collier

Promoter

The regulation of development by Hox proteins is important in the evolution of animal morphology, but how the regulatory sequences of Hox-regulated target genes function and evolve is unclear. To understand the regulatory organization and evolution of a Hox target gene, a wing-specific cis-regulatory element was identified controlling the knot gene, which is expressed in the developing Drosophila wing but not the haltere. This regulatory element contains a single binding site that is crucial for activation by the transcription factor Cubitus interruptus (Ci), and a cluster of binding sites for repression by the Hox protein Ultrabithorax (Ubx). The negative and positive control regions are physically separable, demonstrating that Ubx does not repress by competing for occupancy of Ci-binding sites. Although knot expression is conserved among Drosophila species, this cluster of Ubx binding sites is not. The knot wing cis-regulatory element was isolated from D. pseudoobscura, which contains a cluster of Ubx-binding sites that is not homologous to the functionally defined D. melanogaster cluster. It is, however, homologous to a second D. melanogaster region containing a cluster of Ubx sites that can also function as a repressor element. Thus, the knot regulatory region in D. melanogaster has two apparently functionally redundant blocks of sequences for repression by Ubx, both of which are widely separated from activator sequences. This redundancy suggests that the complete evolutionary unit of regulatory control is larger than the minimal experimentally defined control element. The span of regulatory sequences upon which selection acts may, in general, be more expansive and less modular than functional studies of these elements have previously indicated (Hersh, 2005).

The knot gene is expressed in the developing Drosophila wing imaginal disc at the anteroposterior compartment boundary, but is not expressed in the haltere imaginal disc. Furthermore, knot expression is genetically downstream of Ubx; overexpression of Ubx in clones in the wing causes cell-autonomous loss of knot expression. Because these features make knot a candidate for direct regulation by Ubx, attempts were made to identify the regulatory element that controls knot expression in the wing (Hersh, 2005).

One regulatory element of knot has been identified that drives reporter gene expression in the embryonic head and mesoderm. This element extends ~5 kb from the transcriptional start site of knot. When a knot cDNA was placed under control of this element, embryonic lethality was rescued, but wing vein defects were not, indicating that the wing regulatory element is located elsewhere. The lesion underlying the wing-specific knot^SA2 allele, which is a complex translocation with a breakpoint 10-20 kb 5' of the knot-coding region, suggested the location of a wing-specific regulatory element (Hersh, 2005).

Based on the location of the knot^SA2 lesion, reporter constructs were generated with genomic DNA from the region 5-20 kb 5' of knot. A 6.8 kb region of DNA ~15 kb 5' of knot was identified that drives expression of lacZ in a stripe at the anteroposterior compartment boundary in the wing imaginal disc, consistent with the expression pattern of the Knot protein. No expression of lacZ was observed in the haltere, demonstrating that this large region accurately recapitulates the expression and regulation of the endogenous knot gene. To determine if both wing and haltere regulation was confined to a single region within this 6.8 kb, the activity was further narrowed to a 2.3 kb region that drives appropriate reporter gene expression. All subsequent numbering of constructs is in reference to this 2.3 kb region, kn^Mel1-2330 (Hersh, 2005).

Sequence conservation has been successfully employed in the identification of regulatory elements. Attempts were made to use conservation to direct the further dissection of kn^Mel1-2330 to define a minimal regulatory element. Based on several scattered blocks of sequence conservation between D. melanogaster and D. pseudoobscura, PCR primers were designed to amplify sequence from a more distantly related fruit fly, D. virilis. Three conserved blocks were shared between these three flies, and kn^Mel1-2330 was split into two overlapping constructs, each containing two of the conserved blocks. Though both constructs included the central conserved block that contained several potential sites for regulators, only the 1.3 kb kn^Mel701-1991 construct was capable of driving expression in a stripe in the wing, whereas the kn^Mel224-1426 construct was only weakly expressed in a single small spot at the intersection of the DV and AP axes in the wing. Therefore, the 1.3 kb region accurately recapitulates the knot expression pattern in both the wing and haltere, and must contain binding sites for the regulators that generate this pattern (Hersh, 2005).

Expression of the knot gene is dependent on Hedgehog (Hh) activity, and overexpression of Hh can trigger ectopic knot expression in the wing. The transcriptional effector of Hh signaling is the Cubitus interruptus (Ci) protein. Ci is a zinc-finger transcription factor of the Gli family, and binds a 9 bp consensus sequence TGGG(T/A)GGTC. In the 1.3 kb kn^Mel701-1991 fragment, three potential Ci-binding sites were identified that matched at least seven out of nine consensus residues and that are conserved in D. pseudoobscura. Two additional potential sites were present, but were not conserved in D. pseudoobscura. The three conserved binding sites were independently mutagenized, converting a crucial guanine to an adenine, and the mutagenized element was re-introduced into flies. Changes at two of the three candidate sites had no effect on reporter gene expression, whereas the mutation of site Ci1680 almost completely abolishes reporter expression. Mutation of all three sites did not have a more severe effect than mutation of Ci1680 alone. These results indicate that activation of the wing-specific enhancer element by Hh signaling is dependent primarily on a single Ci-binding site at position 1680 in the kn^Mel701-1991 element (Hersh, 2005).

Because the kn^Mel701-1991 element drives expression in the wing, but not the haltere, it was postulated that this sequence integrates information from Hh signaling and the homeotic regulator, Ubx. Therefore, attempts were made to identify possible binding sites for Ubx within this element. Isolated Ubx homeodomain binds optimally in vitro to the sequence TTAATGG, but binding sites in characterized Ubx-responsive regulatory elements often are not exact matches to this optimal sequence. Therefore, the TAAT core sequence commonly bound by homeodomain proteins was sought. The kn^Mel701-1991 fragment contains clusters of TAAT core sequences near both its 5' and 3' limits that might mediate knot repression in the haltere. In addition, there is a single TAAT core sequence located within 10 bp of the crucial Ci-binding site and in a conserved block of sequence, suggesting that it may be important for repression by Ubx (Hersh, 2005).

