collier


REGULATION

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 knotSA2 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 knotSA2 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, knMel1-2330 (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 knMel1-2330 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 knMel1-2330 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 knMel701-1991 construct was capable of driving expression in a stripe in the wing, whereas the knMel224-1426 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 knMel701-1991 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 knMel701-1991 element (Hersh, 2005).

Because the knMel701-1991 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 knMel701-1991 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 knMel701-1991 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 (knMel701-1835), caused the reporter to be expressed at the AP compartment boundary in both the wing and the haltere. Therefore, knMel701-1991 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 knMel701-1991, suggesting that additional repressor binding sites important for appropriate wing expression may have been removed in knMel701-1835. 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 (knMel1599-1991) 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 knMel701-1991, 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 knMel701-1835 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 (knMel701-1991Ubx1KO) 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 knMel701-1991 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 knMel701-1991, and this construct (knMel701-1991KO) 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, knMel701-1835. 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 knMel701-1835 construct was restored. Addition of 43 bp (knMel701-1878) 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 (knMel701-1991Delta) resulted in very weak, inconsistent de-repression in the haltere. By contrast, point mutations introduced at positions 1834-1837 (knMel701-1991mut), the boundary of the derepressed knMel701-1835 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 (knMel701-1991KOmut) 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 knMel1835-1991. The DNA sequence at knMel1834-1837 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 (knPse1-1935) that roughly corresponded to the knMel1-2330 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 knPse1-1935 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 knPse1-1935 was generated that eliminated the TAAT core sequences. This knPse1-1643 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 knMel1835-1991 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 knMel701-1835 construct. Addition of 222 nucleotides (knMel2499-2722), homologous to the D. pseudoobscura sequence necessary for repression, to knMel701-1835 (to generate knMelcomposite) restored repression in the haltere. Therefore, D. pseudoobscura has a single element (located between knPse1643-1935) 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 knMel1835-1991), 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 knMel2499-2722 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 knMel2499-2722KO sequence was attached to the de-repressed knMel701-1835 (generating knMelcompositeKO). Whereas mutation of Ubx sites alone in knMel701-1991KO did not fully de-repress in the haltere, mutation of Ubx sites in knMelcompositeKO was sufficient for complete de-repression in the haltere. Thus, the knMel2499-2722 and knMel1835-1991 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 knPse1643-1935, but also possess sequence similar to knMel1835-1991 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.3.4clvir corresponds to D. mel P2.6cl, whereas P3.4-1.3clvir 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).

Multi-step control of muscle diversity by Hox proteins in the Drosophila embryo

Hox transcription factors control many aspects of animal morphogenetic diversity. The segmental pattern of Drosophila larval muscles shows stereotyped variations along the anteroposterior body axis. Each muscle is seeded by a founder cell and the properties specific to each muscle reflect the expression by each founder cell of a specific combination of 'identity' transcription factors. Founder cells originate from asymmetric division of progenitor cells specified at fixed positions. Using the dorsal DA3 muscle lineage as a paradigm, this study shows that Hox proteins play a decisive role in establishing the pattern of Drosophila muscles by controlling the expression of identity transcription factors, such as Nautilus and Collier (Col), at the progenitor stage. High-resolution analysis, using newly designed intron-containing reporter genes to detect primary transcripts, shows that the progenitor stage is the key step at which segment-specific information carried by Hox proteins is superimposed on intrasegmental positional information. Differential control of col transcription by the Antennapedia and Ultrabithorax/Abdominal-A paralogs is mediated by separate cis-regulatory modules (CRMs). Hox proteins also control the segment-specific number of myoblasts allocated to the DA3 muscle. It is concluded that Hox proteins both regulate and contribute to the combinatorial code of transcription factors that specify muscle identity and act at several steps during the muscle-specification process to generate muscle diversity (Enriquez, 2010).

Eve expression in the DA1 muscle lineage provided the first paradigm for studying the early steps of muscle specification. Detailed characterization of an eve muscle CRM showed that positional and tissue-specific information were directly integrated at the level of CRMs via the binding of multiple transcription factors, including dTCF, Mad, Pnt, Tin and Twi. Based on this transcription factor code and using the ModuleFinder computational approach, this study has identified a CRM, CRM276, that precisely reproduces the early phase of col transcription. This CRM also drove expression in cells of the lymph gland, another organ that is issued from the dorsal mesoderm where col is expressed. Parallel to this study, two col genomic fragments were selectively retrieved in chromatin immunoprecipitation (ChIP-on-chip) experiments designed to identify in vivo binding sites for Twi, Tin or Mef2 in early embryos. One fragment overlaps with CRM276. Based on this overlap and interspecies sequence conservation, a 1.4 kb subfragment of CRM276 that retained most of the transcription factor binding sites identified by ModuleFinder was tested, and it was found to specifically reproduced promuscular col expression. This in vivo validation shows that intersecting computational predictions and ChIP-on-chip data should provide a very efficient approach to identify functional CRMs on a genome-wide scale (Enriquez, 2010).

The eve and col early mesodermal CRMs are activated at distinct A/P and D/V positions. It is now possible to undertake a comparison of these two CRMs, in terms of the number and relative spacing of common activator and repressor sites and their expanded combinatorial code, in order to understand how different mesodermal cis elements perform a specific interpretation of positional information (Enriquez, 2010).

A progenitor is selected from the Col promuscular cluster in T2 and T3 but not T1. One cell issued from the Col-expressing promuscular cluster in T1 nevertheless shows transiently enhanced Col expression, suggesting that the generic process of progenitor selection is correctly initiated in T1. This process aborts, however, in the absence of a Hox input, as shown by the loss of progenitor Col expression and DA3 muscle in specific segments in Hox mutants. The similar changes in Nau and Col expression observed under Hox gain-of-function conditions leads to the conclusion that the expression of 'identity' transcription factor iTFs is regulated by Hox factors at the progenitor stage. The superimposition of Hox information onto the intrasegmental information thereby implements the iTF code in a segment-specific manner and establishes the final muscle pattern. Unlike DA3, a number of specific muscles are found in both T1 and T2-A7, such as the Eve-expressing DA1 muscle; other muscles form in either abdominal or thoracic segments, as illustrated by the pattern of Nau expression in stage 16 embryos. This diversity in segment-specific patterns indicates that Hox regulation of iTF expression is iTF and/or progenitor specific (Enriquez, 2010).

