pannier

Promoter

During Drosophila embryogenesis, the homeobox gene tinman is expressed in the dorsal mesoderm where it functions in the specification of precursor cells of the heart, visceral, and dorsal body wall muscles. The GATA factor gene pannier is similarly expressed in the dorsal-most part of the mesoderm where it is required for the formation of the cardial cell lineage. Despite these overlapping expression and functional properties, potential genetic and molecular interactions between the two genes remain largely unexplored. pannier has been shown to be a direct transcriptional target of Tinman in the heart-forming region. The resulting coexpression of the two factors allows them to function combinatorially in the regulation of cardiac gene expression, and a physical interaction of the proteins has been demonstrated in cultured cells. Functional domains of Tinman and Pannier have been described that are required for their synergistic activation of the D-mef2 differentiation gene in vivo. Together, these results provide important insights into the genetic mechanisms controlling heart formation in the Drosophila model system (Gajewski, 2001).

Around the time of heart precursor cell specification, pnr RNA is detected in the dorsal mesoderm in cells that also express the tin gene. A pnr enhancer has been identified that is active in this heart-forming region and maps to a 457-bp DNA immediately upstream of the gene. Because the enhancer contains two putative Tin recognition sites, the binding of Tim to this DNA was investigated. A GST-Tin fusion protein was used in a gel-shift assay to test its ability to bind to the Tin1 sequence TCAAGTG, a known recognition element of the homeodomain protein in mesodermal enhancers of the D-mef2, tin, and b3 tubulin genes. Tin can bind specifically to the Tin1 consensus but not to a mutant version of the sequence. DNase I protection assays were also performed with the fusion protein on the pnr DNA, and two separate footprints were obtained that correspond to the Tin1 and Tin2 sequences. These in vitro experiments demonstrate that Tin can recognize and bind to two sites within the pnr dorsal mesoderm enhancer, suggesting the regulatory DNA might be a direct target of Tin transcriptional activity (Gajewski, 2001).

The defined pnr enhancer functions in the dorsal-most cells of the mesoderm and in cells of the amnioserosa. To determine whether its activity is regulated by Tin around the time of heart cell formation, the expression of a pnr enhancer-lacZ fusion gene was monitored in tin gain and loss of function embryos. Initially, the Gal4/UAS binary system was used to express tin throughout the mesoderm and mesectoderm under the control of the twi-Gal4 driver. An expanded function of the enhancer was observed within the mesoderm, coupled with ectopic activity in midline cells of the central nervous system (CNS) due to the forced expression of Tin. Conversely, in tin null embryos, a complete absence of beta-galactosidase expression is observed, which demonstrates a requirement of Tin function for enhancer activity (Gajewski, 2001).

pannier encodes two structurally related isoforms that are differentially expressed during Drosophila development and display distinct functions during thorax patterning

pannier encodes a GATA transcription factor that is involved in various biological processes, including heart development, dorsal closure during embryogenesis as well as neurogenesis and regulation of wingless (wg) expression during imaginal development. This study demonstrates that pnr encodes two highly related isoforms that share functional domains but are differentially expressed during development. Moreover, two genomic regions of the pnr locus are described that drive expression of a reporter in transgenic flies in patterns that recapitulate essential features of the expression of the isoforms, suggesting that these regions encompass crucial regulatory elements. These elements contain, in particular, sequences mediating regulation of expression by Decapentaplegic (Dpp) signaling, during both embryogenesis and imaginal development. Analysis of pnr alleles reveals that the isoforms differentially regulate expression of both wg and proneural achaete/scute (as/sc) targets during imaginal development. Pnr function has been demonstrated to be necessary both for activation of wg and, together with U-shaped (Ush), for its repression in the dorsal-most region of the presumptive notum. Expression of the isoforms define distinct longitudinal domains and, in this regard, it is shown that the dual function of pnr during regulation of wg is achieved by one isoform repressing expression of the morphogen in the dorsal-most region of the disc while the other laterally promotes activation of the notal wg expression. This study provides novel insights into pnr function during Drosophila development and extends knowledge of the roles of prepattern factors during thorax patterning (Fromental-Ramain, 2008).