To determine which TAAT sequences might be functionally important for Ubx repression, sequences were removed from each end of kn^Mel701-1991 and the effect on reporter gene expression in vivo was observed. Removal of the 5' end, with its small cluster of four core sequences, had no effect on expression. By contrast, removal of 156 bp from the 3' end, including nine putative Ubx-binding sites (kn^Mel701-1835), caused the reporter to be expressed at the AP compartment boundary in both the wing and the haltere. Therefore, kn^Mel701-1991 does appear to be directly negatively regulated by Ubx in the haltere, and removal of Ubx-binding sites relieves repression in the haltere. In addition to the ectopic activation of expression in the haltere, it was noted that the expression level in the wing is also elevated compared with kn^Mel701-1991, suggesting that additional repressor binding sites important for appropriate wing expression may have been removed in kn^Mel701-1835. Importantly, the response to local spatial information within the wing field (encompassing both the wing and haltere) was maintained; expression was appropriately observed at the AP compartment boundary in both tissues. Because the single deletion preserved the response to spatial information within the dorsal appendage wing field but altered the response to spatial information along the anteroposterior axis, it is suggested that activation by Ci and repression by Ubx are mediated through physically separable sites within knot cis-regulatory sequences (Hersh, 2005).

To identify which potential binding sites could be occupied by Ubx in vitro, DNaseI footprinting was performed on a 392 bp fragment (kn^Mel1599-1991) that includes the functional Ci site and the 156 bp required for repression in the haltere. This fragment is itself capable of driving expression in the wing, although at a significantly lower level than that driven by the full kn^Mel701-1991, and is repressed in the haltere. Four regions protected from DNaseI digestion by binding of Ubx were identified. These four regions include all TAAT core sequences present in the 392 bp fragment (10 in total) (Hersh, 2005).

Although Ubx site 1 is located only 4 bp from the Ci-binding site, it is still present in the kn^Mel701-1835 construct that is derepressed in the haltere, so this site alone is not sufficient to mediate repression by Ubx. To determine whether this site is necessary for repression by Ubx, Ubx site 1 alone (kn^Mel701-1991Ubx1KO) was mutated and no derepression of reporter gene expression in the haltere was observed. Therefore, Ubx Site 1, unlike individual Ubx-binding sites in the spalt enhancer, does not appear to contribute significantly to repression of this element by Ubx. Of the other regions protected by Ubx, the largest spans six TAAT core sequences and ~24 bp of sequence, and is located ~250 bp from the Ci binding site. Therefore, the DNA sequences necessary for repression in the haltere appear to be comprised of multiple, functional Ubx-binding sites that do not overlap with the activating Ci-binding site. This organization suggests that Ubx does not repress knot in the haltere by competing for activator binding sites (Hersh, 2005).

Individual Ubx-binding sites can additively contribute to repression in the haltere of the sal wing-specific regulatory element. To determine how individual Ubx binding sites in the knot element contribute to repression in the haltere, TAAT core sequences in Ubx site 1, and Ubx site 4 were independently mutated in kn^Mel701-1991 and these mutated constructs were introduced into flies. Elimination of these individual sites had no detectable effect on reporter gene expression in the haltere, so all 10 TAAT core sequences were mutated in kn^Mel701-1991, and this construct (kn^Mel701-1991KO) was introduced into flies. Elimination of all Ubx sequences results in de-repression in the haltere, demonstrating that some combination of these sites is required for repression in vivo. However, it is noted that the level of expression of this construct is lower than that observed in the deletion construct, kn^Mel701-1835. This difference was not expected and suggests the presence of additional regulatory sequences that contribute to repression in the haltere (Hersh, 2005).

To determine where additional potential regulatory sequences are located, sequence 3' of the kn^Mel701-1835 construct was restored. Addition of 43 bp (kn^Mel701-1878) was sufficient to partially restore repression in the haltere, suggesting the additional regulatory information was contained within this region. Deletion of this block of sequence (kn^Mel701-1991Delta) resulted in very weak, inconsistent de-repression in the haltere. By contrast, point mutations introduced at positions 1834-1837 (kn^Mel701-1991mut), the boundary of the derepressed kn^Mel701-1835 construct, resulted in consistent, partial, de-repression. Since this position is not a Ubx site, this result suggests that at least one transcription factor acts in addition to Ubx to repress knot in the haltere through this regulatory element. Mutation of both positions 1834-1837 and all Ubx TAAT core sequences (kn^Mel701-1991KOmut) resulted in full de-repression in the haltere, suggesting that Ubx and another repressor act together to reduce expression in the haltere through sequences located between kn^Mel1835-1991. The DNA sequence at kn^Mel1834-1837 does not clearly match any binding sites archived in transcription factor databases, and as the identity of the factor that may act with Ubx to repress knot in the haltere is not known (Hersh, 2005).

To understand how Ubx-regulated target gene networks evolve, it is crucial to determine how Ubx regulation of individual target genes evolves. Dissection of the knot wing regulatory element was combined with comparative genomics within Drosophila to establish how Ubx-responsive regulatory sequences in knot have evolved. The 156 bp knot repressor element from D. melanogaster was compared to D. pseudoobscura sequence, and neither significant sequence conservation nor a comparable cluster of potential Ubx-binding sites was observed in D. pseudoobscura. Because the expression pattern of knot is the same between these two species, these significant sequence differences suggest that regulation by Ubx is mediated through different regulatory sequences in D. pseudoobscura. Therefore, attempts were made to identify a functional regulatory element from D. pseudoobscura that could regulate reporter expression in the appropriate pattern (Hersh, 2005).

Using blocks of sequence identity as relational anchor points, a fragment from D. pseudoobscura (kn^Pse1-1935) that roughly corresponded to the kn^Mel1-2330 D. melanogaster fragment was amplified. This fragment was introduced into D. melanogaster and it was found that it could properly drive expression in the wing while repressing expression in the haltere. The kn^Pse1-1935 construct contained at its 3' end a cluster of 12 TAAT Ubx core binding sites. To determine if this region is important for repression by Ubx in D. pseudoobscura, a truncation of kn^Pse1-1935 was generated that eliminated the TAAT core sequences. This kn^Pse1-1643 construct appropriately drove expression in the wing, but now also drove haltere expression. Therefore, the region containing these putative Ubx-binding sites acts as a repressor element in the haltere (Hersh, 2005).

Interestingly, this functional cluster of Ubx-binding sites is conserved between D. pseudoobscura and D. melanogaster, and is located ~500 bp 3' of the kn^Mel1835-1991 sequence necessary for repression, just 3' of the limit of the 6.8 kb fragment originally isolated that contains the functional D. melanogaster knot regulatory element. Therefore, the knot regulatory region in D. melanogaster could potentially contain two sets of functional repressor input sites. To determine whether this second, conserved block can also function to repress the D. melanogaster knot regulatory element, the D. melanogaster sequence was attached to the de-repressed kn^Mel701-1835 construct. Addition of 222 nucleotides (kn^Mel2499-2722), homologous to the D. pseudoobscura sequence necessary for repression, to kn^Mel701-1835 (to generate kn^Melcomposite) restored repression in the haltere. Therefore, D. pseudoobscura has a single element (located between kn^Pse1643-1935) that represses expression of knot in the haltere, and this element is shared with D. melanogaster. However, D. melanogaster possesses a second element (located between kn^Mel1835-1991), not shared with D. pseudoobscura, that also functions to repress expression in the haltere (Hersh, 2005).