As early as 1994, Hox proteins were proposed to regulate the segment-specific expression of iTFs. Seven years later, an apterous mesodermal enhancer (apME680) active in the LT1-4 muscles was characterized and itwas proposed that regulation by Antp was direct. However, mutation of the predicted Antp binding sites present in apME680 abolished its activity also in A segments, suggesting that some of the same sites were bound by Ubx/AbdA. Evidence is now available that the regulation of col expression by Ubx/AbdA in muscle progenitors is direct and involves a single Hox binding site. However, regulation by Antp does require other cis elements. It remains to be seen whether regulation by Antp is also direct. Since Antp, Ubx and AbdA display indistinguishable DNA-binding preferences in vitro, the modular regulation of col expression by different Hox paralogs suggests that other cis elements and/or Hox collaborators contribute to Hox specificity. Direct regulation of col by Ubx has previously been documented in another cellular context, that of the larval imaginal haltere disc, via a wing-specific enhancer. In this case, Ubx directly represses col expression by binding to several sites, contrasting with col-positive regulation via a single site in muscle progenitors. This is the second example, in addition to CG13222 regulation in the haltere disc, of direct positive regulation by Ubx via a single binding site. Hox 'selector' proteins collaborate on some cis elements with 'effector' transcription factors that are downstream of cell-cell signaling pathways. In the DA3 lineage, it seems that Dpp, Wg and Ras signaling act on one col cis element and the Hox proteins on others. The regulation of col expression by Hox proteins in different tissues via different CRMs provides a new paradigm to decipher how different Hox paralogs cooperate and/or collaborate with tissue- and lineage-specific factors to specify cellular identity (Enriquez, 2010).

The DA3 muscle displays fewer nuclei in T2 and T3 than in A1-A7, an opposite situation to that described for an aggregate of the four LT1-4 muscles. It has been proposed that the variation in the number of LT1-4 nuclei was controlled by Hox proteins. These studies of the DA3 muscle extend this conclusion by showing that the variations due to Hox control are specific to each muscle and are exerted at the level of FCs. Since the number of nuclei is both muscle- and segment-specific, Hox proteins must cooperate and/or collaborate with various iTFs to differentially regulate the nucleus-counting process. As such, Hox proteins contribute to the combinatorial code of muscle identity. Identifying the nature of the cellular events and genes that act downstream of the iTF/Hox combinatorial code and that are involved in the nucleus-counting process represents a new challenge (Enriquez, 2010).

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 rhove 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).

The role of Parafibromin/Hyrax as a nuclear Gli/Ci-interacting protein in Hedgehog target gene control: Genetic impairment of hyx decreased the expression of the high-threshold Hh target gene knot/collier

The Hedgehog (Hh) pathway, an evolutionarily conserved key regulator of embryonic patterning and tissue homeostasis, controls its target genes by managing the processing and activities of the Gli/Ci transcription factors. Little is known about the nuclear co-factors the Gli/Ci proteins recruit, and how they mechanistically control Hh target genes. This study provides evidence for the involvement of Parafibromin/Hyx as a positive component in Hh signaling. hyx RNAi impaired Hh pathway activity in Drosophila cell culture. Consistent with an evolutionarily conserved function in Hh signaling, RNAi-mediated knockdown of Parafibromin in mammalian cell culture experiments diminished the transcriptional activity of Gli1 and Gli2. In vivo, in Drosophila, genetic impairment of hyx decreased the expression of the high-threshold Hh target gene knot/collier. Conversely, hyx overexpression ameliorated the inhibitory activity of Ptc and Ci(75) misexpression during Drosophila wing development. It was subsequently found that Parafibromin can form a complex with all three Glis, and evidence is provided that Parafibromin/Hyx directly binds Region 1, the Su(fu) interaction domain, in the N-terminus of all Glis and Ci. Taken together, these results suggest a target gene-selective involvement of the PAF1 complex (see Drosophila Paf1) in Hh signaling via the Parafibromin/Hyx-mediated recruitment to Gli/Ci (Mosimann, 2009).

knot encodes a Col/Olf-1/EBF (COE) family helix-loop-helix-containing transcription factor controlling specification of the intervein region between L3 and L4. Compared to dpp, ptc, and en, the Hh-dependent transcriptional regulation of kn is less well analyzed. kn can be induced in the normally Ci-devoid P-compartment by Ci155 misexpression. Additionally, cells with amorphic PKA alleles in the wing pouch upregulate kn cell-autonomously when not situated close to the D/V compartment boundary, while ptc loss of function clones induce kn expression irrespective of their location in the anterior wing pouch. Unfortunately, these findings do not allow the drawing of an unambiguous picture of kn control by Ci at the A/P boundary, as no study involved complete ci loss of function. However, in the developing wing pouch, the expression of kn is clearly induced by highest Hh output. The selective effect of Hyx impairment on kn expression suggests that Hyx is a context-dependent co-factor of Ci required for selective target genes (Mosimann, 2009).

One potential caveat is that the hypomorphic allele hyxEY6895, which was used in most of the experiments, does not reduce Hyx levels sufficiently to detectably affect expression of lower threshold targets such as ptc or dpp. Arguing against this is that ptc expression was also not affected in a stronger hyx loss of function situation using the hyx2 allele (Mosimann, 2009).

Interestingly, Hyx is not the first reported seemingly target gene-restricted Ci co-factor. The Mediator complex subunits Skuld (Skd) and Kohtalo (Kto) are involved in the control of cell affinity-regulating genes by Ci155, yet not ptc and dpp transcription (Janody, 2003). It remains to be seen if Skd or Kto are also involved in kn control and if they directly interact with Ci. In contrast, the histone acetyl-transferase CBP is required for ptc expression and has been suggested to be an obligate Ci155 partner, but in-depth genetic analysis is hampered by its broad involvement as general transcriptional co-factor (Akimaru, 1997). Together with these results, the current findings strongly suggest that during development, Ci155 assembles differential sets of co-factors dependent on the respective target gene context (Mosimann, 2009).

