 1990 ; WALTZER and BIENZ 1998 ). Females bearing GLC were generated using the FLP-DFS system (CHOU and PERRIMON 1992 ). To determine whether Med¹ mutations dominantly enhance the effect of arm and nej mutations on dpp^F11 expression, we generated arm⁴ and nej³ zygotic mutant embryos that were also heterozygous for Med¹. These embryos were derived from crosses between males that carried dpp^F11 and Med¹ and females heterozygous for arm⁴ or nej³.

Gene expression:
Histochemical staining for ß-galactosidase (lacZ) activity in embryos was conducted as described by NEWFELD et al. 1996 . We utilize histochemical staining for the following reasons: (1) The strong catalytic ability of lacZ significantly amplifies weak signals (such as those seen in the germline clone embryos) well above that obtainable with antibodies to lacZ, and (2) histochemical staining is the only method capable of detecting lacZ activity after cuticle deposition during stage 16 (ASHBURNER 1989 ). For consistency, histochemical staining is reported for all embryos. Processing of all embryos shown in the same figure was conducted in parallel to minimize variation between staining reactions. Histochemical staining for ß-galactosidase (lacZ) activity in imaginal discs was conducted as described by MARQUEZ et al. 2001 . RNA in situ hybridization with the dpp cDNA H1 was conducted as described by RAY et al. 1991 .

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

dpp^F11 lacZ expression accurately depicts dpp mRNA expression in leading edge cells of the dorsal ectoderm:
dpp^F11 is a unique haplolethal allele (maintained in stock with a duplication of dpp) that carries a hobo enhancer trap construct inserted in the dpp transcription unit. Restriction fragment length polymorphism data initially suggested that the dpp^F11 transgene is inserted into intron 2 (NEWFELD and TAKAESU 1999 ). Subsequently, plasmid rescue of genomic sequences flanking the dpp^F11 transgene showed a precise insertion between nucleotides 13434 and 13435 of the dpp genomic sequence (GenBank accession no. U63857). This places the insertion 40 nucleotides upstream of the exon 2 splice acceptor and between the codons for leucine 276 and threonine 277 in the dpp open reading frame. Given the proximity of the dpp^F11 enhancer trap insertion to intron 2, we wondered if dpp^F11 lacZ expression was under the influence of an intronic enhancer. Previous studies have shown that there are tissue-specific enhancers and repressors in intron 2 (HUANG et al. 1993 ) as well as numerous conserved sequences of unknown function (NEWFELD et al. 1997 ).

Histochemical examination of embryos revealed that the dpp^F11 transgene expresses lacZ exclusively in leading edge cells of the dorsal ectoderm. lacZ expression begins during germband retraction (stage 12, Fig 1A) and continues strongly during the leading edge cell movements known as dorsal closure (stage 14, Fig 1C). After dorsal closure, leading edge cells from both sides of the embryo form the dorsal midline and dpp^F11 expression is still strong (stage 17, Fig 1E). A side-by-side comparison shows that lacZ expression from the dpp^F11 transgene accurately reflects dpp mRNA expression in leading edge cells (Fig 1B, Fig D, and Fig F). This is true up to the limit of detection for RNA in situ hybridization experiments (stage 16, due to cuticle deposition).

View larger version (84K): In this window In a new window Download PPT slide   Figure 1. Comparison of wild-type dpp mRNA and dppF11 lacZ expression. Staged embryos are shown and arrowheads indicate expression in leading edge cells of the dorsal ectoderm. (A and B) dppF11 lacZ and dpp mRNA are strongly expressed in leading edge cells. (C and D) dppF11 lacZ and dpp mRNA expression in these cells is maintained at high levels through dorsal closure. (E and F) dppF11 lacZ and dpp mRNA expression are visible in cells along the dorsal midline. dppF11 lacZ expression accurately reflects dpp mRNA expression in leading edge cells of the dorsal ectoderm.

We also examined dpp^F11 lacZ expression in wing and leg imaginal discs. We did not detect any expression in leg discs. In wing discs, dpp^F11 expression was visible just anterior to the anterior-posterior compartment boundary (data not shown) in a pattern that accurately reflects dpp expression (BLACKMAN et al. 1991 ).

