drifter

Promoter Structure

The POU domain of VVL and of other POU domain transcription factors is a DNA binding motif which interacts with promoter sequence elements based on the octamer consensus element ATGCAAAT. An autoregulatory enhancer of vvl, serving to regulate VVL interaction with its own promoter, consists a 514-bp minimal enhancer located 4.5 kb upstream of the vvl transcriptional initiation site. Two response elements, separated from each other by over 200 base pairs, VVLE1 and VVLE2, consist of a divergent TAATGATATGC element and a consensus element respectively. Disruption of VVLE1 with point mutations block VVL protein binding to this element and show no detectable expression in medial glia of the midline. Expression in trachea and oenocyte clusters are normal. Disruption of VVLE2, on the other hand, gives glial expression but no expression in trachea, while oenocyte expression is normal. It is suggested that distinguishable alterations in the accessable surface of the VVL POU domain caused by flexable recognition of the variant elements VVLE1 and VVLE2 may influence interactions between DNA-bound VVL protein and tissue-specific coactivators or coregulators. Alternatively the separable functions could be due to the proximity of each element to distinct tissue-specific cis-regulatory elements (Certel, 1996).

Transcriptional Regulation

vvl regulation must be complex, because it is expressed through the activity of several pathways. vvl is genetically downstream of single minded in midline neural cells, and is a potential target of that basic HLH transcription factor. It is possible, however that vvl would be normally expressed in cells of single minded mutants, had they been viable, but they are not (Billin and Poole, 1995). vvl expression in the wing imaginal disc and trachea is dependent on coordinate activities of signaling molecules such as DPP, WG and HH. In these cases, vvl appears to be downstream of the torpedo EGF receptor signaling (de Celis, 1995). This gene is obviously a good candidate for promoter analysis.

Formation of the trachea occurs by the migration and fusion of clusters of ectodermal cells specified in each side of ten embryonic segments. Morphogenesis of the tracheal tree requires the activity of many genes, among them breathless (btl) and ventral veinless (vvl), whose mutations abolish tracheal cell migration. Activation of the btl receptor by branchless (bnl), its putative ligand, exerts an instructive role in the process of guiding tracheal cell migration. decapentaplegic determines vvl expression along the embryonic dorsoventral axis; expansion of dpp expression results in an increased recruitment of cells to express vvl. These cells are allocated in the expanded tracheal placodes, indicating that expansion of dpp expression causes a concomitant enlargement of the traceal placodes and of vvl expression. vvl is also required for the maintenance of btl expression during tracheal migration (Llimargas, 1997).

The role of Castor in regulating pdm genes raises the possibility that it may regulate expressions of other POU genes. To test this, the expression domains of Cas and Drifter/Ventral veins lacking were examined. Drf expression was examined in cas- embryos. In addition to its established role in midline glia and tracheal development, Drf is also expressed in a subset of NB progeny in both the developing brain and ventral cord. Many Cas-expressing NB sublineages also express Drf. Thus, it appears that Cas does not repress drf expression: to the contrary, a marked reduction in late-lineage Drf expression is observed in cas- embryos, suggesting Cas either directly or indirectly plays a role in activating and/or sustaining drf expression in these sublineages. Ectopically activated Cas has no effect on Drf expression. In the absence of castor function, I-POU expression is lost in a subset of ventral cord cells, but ectopic Cas has no effect on the I-POU wild-type expression pattern. It is not known if Cas is a direct activator of drf and/or I-POU. However, the data indicate that if Cas is playing a direct activator role, it most likely requires co-factors that are not expressed outside of its normal domain (Kambadur, 1998).

Ken & barbie selectively regulates the expression of a subset of Jak/STAT pathway target genes

A limited number of evolutionarily conserved signal transduction pathways are repeatedly reused during development to regulate a wide range of processes. A new negative regulator of JAK/STAT signaling is described and a potential mechanism identified by which the pleiotropy of responses resulting from pathway activation is generated in vivo. As part of a genetic interaction screen, Ken & Barbie (Ken), which is an ortholog of the mammalian proto-oncogene BCL6, has been identified as a negative regulator of the JAK/STAT pathway. Ken genetically interacts with the pathway in vivo and recognizes a DNA consensus sequence overlapping that of STAT92E in vitro. Tissue culture-based assays demonstrate the existence of Ken-sensitive and Ken-insensitive STAT92E binding sites, while ectopically expressed Ken is sufficient to downregulate a subset of JAK/STAT pathway target genes in vivo. Finally, endogenous Ken is shown specifically represses JAK/STAT-dependent expression of ventral veins lacking (vvl) in the posterior spiracles. Ken therefore represents a novel regulator of JAK/STAT signaling whose dynamic spatial and temporal expression is capable of selectively modulating the transcriptional repertoire elicited by activated STAT92E in vivo (Arbouzova, 2006).

Analysis of phenotypes associated with mutations in Drosophila JAK/STAT pathway components have identified a wide variety of requirements for the pathway during embryonic development and in adults. What is less clear is how the repeated stimulation of a single pathway is able to generate this pleiotropy of developmental functions. In order to identify modulators of JAK/STAT signaling that may be involved in this process, a genetic screen was undertaken for modifiers of the dominant phenotype caused by the ectopic expression of the pathway ligand Unpaired (Upd) in the developing eye imaginal disc. Such misexpression by GMR-updΔ3′ results in overgrowth of the adult eye, a phenotype sensitive to the strength of pathway signaling activity. With this assay, one genomic region, defined by Df(2R)Chi^g320, was found to enhance the GMR-updΔ3′-induced eye overgrowth phenotype. Of the genes deleted by Df(2R)Chi^g320, only mutations in ken showed consistent and reproducible enhancement of the phenotype. In addition, other dominant phenotypes induced by transgene expression from the GMR promoter are not modulated by ken mutations, indicating that Ken is unlikely to interact with the misexpression construct used (Arbouzova, 2006).