When Hyx overexpression was analyzed in genetic systems sensitized for Hh signaling, it was found that Hyx partially counter-acts the strong effect caused by ptc misexpression on the developing wing. Anticipating a nuclear function together with Ci, an effect on phenotypes mediated by direct Ci overexpression was subsequently assayed. It was found that Hyx severely attenuates the effects of Ci75 overproduction, but has no effect on overexpressed CiPKA or overexpressed wild-type Ci, which only shows transactivating activity upon Hh input (Mosimann, 2009).

This finding is interpreted as an indication that overexpressed Hyx dominant-negatively interferes with the repressive activity of overexpressed Ci75. Surplus Ci75 may act primarily by occupying the promoters of Hh target genes, and overexpressed Hyx interferes with this property. In contrast, in a wild-type situation the negative activity of endogenous Ci75 may be mediated by the binding of repressive co-factors. This binding is not effectively competed off by additional Hyx, explaining the lack of a detectable effect in a wild-type background (Mosimann, 2009).

Region 1 of Ci/Gli has never revealed any autonomous transactivation potential when tethered to DNA, in contrast to C-terminal Gli fragments. Parafibromin/Hyx binding to Region 1 would not necessarily stimulate transcription on its own, as DNA-tethered Hyx shows no detectable transactivation effect, suggesting that it is not sufficient for triggering RNAPII-mediated transcription. Instead, in agreement with these results, the recruitment of Hyx to Hh target genes by binding to Region 1 probably helps to ensure efficient reoccurring transcription. This function might be particularly important for certain genes induced at high Hh levels and might involve particular chromatin modifications dependent on the PAF1 complex (Mosimann, 2009).

Region 1 is also the minimal interaction site for Su(fu). While competitive Su(fu) binding is an intriguing possibility, the idea of consecutive binding is favored since Parafibromin/Hyx appears to be principally required for high signal output -- conditions under which, due to Fu action, Su(fu) binding is believed not to occur. Su(fu) plays a critical negative regulatory role in the Hh pathway, especially in mammals. How this factor functions is unclear, but it may regulate Gli processing, act as a co-repressor, and/or regulate Gli/Ci localization. The finding that positive and negative regulators bind to Region 1 may explain why its deletion in Ci only had a minor effect (Mosimann, 2009).

In Wnt/Wg signaling, Parafibromin/Hyx seems to participate in a sequence of co-factor exchanges that occurs on β-catenin/Armadillo. This potentially reflects the need for priming chromatin remodeling steps before PAF1 complex function. Interestingly, β-catenin/Arm has overlapping binding sites for its co-activators such as CBP, Brg-1/Brahma (Brm), and Parafibromin/Hyx. This contrasts with Gli/Ci, on which Parafibromin/Hyx occupies a different binding site than CBP. Gli/Ci therefore could organize multiple recruitment steps for auxiliary components via separate domains rather than solely by sequential binding (Mosimann, 2009).

Considering the impact of Hyx impairment on the analyzed Hh target genes in vivo, combined with the overexpression data and RNAi results, it is predicted that Parafibromin/Hyx is a factor involved in maximal Gli/Ci target gene induction. Parafibromin/Hyx, as part of the PAF1 complex, could implement efficient RNAPII control at Hh target genes when sustainable transcriptional induction is needed. On other targets, such as ptc, this process might be redundant with other ways to guide RNAPII. One possibility could be recruitment of the PAF1 complex by a module other than Parafibromin/Hyx, or potentially even via another transcription factor that binds in the vicinity of the Gli/Ci binding site (Mosimann, 2009).

Seven up acts as a temporal factor during two different stages of neuroblast 5-6 development

Drosophila embryonic neuroblasts generate different cell types at different time points. This is controlled by a temporal cascade of Hb->Kr->Pdm->Cas->Grh, which acts to dictate distinct competence windows sequentially. In addition, Seven up (Svp), a member of the nuclear hormone receptor family, acts early in the temporal cascade, to ensure the transition from Hb to Kr, and has been referred to as a 'switching factor'. However, Svp is also expressed in a second wave within the developing CNS, but here, the possible role of Svp has not been previously addressed. In a genetic screen for mutants affecting the last-born cell in the embryonic NB5-6T lineage, the Ap4/FMRFamide neuron, a novel allele of svp was isolated. Expression analysis shows that Svp is expressed in two distinct pulses in NB5-6T, and mutant analysis reveals that svp plays two distinct roles. In the first pulse, svp acts to ensure proper downregulation of Hb. In the second pulse, which occurs in a Cas/Grh double-positive window, svp acts to ensure proper sub-division of this window. These studies show that a temporal factor may play dual roles, acting at two different stages during the development of one neural lineage (Benito-Sipos, 2011).

This study has found that Svp is expressed in two pulses and plays two different roles in the NB5-6T lineage. Initially, Svp is expressed briefly in the early part of this lineage, where it acts to control the downregulation of the first temporal factor, Hb. Subsequently, Svp is expressed in the late part of this lineage, in the Ap window, in a highly dynamic fashion: initiated in all four Ap neurons, to be downregulated in the first- and last-born Ap cells. In the second expression phase, Svp acts to suppress Col and Dimm, thereby preventing the first-born Ap neuron fate, Ap1/Nplp1, from being established in the subsequently born Ap2 and Ap3 neurons. Misexpression studies further indicate that Svp also suppresses the last-born Ap neuron fate, Ap4/FMRFa, from being established in Ap2/3 (Benito-Sipos, 2011).

Previous studies of Svp demonstrated that it is expressed in a brief pulse in the majority of early embryonic neuroblasts, where it acts to suppress Hb, thereby allowing for the switch to the next stage of temporal competence. Recently, studies have identified additional factors involved in the downregulation of Hb: the pipsqueak-domain proteins Distal antenna and Distal antenna-related (herein referred to collectively as 'Dan'). Dan is expressed somewhat earlier than Svp, and is also maintained in a longer pulse. svp and dan do not regulate each other, and although they can be activated by ectopic hb expression, neither Svp nor Dan expression is lost in hb mutants. This raises the intriguing questions of how Svp and Dan are activated during early stages of lineage progression, and how they become downregulated at the appropriate stage (Benito-Sipos, 2011).