The correspondence of dpp mRNA expression and lacZ expression from dpp^F11 in leading edge cells suggested that an analysis of dpp^F11 regulation would reveal factors regulating dpp mRNA expression in this tissue. Given dpp's highly dynamic expression pattern, the ability to focus on the regulation of just one aspect of dpp expression using the dpp^F11 enhancer trap simplifies the analysis tremendously. dpp^F11 is the only transgene that mimics just this aspect of dpp expression. The region where dpp^F11 is inserted is refractory to P-element enhancer trap insertion (NEWFELD and TAKAESU 1999 ) and dpp leading edge expression is not recapitulated by any existing reporter gene (FLYBASE 2002 ). Finally, the regulatory sequences that drive dpp mRNA expression in leading edge cells have not yet been identified. Thus, the dpp^F11 hobo transgene insertion appears to provide a unique opportunity to further illuminate mechanisms of dpp regulation in leading edge cells.

dpp^F11 expression is not fully maintained in kay, arm, Med, or nej zygotic mutants:
If studies of dpp^F11 regulation are to provide new insight into the regulation of dpp mRNA expression in leading edge cells, then dpp^F11 must mimic dpp mRNA expression in wild-type and mutant embryos. To test this premise, we analyzed dpp^F11 expression in Jun amino-terminal kinase (JNK), Wg and Dpp signaling pathway mutants with demonstrated effects on dpp mRNA expression. We examined embryos with zygotic mutations in the following genes: kay (dFos), a transcription activator in the JNK pathway; arm, a transcription activator in the Wg pathway; and Med, a transcription activator in the Dpp pathway. dpp mRNA expression in leading edge cells is not maintained in kay mutants (RIESGO-ESCOVAR and HAFEN 1997 ), arm mutants (MCEWEN et al. 2000 ), or Dpp pathway mutant embryos (TORRES-VAZQUEZ et al. 2001 ). If dpp^F11 expression is an accurate reflection of dpp mRNA expression, then dpp^F11 expression should not be maintained in leading edge cells in these mutants.

In embryos younger than stage 15, we observed relatively normal expression of dpp^F11 in each mutant background. This is likely due to the fact that kay, arm, and Med have a maternal component that sustains dpp^F11 expression in these embryos (FLYBASE 2002 ). In late-stage embryos, dpp^F11 expression in leading edge cells was well below wild-type levels in kay¹, arm⁴, and Med¹ null mutant backgrounds (Fig 2A, Fig B, and Fig D). In stage 17 embryos, each mutant's effect on dpp^F11 expression matches the severity of its mutant phenotype. kay¹ and arm⁴ zygotic mutants have "dorsal open" phenotypes with extensive defects in tissues derived from the dorsal ectoderm (PEIFER and WIESCHAUS 1990 ; RIESGO-ESCOVAR and HAFEN 1997 ). Dorsal defects are seen only occasionally in Med¹ zygotic mutants (S. NEWFELD, unpublished observations). The data for dpp^F11 agree with previous studies that showed that the JNK pathway, the Wg pathway, and the Dpp pathway are required to maintain dpp mRNA expression in leading edge cells. This correspondence supports the use of dpp^F11 in further studies of Wg and Dpp pathway regulation of dpp expression in leading edge cells.

View larger version (68K): In this window In a new window Download PPT slide   Figure 2. dppF11 expression is not fully maintained in kay, arm, nej, or Med zygotic mutants. Stage 17 embryos are shown. lacZ expression from dppF11 is shown in kay1 (A), arm4 (B), nej3 (C), and Med1 zygotic mutant embryos (D). dppF11 expression is below wild-type levels (see Fig 1E) in all embryos.

We then examined lacZ expression from dpp^F11 in nej zygotic mutant embryos. dpp mRNA expression in leading edge cells has not been studied in nej mutants. nej is the Drosophila homolog of the mammalian transcription coactivator CBP/p300 (AKIMARU et al. 1997 ). We utilized nej mutants for two reasons. First, two studies have shown that nej can participate in the Dpp signaling pathway. Expression from a Dpp-responsive midgut enhancer is reduced in nej³ zygotic mutant embryos (WALTZER and BIENZ 1999 ) and dorsal-ventral patterning genes requiring maximal levels of Dpp signaling (e.g., hindsight) are not expressed in nej¹ germline clone mutants (ASHE et al. 2000 ). Second, nej was shown to antagonize Wg signaling in the midgut mesoderm (WALTZER and BIENZ 1998 ). If the Dpp pathway and the Wg pathway are both required for dpp^F11 expression in leading edge cells, then we wondered if Nej (to our knowledge, the only gene shown to influence both pathways) was somehow involved.