The enhancement of the GMR-updΔ3′ phenotype after removal of one copy of ken implies that Ken normally functions antagonistically to JAK/STAT signaling. Therefore phenotypes associated with mutations in other pathway components were tested to establish the reliability of this initial observation. Consistent with this, genetic interaction assays between ken mutations and the hypomorphic loss-of-function allele stat92E^HJ show a reduction in the frequency of wing vein defects normally associated with this stat92E allele. Moreover, the degree of suppression is consistent with the strength of ken alleles tested. Similarly, the frequency of “strong” posterior spiracle phenotypes caused by the dome³⁶⁷ allele of the pathway receptor is also reduced when crossed to ken alleles or the Df(2R)Chi^g320 deficiency, with a concomitant increase in “weak” phenotypes (Arbouzova, 2006).

Thus, multiple independent ken alleles all modify diverse phenotypes caused by both gain- and loss-of-function mutations in multiple JAK/STAT pathway components. Each of these components acts at different levels of the signaling cascade and show interactions indicating that Ken consistently acts as an antagonist of the pathway (Arbouzova, 2006).

The ken locus contains three exons encoding a 601 aa protein. Ken possesses an N-terminal BTB/POZ domain between aa 17 and 131 and three C-terminal C2H2 zinc finger motifs from aa 502 to 590. Strikingly, a number of Zn finger-containing proteins that also contain BTB/POZ domains have also been shown to function as transcriptional repressors—often via the recruitment of corepressors such as SMRT, mSIN3A, N-CoR, and HDAC-1 (Arbouzova, 2006).

Searches for proteins similar to Ken identified homologs in Drosophila pseudoobscura and the mosquito Anopheles gambiae. In vertebrates, human B-Cell Lymphoma 6 (BCL6) was the closest full-length homolog. Drosophila Ken and human BCL6 share the same domain structure and show 20.3% overall identity. Proteins listed as potential vertebrate homologs of Ken in Flybase are more distantly related (Arbouzova, 2006).

Expression of ken was also examined during development, where it is detected in a dynamic pattern from newly laid eggs, throughout embryogenesis, and in imaginal discs. As such, endogenous Ken is present in all tissues and stages in which genetic interactions were observed (Arbouzova, 2006).

Given the presence of potentially DNA binding Zn finger domains and the nuclear localization of GFPKen, the DNA binding properties of Ken was determined by using an in vitro selection technique termed SELEX (systematic evolution of ligands by exponential enrichment). With a GST-tagged Ken Zn finger domain and a randomized oligonucleotide library, ten successive rounds of selection were undertaken. Sequencing of the resulting oligonucleotide pool and alignment of 43 independent clones showed that all recovered plasmids were unique and each contained one, or occasionally two, copies of the motif GNGAAAK (K = G/T) (Arbouzova, 2006).

To confirm the SELEX results, GFPKen was expressed in tissue culture cells and these were used for electromobility shift assays (EMSA). A radioactively labeled probe containing the wild-type (wt) consensus binding site GAGAAAG gives a specific band, which can be supershifted by an anti-GFP antibody and therefore represents a GFPKen/DNA complex. In order to identify positions essential for binding, a competition assay was used in which unlabeled oligonucleotides containing single substitutions in each position from 1 to 7 were added to binding reactions. 10-fold excess of unlabeled wild-type consensus oligonucleotide greatly diminished the intensity of the GFPKen band, while 50- and 100-fold excess totally blocked the original signal. By contrast, competition with unlabeled m3 oligonucleotides containing a G to A substitution at position 3 failed to significantly reduce the intensity of the band even at 100-fold excess. With this approach, the positions 1 and 7 are found dispensable for DNA binding, whereas the central GAAA core is absolutely required. Similar results were obtained with the converse experiment with labeled mutant probes, although in this case the wt probe produces a stronger signal than the m1 and m7 mutant oligonucleotides. Taken together, these experiments not only define the core sequence for Ken binding, but also demonstrate the specificity of Ken as a site-specific DNA binding molecule. Interestingly, the core consensus bound by Ken is very similar to that identified for human BCL6, with the Zn fingers of the latter binding to a DNA sequence containing a core GAAAG motif (Arbouzova, 2006).

One initial observation made is that the core GAAA essential for Ken binding overlaps the sequence recognized by STAT92E. Consistent with this overlap, a 100-fold excess of unlabeled oligonucleotide containing the STAT92E consensus is sufficient to fully compete for Ken in EMSA assays. Given this finding, it is hypothesized that the negative regulation of JAK/STAT signaling by Ken observed in genetic interaction assays may occur via a mechanism of competitive DNA binding site occupation. Due to the incomplete overlap between the STAT92E and Ken core sequences, this hypothesis also implies the existence of STAT92E DNA binding sites to which both STAT92E and Ken could bind (STAT⁺/Ken⁺) as well as sites with which Ken cannot associate (STAT⁺/Ken⁻) (Arbouzova, 2006).