Next, attempts were made to determine whether Ubx-binding sites in the kn^Mel2499-2722 conserved element are sufficient to repress reporter expression, or whether this element also requires the action of a collaborating repressor. All Ubx core binding sites were mutated in this sequence and the mutated kn^Mel2499-2722KO sequence was attached to the de-repressed kn^Mel701-1835 (generating kn^MelcompositeKO). Whereas mutation of Ubx sites alone in kn^Mel701-1991KO did not fully de-repress in the haltere, mutation of Ubx sites in kn^MelcompositeKO was sufficient for complete de-repression in the haltere. Thus, the kn^Mel2499-2722 and kn^Mel1835-1991 repressor elements appear to be organized differently -- the former with input only from Ubx, and the latter with input from Ubx and an additional trans-acting factor (Hersh, 2005).

Does the presence of two elements in D. melanogaster indicate the acquisition of a new element in this lineage or the loss of an element in D. pseudoobscura? To analyze the distribution of these two regulatory elements in other drosophilids, the knot regulatory region was amplified from three additional Drosophila species – D. mauritiana, D. biarmipes and D. malerkotliana – phylogenetically intermediate between D. melanogaster and D. pseudoobscura. All three species have sequence similar to kn^Pse1643-1935, but also possess sequence similar to kn^Mel1835-1991 in varying degrees. For example, the core TAAT of Ubx site 3 is shared by all three additional species (though sequence surrounding the core is non-identical), whereas Ubx site 2 is found only in D. mauritiana. The most interesting pattern is observed for Ubx site 4. D. malerkotliana has only a single core Ubx sequence conserved with D. melanogaster; D. biarmipes has two conserved core sequences and two additional core sequences that are unique, and D. mauritiana has five of the six core sequences present in D. melanogaster. Therefore, in this sample of five drosophilid species, the pattern of an apparent accretion of Ubx-binding sites in this region is observed in the evolution of the D. melanogaster lineage (Hersh, 2005).

Thus, a wing-specific cis-regulatory element was identified for the gene knot. This regulatory element is activated in the wing by direct input from Ci and is repressed in the haltere by direct input from Ubx. The regulatory sequences governing activation and repression are physically separable, and the repression element was found not to be shared with D. pseudoobscura. A distinct functional repression element was identified in D. pseudoobscura that is shared with D. melanogaster, indicating that the entire knot wing regulatory region in D. melanogaster contains two apparently redundant repressor elements. One element appears to have been acquired in the course of the evolution of the D. melanogaster lineage. These results suggest that complete functional cis-regulatory elements, the units of function that selection is operating upon, may be larger and more diffuse than the minimal functional sequences typically defined by molecular dissection (Hersh, 2005).

collier transcription in a single Drosophila muscle lineage: the combinatorial control of muscle identity

Specification of muscle identity in Drosophila is a multistep process: early positional information defines competence groups termed promuscular clusters, from which muscle progenitors are selected, followed by asymmetric division of progenitors into muscle founder cells (FCs). Each FC seeds the formation of an individual muscle with morphological and functional properties that have been proposed to reflect the combination of transcription factors expressed by its founder. However, it is still unclear how early patterning and muscle-specific differentiation are linked. This question was addressed using Collier (Col; also known as Knot) expression as both a determinant and read-out of DA3 muscle identity. Characterization of the col upstream region driving DA3 muscle specific expression revealed the existence of three separate phases of cis-regulation, correlating with conserved binding sites for different mesodermal transcription factors. Examination of col transcription in col and nautilus (nau) loss-of-function and gain-of-function conditions showed that both factors are required for col activation in the 'naive' myoblasts that fuse with the DA3 FC, thereby ensuring that all DA3 myofibre nuclei express the same identity programme. Together, these results indicate that separate sets of cis-regulatory elements control the expression of identity factors in muscle progenitors and myofibre nuclei and directly support the concept of combinatorial control of muscle identity (Dubois, 2007).

col belongs to the class of Drosophila regulatory genes with numerous introns, large amounts of flanking sequence and multiple expression sites. During embryogenesis, col is expressed in the MD2/PS0 head region, the somatic DA3 muscle, precursor cells of the lymph gland, a small set of multidendritic (md) neurons of the peripheral nervous system and specific neurons of the central nervous system (CNS). A lacZ reporter transgene (P{5col::lacZ}, abbreviated P5cl, contains 5 kb of col upstream DNA, which faithfully reproduced col transcription both in the MD2/PS0 and the DA3 muscle, starting at the progenitor stage and not in promuscular cluster(s). To identify the missing cis-regulatory information, a longer construct was tested containing the entire 9 kb region separating col from CG10200, the next predicted upstream gene. In addition to the head and DA3 muscle, P9cl expression reproduced col expression in md neurons and a subset of neurons in the CNS. A DNA fragment located further upstream, between CG10200 and the next predicted gene CG10202, was independently shown to drive col expression in the anteroposterior organiser of the wing imaginal disc (Hersh, 2005). However, neither this construct nor P9cl reproduced Col expression in promuscular clusters. The col transcription unit is immediately flanked at its 3' end by another gene, BEAF32, making rather unlikely the presence of cis-regulatory elements within this region. However, it contains ten different introns, of total length around 30 kb, the cis-regulatory content of which remains to be assessed (Dubois, 2007).

To delineate more precisely the CRM driving col expression in the DA3 muscle, a series of constructs was tested containing 2.6, 2.3, 1.6 and 0.9 kb of DNA upstream of the col transcription start site, respectively. P2.6cl retained the information necessary for col expression in MD2/PS0 and the DA3 progenitor and muscle, although it was noted that P2.6cl expression in muscle progenitors was less robust than P9cl. P2.3cl was also activated in MD2/PS0 at stage 6 and the DA3 muscle. However, unlike P9cl or P2.6cl, P2.3cl was not activated in the DA3/DO5 progenitor but only at the FC stage; ectopic lacZ expression was observed in clusters of neuroectodermal cells at embryonic stage 11). This difference indicated that cis-regulatory elements required for col expression in the DA3/DO5 progenitor reside between positions -2.6 and -2.3 and act separately from those required for expression in the DA3 FC and muscle. P1.6cl was active only in MD2/PS0, whereas no expression at all could be detected with P0.9cl. Together, expression data from this series of reporter constructs allowed the mapping of the CRM required for col-specific expression in the DA3/DO5 muscle progenitor and DA3 FC/myofibre to a DNA fragment between positions -2.6 and -1.6 upstream of the col transcription start (Dubois, 2007).