In nej³ null mutants, we observed relatively normal expression of dpp^F11 in embryos younger than stage 15 because nej also has a maternal component (AKIMARU et al. 1997 ). In stage 17 embryos, dpp^F11 expression in the leading edge was below wild-type levels in nej³ null mutants (Fig 2C). In these embryos, nej's effect on dpp^F11 expression matches the severity of its mutant phenotype. Dorsal ectoderm defects are seen only rarely in nej³ zygotic mutants (M. BIENZ, personal communication). Overall, the zygotic mutant data suggest that the JNK pathway via kay, the Wg pathway via arm, and the Dpp pathway via Med and nej are all required to maintain dpp^F11 expression in leading edge cells.

An arm nej zygotic double mutant shows synergystic effects on dpp^F11 expression:
Interestingly, arm and nej zygotic mutants both reduce the level of dpp^F11 expression. In leading edge cells, nej does not appear to antagonize Wg signaling as it does in the midgut mesoderm (WALTZER and BIENZ 1998 ). A positive role for nej in Wg signaling has not been shown previously in Drosophila. This possibility does have a precedent in vertebrates. In Xenopus, CBP (nej homolog) synergized with ß-catenin (arm homolog) to stimulate the transcription of Wnt target genes (TAKEMARU and MOON 2000 ). Alternatively, the reduction in dpp^F11 expression in nej mutants may be due to nej playing a positive role in Dpp signaling. To date, nej has not been reported to participate in the JNK pathway and we have preliminary data, discussed below, suggesting that JNK regulation of dpp expression in leading edge cells is independent of the Wg and Dpp pathways.

We tested the hypothesis that Nej plays a positive role in the Wg signaling pathway in the regulation of dpp expression in leading edge cells. We examined dpp^F11 expression in arm² nej³ zygotic double-mutant embryos and looked for additive effects. arm² is a moderate hypomorphic allele and arm² zygotic mutant embryos do not have dorsal defects (PEIFER and WIESCHAUS 1990 ). We reasoned that if arm and nej were acting synergistically in the Wg pathway, then the effect of the zygotic double mutant would be more severe than that of either zygotic single mutant alone. Alternatively, if nej were acting in the Dpp pathway, then the effect of the double mutant should be similar to the effect of each single mutant.

dpp^F11 expression is affected much more severely in an arm² nej³ zygotic double mutant than in either single mutant. In the double mutant, dpp^F11 expression is virtually absent in late-stage embryos (Fig 3F) whereas dpp^F11 expression is clearly visible in arm² (Fig 3E) and nej³ (Fig 2C) single mutants. The presence of nej³ clearly enhances (not antagonizes) the effect of arm² on dpp^F11 expression in double-mutant embryos. This synergistic effect, the significant reduction of dpp^F11 expression in arm² nej³ zygotic double mutants, supports the hypothesis that nej acts positively in the Wg pathway to maintain dpp^F11 expression.

View larger version (101K): In this window In a new window Download PPT slide   Figure 3. An arm nej zygotic double mutant shows synergystic effects on dppF11 expression. Staged embryos are shown. lacZ expression from dppF11 is shown in arm2 zygotic mutants (A, C, and E) and arm2 nej3 zygotic double-mutants embryos (B, D, and F). The effect of arm2 nej3 zygotic double mutants on dppF11 initiation and maintenance is much more severe than that seen in arm2 or nej3 (see Fig 2C) zygotic single mutants.

We also noted that dpp^F11 expression does not initiate at wild-type levels in arm² nej³ zygotic double mutants and expression remains below wild-type levels even in mid-stage embryos (Fig 3B and Fig D). In arm² embryos younger than stage 15, we observed relatively normal expression of dpp^F11 (Fig 3A and Fig C). The initiation of dpp^F11 expression may be affected in double-mutant embryos because the female parent is heterozygous for the double-mutant chromosome. Heterozygosity of the female parent for arm² or nej³ single-mutant chromosomes had no effect on dpp^F11 initiation in these mutant embryos. Again, the presence of nej³ enhances (not antagonizes) the effect of arm² on dpp^F11 expression in double-mutant embryos. This second synergistic effect, the inability to fully initiate dpp^F11 expression, suggests that arm and nej as part of the Wg pathway are required for the initiation of dpp expression in leading edge cells.

dpp^F11 expression does not properly initiate in arm or nej GLC mutants:
We tested the hypothesis that arm and nej are required for the initiation of dpp^F11 expression. We examined embryos lacking maternal and zygotic gene function derived from females bearing arm² or nej¹ GLC. The hypomorphic alleles arm² and nej¹ were used to make GLC because the null alleles arm⁴ and nej³ do not come through the germline (PEIFER and WIESCHAUS 1990 ; WALTZER and BIENZ 1998 ).