To test this hypothesis, a cell culture-based assay was set up by using a luciferase-expressing reporter containing four STAT92E binding sites originally identified in the promoter of the Draf locus. In addition to this STAT⁺/Ken⁺ wild-type reporter, STAT⁺/Ken⁻ and STAT⁻/Ken⁻ variants identical but for the binding sequences were generated. When transfected into the hemocyte-like Kc₁₆₇ Drosophila cell line, both STAT⁺/Ken⁺ and STAT⁺/Ken⁻ reporters showed strong stimulation upon coexpression with the pathway ligand Upd, an assay previously shown to require an intact JAK/STAT cascade. When cotransfected with KenGFP, the activity of the STAT⁺/Ken⁺ reporter was reduced, an effect reproduced in three independent experiments with both KenGFP and Ken. While the reduction in reporter activity for the STAT⁺/Ken⁺ assay shown is statistically significant, the STAT⁺/Ken⁻ reporter was unaffected by the coexpression of Ken. Reporters containing binding sites mutated to prevent binding of both STAT92E and Ken (STAT⁻/Ken⁻) showed no activation after pathway stimulation and did not respond to Ken (Arbouzova, 2006).

These results indicate that Ken functions as a transcriptional repressor in this cell-culture system and shows that this effect is specific to the DNA sequence determined by SELEX and EMSA. This result is also consistent with a recent whole-genome RNAi-based screen, which used a reporter containing STAT⁺/Ken⁺ binding sites and includes Ken among the list of JAK/STAT regulators identified. In addition, recent reports have also demonstrated BCL6 binding to STAT6 sites in vitro and have shown that BCL6 can act as a repressor of STAT6-dependent target gene expression in cell culture. Although this repression is mediated by the binding to corepressors to the BTB/POZ domain of BCL6, no link between BCL6 and STAT activity has been demonstrated in vivo (Arbouzova, 2006).

Finally, it should also be noted that both the STAT⁺/Ken⁺ and STAT⁺/Ken⁻ reporters contain additional GAAA sequences that are not part of the characterized STAT92E binding sequences. However, despite the presence of these potential Ken binding sites within 15 bp of the STAT92E site, Ken expression did not affect the STAT⁺/Ken⁻ reporter, suggesting that Ken may require STAT92E to influence gene expression. Although no direct association between Ken and STAT92E has been demonstrated, this possibility cannot be excluded, and further analysis remains to be undertaken (Arbouzova, 2006).

Having established that Ken functions at the level of DNA binding in cell culture, it was asked whether Ken also acts as a transcriptional repressor of JAK/STAT pathway target genes in vivo. For this, the effect of ectopically expressed Ken on the expression of putative JAK/STAT pathway target genes was examined and, given the high levels of maternally loaded STAT92E present at blastoderm stage, focus was placed on targets expressed later in embryogenesis. These include the hindgut-specific expression of vvl, the expression of trachealess (trh) and knirps (kni) in the tracheal placodes, and the dynamic expression of socs36E throughout the embryo (Arbouzova, 2006).

First, the effect of Ken was addressed on trh, whose expression precedes the formation of the tracheal pits in the embryonic segments T2 to A8. Levels of trh are greatly reduced in embryos uniformly misexpressing Ken driven by the daughterless-GAL4 (da-GAL4) line. Many tracheal placodes express little or no trh, and tracheal pits fail to form even in the presence of residual trh. Similar effects are seen in upd^OS1A mutant embryos lacking all pathway activity. Likewise, downregulation of Kni expression is also observed in embryos misexpressing ken. These results show that both endogenous trh and kni are downregulated by ectopically expressed Ken (Arbouzova, 2006).

Whether Ken can modulate the expression of socs36E, a Drosophila homolog of mouse SOCS-5, was tested. socs36E expression closely mirrors that of upd, showing JAK/STAT pathway-dependent upregulation in segmentally repeated stripes, tracheal pits, and the hindgut. By contrast to trh and kni, ectopically expressed Ken does not affect any aspect of socs36E transcription. However, controls expressing a dominant-negative form of the pathway receptor DomeΔCyt, using the same Gal4 driver line, show a strong downregulation of socs36E, an effect reproduced by the complete removal of all JAK/STAT pathway activity by the upd^OS1A allele. Taken together, these results illustrate that ectopic expression of Ken during Drosophila development is sufficient to downregulate the expression of only a subset of putative JAK/STAT pathway target genes (Arbouzova, 2006).

As part of this analysis, modulation of vvl by Ken was tested. In wild-type embryos, vvl is expressed in the developing trachea and lateral ectoderm (in a JAK/STAT-independent manner) and in the hindgut of stage 12–14 embryos, where it requires JAK/STAT signaling. In upd^OS1A mutants, no vvl expression in the hindgut can be detected, indicating that this locus is a target of pathway activation. When Ken is uniformly misexpressed throughout the embryo, vvl expression is no longer detectable in the hindgut. Thus vvl, like trh and kni, can be a target of Ken-mediated repression (Arbouzova, 2006).

Having established that ectopic Ken is sufficient to downregulate vvl in the hindgut, whether endogenous Ken performs a similar role was determined. One overlap between ken expression and regions known to require JAK/STAT signaling are the developing posterior spiracles, structures in which both the pathway ligand upd and ken are simultaneously expressed. However, vvl is never detected in the posterior spiracle primordia in wild-type embryos, despite JAK/STAT pathway activity induced by upd expression in these tissues. Intriguingly, in a heteroallelic combination of the strongest ken^k11035 allele and Df(2R)Chi^g320, vvl transcript was detected not only in its normal expression domain within the hindgut but also in the posterior spiracles. This ectopic expression is initially detected from late stage 13 and rapidly strengthens during stage 14–15. When ken^k11035/Df(2R)Chi^g320 embryos simultaneously mutant for the amorphic upd^OS1A allele were analyzed, upregulation of vvl in the presumptive posterior spiracles was never observed at the stage by which ectopic vvl expression was first detected in the ken mutant embryos. At later stages, JAK/STAT pathway activity is required for posterior spiracle morphogenesis, posterior spiracles do not form, and upregulated vvl is not present (Arbouzova, 2006).