Another interesting complexity with respect to Svp expression and function pertains to the fact that the Hb window is of different size in different lineages. For example, in NB6-4T and NB7-3, Hb is downregulated in the neuroblast immediately after the first division, whereas in NB5-6T, Hb expression is evident during three divisions. In line with this, no Svp expression is observed in NB5-6T until stage 10, when the neuroblast has already gone through two rounds of division. How the on- and offset of Svp, and perhaps Dan, expression is matched to the specific lineage progression of each unique neuroblast lineage, to thereby allow for differing Hb window sizes, is an interesting topic for future studies (Benito-Sipos, 2011).

Svp is re-expressed in the NB5-6T lineage in a second pulse. In contrast to the early pulse of Svp expression, where there is no evidence for temporal genes controlling Svp, it was found that the second pulse of Svp expression is regulated by the temporal genes cas and grh. However, it was not found that svp is important for the expression of Cas or Grh. Instead, svp participates in the sub-division of the Cas/Grh temporal window, i.e. the Ap window. Based upon the idea that Svp is regulated by temporal genes, and acts to sub-divide a broader temporal window, it could be referred to as a 'sub-temporal' factor in the latter part of the NB5-6T lineage (Benito-Sipos, 2011).

The expression of Svp is dynamic also in the second pulse of expression, commencing in the neuroblast at stage 14 -- after the three first Ap neurons are born -- and being maintained in the neuroblast until it exits the cell cycle at stage 15. At late stage 14 and 15, Svp expression becomes evident in all four Ap neurons, but it is rapidly downregulated from Ap1 and Ap4 during stages 16 and 17. Svp is, however, maintained in the Ap2 and Ap3 neurons into late embryogenesis. The role of svp in the Ap window appears to be to ensure proper specification of the Ap2/3 interneurons, generated in the middle of the Ap window. This is achieved by svp suppressing the first- and last-born Ap neuron fates: the Ap1/Nplp1 and Ap4/FMRFa fates. With regard to the suppression of the Ap1 fate, one important role for svp is to suppress Col expression in Ap2/3. Importantly, the temporal delay in Svp expression when compared to Col -- commencing two stages after Col in the Ap neurons -- allows for col to play its critical early role in Ap neuron specification: activating ap and eya. The timely suppression of Col in Ap2/3 is mediated also by sqz and nab, and the loss of Nab expression in svp mutants may be a contributing factor to the failure of Col downregulation in svp. However, the potent function of svp in suppressing Ap1/Nplp1 fate when misexpressed postmitotically from apGal4 does not appear to require Nab, as Nab is not ectopically expressed in these experiments. Thus, svp may act via several routes to prevent Ap1/Nplp1 fate from being established in the Ap2/3 cells: by suppressing Col and by activating Nab (Benito-Sipos, 2011).

Regarding the second role of svp in the Ap window -- the suppression of the Ap4/FMRFa fate -- it is less clear what the target(s) may be. However, a common denominator for both the Ap1/Nplp1 and the Ap4/FMRFa neurons is the expression of Dimm. Dimm, a basic-helix-loop-helix protein, is a critical determinant of the neuropeptidergic cell fate, and also controls high-level neuropeptide expression in many neuropeptide neurons. Both svp loss and gain of function results in robust effects upon Dimm expression in the NB5-6T lineage, indicating that Dimm is an important target for svp. However, dimm mutants show only reduced levels of FMRFa expression, and thus svp is likely to regulate additional targets to prevent the Ap4/FMRFa cell fate in the Ap2/3 neurons (Benito-Sipos, 2011).

Another interesting phenotype in svp mutants, pertaining to the second pulse of Svp expression in the NB5-6T lineage, is the finding of one to two extra Ap neurons. This indicates that the NB5-6T neuroblast undergoes one to two extra rounds of division, and that the expression of Svp in the neuroblast during stage age 14-16 is important for precise cell cycle exit. Interestingly, the other temporal (cas and grh) and sub-temporal (sqz and nab) genes acting in the latter part of the NB5-6T lineage also play roles in controlling cell cycle exit. Moreover, studies of neuroblast cell cycle exit in other neuroblasts, both embryonic and postembryonic, have also shown roles for grh and svp in these decisions. Thus, a picture is emerging in which late temporal and sub-temporal genes may be broadly involved in controlling timely cell cycle exit of many neuroblasts (Benito-Sipos, 2011).

The early role of svp, in its first expression pulse, is to suppress Hb expression. Svp is expressed transiently by most if not all neuroblasts, and the regulation of Hb also appears to be a global event. Similarly, the second pulse of Svp expression has been observed in many lineages, although the role for svp in this later pulse was hitherto unknown. The findings of a role for svp as a sub-temporal gene in the latter part of the NB5-6T lineage indicates that svp may play such roles in many lineages. However, it should be noted that global changes in Col, Dimm and Eya expression in the embryonic central nervous system (CNS) are not seen. Thus, unlike the more universal role of svp in regulating Hb during the first pulse, the putative sub-temporal function of the second pulse of svp expression in other lineages must be highly context-dependent and involve other targets (Benito-Sipos, 2011).

In mammals, the svp orthologues COUP-TFI and -II are expressed dynamically in the developing CNS. Functional studies reveal a number of important roles for COUP-TFI/II during nervous system development, and mutant mice display aberrant neuro- and gliogenesis, accompanied by axon pathfinding defects. Intriguingly, recent studies have revealed that COUP-TFI/II acts in a temporal manner to control the timing of generation of sub-classes of neurons and glia in the developing mouse brain. Given that the other genes described in this study are also conserved, it is tempting to speculate that temporal and sub-temporal cascades similar to those outlined in this study are also used in the mammalian CNS during development (Benito-Sipos, 2011).

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).