Weak dpp^F11 expression is seen at stage 12 in arm² GLC embryos (Fig 4A). No lacZ expression is seen at later stages in arm² GLC mutant embryos (Fig 4C and Fig E). dpp^F11 expression does not initiate during stage 12 in nej¹ GLC mutant embryos (Fig 4B). Faint lacZ expression is seen at later stages in nej¹ GLC mutant embryos (Fig 4C and Fig F). dpp^F11 expression in these embryos is likely due to the fact that arm² and nej¹ are not null alleles. In stage 17 embryos, each mutant's effect on dpp^F11 expression matches the severity of its mutant phenotype. nej¹ GLC and arm² GLC mutant embryos have extensive dorsal defects (PEIFER and WIESCHAUS 1990 ; ASHE et al. 2000 ). Taken together, our analyses of three classes of arm and nej mutants (zygotic single, zygotic double, and GLC) suggest that the Wg pathway is required for the initiation and maintenance of dpp expression in leading edge cells.

View larger version (124K): In this window In a new window Download PPT slide   Figure 4. dppF11 expression does not initiate properly in arm or nej GLC mutants. Staged, hemizygous GLC mutant embryos (those without maternal or zygotic gene function) are shown. lacZ expression from dppF11 is shown in arm2 GLC (A, C, and E) and nej1 GLC mutant embryos (B, D, and F). The effect of arm2 and nej1 GLC mutants on dppF11 expression is more severe than that of arm2 nej3 zygotic double-mutant embryos (see Fig 3B, Fig D, and Fig F).

Med¹ is a dominant enhancer of arm⁴ and nej³ effects on dpp^F11 expression:
We formally tested the hypothesis that the Wg pathway and the Dpp pathway act synergistically in the maintenance of dpp expression in leading edge cells. We assayed for dominant interactions between components of these pathways. Specifically, we examined lacZ expression from dpp^F11 in arm⁴ or nej³ zygotic mutant embryos that were also heterozygous for Med¹. We reasoned that if the two pathways were acting independently, then heterozygosity for Med¹ (a recessive null allele) would have no effect on arm⁴ or nej³ regulation of dpp^F11 expression. However, if there were a synergistic interaction between the pathways, then the dosage of Med could influence the affect of arm⁴ or nej³ on the maintenance of dpp^F11 expression.

The initiation of lacZ expression from dpp^F11 in leading edge cells is largely unaffected in all embryos due to maternal contributions from each gene. However, dpp^F11 expression is well below wild-type levels in both Med-enhanced zygotic mutant backgrounds at stage 17 (compare Fig 5A and Fig B, with Fig 1E). Of greater importance, the effect of arm⁴ or nej³ on dpp^F11 expression is more severe in the absence of one functional copy of Med than in their respective zygotic single mutants. To see the effect of heterozygosity for Med¹, compare Fig 5A to Fig 3C for nej³ and compare Fig 5B to Fig 2B for arm⁴. Dominant enhancement of arm⁴ and nej³ zygotic mutant phenotypes by Med¹ strongly suggests that the Dpp pathway synergizes with the Wg pathway to maintain dpp expression in leading edge cells. Further, the data indicate that the transcriptional coactivator Nej, with its positive roles in both Wg signaling (Fig 3) and Dpp signaling (WALTZER and BIENZ 1999 ), may act to bridge the pathways.