Advantage was taken of the recently available genome sequences of several Drosophila species to search for conserved motifs in the col upstream DNA, as it has often proven to be effective to identify functionally important cis-regulatory elements. Among these species, D. virilis (D. vir) is the most distant from D. melanogaster (D. mel). It was first verified that Col expression in D. vir was similar to that in D. mel embryos and could infer from this that the regulatory information controlling col transcription in the DA3 muscle lineage has been conserved. Sequence comparison of 9 kb of the col upstream region between D. mel, D. vir and four other Drosophila species, D. yakuba, D. ananassae, D. pseudoobscura and D. mojavensis revealed numerous stretches of high sequence conservation, of sizes up to 100 bp. Ten conserved motifs of size >20 bp, numbered 1 to 10 from 5' to 3', were found in the same order and at the same relative position between position -2.6 and the start of transcription in all six Drosophila species. To test the relevance of this conservation, lacZ reporter constructs were created containing either D. vir or D. mel DNA (Dubois, 2007).

P.3.4cl^vir corresponds to D. mel P2.6cl, whereas P3.4-1.3cl^vir and P2.6-0.9cl are truncated versions covering motifs 1 to 10. All four reporter genes showed expression in the DA3 muscle, starting at the progenitor stage, confirming the evolutionary conservation of a DA3-muscle-specific CRM. A Gal4 driver line containing only the -2.6 to -1.6 region (P2.6-1.6cG), harbouring only motifs 1 to 7, was also specifically expressed in the DA3 muscle. This confirmed that the DA3 muscle CRM is located between positions -2.6 and - 1.6. It was noticed, however, that expression of P2.6-1.6cG was weaker and more sporadic than P2.6-0.9cl, suggesting the existence of cis-regulatory element(s) between positions -1.6 and -0.9 contributing to robust DA3 muscle expression. The conserved motifs 1 to 10 were searched for consensus binding sites of known TFs that could account for col activation in the DA3 muscle. This identified a binding site for the mesodermal basic helix-loop-helix (bHLH) protein Twi (within motif 2), correlating well with the position of the muscle progenitor cis-element and a potential EBF/Col-binding site within motif 7. Further visual inspection of the sequence alignments identified other conserved TF-binding sites, including one Mef2-binding site within the -1.6 to -0.9 fragment and one consensus binding site for Nau (Huang, 1996; Kophengnavong, 2000). In contrast, the position of the Mef2 site correlated well with the requirement of the -1.6 to -0.9 fragment for robust DA3 muscle expression. The presence of a Nau-binding site was particularly intriguing since Nau is required for DA3 muscle formation. Potential binding sites for other TFs could be found in the DA3 CRM, but the annotation to the conserved sites. The relative paucity of known TF-binding sites in the conserved sequence motifs found in the DA3 muscle CRM leaves largely open the question of the roles of these motifs in col regulation (Dubois, 2007).

Functional dissection of the DA3 muscle CRM present in the col upstream region showed that col expression in the DA3 FC can be separated from its expression in the DA3/D05 progenitor and the promuscular cluster. It thus revealed the existence of three steps in the transcriptional control of muscle identity. That col expression in the DA3/D05 progenitor could be uncoupled from that in promuscular clusters was in apparent contradiction with the previous conclusion from pioneering studies on Eve expression in dorsal muscle progenitors that this expression issued from Eve activation in promuscular clusters. Restriction of Eve expression to progenitors was considered a secondary step, mediated by N-signalling during progenitor selection by lateral inhibition. To reconcile these data and this model, it is proposed that the muscle DA3 CRM is active only in the DA3/D05 progenitor because it lacks some positively acting cis-elements necessary to counteract N-mediated repression of col transcription. It has been shown that col transcription is repressed by N during the progenitor selection process. It is also noted that a Twi-binding site is present in the 'progenitor' subdomain of the DA3 CRM. The functional importance of this site is supported by its in vivo occupancy in 4- to 6-hour-old embryos when selection of the DA3/DO5 progenitor takes place (Sandmann, 2007). Together, Twi in vivo binding and the col/P2.6cl/P2.3cl expression data suggest that Twi activity contributes to col expression in the DA3/DO5 progenitor but may not be sufficient to override N repression of col transcription before progenitor selection. Additional binding sites for Twi present in the col upstream region, between positions -8.7 and -8.3, are also bound by Twi in vivo (Sandmann, 2007) and probably contribute to the robustness of P9cl expression in progenitor cells, but the question of which cis-regulatory elements mediate col activation in promuscular clusters remains open. From Eve expression studies, a computational framework has been developed to identify other FC-specific genes (Estrada, 2006; Philippakis, 2006). This framework, named Codefinder, integrates transcriptome data and clustering of combinations of binding sites for five different TFs (Pnt, dTCF, Mad, Twi and Tin). col/kn was selected by Codefinder owing to the presence of five clusters of binding sites, four of which are located within introns (Philippakis, 2006). It remains to be determined which of these could be responsible for col activation in promuscular clusters, but it is interesting to note that another in vivo Twi-binding site in 4-6-hour-old embryos correlates with the 3'-most cluster (Sandmann, 2007). In addition to Twi, conserved binding sites for Nau and Mef2 are found within the DA3 CRM. The Mef2 binding site is located in a region required for robust DA3-muscle expression of a reporter gene. A direct control of col transcription by Mef2 during the muscle fusion process is further supported by the recent finding (Sandmann, 2006) that Mef2 binds in vivo to the col upstream region between 6 and 8 hours of embryonic development (Dubois, 2007).