Second order regulator Collier directly controls intercalary-specific segment polarity gene expression

In Drosophila, trunk metamerization is established by a cascade of segmentation gene activities: the gap genes, the pair rule genes, and the segment polarity genes. In the anterior head, metamerization requires also gap-like genes and segment polarity genes. However, because the pair rule genes are not active in this part of the embryo, the question of which gene activities fulfill the role of the second order regulators still remains to be solved. This study provides first molecular evidence that the Helix-Loop-Helix-COE transcription factor Collier fulfills this role by directly activating the expression of the segment polarity gene hedgehog in the posterior part of the intercalary segment. Collier thereby occupies a newly identified binding site within an intercalary-specific cis-regulatory element. Moreover, a direct physical association has been identified between Collier and the basic-leucine-zipper transcription factor Cap'n'collar B, which seems to restrict the activating input of Collier to the posterior part of the intercalary segment and to lead to the attenuation of hedgehog expression in the intercalary lobes at later stages (Ntini, 2011b).

In the context of an analysis to identify cis-regulatory elements controlling expression of segment polarity genes in the embryonic head, an intercalary-specific cis-regulatory element of hhic-CRE—was isolated within the upstream 6.43 kb region (Ntini, 2011a). The ~ 1 kb enhancer fragment (− 4085 to − 3077 bp) mediates reporter expression in the hh expressing cells of the posterior part of the intercalary segment, when combined with the endogenous hh promoter (− 120 to + 99 bp;). Further functional dissection of this element showed that the 450 bp ?1mF5 subfragment (− 3914 to − 3465 bp) mediates the intercalary-specific expression with slightly delayed onset, while the 335 bp F5_R4 subfragment (− 3799 to − 3465 bp) constitutes the minimum sequence required for the intercalary expression, but mediates an additional spotty metameric pattern in the trunk (Ntini, 2011). Because a high degree of phylogenetic conservation in non-coding DNA sequence implicates a functional role in vivo, such as recognition and DNA-binding by sequence-specific transcription factors, the sequence of the ic-CRE was subjected to phylogenetic conservation analysis within the genome of twelve Drosophila species, and different in silico analyses were performed to detect putative transcription factor binding sites. The minimum 335 bp ic-CRE consists of six highly conserved sequence blocks. A series of complete block deletions designed in the context of the minimum ic-CRE in combination with the endogenous hh promoter resulted in non-functional elements. This could be either because individual binding motifs were disrupted or inter-motif distances crucial for transcription factor binding and operation were disturbed. A point mutagenesis screen was conducted in the context of the 450 bp ic-CRE to extract crucial cis-regulatory information in respect to the conserved in silico identified transcription factor binding sites (Ntini, 2011b).

The ic-CRE responds to the homeotic transformation of the mandibular into an intercalary segment resulting from ectopic ems expression by a duplication of its expression pattern. However, despite this and the fact that the Hox gene labial is active in the intercalary segment, disrupting the homeodomain binding sites in conserved sequence blocks III or IV by point mutations did not abolish the ic-CRE mediated reporter expression. In contrast, disrupting a putative binding site for the fork head transcription factor Sloppy paired 1 (Slp1) in block IV eliminated the ic-CRE-mediated reporter expression. This is consistent with the reduced reporter expression in an RNAi-mediated knock-down of slp1, which is a proposed head gap-like and pair rule segmentation gene (Ntini, 2011b).

Another in silico prediction was found in conserved block II at position − 3771 to − 3755 bp that scores the binding matrix of the mammalian COE factor Olf1. Disrupting this site by point-mutation resulted in the complete abolishment of the ic-CRE mediated reporter expression, indicating that the site is absolutely required for the function of the 450 bp ic-CRE. Olf1 is the mammalian COE homolog of Collier and the endogenous hh expression in the intercalary segment is abolished in a col loss-of-function mutant (col1. Likewise, the ic-CRE-mediated expression pattern is abolished in col1 or col knock-down. In addition, the DNA-binding domain of Collier displays a high degree of primary sequence identity (86%) to the mammalian homolog. High degree of primary sequence identity in the DNA-binding domain, shared among the members of the COE family allows for a similar DNA-binding specificity: both Collier and the Xenopus homologs recognize the mammalian DNA target sequences in vitro. Therefore, the Olf1 prediction identified in silico within the ic-CRE is regarded as a putative Collier binding site and referred to as a Collier recognition site (Ntini, 2011b).

Apart from this functionally required Collier recognition site at − 3773 to − 3751 bp, scanning in silico the 6.43 kb upstream hh enhancer using MatInspector with a similarity cut-off of 1, 0.8 (core, matrix) identifies one more Olf1 prediction within the ic-CRE at position − 3967 to − 3945 bp. The 6.43 kb upstream enhancer of hh was also submitted to rVISTA using the nucleotide positions 3–19 of the binding matrix of Olf1. When setting the highest possible similarity cut-off 0.95, 0.85 (core, matrix), so that at least one prediction is generated, then only the functionally required Collier recognition site CAATTCCCCAATGGCAT (at − 3771 to − 3755) within the ic-CRE is detected. Lowering the matrix similarity threshold by 0.05, using cut-off 0.95, 0.8, generates three additional predictions. These are two distant sites, GAGACACTTGGGATGAG at − 3963 to − 3947 and CACACCACGGGGAAGCG at − 2872 to − 2856, and one promoter-proximal site CACTTCCCTTGCGCATA at − 212 to − 196. The first distant site is within the ic-CRE, 190 bp upstream of the functionally required Collier recognition site, and is also predicted by the MatInspector. Interestingly, in contrast to the functionally required Collier recognition site within the ic-CRE, none of the other predicted sites are phylogenetically conserved among the twelve Drosophila species. Considering the displayed short-range homotypic clustering (within 200 bp), it is, however, possible that the weaker predictions may contribute to the transcriptional outcome of the ic-CRE, even though they might be recognized with minor affinity by Collier in vivo (Ntini, 2011b).

In order to verify that the in silico identified and functionally required Collier recognition site within the ic-CRE is indeed occupied by Collier in vivo, chromatin immunoprecipitations (ChIP) from Drosophila embryonic nuclear extracts were performed with an antibody against Collier. In the anti-Col ChIPs, the functionally required Collier binding site within the ic-CRE was specifically enriched in comparison to mock ChIPs, which indicates that the site is indeed occupied by Collier in vivo (Ntini, 2011b).