Focus was placed on reporter expression in the wing disc where pnr is necessary for the development of thoracic macrochaetae. A DNA fragment, 15.7 kilobases (kb) in length and including the 5′ untranslated sequences of exon 1 (construct A15.7), directs expression of lacZ in the dorsal-most domain of the disc. The 15.7 kb DNA fragment was dissected by 5′-end deletion, it was observed that the genomic sequences contain two distinct regions responsible for reporter expression. The 3.2 kb DNA fragment adjacent to pnr (construct E3.2) drives expression of the reporter along the A/P border of the notal region of the disc where Dpp is expressed, and also in a central cluster of cells. Expression remains similar in lines carrying the reporter under the control of the 9.3 kb fragment (construct C9.3), suggesting that the supplementary 6.1 kb DNA fragment does not contain essential regulatory sequences. When the DNA fragment inserted upstream of the reporter is the 12 kb fragment (construct B12), lacZ expression is reinforced in comparison of expression seen with lines carrying construct C9.3. Expression of the reporter fully covers dorsal domain of the disc when the promoter sequences include the distal DNA fragment (construct A15.7). Thus, a second domain responsible for expression in the disc appears to be located in the distal region of construct A15.7 (Fromental-Ramain, 2008).

It is concluded that reporter expression depends on activity of two domains, a proximal one located in the 3.2 kb fragment adjacent to pnr (construct E3.2) and a distal one corresponding to the 5′-end of construct A15.7. These observations are reinforced by the fact that both the distal fragment (construct H6.4) and the proximal fragment (construct J1.8) inserted in front of an heterologous hsp43 (heat shock protein43) minimal promoter direct reporter expression in the wing disc. In contrast, the intervening fragment (construct I6.1) does not promote expression when placed in front of this heterologous promoter (Fromental-Ramain, 2008).

Interestingly, the location of the two domains suggests that they may correspond to alternate promoters of the pnr isoforms. Indeed, sequence analysis of the pnr locus and characterization of the mRNAs expressed during development led to the prediction that pnr may encode two isoforms. Isoform-α (pnr-α) encodes the Pnr protein as it has been identified, whereas the putative isoform-β (pnr-β) encodes a truncated version of the Pnr-α protein, lacking the 52 N-terminal amino acids. However, Pnr-α and Pnr-β share functional domains and the N-terminus of Pnr-α does not contain any obvious functional signature. In vitro experiments revealed that both Pnr-α and -β associate with Ush and equivalently activate a reporter driven by promoter sequences including GATA sites in a cultured cell line (Fromental-Ramain, 2008).

Several reports have implicated Pnr as a key transcriptional regulator during expression of both ac/sc and wg in the presumptive notum. The current study extends previous work and importantly demonstrates that Pnr function is achieved by two structurally related isoforms with distinct expression domains. Moreover, the isoforms display distinct transcriptional activities, including antagonism during regulation of wg expression (Fromental-Ramain, 2008).

In the presumptive notum of Drosophila, wg expression is regulated by different mechanisms, acting downstream of Dpp. It was shown that expression of pnr and ush are activated by Dpp signaling. pnr RNA is expressed in the dorsal-most domain of the disc, including the authentic domain of wg expression, whereas ush RNA appears restricted to the future medial notum, abutting on the authentic domain of wg expression. ush appears to serve as a negative factor for wg expression since wg was misexpressed in mutant cells lacking ush activity and induced in the dorsal-most territories of the disc. Thus, it was proposed that Ush-unbound Pnr activates wg in its authentic domain of expression, while Pnr-Ush complexes repress wg in the dorsal-most domain of the disc. Moreover, mosaic clones lacking pnr function and induced in the dorsal-most area of the disc, exhibit mis-expressed wg expression, suggesting that wg can be activated by a Pnr-independent mechanism (Fromental-Ramain, 2008).