View larger version (41K): In this window In a new window Download PPT slide   Figure 5. Dominant enhancement of arm and nej zygotic mutants by Med1. Stage 17 embryos are shown. lacZ expression from dppF11 is shown in nej3 (A) and arm4 zygotic mutant embryos (B) that are also heterozygous for Med1. Heterozygosity for Med1 significantly enhances the effect of nej3 (see Fig 2C) and arm4 (see Fig 2B) zygotic mutants on dppF11 expression.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

During early stages of embryogenesis, wg and dpp are expressed in undifferentiated dorsal ectoderm. wg mRNA expression, in 15 stripes along the entire dorsal-ventral axis of the embryo (including the dorsal ectoderm), begins at stage 8. wg expression persists in this striped pattern through stage 17 (BAKER 1987 ). dpp mRNA is expressed on the dorsal side of the embryo along the entire anterior-posterior axis, beginning at stage 4. dpp mRNA expression persists in a large portion of the dorsal ectoderm through stage 8 and resolves into leading edge cell-specific expression in stage 12 embryos (RAY et al. 1991 ). At this time the embryonic expression pattern of nej has not been reported. However, some information can be inferred from nej mutant phenotypes. nej zygotic mutant embryos show visible defects in the tracheal system at stage 12 (WALTZER and BIENZ 1999 ). The tracheal system is derived from the dorsal ectoderm, suggesting that nej is expressed in this tissue prior to stage 12.

Our analysis of dpp^F11 suggests that dpp expression in leading edge cells is initiated by prior episodes of wg and dpp expression in the undifferentiated dorsal ectoderm. The maintenance of dpp expression in leading edge cells appears to require continuous input from wg and from a dpp feedback loop. The initiation and maintenance of dpp expression in leading edge cells also require continuous nej activity. Overall, our data are consistent with the following combinatorial signaling model (Fig 6): Med (signaling for the Dpp pathway) interacts with Arm (signaling for the Wg pathway) via the transcriptional coactivator Nej. This multimeric complex initiates and, with continuous signaling, maintains dpp expression in leading edge cells.

View larger version (23K): In this window In a new window Download PPT slide   Figure 6. Combinatorial model for the regulation of dpp expression in leading edge cells. In leading edge cells, Dpp signals are carried from the cytoplasm to the nucleus by a complex that includes Med. Wg signals are carried by a complex that includes Arm. In the nucleus, Dpp and Wg signals are integrated via a multimeric transcription factor complex that includes Arm, Med, and the coactivator Nej. This complex initiates dpp expression. With continuous Wg and Dpp signaling, complexes of this type are constantly formed and they act to maintain dpp expression in these cells.

Our data extend previous studies of dpp expression in leading edge cells and Dpp signaling in several ways. MCEWEN et al. 2000 suggest a role for Wg signaling in the regulation of dpp expression in the leading edge. Their data are consistent with ours. We both show that dpp leading edge expression is not maintained in arm² zygotic mutants and does not initiate in arm² germline clones. We extend their study by demonstrating the involvement of nej and Med in the regulation of dpp expression in leading edge cells. WALTZER and BIENZ 1999 report that nej participates in Dpp signaling. Their data are consistent with ours. While they show that nej³ enhances dpp wing phenotypes, we show that Med¹ enhances nej³ embryonic phenotypes. We extend their study by suggesting a role for nej in mediating combinatorial signaling by the Wg and Dpp pathways.

Several questions remain about the combinatorial regulation of dpp expression by Wg, Dpp, and Nej. One question is, how is Nej recruited to bridge the two pathways? Numerous studies have shown that p300/CBP transcriptional coactivation functions are stimulated by its phosphorylation but the site of phosphorylation has never been mapped (GOODMAN and SMOLIK 2000 ). Perhaps Zeste white3 (a serine-threonine kinase in the Wg pathway) or Thickveins (a serine-threonine kinase in the Dpp pathway) are involved in recruiting Nej to participate in combinatorial signaling.

A second question concerns the nature of the enhancer element that directs dpp expression in leading edge cells. Using reporter genes, we have identified a 54-nucleotide candidate enhancer near the dpp^F11 transgene insertion that drives lacZ expression in a subset of leading edge cells (TAKAESU et al. 2002 ). The region contains two sets of conserved, overlapping consensus-binding sites for dTCF (a transcriptional partner for Arm in the Wg pathway) and Mad (a transcriptional partner for Med in the Dpp pathway). No JNK-pathway-binding sites are in the region, suggesting that JNK regulation of dpp expression in leading edge cells is independent of Wg and Dpp signaling.