In the case of the mammalian COE homolog of Collier, it was previously deciphered that the mouse transcription factor EBF contains two distinct and functionally independent transcription activation domains, the second one within the C-terminal region. Although Drosophila Collier has been genetically implicated as an activator of downstream segment polarity gene expression, its transcriptional activation potential had not yet been analyzed. In Drosophila two Collier isoforms are expressed from the col gene locus. The cDNAs encoding Collier A (also termed Col2) and Collier B (Col1) differ from each other by 465 bp due to alternative splicing. The two protein isoforms share the same first 528 N-terminal amino acids and differ in the C-terminal 29 amino acids for Collier A and 47 amino acids for Collier B. No specific expression pattern of collier A could be detected by double in situ hybridization using an RNA probe specific for collier B and a probe that hybridizes with both transcripts (Ntini, 2011b).

Therefore the transcriptional activation potential of each of the two Collier isoforms was examined by reporter assays in Drosophila S2 R+ cell transfections. In the reporter construct the functionally required and in vivo occupied Collier site was cloned in a single copy upstream of the endogenous hh promoter (− 120 to + 99 bp) driving luciferase gene expression. Both Collier isoforms activate luciferase expression when independently co-transfected with the reporter construct, indicating that both isoforms possess transcriptional activation potential. A truncated form of ColA lacking the last 23 C-terminal amino acids (ColA 1–534) displays a significantly reduced activation potential (~ 84% decrease), which indicates that a transcriptional activation domain must reside within either C-terminal region of both isoforms. Disrupting the Collier recognition site by point mutations decreased the mediated reporter activation by ~ 48% in the case of Collier A and ~ 44% in the case of Collier B. Taking into consideration that disrupting the Collier binding site in the context of the ic-CRE resulted in a complete abolishment of the mediated reporter expression in vivo, and that the same mutation does not support Collier DNA-binding in vitro, it is assumed that part of the reporter activation assessed in cell transfection may be achieved by Collier transactivating via unknown system-provided DNA-binding activities on the regulatory sequences of the reporter plasmid. Moreover, Collier carries a perfect SUMOylation motif within the N-terminus, predicted with the highest threshold value. The protein sequence TSLKEEP at amino acid position 44-50 matches the SUMOylation motif. Additional members of the COE transcription factor family contain also a SUMOylation motif at this conserved position. Apart from antagonizing ubiquitin-mediated degradation, sumoylation has been implicated in modifying transcriptional activation/repression potential of transcription factors. Mutant versions of Collier A and Collier B where the K within the SUMOylation motif is mutated towards R (ColA RK and ColB RK) display reduced activation potential, implying a possible role for sumoylation in regulation of Collier transcriptional activity (Ntini, 2011b).

Data is presented consistent with the cap-n-collar isoform CncB performing as a sequestering factor or inhibitor of Collier DNA-binding to its cognate site found within the ic-CRE. Furthermore, fluorescent immunostaining revealed that only a small fraction of the expressed Collier protein is nuclear localized in vivo. Conversely, CncB protein greatly accumulates in the nuclei. Prediction of nuclear localization signals (NLS) in silico generates no results for Collier, while CncB contains an NLS within the bZIP domain (aa 617–680). Interestingly, Collier carries a perfect SUMOylation motif in the very N-terminus, predicted with the highest threshold value. Apart from antagonizing ubiquitin-mediated degradation and modifying transcriptional activation/repression potential of transcription factors, sumoylation has also been implicated in protein nucleo-cytoplasmic translocation. Alternatively, in the absence of a nuclear localization signal, Collier import in the nucleus may be realized by heterodimerization with a protein that carries an NLS. This would increase the probability that Collier is recruited into combinatorial control mechanisms, which has already been implicated in muscle specification. Furthermore, nuclear accumulation of CncB, in converse to a relatively low concentration of nuclear Collier protein, indicated by the fluorescent immunostainings, may facilitate the sequestering function of CncB to antagonize and overcome the DNA-binding activity of Collier on the ic-CRE in the cells of the anterior most part of the mandibular segment during the establishment of procephalic hh expression, and at later stages in the hh expressing cells of the intercalary lobes (Ntini, 2011b).

In this respect it is interesting to note that despite the intrinsic transcriptional activation properties of the Cnc homologs, CncB acts to suppress both the expression and the homeotic selector (maxillary structures promoting) function of Deformed (Dfd) in the mandibular segment. In particular, CncB represses the maintenance phase of Dfd transcription in the mandibular cells, most probably by interfering with the positive regulatory function of Deformed within the Dfd autoactivation circuit. Overexpression of CncB partially represses Dfd-responsive transcriptional target elements in vivo. Interestingly, interaction between CncB and Dfd proteins has been reported. Perhaps the negative regulation of Dfd expression and function caused by CncB results from CncB interfering with Dfd binding to its cognate target cis-regulatory elements in vivo, as a consequence of a direct physical interaction at protein level with a sequestering effect similar to the interaction with Collier reported in this study (Ntini, 2011b).

The isolation of an intercalary-specific cis-regulatory element from the hh upstream region supports a unique mode for anterior head segment-specific transcriptional control of segment polarity gene expression. Thus, not only cross-regulatory interactions among segment polarity genes during the maintenance phase, but also the initial establishment of procephalic segment polarity gene expression seems to be unique for each of the anterior head segments. The previously proposed mode of second order regulation in anterior head patterning, resulting in activation of hh in the posterior part of the intercalary segment, is mediated by the HLH-COE factor Collier evidently via direct DNA binding. The reported physical interaction between Collier and CncB is likely to attenuate the activating function of Collier in the hh expressing cells of the posterior part of the intercalary segment at a later developmental stage, and it might also be involved in eliminating the potential of target activation by Collier in the anterior most part of the mandibular segment where the two factors are co-expressed (Ntini, 2011b).