This study demonstrates that pnr encodes two isoforms which are differentially expressed during development and are likely regulated by Dpp signaling in embryos and/or wing discs. Isoform-β is expressed in the dorsal-most area of the presumptive notum in a pattern similar to that of both the UASGFP reporter driven by pnr^Gal4 and the reporter present in lines carrying construct G5.6. Isoform-β expression, visualized using the reporter present in lines carrying construct G5.6, delimits the authentic domain of wg expression revealed by in situ hybridization. This observation slightly differs from a previous report where overlapping expression is described of both UASGFP driven by pnr^Gal4 and Wg (Garcia-Garcia, 1999). However, overexpression of UASGFP driven by pnr^Gal4 probably led to an overestimate of the overlap of pnr and wg expression. In the current study, pnr-β expression is visualized with a reporter driven by regulatory sequences from the pnr locus and is compared with wg mRNA expression. The distinct experimental approaches used to detect pnr and wg expression may explain these differences. Nevertheless, this study demonstrates that pnr-β expression is identical to that of the UASGFP reporter driven by pnr^Gal4. Moreover, pnr-α expression laterally extends with respect to the domain of pnr-β expression and functional analysis of pnr alleles revealed that pnr-α mediates wg activation (Fromental-Ramain, 2008).

The pnr-α isoform corresponds to pnr as it was previously identified. It is weakly expressed in the dorsal territory of the wing disc, with stronger accumulation along the A/P border of the notal region of the disc and in a central cluster of cells. These features of pnr-α expression are reproduced in lines carrying construct C9.3. Doubly-stained wing discs for wg RNA and reporter expression driven by construct C9.3 revealed that the lateral domain of reporter expression coincides with the authentic domain of wg expression, suggesting that isoform-α may activate wg during imaginal development. In addition, the expression domains of reporters driven by construct C9.3 or G5.6 are very similar to those of pnr-α and pnr-β. Together, they define an expression domain similar to that of the pnr RNA as described by in situ hybridization with a cDNA probe detecting both isoform (Fromental-Ramain, 2008).

Previous analysis have shown that both pnr mutant flies (pnr^Gal4/pnr^VX6) and the homozygous pnr^Gal4 flies exhibit expanded wg expression towards dorsal-most territories of the disc. This correlates with severe reduction of pnr-β accumulation, while pnr-α accumulation is dramatically increased. This suggests that Pnr-α activates wg, while Pnr-β, possibly together with Ush, represses expression in the dorsal-most domain. Finally, analysis of the pnr^V1 allele also supports the hypothesis of antagonistic roles of pnr isoforms during wg expression. Homozygous pnr^V1 flies exhibit increased pnr-α and wild-type pnr-β, associated with expansion of wg expression towards the dorsal-most territories of the wing disc. As the pnr^V1 allele likely encodes functional proteins, it is concluded that Pnr-α is a crucial factor during wg activation and that Pnr-β antagonizes Pnr-α function (Fromental-Ramain, 2008).

Further evidence for antagonistic activities of the pnr isoforms during thorax patterning is provided by comparison of wg expression between homozygous (pnr^V1) and (pnr^Gal4/pnr^VX6) flies. Indeed, pnr-β expression is not affected in pnr^V1, although it is severely impaired in the (pnr^Gal4/pnr^VX6) combination, while wg expression fully covers the dorsal-most domain of the disc only in the case of the (pnr^Gal4/pnr^VX6) combination. It is concluded that Pnr-α activates wg whereas Pnr-β, possibly together with Ush, mediates repression in the dorsal-most domain of the disc. Moreover, pnr isoforms display antagonistic activities and the wg expression is regulated by the molecular ratio between activating pnr-α and repressing pnr-β (Fromental-Ramain, 2008).

These conclusions are further supported by experiments where isoforms are ubiquitously overexpressed using the c765 line. Expression of wg is ectopically induced in the lateral domains of the presumptive notum by overexpressed pnr-α (Garcia-Garcia, 1999), whereas this study shows that it is repressed in the medial domain by overexpressed pnr-β. wg expression in the medial domain does not totally depend on pnr, since mutant cells lacking pnr activity exhibit reduced wg expression. The complete repression of wg in its authentic domain after overexpression of isoform-β does not depend on repression of isoform-α by isoform-β, but rather on a competition for the notal wg enhancer between isoform-β and a factor(s) responsible for pnr-independent expression of wg (Fromental-Ramain, 2008).