These results demonstrate that Ken is not only sufficient to downregulate the JAK/STAT pathway-dependent expression of vvl in the hindgut, but its endogenous expression is also necessary for vvl repression in the posterior spiracles. In ken mutants, ectopic vvl expression in the posterior spiracles results from a derepression of endogenous STAT92E activity (Arbouzova, 2006).

The overlap between the consensus sequences bound by STAT92E and Ken, together with the analysis of reporters containing STAT⁺/Ken⁺ and STAT⁺/Ken⁻ binding sites, indicate that Ken is likely to selectively regulate only a subset of JAK/STAT target genes. In this model, some target genes are regulated by binding sites compatible with both STAT92E and Ken, while others contain sequences to which only STAT92E can associate. While the DNA binding site is critical in cell-culture systems, similar proof is more difficult to establish in vivo. In particular, only a limited number of JAK/STAT pathway target genes have been rigorously demonstrated to require STAT92E binding in vivo (Arbouzova, 2006).

Although studied in some detail, the regulatory domains controlling vvl expression in the developing hindgut have not been identified. Therefore, although these results predict that such a domain would contain STAT⁺/Ken⁺ binding sequences, further analysis is required to confirm this hypothesis. By contrast, the regulatory domain of socs36E required to drive gene expression in the blastoderm, tracheal pits, and hindgut comprises a 350 bp region containing three STAT⁺/Ken⁺ and two STAT⁺/Ken⁻ binding sites. Although not conclusive, the presence of STAT92E-exclusive sites in this region may explain the inability of Ken to downregulate socs36E in vivo (Arbouzova, 2006).

The findings also draw a parallel between Drosophila Ken and BCL6. The data presented demonstrate that both proteins show similar abilities to bind DNA and to mediate transcriptional repression with some evidence also linking BCL6 to JAK/STAT signaling as described here. Taken together, these similarities suggest that Ken and BCL6 represent functional orthologs of one another. Given this evolutionary conservation, it is tempting to speculate that the selective regulation of JAK/STAT pathway target genes is also conserved and may represent a general mechanism by which the pathway is modulated to elicit diverse developmental roles in vivo. Although many STAT targets undoubtedly remain to be identified, it will be intriguing to see which may also be coregulated by Ken/BCL6-dependent mechanisms (Arbouzova, 2006).

Targets of Activity

vvl binds to upstream sequences of dopa decarboxylase, which catalyzes the last step in the synthesis of serotonin and dopamine (Johnson, 1990 and Billin, 1991).

Both breathless and drifter are expressed in all tracheal cells and are essential for directed cell migrations. Ubiquitously expressed Breathless protein under control of a heterologous heat-shock promoter is able to rescue the severely disrupted tracheal phenotype associated with drifter loss-of-function mutations. In the absence of Drifter function, breathless expression is initiated normally but transcript levels fall drastically to undetectable levels as tracheal differentiation proceeds. In addition, breathless regulatory DNA contains seven high affinity Drifter binding sites similar to previously identified Drifter recognition elements. These results suggest that the Drifter protein, which maintains its own expression through a tracheal-specific autoregulatory enhancer, is not necessary for initiation of breathless expression but functions as a direct transcriptional regulator necessary for maintenance of breathless transcripts at high levels during tracheal cell migration. This example of a mechanism for maintenance of a committed cell fate offers a model for understanding how essential gene activities can be maintained throughout organogenesis (Anderson, 1996).

vvl is independently required for the specific expression in the tracheal cells of thick veins (tkv) and rhomboid (rho), two genes whose mutations disrupt only particular branches of the tracheal system. Expression in the tracheal cells of an activated form of tkv, the Decapentaplegic receptor, induces shifts in the migration of these cells, asserting the role of the dpp pathway in establishing the branching pattern of the tracheal tree. In addition, by ubiquitous expression of the btl and tkv genes in vvl mutants it is shown that both genes contribute to vvl function. These results indicate that through activation of its target genes, vvl makes the tracheal cells competent to further signaling and suggest that the btl transduction pathway could collaborate with other transduction pathways also regulated by vvl to specify the tracheal branching pattern (Llimargas, 1997).

The Drosophila tracheal system is a network of epithelial tubes that arises from the tracheal placodes, lateral clusters of ectodermal cells in ten embryonic segments. The cells of each cluster invaginate and subsequent formation of the tracheal tree occurs by cell migration and fusion of tracheal branches, without cell division. The combined action of the Decapentaplegic (Dpp), Epidermal growth factor (EGF) and breathless/ branchless pathways are thought to be responsible for the pattern of tracheal branches. It is asked how these transduction pathways regulate cell migration and the consequences on cell behaviour of the Dpp and EGF pathways is examined. rhomboid (rho) mutant embryos display defects not only in tracheal cell migration but also in tracheal cell invagination unveiling a new role for EGF signaling in the formation of the tracheal system. These results indicate that the transduction pathways that control tracheal cell migration are active in different steps of tracheal formation, beginning at invagination (Llimargas, 1999).

Defects in tracheal migration are associated with defects in invagination in rho and vvl mutant embryos, but not in bnl and btl mutant embryos. This is consistent with the observation that EGF-dependent activation of MAP kinase (ERK) in the tracheal placode precedes ERK activation by the Bnl/Btl pathway. Thus the tracheal phenotype of mutations in the EGF pathway, which has been shown to result from impaired activity of the pathway in the trachea, is likely to originate before the onset of migration. It has been proposed that the EGF pathway might be required for tracheal cells in specific branches to follow the leading cell. Tracheal migration defects of rho mutant embryos are due, at least in part, to the failure of some tracheal cells to invaginate (Llimargas, 1999).