Interestingly, there is also a consensus Brinker (Brk) binding site in the candidate enhancer (RUSHLOW et al. 2001 ). Brk is a transcriptional repressor of Dpp target genes and one mechanism by which Dpp signaling activates its target genes is to relieve Brk repression (TORRES-VAZQUEZ et al. 2001 ). Our genetic data cannot discriminate between the possibility that combinatorial signaling by the Wg and Dpp pathways regulates dpp expression in leading edge cells by direct activation or by relief of Brk repression. Using this candidate enhancer, we are preparing to conduct biochemical analyses of DNA-protein interactions that will determine if one or both of these mechanisms are involved.

In addition to advancing our understanding of dpp regulation in leading edge cells, our analysis of dpp^F11 further establishes the value of the hobo genetic system as an analytical tool in Drosophila. Our study shows that (with the caveat that suitable strains must first be identified) the hobo system is capable of a wide range of sophisticated genetic techniques first developed for the P-element system. We demonstrate several technical advances for the hobo genetic system that reflect its versatility. This study is the first to utilize plasmid rescue of sequences flanking hobo transgenes and the histochemical analysis of ß-galactosidase expression from hobo enhancer trap vectors in embryos as analytical tools to address developmental questions. In addition, we describe a set of hobo sequencing primers for the analysis of rescued, flanking genomic DNA and the analysis of ß-galactosidase expression from hobo enhancer traps in imaginal discs.

Like many genetic analyses, our study of dpp^F11 was conducted over several years. This allows us to address important issues about the long-term stability of hobo transgenes in permanent laboratory stocks and during complex crossing schemes as well as the practicality of finding suitable strains for the analysis of one's favorite hobo-associated mutant. Regarding the stability of hobo transgenes in stocks and in crosses, we found absolutely no evidence of instability. In our hands, this issue is no more relevant for hobo than it is for P. The dpp^F11 strain has been successfully maintained in stock for nearly a decade side by side with P transgene strains. During this time there were no alterations to the genetic or molecular characteristics of the dpp^F11 strain. For example, the strain always demonstrates haploinsufficiency when recombinant progeny with the hobo insertion but without the dpp duplication are generated and there have never been any alterations in eye color or lacZ expression pattern.

Regarding the practicality of finding suitable strains for the analysis of one's favorite hobo-associated mutant, we admit that this is more tedious than using the P system. The trade-off is that P and hobo elements have distinct insertion preferences. This was shown in a genome-wide survey (SMITH et al. 1993 ) and in an analysis at the dpp locus (NEWFELD and TAKAESU 1999 ). In addition, there is no a priori reason to believe that strains associated with Dpp signaling, such as those used in this study, are more or less prone to possess endogenous hobo elements than those necessary for the analysis of other genes. Thus, it seems likely that suitable strains can be found for just about any study. We are willing to share strains that are widely applicable for hobo genetics, such as those useful for germline transformation, mutagenesis screens, and blue balancers. See WALDRIP et al. 2001 for a complete list of available strains and a discussion of two large collections of developmental mutants compatible with the hobo genetic system.

From a genome-wide perspective, the majority of predicted genes are not yet mutagenized by P-element insertions (SPRADLING et al. 1999 ). Some well-studied genes appear immune to such insertions. For example, no P-element mutations were found in alcohol dehydrogenase in a database search that identified 106 mutant alleles (ASHBURNER et al. 1999 ). Thus, it seems logical to utilize another element with a well-developed genetic system such as hobo to extend the reach of current mutagenesis methods. It seems likely that the hobo enhancer trap collection of SMITH et al. 1993 , which has not been widely exploited for genetic analyses, contains hits in genes not susceptible to P insertion.

In summary, our study suggests that an expanded use of hobo transgenes will facilitate our understanding of the developmental biology of D. melanogaster. Given their membership in large multigene families, our analysis of the combinatorial regulation of dpp^F11 expression in leading edge cells by Dpp and Wg will likely have wide relevance to TGF-ß and Wnt signaling in many species.

	ACKNOWLEDGMENTS

We thank Brian Calvi for hobo sequencing primers. We thank Esther Siegfried and Mike O'Connor for valuable discussions and Ann Bradley for help with fly stocks. Ray Marquez, Will Sewall, Omar Sultani, and Ross Waldrip assisted with lacZ staining and Aaron Johnson provided assistance with image analysis. We thank Mariann Bienz, Beth Noll, and the Bloomington Stock Center for strains. This research was supported by a Basil O'Connor Starter Scholar Research Award from the March of Dimes, a Research Incentive Award from Arizona State University, and a grant from the National Institutes of Health (CA-95875).