Detailed analysis of col auto-activation revealed a reiterative, two-step process: import of pre-existing Col protein in the fusion competent myoblast nuclei that incorporate into the growing DA3 myofibre precedes activation of col transcription. This process ensures that all incorporated FCM nuclei acquire the same identity. Nau is required for maintaining col transcription in the DA3 muscle precursor and this control is probably direct. The presence of a putative EBF-binding site in the DA3 muscle CRM also correlates with the Col requirement for maintaining its own transcription beyond the FC stage. Thus, despite the failure to detect strong Col binding to this site in vitro, it appears to be essential for col auto-regulation in vivo. This suggests that in vivo binding is potentiated by one or more specific co-factor(s) present in the DA3 muscle. One co-factor is probably Nau, as the ability of Col to activate its own transcription in newly recruited fusion competent myoblasts is dependent upon Nau activity. Nau is not sufficient, however, as many muscles containing both Nau and Col proteins do not activate col transcription. Interestingly, mouse EBF (also known as Ebf1 and Olf1 - Mouse Genome Informatics) and E2A (Tcfe2a - Mouse Genome Informatics), a bHLH protein of the same subgroup as MyoD, have been shown to act on the same target promoter and synergistically upregulate transcription of B-lymphocyte-specific genes, although no direct physical interaction between EBF and E2A could be found in vitro. This suggested that functional interaction of EBF and E2A, similar to Col and Nau, requires yet another factor. Taking into account the restricted pattern of ectopic col activation in hs-col conditions, it is hypothesised that Vg could be another component of the DA3 combinatorial identity. However, we found that Vg is not required for DA3 muscle specification, leaving open the question of which factor may bridge Col and Nau functions (Dubois, 2007).

Unlike col or P2.6cl, P2.3cl is expressed in the DA3 FC and muscle precursor but not the DA3/DO5 progenitor, showing that col transcription in the progenitor and muscle precursor is under separate control. These two phases of col regulation are intimately linked, however, as Col is required for activating its own transcription in the nuclei of FCM recruited by the DA3 FC. This regulatory cascade may explain how pre-patterning of the somatic mesoderm and muscle identity are transcriptionally linked in the Drosophila embryo. As discussed above, the ability of Col to auto-regulate depends upon the presence of Nau, another muscle identity TF. Col and Nau act as obligatory co-factors fo maintenance/activation of Col expression in all nuclei of the DA3 muscle, thus bringing to light a clear case of combinatorial coding of muscle identity (Dubois, 2007).

Transcriptional Regulation

buttonhead might be a 'generic transcriptional activator' required for transcriptional activation of specific target genes, such as even skipped and collier, an ortholog of mammalian early B-cell factor/Olfactory-1. Recent data on the activation of collier in PS0/mitotic domain 2, suggest a possible mechanism by which btd and eve cooperate to pattern PS1. Activation of col requires btd. Conversely, in the absence of eve, col expression is expanded posteriorly to overlap a region roughly corresponding to PS1, indicating that Eve acts as a repressor of col in this parasegment. Likewise, expression of string in mitotic domain 2, which also requires btd, is expanded posteriorly in eve mutant embryos. The current working model is that the activation of eve by btd in anterior PS1 cells allows for differential gene expression between PS0 and PS1. In addition to the control of CF formation, the btd/eve interaction may thus assign separate gene expression and mitotic programs to cells on either side of the pro-cephalon/posterior head border (Crozatier, 1996 and Vincent, 1997).

There is no collier expression in embryos from bicoid mothers or in buttonhead homozygous mutant embryos. In empty spiracles mutant embryos, col expression expands ventrally to include mesodermal precursor cells. These results indicate that col acts downstream of the head gap genes in the transcription regulatory cascade that patterns the anterior part of the head, with buttonhead being absolutely required for col activation. In string mutant embryos, col expression during gastrulation is the same as that in wild-type embryos, indicating that the expression of col is independent of the mitotic program of cells in mitotic domain 2 (Crozatier, 1996).

A dominant interaction between combgap and engrailed/invected mutations that gives rise to a gap in vein L4 strongly suggests that Cg and En/Inv act together to repress posterior cubitus interruptus transcription. Posterior expression of En represses the transcription of ci resulting in anterior specific expression. En has been shown to interact directly with the ci regulatory elements. In cg mutant wing imaginal discs, weak ectopic expression of ci-lacZ reporter constructs are found in posterior cells, thus Cg may act in concert with En to repress posterior ci. Hypomorphic mutants in either cg or en/inv can give rise to the reduction in vein L4 that is characteristic of ectopic ci expression (Svendsen, 2000).

The changes in the A/P pattern observed in cg mutant limbs are caused by the mis-regulation of Hh-responsive genes regulated by the Ci-155 and Ci-75 transcription factors. In cg mutant wing imaginal discs, Ci-155 is ectopically expressed in the posterior compartment and is associated with posterior compartment defects and posterior misexpression of genes such as patched and knot. Ectopic expression of kn is sufficient to suppress vein fate. Thus, the misexpression in the posterior compartment of kn and other genes regulated by high levels of Ci-155 probably leads to the vein defects described in this study. The occurrence of both higher levels of posterior Ci and Kn expression, and higher frequency of posterior compartment defects in cg¹/cg¹ mutant wings supports this explanation. Stronger allelic combinations of cg have lower levels of ectopic Ci and Kn, and lower incidence of posterior compartment wing defects, but they result in a greater reduction in Ci in the anterior compartment and more anterior vein defects. The posterior and anterior vein defects, as well as occasional anterior wing margin bifurcations, resemble the effects of regulatory mutants of ci that cause the ectopic expression of ci in posterior cells and the reduction of ci expression in the anterior compartment (Svendsen, 2000 and references therein).

During Drosophila wing development, Hedgehog (Hh) signaling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signaling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. A functional hierarchy of these signaling pathways is described that highlights the importance of the competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc has revealed that collier/knot (col/kn), patched, and decapentaplegic are repressed at the DV boundary by N signaling. Attenuation of Hh signaling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. This down-regulation depends on activity of the canonical Wg signaling pathway. It is proposed that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. These findings suggest a paradigm of repression of the Hh response by N and/or Wnt signaling that may be applicable to signal integration in vertebrate appendages (Glise, 2002).

Short-range Hh signaling, partly through activation of Col function, is essential for correct AP patterning and differentiation of L3-L4 intervein tissue. N and Wg first define the DV boundary and later subdivide the region near this boundary into a number of distinct subregions that will eventually differentiate into wing margin bristles and vein tissue. These signals overlap spatially and temporally and lead to opposite fates. It is proposed that in and close to the DV boundary, N, Wg, and Hh signaling exist in a delicate balance to allow vein tissue, bristle, and sensory organ differentiation along the adult wing margin (Glise, 2002).