These observations also illustrate the role of vvl in tracheal formation. Since btl expression is normally initiated in vvl mutants, early but not sustained activity of the Btl pathway could cause the tracheal phenotype in vvl mutant embryos. Since vvl is also required for the tracheal expression of tkv and rho, failure to activate the Dpp and EGF pathways could also be the source of the cell shape phenotypes in vvl mutant embryos. This latter possibility is substantiated by the observation that vvl and rho mutant embryos show abnormalities in tracheal invagination that are not present in btl mutant embryos. Finally, the tkv;rho double mutant tracheal phenotype is very similar to the vvl phenotype (Llimargas, 1999).

The Drosophila Vestigial protein has been shown to play an essential role in the regulation of cell proliferation and differentiation within the developing wing imaginal disc. Cell-specific expression of vg is controlled by two separate transcriptional enhancers. The boundary enhancer controls expression in cells near the dorsoventral (DV) boundary and is regulated by the Notch signal transduction pathway, while the quadrant enhancer responds to the Decapentaplegic and Wingless morphogen gradients emanating from cells near the anteroposterior (AP) and DV boundaries, respectively. MAD-dependent activation of the vestigial quadrant enhancer results in broad expression throughout the wing pouch but is excluded from cells near the DV boundary. This has previously been thought to be due to direct repression by a signal from the DV boundary; however, this exclusion of quadrant enhancer-dependent expression from the DV boundary has been shown to be due to the absence of an additional essential activator in those cells. The Drosophila POU domain transcriptional regulator, Drifter, is expressed in all cells within the wing pouch expressing a vgQ-lacZ transgene and is also excluded from the DV boundary. Viable drifter hypomorphic mutations cause defects in cell proliferation and wing vein patterning correlated with decreased quadrant enhancer-dependent expression. Drifter misexpression at the DV boundary using the GAL4/UAS system causes ectopic outgrowths at the distal wing tip due to induction of aberrant Vestigial expression, while a dominant-negative Drifter isoform represses expression of vgQ-lacZ and causes severe notching of the adult wing. In addition, an essential evolutionarily conserved sequence element bound by the Drifter protein with high affinity has been identified and it has been located adjacent to the MAD binding site within the quadrant enhancer. These results demonstrate that Drifter functions along with MAD as a direct activator of Vestigial expression in the wing pouch (Certel, 2000).

Localized activation of the Ras/Raf pathway by epidermal growth factor receptor (Egfr) signalling specifies the formation of veins in the Drosophila wing. However, little is known about how the Egfr signal regulates transcriptional responses during the vein/intervein cell fate decision. Evidence is provided that Egfr signaling induces expression of vein-specific genes by inhibiting the Capicua (Cic) HMG-box repressor, a known regulator of embryonic body patterning. Lack of Cic function causes ectopic expression of Egfr targets such as argos, ventral veinless and decapentaplegic and leads to formation of extra vein tissue. In vein cells, Egfr signaling downregulates Cic protein levels in the nucleus and relieves repression of vein-specific genes, whereas intervein cells maintain high levels of Cic throughout larval and pupal development, repressing the expression of vein-specific genes and allowing intervein differentiation. However, regulation of some Egfr targets such as rhomboid appears not to be under direct control of Cic, suggesting that Egfr signaling branches out in the nucleus and controls different targets via distinct mediator factors. These results support the idea that localized inactivation of transcriptional repressors such as Cic is a rather general mechanism for regulation of target gene expression by the Ras/Raf pathway (Roch, 2002).

There are two key aspects of Cic function as a developmental regulator: its ability to repress specific target genes in defined territories, and its inhibition by the Ras/Raf pathway to allow expression of those targets in complementary positions. In the blastoderm embryo, Cic is required for development of trunk body regions and represses genes mediating differentiation of terminal structures. Torso RTK activation at each pole of the embryo alleviates Cic-dependent repression and initiates the terminal gene expression program. A similar model is proposed for cic function during specification of vein versus intervein fate in the wing. Loss of cic function in the wing causes formation of ectopic vein tissue, implying that Cic mediates intervein specification by restricting vein formation to appropriate regions. In intervein territories, Cic behaves as a repressor of vein-specific genes such as argos and vvl but does not seem to affect directly the expression of blistered, which is required for the specification of intervein fates. Finally, Egfr signaling leads to downregulation of Cic protein levels in vein nuclei, thus relieving Cic-mediated repression and promoting vein development (Roch, 2002).

Vbl regulates decapentaplegic expression during Drosophila wing veins pupal development

Sotillos, S. and de Celis, J. F. (2006). Regulation of decapentaplegic expression during Drosophila wing veins pupal development. Mech. Dev. 123(3): 241-51. 16423512

The differentiation of veins in the Drosophila wing relies on localised expression of decapentaplegic in pro-vein territories during pupal development. The expression of dpp in the pupal veins requires the integrity of the shortvein region (shv), localised 5' to the coding region. It is likely that this DNA integrates positive and negative regulatory signals directing dpp transcription during pupal development. A minimal 0.9 kb fragment has been identified giving localised expression in the vein L5 and a 0.5 kb fragment giving expression in all longitudinal veins. Using a combination of in vivo expression of reporter genes regulated by shv sequences, in vitro binding assays, and sequence comparisons between the shv region of different Drosophila species, binding sites were found for the vein-specific transciption factors Araucan, Knirps and Ventral veinless, as well as binding sites for the Dpp pathway effectors Mad and Med. It is concluded that conserved vein-specific enhancers regulated by transcription factors expressed in individual veins collaborate with general vein and intervein regulators to establish and maintain the expression of dpp confined to the veins during pupal development (Sotillos, 2006).