Manuscript received November 27, 2001; Accepted for publication March 7, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AKIMARU, H., D. HOU, and S. ISHII, 1997  Drosophila CBP is required for dorsal-dependent twist gene expression. Nat. Genet. 17:211-214[Medline].

ASHBURNER, M., 1989 Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

ASHBURNER, M., S. MISRA, J. ROOTE, S. LEWIS, and R. BLAZEJ et al., 1999  An exploration of the sequence of a 2.9-Mb region of the genome of Drosophila melanogaster: the Adh region. Genetics 153:179-219[Abstract/Free Full Text].

ASHE, H., M. MANNERVIK, and M. LEVINE, 2000  Dpp signaling thresholds in the dorsal ectoderm of the Drosophila embryo. Development 127:3305-3312[Abstract].

BAKER, N., 1987  Molecular cloning of sequences from wingless, a segment polarity gene in Drosophila: the spatial distribution of a transcript in embryos. EMBO J. 6:765-773.

BLACKMAN, R., R. GRIMAILA, M. KOEHLER, and W. M. GELBART, 1987  Mobilization of hobo elements residing within the decapentaplegic gene complex: suggestion of a new hybrid dysgenesis system in Drosophila melanogaster. Cell 49:497-505[Medline].

BLACKMAN, R., M. SANICOLA, L. RAFTERY, T. GILLEVET, and W. M. GELBART, 1991  An extensive 3' cis-regulatory region directs the imaginal disk expression of dpp, a member of the TGF-ß family in Drosophila. Development 111:657-665[Abstract].

CHOU, T. and N. PERRIMON, 1992  Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. Genetics 131:643-653[Abstract].

CREASE, D., S. DYSON, and J. GURDON, 1998  Cooperation between the Activin and Wingless pathways in the spatial control of organizer gene expression. Proc. Natl. Acad. Sci. USA 95:4398-4403[Abstract/Free Full Text].

DAS, P., L. MADUZIA, H. WANG, A. FINELLI, and S. CHO et al., 1998  The Drosophila gene Medea demonstrates the requirement for different classes of Smads in Dpp signaling. Development 125:1519-1528[Abstract].

FLYBASE, 2002 FlyBase: a database of the Drosophila genome (http://flybase.bio.indiana.edu/).

GINDHART, J., A. KING, and T. KAUFMAN, 1995  Characterization of the cis-regulatory region of the Drosophila homeotic gene Sex combs reduced.. Genetics 139:781-795[Abstract].

GOODMAN, R. and S. SMOLIK, 2000  CBP/p300 in cell growth, transformation and development. Genes Dev. 14:1553-1577[Free Full Text].

HUANG, J., D. SCHWYTER, J. SHIROKAWA, and A. COUREY, 1993  The interplay between multiple enhancer and silencer elements defines the pattern of dpp expression. Genes Dev. 7:694-704[Abstract/Free Full Text].

KASSIS, J., E. NOLL, E. VAN SICKLE, W. ODENWALD, and N. PERRIMON, 1992  Altering the insertional specificity of a Drosophila transposable element. Proc. Natl. Acad. Sci. USA 89:1919-1923[Abstract/Free Full Text].

KOPP, A., R. BLACKMAN, and I. DUNCAN, 1999  Wg, Dpp and EGF receptor signaling pathways interact to specify dorsoventral pattern in the adult abdomen of Drosophila. Development 126:3495-3507[Abstract].

LECUIT, T. and S. COHEN, 1997  Proximal-distal axis formation in the Drosophila leg. Nature 388:139-145[Medline].

MARQUEZ, R., M. SINGER, N. TAKAESU, W. WALDRIP, and Y. KRAYTSBERG et al., 2001  Transgenic analysis of the Smad family of TGF-ß signal transducers suggests new roles and new interactions between family members. Genetics 157:1639-1648[Abstract/Free Full Text].

MCEWEN, D., R. COX, and M. PEIFER, 2000  The canonical Wg and JNK cascades collaborate to promote dorsal closure and ventral patterning. Development 127:3607-3617[Abstract].

NEWFELD, S. and N. TAKAESU, 1999  Local transposition of a hobo element within the decapentaplegic locus of Drosophila. Genetics 151:177-187[Abstract/Free Full Text].