The Hh target genes col/kn and ptc, in contrast to en, are repressed in a wild type wing in cells corresponding to the presumptive wing margin. It has been demonstrated, using both gain- and loss-of-function experiments, that this repression is mediated by N signaling and that its inhibition results in aberrant morphogenesis of the wing. Hh signaling, achieved either by overexpression of Hh or loss of Ptc activity, is not sufficient to give maximum activation of Hh targets in cells of the prospective wing margin, suggesting that a finely tuned balance of activation and repression is required to achieve the appropriate biological output. However, overexpression of a stabilized form of Ci under the ptc-Gal4 driver results in the activation of Col in the prospective wing margin and defects in wing margin differentiation, indicating that N repression can be overcome by hyperactivity of the Hh signaling pathway. N signaling may lead to the repression of col, ptc, and dpp directly or it may act indirectly by affecting the ability of Ci to act as a transcriptional activator. Since expression of en, which requires the highest level of Hh signaling and Ci activity, appears immune to N repression, the former possibility is favored (Glise, 2002).

The Drosophila wing is a classical model for studying the generation of developmental patterns. Previous studies have suggested that vein primordia form at boundaries between discrete sectors of gene expression along the antero-posterior (A/P) axis in the larval wing imaginal disc. Observation that the vein marker rhomboid (rho) is expressed at the center of wider vein-competent domains led to the proposal that narrow vein primordia form first, and produce secondary short-range signals activating provein genes in neighboring cells. This study examined how the central L3 and L4 veins are positioned relative to the limits of expression of Collier (Col), a dose-dependent Hedgehog (Hh) target activated in the wing A/P organizer. rho expression is first activated in broad domains adjacent to Col-expressing cells and secondarily restricted to the center of these domains. This restriction, which depends upon Notch (N) signaling, sets the L3 and L4 vein primordia off the boundaries of Col expression. N activity is also required to fix the anterior limit of Col expression by locally antagonizing Hh activation, thus precisely positioning the L3 vein primordium relative to the A/P compartment boundary. Experiments using Nts mutants further indicate that these two activities of N can be temporally uncoupled. Together, these observations highlight new roles of N in topologically linking the position of veins to prepattern gene expression (Crozatier, 2003).

Longitudinal vein primordia can be visualized in third instar larval wing discs as a series of stripes of cells expressing provein genes, alternating with domains of D-SRF expression. col activates D-SRF expression in A/P organizer cells and positions L3 vein by limiting L3 vein competence to cells expressing iro-C but not col. Therefore col transcription was examined in N55/+ third instar wing discs; it is expanded toward the anterior by one to two rows of cells. The position of the SOPs were examined, using a neuralised (neu)-lacZ reporter gene (transgenic line A101). Whereas in wild-type, one row of cells separates SOPs from the anterior limit of Col expression, SOPs are found immediately adjacent to cells expressing high levels of Col protein in N55/+ discs. Counterstaining of discs with propidium iodide (which labels all nuclei) confirms that the position of SOPs relative the A/P border (anterior limit of hh/posterior limit of Col expression) is unchanged, leading to the conclusion that reducing N activity in third instar larvae specifically results in anterior expansion of Col expression. Col expression was then examined in clones of N mutant cells generated in a heterozygous N55/+ background and spanning the A border of Col expression; it was found not to be expanded further anteriorly. col expression is established in response to Hh in a dose-dependent manner. The present data indicate that: (1) only one or two rows of cell activate col in response to Hh in the absence of Notch signaling, and (2) the same expansion on col expression results from complete absence of N or 2-fold reduction of N signaling suggesting that col expression is very dose sensitive. Thus, the expansion of col expression observed in N55/+ discs indicates that N signaling locally antagonizes Hh activation of col transcription, to precisely position the posterior limit of the L3 vein primordium. Repression of col transcription by Notch signaling has already been reported in formation of an embryonic muscle and at the wing margin but the molecular mechanisms underlying this expression remain to be determined. iro-C expression is also expanded anteriorly in late 3rd instar larval discs in N55/+ mutants, indicating that the entire L3 vein-competence domain is shifted anteriorly. The opposite, posterior shift of iro-C expression (and consequently L3 vein position) observed in col mutant discs (this correlates with the posterior shift of L3 vein observed in these mutants) is linked to the modified range of Dpp signaling resulting from lack of col activity. Similarly, it is proposed that the anterior expansion of iro-C expression in N55/+ mutant discs reflects a modified range of Dpp signaling induced by anterior extension of Col expression. Thus, in wild-type discs the cross-regulation between Hh, N and Dpp signalling allows the positioning of the L3 vein primordium in register with CS. Next, the question of the relation between the A and P boundaries of Col expression and positions of L3 and L4 veins versus proveins was addressed (Crozatier, 2003).

The current view is that vein primordia form at borders between adjacent A/P sectors of gene expression. According to this view, and based on col expression and requirement in cells along the A/P compartment boundary, the L4 and L3 vein primordia are predicted to edge the Col-expressing domain. It was observed, however, that in late 3rd instar wing discs, the col expression domain is not directly flanked by rho-expressing cells, but is separated from them by one to two rows of cells expressing neither gene and expressing Dl. This led to an examination at an earlier stage. In mid-third instar larvae, the col and rho expressing domains are immediately adjacent to one another, suggesting that the vein-centered-over-provein pattern is established secondarily as the disc continues to grow in size due to cell proliferation. Contrary to wild-type, in N55/+ mutant discs, col and rho expression domains remain juxtaposed, correlating with an increased number of rows of rho-expressing cells. These observations indicate that Notch signaling is involved in restricting rho expression and EGFR signaling to single rows of cells at the center of provein domains, probably via lateral inhibition. This process therefore operates already in mid-third instar larvae and results in a displacement of one to two cells between the positions of L3 and L4 vein primordia and the boundaries of Col expression. This displacement offers an explanation for the observation that the adult L4 vein is separated by several rows of intervein cells from the A/P compartment boundary. Although rho expression at the center of proveins in late third instar larvae likely prefigures the position of adult veins, the provein into vein and intervein resolution process can be initiated or, conversely abort later during pupal development, as shown by analysis of various mutants including Nts mutants. While consonant with the view that different A/P boundaries of prepattern gene expression in the wing primordium define the positions where provein domains are specified, the data do not support the suggestion that secondary short-range signals organize proveins around vein primordia. They rather support a sequential induction mechanism in which activation of the EGFR pathway defines vein-competent groups of cells in early 3rd instar larvae as well as promote the expression of Dl; in turn, Dl activation initiates lateral inhibitory signaling and restricts EGFR signaling to cells at the center of vein-competent domains, through a feed-back regulatory loop requiring Notch. This mechanism is consistent with the loss of the L3 and L4 stripes of Dl expression in rho^ve vn¹ (vn, an EGFR ligand) mutants, indicating that Dl expression is dependent on EGF-R signaling. A similar EGFR-->Dl sequential induction model has recently been proposed to operate in differentiating photoreceptor cells in the developing eye of Drosophila. In conclusion, these observations highlight the importance of cross-talk between the Hh and N signaling pathways in assigning overlapping A/P positions to the L3 vein and associated sensory organs and the role of N in precisely positioning vein primordia, thus intimately linking prepattern to the vein resolution process (Crozatier, 2003).