The expression of dpp in the wing disc is restricted to a narrow stripe of anterior cells localised at the anterior/posterior compartment boundary. This expression is regulated by sequences localised 3′ to the dpp coding region, and the function of the gene at this stage is required for the growth and patterning of the wing. The expression of dpp is still detected at the A/P boundary during the 8 h of pupal development. Later, at 14 h APF novel domains of dpp expression appear corresponding to the developing wing veins. The function of dpp during pupal development requires the integrity of the shv region, which is localised 5′ to the dpp coding region. There are two different transcripts expressed during pupal development, transcripts dpp-RA and dpp-RC, whose promoters (P5 and P3, respectively) are separated by approximately 20 kb of DNA. This DNA includes the first exon of transcript dpp-RC and corresponds to the place where all dpp^s alleles map. Because the strength of dpp^s alleles correlates with their distance to the P3 promoter, it is likely that dpp function in pupal development is mediated mainly by transcript dpp-RC. This suggests that dpp^s mutations affect regulatory sequences necessary to drive dpp expression in presumptive vein territories during pupal development. This possibility was confirmed by analysing the expression of a 8.5 kb construct containing most of the shv region fused to the reporter gene lacZ (shv^8.5–lacZ). The expression of βGal in shv^8.5–lacZ is detected exclusively in the pupal veins, indicating that this region includes all dpp wing veins regulatory regions (Sotillos, 2006).

Several constructs were made using different sub-fragments from the original 8.5 kb dpp^s DNA to identify with more precision the sequences that regulate dpp expression during pupal development. These fragments were cloned in front of a dicistronic lacZ–Gal4 reporter gene and the activity of these constructs was analysed by looking at the expression of βGal in pupal wings from transgenic flies. In addition, to amplify the signal of the dicistronic lacZ–Gal4 gene, the expression was monitored of a reporter gene regulated by UAS sequences. This expression should reveal all places where the Gal4 protein is present. Several regulatory regions were detected that control dpp expression in the veins during pupal development. One regulatory sequence is localised in a 1.1 kb fragment localised 6.5 kb from P3, and drives high levels of expression in most pupal veins and low levels of expression in some interveins. Additional regulatory sequences that efficiently drive expression in most veins are localised in an adjacent 0.5 kb fragment, and further vein-specific regulatory sequences for L5 are localised in the 0.9 kb SalI/KpnI fragment (Sotillos, 2006).

The expression of dpp during embryogenesis is highly dynamic and several independent regulatory regions controlling embryonic dpp expression have already been identified. The shv constructs included in the 8.5 kb EcoRI fragment drive reporter expression during embryonic development from stage 12/13 mainly in three regions of the mesoderm: the oesophagus, gastric caeca and midgut. Regulatory regions controlling dpp expression in the oesophagus appear to be duplicated, because they are localised in the 2.7 kb EcoRI/SalI fragment and also in the 3 kb KpnI/KpnI fragment. Similarly, regions controlling dpp expression in the gastric caeca are also present in the two adjacent fragments 0.9 kb SalI/KpnI and 3 kb KpnI/KpnI. The regions driving reporter expression in the gut are localised in the 3′ end of the shv region (Sotillos, 2006).

To better understand the regulation of dpp expression during vein development, the interactions were analyzed between a 2.5 kb region including wing veins pupal enhancers and several proteins involved in the regulatory network controlling the formation of veins. For this purpose, the 2.5 kb region was subdivided into overlapping fragments of 250-300 bp used as probes to detect the binding of different transcription factors by Electrophoretic Mobility-Shift Assays (EMSAs). Both prepattern specific genes that control vein development, such as Ventral veinless (Vvl) and the Araucan protein (Ara), and transcription factors belonging to the Dpp pathway (Mad and Medea) were analyzed (Sotillos, 2006).

The POU-Homeodomain protein Vvl is expressed in all vein territories throughout pupal development and is required for vein differentiation. Vvl was able to bind all the probes included in the 2.5 kb SalI/SacII region. The Vvl binding was competed by cold DNA as well as by specific oligonucleotides previously described to compete Vvl binding to the vestigial quadrant enhancer. To further characterise the binding of Vvl to the shv enhancer, focus was placed on the 0.5 kb SacII fragment, which drives expression in all longitudinal veins. This fragment was subdivided into two overlapping probes (S9 and S10) and both of them bind Vvl specifically. These bindings were competed using oligonucleotides covering these probes. Vvl-binding regions were found in S9 and in S10. Interestingly, these sequences contain previously identified consensus sequences for Vvl binding. These data suggest that Vvl participates in vein formation during pupal development through the regulation of dpp expression via the shv enhancers (Sotillos, 2006).