Tup/Islet1 integrates time and position to specify muscle identity in Drosophila

The LIM-homeodomain transcription factor Tailup/Islet1 (Tup) is a key component of cardiogenesis in Drosophila and vertebrates. This study reports an additional major role for Drosophila Tup in specifying dorsal muscles. Tup is expressed in the four dorsal muscle progenitors (PCs) and tup-null embryos display a severely disorganized dorsal musculature, including a transformation of the dorsal DA2 into dorsolateral DA3 muscle. This transformation is reciprocal to the DA3 to DA2 transformation observed in collier (col) mutants. The DA2 PC, which gives rise to the DA2 muscle and to an adult muscle precursor, is selected from a cluster of myoblasts transiently expressing both Tinman (Tin) and Col. The activation of tup by Tin in the DA2 PC is required to repress col transcription and establish DA2 identity. The transient, partial overlap between Tin and Col expression provides a window of opportunity to distinguish between DA2 and DA3 muscle identities. The function of Tup in the DA2 PC illustrates how single cell precision can be reached in cell specification when temporal dynamics are combined with positional information. The contributions of Tin, Tup and Col to patterning Drosophila dorsal muscles bring novel parallels with chordate pharyngeal muscle development (Boukhatmi, 2012).

The pattern of rp298lacZ (a general marker of PCs/FCs) expression and the three-dimensional arrangement of founder cells (FCs) distinguished four groups: dorsal, dorsolateral, lateral and ventral. Whether this topology reflects specific genetic programs has remained unclear. Tup and Col are expressed in the four dorsal and three DL PCs, respectively, supporting the notion of DV regionalization of the somatic mesoderm. This notion was evoked by regional Pox meso (Poxm) expression in most ventral and lateral FCs. As other known iTFs are only expressed in subsets of dorsal PCs/FCs, it raised the possibilty that Tup could reside at the top of dorsal 'identity' transcription factor (iTF) cascades. The data show that this is not the case, as Tup, although required for Kr expression in the DO1 PC and for Col repression in the DA2 PC, is not required for expression of Eve, Runt and Vg in the DA1, DO2 and DA2 and DA1 lineages, respectively. Likewise, Col is required for expression of some iTFs but not others in DL PCs. Together, the patterns of Col, Eve, Kr, Poxm, Runt, Tup and Vg expression in wild-type and tup or col mutant conditions underline the intertwined, combinatorial nature of transcriptional regulatory networks specifying muscle identity. The DA2 PC gives rise to the DA2 muscle/DL adult muscle precursor (AMP) mixed lineage. Each abdominal hemisegment features six AMPs at stereotypical positions. Other AMPs originate from mixed lineages, e.g. the ventral VA3/AMP and lateral SBM/AMP lineages. The VA3/AMP and SBM/AMP PCs express Poxm and S59, and Lb, respectively. Tup expression in the DA2/AMP lineage confirms that different AMPs express different iTFs at the time of specification. Whether, as for somatic muscles, the iTF code confers specific properties to each AMP remains an unresolved issue (Boukhatmi, 2012).

How PCs born at similar positions in the somatic mesoderm come to express different combinations of iTFs and acquire distinct identities has remained elusive. For example, what distinguishes the fate of the two Eve-expressing PCs, which are sequentially born from the same dorsal cluster, is unknown. One other example is the expression of S59 and Lb, each in one of two abutting ventrolateral PCs: the LO1/VT1 and SBM PCs. Activation of both Lb and Slo expression in the two PCs is controlled by the same upstream regulator, Org-1 (Schaub, 2012). Subsequent reciprocal secondary cross-repression results in exclusive S59 or Lb expression, but the nature of the presumed positional bias responsible for the oriented resolution of this cross-repression has not been explored. In the case of the adjacent DA2 and DA3 PCs, this study shows that Tup activation by Tin in the DA2 PC is instrumental in distinguishing between DA2 and DA3 identities. The DA2 PC is selected from a small group of cells at the intersection between Tin and Col expression domains. Thus, the relative registers of tin and col expression along the DV axis provide precise positional information. Another key is timing. The overlap between Tin and Col expression is only transient, such that only the earlier-born Col-expressing PC expresses Tin. This provides a unique temporal window for Tup activation and Col repression. The transient overlap is due to the dorsal restriction of Tin expression to cardial cells during stage 11 This dynamic process is controlled by JAK-STAT signalling activity in the mesoderm, which is itself modulated by Tin activity. The key function of Tup in the DA2 PC, which is to distinguish between two muscle identities, illustrates how cell identity can be specified with single-cell precision when temporal dynamics are combined with positional information (Boukhatmi, 2012).

Some iTFs are expressed during all steps of myogenesis, from promuscular stage to muscle attachment. Schematically, two major phases of expression can be distinguished: (1) PC specification when multiple iTFs are expressed in different PCs and extensive cross-regulation occurs, leading to FC-specific iTF patterns; and (2) muscle differentiation when the FC pattern is maintained and propagated into the syncytial fibre via transcriptional activation of the iTF code in newly fused FCMs . Analysis of col regulation in the DA3 lineage showed that these two phases rely on two separate, early (CRM276) and late (4_0.9) CRMs, the activity of the late CRM requiring Col provided under the control of the early CRM. This auto-regulatory loop has been termed a CRM handover mechanism. This study has provided evidence that tup transcriptional regulation in the DA2 muscle follows the same rule. On the one hand, tup activation by Tin is mediated by an early CRM, IsletH; on the other, tup expression is maintained in differentiating muscles via a late CRM, DME, the activity of which depends upon Tup. It is proposed that this handover relay mechanism could be a widespread mode of iTF regulation, as it efficiently links early steps of muscle specification in response to positional information with final muscle identity (Boukhatmi, 2012).