Targets of Activity

The secreted Hedgehog (Hh) proteins have been implicated as mediators of positional information in vertebrates and invertebrates. A gradient of Hh activity contributes to antero-posterior (A/P) patterning of the fly wing. In addition to inducing localised expression of Decapentaplegic (Dpp), which in turn relays patterning cues at long range, Hh directly patterns the central region of the wing. Short-range, dose-dependent Hh activity is mediated by activation of the transcription factor Collier (Col). In the absence of col activity, longitudinal veins 3 and 4 (L3 and L4) are apposed and the central intervein is missing. Hh expression induces col expression in a narrow stripe of cells along the A/P boundary through a dual-input mechanism: inhibition of proteolysis of Cubitus-interruptus (Ci) and activation of the Fused (Fu) kinase. Col, in cooperation with Ci, controls the formation of the central intervein by activating the expression of blistered (bs), which encodes the Drosophila serum response factor (D-SRF). D-SRF activity is required for the adoption and maintenance of the intervein cell fate. Furthermore, col is allelic to knot, a gene involved in the formation of the central part of the wing. This finding completes an understanding of the sectorial organization of the Drosophila wing. It is concluded that Col, the Drosophila member of the COE family (Col/Olf-1/EBF) of non-basic, helix-loop-helix (HLH)-containing transcription factors, is a mediator of the short-range organizing activity of Hh in the Drosophila wing. These results support the idea that Hh controls target gene expression in a concentration-dependent manner and highlight the importance of the Fu kinase in this differential regulation. The high degree of evolutionary conservation of the COE proteins and the diversity of developmental processes controlled by Hh signaling raises the possibility that the specific genetic interactions depicted here may also operate in vertebrates (Vervoort, 1999).

Because kn encodes a transcription factor, it was of interest to determine whether kn is required for the proper regulation of segment-specific homeotic genes. Notably, kn mutant embryos (zygotic + maternal) show an altered expression of cnc in the early embryo. At early gastrulation, the mandibular stripe of cnc expression is approximately one cell narrower than normal. In stage 10, cnc expression is normally found throughout the anterior compartment of the mandibular segment. However, in kn mutant embryos at stage 10, mandibular cnc expression is restricted to the posterior portion of the anterior compartment of the mandibular segment (which later gives rise to the mandibular lobe) and absent from the anterior-most portion. In wild-type stage 12 embryos cnc is found in both the hypopharyngeal and mandibular lobes. In kn mutant embryos at stage 12, mandibular cnc expression is restricted to the mandibular lobes, while the hypopharyngeal lobes fail to form. Unfortunately, because the cells that normally form the hypopharyngeal lobe are no longer marked with cnc in the kn mutant embryos, it is not possible to determine their new, alternative fate. This early alteration of the homeotic gene cnc expression, prior to the manifestation of the hypopharyngeal lobe, indicates that kn functions in establishing the cell fate of hypopharyngeal lobe. In contrast, no alteration in the expression of the two HOX genes, lab and Dfd, expressed in flanking ectodermal regions was observed. In addition, no altered expression was found in the mandibular region of kn mutant embryos of kn itself prior to stage 11, when mandibular-specific expression of kn ceases and kn becomes expressed in a segmentally reiterated pattern (Seecoomar, 2000).

While the predominant source of mesoderm in the early embryo derives from the ventral furrow invagination, ingressing cells from the cephalic furrow give rise to at least part of the cephalic mesoderm. In early embryogenesis, kn expression is restricted ventrally and does not overlap the ventral furrow. In stage 11, kn expression is observed in the cephalic mesoderm and presumably derives from cells ingressing in the cephalic furrow. The expression pattern of the mRNA corresponding to the CK01299 EST, with significant similarity to mammalian IP3 5-phosphatase, appears to specifically mark the cephalic mesoderm in the early embryo. CK01299 expression, first detected in the cephalic furrow during stage 8, is restricted to the cephalic mesoderm during stages 10 and 11, and disappears in early stage 12. In kn mutant embryos (zygotic + maternal), no CK01299-related expression is observed indicating that kn activity is required for at least this aspect of proper cephalic mesoderm activity (Seecoomar, 2000).

Hedgehog (Hh) signaling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely unknown. The role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary (the AP organizer in the imaginal wing disc) has been examined. col mutant wings are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. These phenotypes are linked to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation, the EGFR ligand Vein and the intervein determination gene blistered. Attenuation of Dpp signaling in the AP organizer is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signaling pathways in the wing AP organizer is presented (Crozatier, 2002).

The observation that col¹/kn¹ wings display both an intervein L2-L3 of normal size and a reduced L3-L4 intervein suggests that the loss of the L3-L4 intervein and the displacement of L3 vein observed in the col¹ mutant are not necessarily linked. The specific loss or reduction of the L3-L4 intervein territory in col adult wings could be due to local programmed cell death and/or reduced cell proliferation. To distinguish between these possibilities, wild-type and col¹ pupal wing discs were stained with a probes for reaper (rp), a marker for apoptotic cells, and an antibody against a phosphorylated form of histone H3 (H3P), which reveals mitosis. No difference in rp expression was detected between wild-type and col¹ mutant wings, which excluded a major contribution from apoptosis and suggested instead defective proliferation. In wild-type pupal wings, a wave of mitosis takes place in each intervein primordium between 15 and 21 hours APF. H3P staining of wild-type pupal wings 18-20 hours APF allowed the visualization of the proliferation of intervein cells along the proveins. Staining was specifically absent from the central region of either col¹ or col¹/kn¹ mutant wings, correlating with changes from intervein to provein fate. However, the reduced size of the medial region of the wing already detectable at this early pupal stage probably reflects an earlier proliferation defect. Double staining with Blistered and propidium iodide (PI), which labels all nuclei, enabled counting the number of rows of cells that separate the L2-L3 and L4-L5 intervein primordia in wild-type and col¹ larval discs. Ten independent measurements gave average numbers of 10.5 and 7.5 rows, respectively, indicating a significant reduction in col¹ mutants. The expressions of extramacrochaetae (emc), which encodes a helix-loop-helix (HLH) protein lacking a basic motif, and Notch (N), were examined because both genes have been shown to be involved in the control of cell proliferation in the wing. In third instar larvae, emc is expressed at a low level throughout the wing disc and at a higher level in two stripes of cells corresponding to the prospective A margin and the AP organizer. Unmodified at the A margin, emc expression is completely lost from the AP organizer cells in either col¹ or col¹/kn¹ mutant discs, showing that Col is required for emc transcription in the L3-L4 intervein primordium. Levels of N protein are high in intervein regions and low in presumptive vein territories in late third instar. In col¹ mutants, N is downregulated in the L3m provein domain. col requirement for emc and N upregulation in the AP organizer cells is consistent with the reduced cell number in the central region of col¹ mutant discs (Crozatier, 2002).