The pattern of four longitudinal veins is very similar in all Drosophilids despite the differences in wing size and pigmentation that exist between species. This conservation suggests that the mechanisms underlying vein pattern formation are conserved. The availability of the genomic sequence for different Drosophila species allows a direct comparison between their dpp^s regions. Two Drosophila species from the melanogaster group (D. melanogaster and D. ananassae), one Drosophila from the obscura group (D. pseudoobscura) and D. virilis from the virilis group were compared. It is expected that sequence similarity in non-coding regions corresponds to functional regulatory DNA. In the 2.5 kb region analysed several clusters of sequence conservation were found including most of the binding sites identified by in vitro analysis. Thus, there are four highly conserved regions corresponding to the S1, S4-5, S7-8 and S9-S10 probes containing conserved binding sequences for Vvl, Mad, Med and Ara. This conservation reinforces the importance of these regions to regulate the expression of dpp in the pupal veins. In the case of Vvl specific DNA binding to all probes was shown. However, the putative Vvl binding sequences localised in the conserved regions are only in S1, S3, S7, S8 and S10. In the case of the Dpp pathway transcription factors Mad and Med, putative binding sites are present throughout the enhancer, and accordingly binding of them to all probes was shown. However, only the S5 and S10 probes contain putative binding sites in regions of high sequence conservation. Interestingly, these conserved Mad/Med binding regions contain overlapping binding consensus for the Brinker repressor. This suggests a competition mechanism between Mad/Med and Brinker for binding to the shv enhancer. Competition mechanisms between activator and repressor also occur in several Dpp-downstream genes such as zen and omb. Four consensus binding sequences were found for the transcription factors of the Knirps-complex. The kni genes are expressed in the L2 vein, where they are required for its formation. Three Kni-binding sites were found in the 1.1 kb KpnI/SacII enhancer and one in the 0.5 SacII regions. Only two of the sequences located in the 1.1 kb KpnI/SacII enhancer present some conservation between Drosophilids. Interestingly, the 0.9 kb SalI/KpnI enhancer responsible of dpp expression in the L5 veins does not contain any putative Knirps binding sequence. Although whether Kni binds directly to the shv enhancer has not been analyzed, the presence of Kni-binding sites in conserved regions of the enhancer suggests that, in addition to its role during imaginal development, Kni might also activate dpp transcription during pupal development (Sotillos, 2006).

Therefore, regulatory sequences that drive dpp expression in the pupal veins in 2.5 kb of the dpp^s region have been found. This regulatory DNA can be subdivided into three fragments, a 1.1 kb fragment that recapitulates almost completely the pupal expression of dpp, a 0.9 kb upstream fragment, which drives expression in the proximal part of L5, and a 0.5 kb fragment that directs expression in all veins. Binding sites were found in these fragments for general transcription factors involved in the development of all veins (Vvl) and for the downstream activators of the dpp pathway, Mad and Medea. The regulatory region also contains binding sites for transcription factors expressed and required only in specific veins, such as Ara (L3 and L5) and Kni (L2). Most of these sequences are located in highly conserved regions of the dpp gene in different Drosophila species, indicating a general conservation of dpp regulation in the Drosophilids (Sotillos, 2006).

Protein Interactions

During Drosophila embryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells. CNS midline development is dependent on Single-minded, a basic-helix-loop-helix (bHLH)-PAS transcription factor. Fish-hook/Dichaete, a Sox HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene expression. single-minded, Dichaete, and drifter are all expressed in developing midline cells, and both loss- and gain-of-function assays reveal genetic interactions between these genes. The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from the slit gene and regulate the activity of this enhancer in cultured Drosophila Schneider line 2 cells. Dichaete directly associates with the PAS domain of Single-minded and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast. In addition, Dichaete can form homodimers and also associates with other bHLH-PAS and POU proteins. These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription factors. Functional interactions between members of these protein families may be important for numerous developmental and physiological processes (Ma, 2000).

To address whether the sim, Dichaete, and dfr genes might functionally interact to regulate development of the embryonic CNS midline, whether they exhibit overlapping expression in developing midline cells was examined. This was accomplished using anti-Dichaete and anti-Dfr sera, as well as a P[3.7sim-lacZ] marker that mimics sim midline expression. P[3.7sim-lacZ] embryos were immunostained using anti-ß-gal and either anti-Dichaete or anti-Dfr sera. Prominent overlapping expression was detected between Sim and Dichaete in developing CNS midline cells from stage 8 throughout the remainder of germ band extension. Overlap was also detected in a subset of prospective foregut cells. Similar overlapping expression was also detected between Sim and Dfr. Midline coexpression of Dichaete and Dfr was detected by immunostaining wild-type embryos with anti-Dichaete and anti-Dfr sera. Both genes are expressed together in the CNS midline throughout germ band extension. In germ band-retracted embryos, Dichaete exhibits overlapping expression with Sim and Dfr in the midline glia. Dichaete and Dfr are also detected together in lateral cells of the thoracic ganglia and a subset of ventral epidermal cells. These analyses indicate that sim, Dichaete, and dfr are coexpressed in developing CNS midline cells. The midline expression of these three genes also overlaps that of the slit gene, which is a downstream target of Sim (Ma, 2000).

Both loss-of-function and gain-of-function assays were used to detect genetic interactions between sim, Dichaete, and dfr. Mutants are known to show genetic interactions in CNS midline differentiation and in Slit protein expression. Potential cooperative interactions between sim, Dichaete, and dfr in regulating slit gene transcription were examined through the use of a P[1.0slit-lacZ] marker. This reporter contains a portion of a slit intron that drives lacZ expression mimicking that of the native slit gene in developing midline glia; P[1.0slit-lacZ] expression is first detected in germ band-extended stage 11 embryos and is maintained throughout the remainder of embryogenesis. Dichaete null mutant embryos exhibit a misplacement and loss of midline glia, as detected via anti-ß-gal immunostaining. P[1.0slit-lacZ] is expressed normally in stage 11 Dichaete mutant embryos, but during germ band retraction the number of midline glia becomes reduced from wild type, and many cells are located at aberrant ventral positions within the nerve cord. Similar, although less severe, defects are observed in dfr mutant embryos, where some midline glia are displaced from their normal positions. Notably, ß-gal-expressing midline glia are still detected in both Dichaete and dfr mutants, indicating that unlike Sim, Dichaete and Dfr are not absolutely required for P[1.0slit-lacZ] expression or midline glial development (Ma, 2000).