NEWFELD, S., E. CHARTOFF, J. GRAFF, D. MELTON, and W. GELBART, 1996  Mothers against dpp encodes a conserved cytoplasmic protein required in DPP/TGF-ß responsive cells. Development 122:2099-2108[Abstract].

NEWFELD, S., R. PADGETT, S. FINDLEY, B. RICHTER, and M. SANICOLA et al., 1997  Molecular evolution at the decapentaplegic locus in Drosophila. Genetics 145:297-309[Abstract].

NEWFELD, S., R. WISOTZKEY, and S. KUMAR, 1999  Molecular evolution of a developmental pathway: phylogenetic analyses of TGF-ß family ligands, receptors and Smad signal transducers. Genetics 152:783-795[Abstract/Free Full Text].

NISHITA, M., M. HASHIMOTO, S. OGATA, M. LAURENT, and N. UENO et al., 2000  Interaction between Wnt and TGF-ß signaling during formation of Spemann's organizer. Nature 403:781-785[Medline].

PEIFER, M. and E. WIESCHAUS, 1990  The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin. Cell 63:1167-1178[Medline].

POLAKIS, P., 2000  Wnt signaling and cancer. Genes Dev. 14:1837-1851[Free Full Text].

RAY, R., K. ARORA, C. NUSSLEIN-VOLHARD, and W. GELBART, 1991  The control of cell fate along the dorsal-ventral axis of the Drosophila embryo. Development 113:35-54[Abstract].

RIESGO-ESCOVAR, J. and E. HAFEN, 1997  Common and distinct roles of dFos and dJun during Drosophila development. Science 278:669-672[Abstract/Free Full Text].

RUSHLOW, C., P. COLOSIMO, M. LIN, M. XU, and N. KIROV, 2001  Transcriptional regulation of the Drosophila gene zen by competing Smad and Brinker inputs. Genes Dev. 15:340-351[Abstract/Free Full Text].

SHULMAN, J., N. PERRIMON, and J. AXELROD, 1998  Frizzled signaling and the developmental control of cell polarity. Trends Genet. 14:452-458[Medline].

SMITH, D., J. WOHLGEMUTH, B. CALVI, I. FRANKLIN, and W. GELBART, 1993  hobo enhancer trapping mutagenesis in Drosophila reveals an insertion specificity different from P elements. Genetics 135:1063-1076[Abstract].

SPRADLING, A., D. STERN, A. BEATON, E. RHEM, and T. LAVERTY et al., 1999  The Berkeley Drosophila genome project gene disruption project: single P-element insertions mutating 25% of vital Drosophila genes. Genetics 153:135-177[Abstract/Free Full Text].

TAKAESU, N., O. SULTANI, A. JOHNSON, and S. NEWFELD, 2002  Combinatorial signaling by an unconventional Wg pathway and the Dpp pathway requires Nejire (CBP/p300) to regulate dpp expression in posterior tracheal branches. Dev. Biol. in press.

TAKEMARU, K. and R. MOON, 2000  The transcriptional coactivator CBP interacts with ß-catenin to activate gene expression. J. Cell Biol. 149:249-254[Abstract/Free Full Text].

TORRES-VAZQUEZ, J., S. PARK, R. WARRIOR, and K. ARORA, 2001  The transcription factor Schnurri plays a dual role in mediating Dpp signaling during embryogenesis. Development 128:1657-1670[Abstract].

WALDRIP, W., N. TAKAESU, and S. NEWFELD, 2001  Identification of blue balancers and mutant collections compatible with hobo element transgenes. Dros. Inf. Serv. 84:169-172.

WALTZER, L. and M. BIENZ, 1998  Drosophila CBP represses the transcription factor TCF to antagonize Wingless signaling. Nature 395:521-525[Medline].

WALTZER, L. and M. BIENZ, 1999  A function of CBP as a transcriptional coactivator during Dpp signaling. EMBO J. 18:1630-1641[Medline].

WISOTZKEY, R., A. MEHRA, D. SUTHERLAND, L. DOBENS, and X. LIU et al., 1998  Medea is a Drosophila Smad4 homolog that is differentially required to potentiate Dpp responses. Development 125:1433-1445[Abstract].

WRANA, J., 2000  Regulation of Smad activity. Cell 100:189-192[Medline].

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