An unanticipated intricacy of the independent, versus Dpp-mediated, Hh patterning activity in the wing arose from comparison of the gradient of Dpp activity with dpp expression. It revealed that Dpp signaling is downregulated in the AP organizer and upregulated in both A and P flanking cells. This downregulation of Dpp activity was shown to result from the localized transcriptional repression of tkv, which is itself due to activation of the transcriptional repressor Master of thick vein (Mtv: Scribbler) in response to Hh signaling. Col activity is required for the downregulation of Dpp signaling in the AP organizer and this regulation is linked to Col requirement for positioning L3 vein and formation of L4 vein. The relative functions of these two genes remain to be examined in detail, although the observation that mtv transcription is downregulated in col mutant discs and tkv is upregulated in clones of col mutant cells suggests that col may act upstream of mtv in the regulatory cascade, thereby attenuating Dpp signaling in the AP organizer (Crozatier, 2002).

It has been been proposed that Hh does directly control the position of L3 vein, although the molecular mechanisms of this control have not been firmly established. In both col and mtv mutant clones, the position of L3 vein is shifted posteriorwards. That both col and mtv control the position of L3 vein suggests that this position is defined by Hh signaling through the modulation of Dpp signaling. iro is required for rho activation in the L3 primordium and formation of L3 vein. iro is activated by both Dpp and Hh signaling and its anterior border of expression is under control of sal/salr, a target of Dpp. The patterns of col, iro and rho expression are intimately connected. Both an increased number of cells expressing rho and a posterior shift of the anterior border of iro expression are observed in col¹ mutant discs. This posterior shift is interpreted as reflecting a modified range of Dpp signaling relayed, at least in part, by sal/salr activity. The increased number of rho-expressing cells, for its part, indicates that Col is able to antagonize rho activation by iro in cells, which express both iro and Col. This correlates well with the wing phenotype – anteriorwards shift of the L3 vein, together with gaps in its distal region – which results from anterior extension of Col expression, in UAS-Col/dpp-Gal4 wing discs. The distal gaps could reflect the complete absence of rho expression close to the DV border, because of the complete overlap between col and iro expression where iro expression is narrower. From col loss- and gain-of function experiments, it is therefore concluded that the primordium of L3 vein corresponds to cells that express iro but not col. Col thus appears to play a dual role in defining the position and width of L3 vein: activating Blistered and repressing EGFR in the wing AP organizer cells, endows these cells with an intervein fate, while attenuating Dpp signaling indirectly positions the anterior limit of iro expression domain, and L3 vein competence anterior to the AP organizer (Crozatier, 2002).

Col regulates vn transcription in the AP organizer. This expression of vn is required for formation of L4 vein. The loss of this vein in col¹ mutants could not, however, be rescued by expressing high level of Vn in the AP organizer, suggesting that a second signal dependent upon Col is also required. The specific loss of L4 vein is observed in conditions of reduced levels of Dpp signaling caused by ubiquitous expression of either Tkv or TkvDN. Together with this, the col¹ wing phenotype and Col requirement for downregulation of Dpp signaling in the AP organizer suggests a role for Dpp signaling in formation of L4 vein. Indeed, expressing a dominant-negative form of Tkv (TkvDN) in the AP organizer results in L4 loss. At first sight, it may appear contradictory that either upregulation (in col mutants) or downregulation (by expressing TkvDN) of Dpp signaling in the AP organizer leads to the preferential loss of posterior L4 vein. In both cases, however, there is increased sequestering of Dpp in the AP organizer, which limits its range of diffusion and signaling in posterior cells. Therefore attenuation of Dpp signaling in the AP organizer and increased signaling in posterior flanking cells appears to be required in addition to Vn activity for formation of L4 vein. By modulating Dpp signaling and vn transcription in cells receiving high doses of Hh, Col thus links Hh short-range activity to both positioning of the anterior L3 vein and formation of the posterior L4 vein (Crozatier, 2002).

In both null and hypomorphic col mutants, the wing blades are 20% smaller than wild type, owing to a reduced number of cells, both in the L3-L4 intervein and the posterior compartment. Reduced proliferation in the posterior compartment is a cell non-autonomous effect that is probably linked to the decreased range of Dpp diffusion that results from the upregulation of Tkv in the AP organizer cells. By contrast, the reduced size or the lack of L3-L4 intervein in col¹/kn¹ and col¹ mutants, respectively, is an independent defect since Dpp signaling is up- rather than down-regulated in this region. Defective cell proliferation in the central region of col wings in pupae could be linked to Col requirement for upregulation of emc and N, previously shown to be required for cell proliferation. Thus, the size reduction of col¹ mutant wings therefore reflects the cumulative effect of changes in Dpp signaling and decreased expression of emc and/or N in the AP organizer (Crozatier, 2002).

Col was initially characterised for its function in the segmentation of the embryonic head, acting upstream of Hh in this process. Its key role in mediating short-range Hh activity in the wing was therefore unanticipated. Col role in the wing illustrates the pivotal importance of tissue-specific mediators of Hh and Hh-->Dpp signaling pathways in patterning different appendages. The concentration of Shh is the primary determinant of AP polarity in the vertebrate limb. In this case, as in Drosophila imaginal discs, a signaling relay involving Bmps is implicated. Col/Kn is the single Drosophila member of the family of COE transcription activators that includes products of the vertebrate ebf/olf-1/coe genes and C. elegans unc-3. It remains to be investigated whether Col function in mediating tissue-specific interpretation of Hh signaling in Drosophila has some equivalent in vertebrates or corresponds to the co-option of Col in a highly derived patterning process (Crozatier, 2002).

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