A dfr-Dichaete double mutant strain was used to examine whether Dichaete and dfr might act together to regulate midline gene expression. Embryos mutant for both Dichaete and dfr exhibit much more severe defects in P[1.0slit-lacZ] expression than either Dichaete or dfr single mutants. Although P[1.0slit-lacZ] is activated normally in stage 11 dfr-Dichaete double mutant embryos, there is a striking loss of midline P[1.0slit-lacZ] expression during germ band retraction. This synergistic effect strongly suggests that Dichaete and Dfr function together to regulate slit transcription. These functions may be mediated directly through Dichaete and Dfr binding sites present in the slit 1 kb regulatory region. Another, nonexclusive possibility is that Dichaete and Dfr might indirectly control slit transcription by regulating the expression of sim. To address this possibility P[3.7sim-lacZ] expression was examined in wild-type and dfr-Dichaete embryos. Compared with wild-type embryos, dfr-Dichaete double mutants exhibit a severe decrease in P[3.7sim-lacZ] expression, a phenotype that first becomes apparent during germ band retraction. Thus, Dichaete and dfr also influence sim expression and hence may indirectly influence the expression of a wide array of midline genes (Ma, 2000).

Analysis of a 380 bp slit midline regulatory fragment has indicated the presence of a single CNS midline element (CME), through which Sim::Tgo heterodimers act. The CME is located within 300 bp of the distal end (farther from the promoter in the native slit gene) of this fragment. An inverted TTCAAT repeat (TTCAATTTCATTGAA) is located 20 bp proximal to the CME. This sequence resembles a (A/T)(A/T)CAAT consensus binding site for Sox proteins, although binding of Sox proteins to a TTCAAT sequence has not been reported. Because sequences present in an extended 1 kb slit DNA fragment are required for normal levels of slit expression in vivo, additional DNA sequences have been obtained. This analysis indicated that no other CMEs are present in the 1 kb slit DNA fragment. However, two perfect Dfr consensus binding sites, ATGCAAAT and CATAAAT, located within 500 bp of DNA proximal to the CME were identified. These two Dfr binding sites are separated by ~150 bp and flank a consensus Dichaete binding site, TACAAT. These data suggest that Dichaete, Sim, and Dfr may all bind to sites present in the 1 kb slit regulatory DNA fragment. To test this possibility, DNA gel mobility shift assays were performed using the Dichaete HMG domain and full-length Dfr protein on double-stranded oligonucleotide probes corresponding to sequences from the slit 1 kb fragment. The Dichaete HMG domain binds strongly to a 26 mer probe containing the TACAAT site. In contrast, Dichaete does not bind consistently to a 26 mer probe containing both TTCAAT sites, suggesting that Dichaete can distinguish between closely related DNA sequences. Dfr protein binds very strongly to a 33 mer probe that contains the ATGCAAAT site, and less strongly to a 32 mer probe containing the CATAAAT site. Dfr binds the ATGCAAAT site both as an apparent monomer and a dimer, because two distinct bands with reduced mobilities are detected. The 1 kb slit fragment thus may integrate the actions of at least three different types of regulatory proteins, represented by Sim, Dichaete, and Dfr (Ma, 2000).

The ability of Dichaete, Dfr, Sim, and Tgo to directly control slit transcription was examined using transient transcription assays in cultured Drosophila S2 cells. The P[1.0slit-lacZ] construct was used as a reporter with various combinations of plasmids that express Dichaete, Dfr, Sim, or Tgo. Dichaete modestly activates P[1.0slit-lacZ] transcription, indicating that in both yeast and fly cells, Dichaete can function as a direct transcriptional activator. Dfr results in little if any activation of P[1.0slit-lacZ], and Dfr and Dichaete together do not exhibit any increased activation over the levels observed for Dichaete alone. Neither Sim nor Tgo alone is able to activate the P[1.0slit-lacZ] reporter, because only background levels of expression are detected. Furthermore, Sim and Tgo together yield only minimal activation. These results imply that although Sim::Tgo heterodimers strongly activate expression of a P[6XCME-lacZ] reporter (>150 units) that contains six multimerized CMEs, additional factors are required to achieve high levels of reporter expression. Significantly, the combination of either Dichaete and Sim::Tgo or Dfr and Sim::Tgo both result in relatively high levels of activation. Thus, both Dichaete and Dfr strongly enhanced the ability of Sim::Tgo heterodimers to activate slit transcription. Comparable levels of activation are observed when all four proteins are expressed together. Taken together, the DNA binding and transcriptional activation assays provide additional evidence that regulation of slit expression in the midline glia requires functional interactions between Dichaete, Dfr, Sim, and Tgo (Ma, 2000).

Functional interactions between Sim, Dichaete, and Dfr may also regulate the midline expression of other genes, including sim and breathless (btl). Thus, sim has autoregulatory functions, and the combined functions of dfr and Dichaete are also required for sustained midline sim expression. In addition, a 2.8 kb interval in the P[3.7sim-lacZ] transgene used in this study contains six evolutionarily conserved CMEs as well as several consensus Dichaete and Dfr binding sites. btl encodes an FGF receptor homolog whose expression in the CNS midline and tracheal cells has been shown to depend, respectively, on Dfr as well as Sim and Tgo, or Trh and Tgo. A 200 bp btl midline/tracheal regulatory region contains three evolutionarily conserved CMEs. Inspection of this region also revealed the presence of a conserved consensus ATCAAT Dichaete binding site located in a 40 bp interval between CME2 and CME3, as well as a conserved consensus GATAAAT Dfr binding site located 40 bp downstream of CME3. Thus, functional interactions between Sim, Dichaete, and Dfr could be a general mechanism to regulate gene transcription during CNS midline development (Ma, 2000).

I-POU does not form a complex with Drifter as reported previously (Treacy, 1992 and Turner, 1996).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.