Tup and Tin are key components of the transcriptional regulatory cascade that controls early cardiogenesis, with Tin acting to activate Tup, the expression of which then persists after Tin has ceased to be expressed. This study now showns that a similar cascade operates in the somatic muscle mesoderm. Tup and Tin expression in both the heart and dorsal somatic muscles recalls Nkx2.5 (Tin ortholog) and Islet1 expression in the pharyngeal mesoderm, which contributes to some head muscles and part of the vertebrate heart. Nkx2.5 is required for deployment of the second heart field (SHF) and Islet1 marks SHF progenitors that contribute both to the right ventricle and the arterial pole of the forming heart and a subset of skeletal pharyngeal muscles. Similarly, in the simple chordate Ciona intestinalis, Nk4 (Tin/NKx2.5) marks the cardio-pharyngeal mesoderm at the origin of the heart and atrian siphon muscles (ASMs) that are evocative of vertebrate pharyngeal muscles. Islet-expressing cells also contribute to ASMs, suggesting an evolutionarily conserved link between cardiac and pharyngeal muscle development. Interestingly, the ascidian Col/EBF ortholog Ci-COE, is expressed in ASM precursors and is a crucial determinant of the ASM fate, reminiscent of Xenopus XCoe2 expression and requirement in pharyngeal arches for aspects of jaw muscle development. It is now well established that distinct genetic networks govern skeletal myogenesis in the vertebrate head and trun. The repertoire of TFs differentially deployed in the head mesoderm includes Tbx1, the Drosophila ortholog of which, Org-1, has recently been shown to act as a muscle iTF . Tin/Nkx2.5, Tup/Isl1, Org-1/Tbx1 and Col/EBF may thus be part of a repertoire of transcription factors co-opted and diversified to regulate muscle patterning in Drosophila trunk and head muscle patterning in chordates (Boukhatmi, 2012).

Sensory-neuron subtype-specific transcriptional programs controlling dendrite morphogenesis: genome-wide analysis of Abrupt and Knot/Collier

The transcription factors Abrupt (Ab) and Knot (Kn) act as selectors of distinct dendritic arbor morphologies in two classes of Drosophila sensory neurons, termed class I and class IV, respectively. Binding-site mapping and transcriptional profiling of these isolated neurons were performed in this study. Their profiles were similarly enriched in cell-type-specific enhancers of genes implicated in neural development. A total of 429 target genes were identified, of which 56 were common to Ab and Kn; these targets included genes necessary to shape dendritic arbors in either or both of the two sensory subtypes. Furthermore, a common target gene, encoding the cell adhesion molecule Ten-m, was expressed more strongly in class I than class IV, and this differential was critical to the class-selective directional control of dendritic branch sprouting or extension. These analyses illustrate how differentiating neurons employ distinct and shared repertoires of gene expression to produce class-selective morphological traits (Hattori, 2013).

The transcriptional programs studied in this paper were predicted to be more specialized for controlling neuronal terminal differentiation at postmitotic stages, compared to those in which proneural genes, such as Asense and its vertebrate homolog Ascl1 (Mash1), regulate cell proliferation or cell-cycle arrest and also promote differentiation. Indeed, predicted molecular functions of Ab and Kn target-coded proteins are diverse, ranging from transcriptional control to cell adhesion, membrane trafficking, Ca2+ entry, and cytoskeleton regulation. Then how do these targets contribute to shaping dendrite arbors in a class-selective fashion? This genome-wide study strongly supports the notion that the class selectors do indeed control transcription of target genes selectively. In contrast, both TFs have chromatin features of the binding sites in common and show the same directional (up or down) regulation of every common target. To explain these findings, the possibility was intriguing that some common targets might be regulated by the two TFs in quantitatively differential fashions. As a precedent, Cut (Ct) is differentially expressed among the three classes (class II-IV), which controls formation of the different branching patterns and the growth of dendritic arbors of individual classes. In this study, compelling data for the above hypothesis was obtained by analyses of a common target, Ten-m. Its high-level expression in class I ddaE endowed its branches with the capability to respond to the decreasing level of Ten-m in the epidermis, thus setting the directional preference of branch sprouting. In contrast, a much lower expression in class IV ddaC ensured the directing of terminal branches rather radially in the distal area of each arbor, where the overlying epidermal Ten-m expression is low. These level-dependent roles of Ten-m could be related or analogous to a role of mouse Ten-m3 in navigating Ten-m3-high retinal projections to the high target region and those of tenurins in instructing synaptic partner matching in the Drosophila olfactory map. It awaits further study to reveal how the differential levels of Ten-m produce the class-selective directional properties of branch patterning, possibly by way of organization of cytoskeletons and membranes (Hattori, 2013).

The other experimental results are also consistent with the critical role of the quantitative control of target gene expression. First, the amount of Lola, one common target-gene product, was higher in class I ddaE than class IV ddaC in a wild-type background. Second, results of knockdown and overexpression of IGF-II mRNA-binding protein (Imp) and lola indicate that their expression levels must be strictly controlled to determine the arbor complexity. Third, there were superficially puzzling findings about downregulated targets (those with decreased expression upon ab or kn misexpression). In the narrowed-down list of Ab target genes, ten targets were downregulated by ab misexpression, and their knockdown in class I (which expresses Ab endogenously) yielded obvious abnormal phenotypes. Ab may keep the transcription of the downregulated targets weakly active and does not totally shut down the expression; moreover, this low-level expression may be required for normal class I development. To test these hypotheses, what would be required is class-selective quantitative expression profiling, ideally at multiple developmental stages, including the onset of primary dendrite formation and a subsequent branch growing phase (Hattori, 2013).

A total of 85% or more of the bound genes were not identified as exclusively Ab and/or Kn-dependent genes; and it could be that Ab and Kn may be able to control transcription of some of those in conjunction with other TFs. Candidate-bound genes of this group include Kn-bound genes Ubx and abd-A that are silenced in class IV by PcG proteins, which showed a similar binding profile with Kn, while an unknown transcriptional coactivator may drive expression of turtle, which is an Ab/Kn-bound gene necessary in class I and class IV. Furthermore, with respect to physiological functions of proprioceptive class I and multimodal nociceptive class IV, it should be mentioned that Gr28b encoding a bright blue light sensor was a Kn-bound gene. Additional profiling data sets, such as that in the copresence of Kn and Ct, will deepen understanding of the intricate transcription codes, with the ultimate goal of identifying the molecular links between the codes and the diverse architectures of dendritic arbors and neuronal functions (Hattori, 2013).


collier: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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