Identification of the isoforms led to revisiting the role of pnr during regulation of ac/sc and ush targets in the wing disc. Both overexpressed pnr-α and overexpressed pnr-β lead to activation of proneural expression and development of ectopic sensory bristles suggesting that the isoforms may act as subunits of the multiprotein proneural complex as it has been previously identified. However, the current analysis of the pnr^V1 and pnr^Gal4 alleles do not argue in favor of such a model during regulation of ac/sc expression. Both the reduced pnr-β expression associated with homozygous pnr^Gal4 animals and the increased pnr-α expression observed in homozygous pnr^V1 animals exhibit a loss of DC bristles and impaired proneural expression at the DC site of the wing/thorax discs. As the domains of isoform expression stay the same in mutants animals, this suggest that the mutant phenotypes result from antagonistic activities of the Pnr proteins. This hypothesis is reinforced by the observation that proneural expression is reduced in both (pnr^Gal4/+) and (pnr^V1/+) animals and is totally abolished in homozygous mutant animals. Thus, proneural expression at the DC site of the imaginal disc relies on the stoichiometry between Pnr-α/Pnr-β. Additional evidence is provided by molecular analysis of the vertebrate complex, homologous to the proneural complex encompassing Pnr, Chip and the heterodimer (Ac/Sc)-Da. Indeed, the vertebrate hematopoietic-specific complex contains only one GATA molecule, that does not support the notion that both the Pnr-α and Pnr-β isoforms simultaneously belong to the Drosophila complex necessary for ac/sc activation during Pnr-driven proneural development (Fromental-Ramain, 2008).

Previous analysis have shown that ush expression is also regulated by Pnr. ush expression is abolished in the dorsal-most domain of (pnr^VX6/pnr^V1) disc. Since the (pnr^VX6/pnr^V1) combination was predicted to correspond to a loss of pnr function, it was postulated that Pnr mediates activation of notal ush expression. It has also been reported that ush expression is lost in (pnr^D1/pnr^V1) disc, except at the A/P border of the notal region. Since pnr^D1 encodes a mutant protein carrying a single amino acid exchange in the DNA binding domain that disrupts interaction with the negative regulator Ush, it was hypothesized that the (Pnr-Ush) complex serves as a transcriptional activator of ush expression. However, the current analysis revealed a strong induction of pnr-α expression at the A/P border of the disc while pnr-β expression is not modified. Hence, expression of the (Pnr^D1-α) protein is induced at the A/P border in (pnr^D1/pnr^V1) discs and it is suggested that Pnr-α-Ush is involved in the repression of ush expression. Moreover, it is also suggested that the ush expression depends on the stoichiometry between Pnr-α and Pnr-β since ush expression is abolished in the dorsal-most domain of the pnr^D1/pnr^V1 discs outside the A/P border. The pnr^V1/pnr^D1 combination is consequently characterized by ectopic sensory bristles and increased proneural expression in the DC area (Fromental-Ramain, 2008).

Pnr is involved in regulation of both the ac/sc and ush targets during neural development and the stoichiometry of the isoforms is a crucial determinant during regulation of gene expression. These characteristics may explain the paradoxical observations that increased pnr-α expression in homozygous pnr^V1 displays reduced ac/sc expression and loss of DC bristles whereas overexpressed pnr-α in (pnr^Gal4/UAS pnr α) leads to activated ac/sc expression and additional macrochaetae. The DC enhancer would require lower Pnr-α concentration for repression than the notal ush enhancer, probably reflecting different affinities of the binding sites for the Pnr-α-Ush effector. At low concentration, the Pnr-α-Ush heterodimer antagonizes Pnr-β activity, leading to reduced ac/sc expression at the DC site and loss of DC bristles. Overexpressed pnr-α mediates repression of ush, leading to reduced concentration of the Pnr-α-Ush heterodimer and consequently, ac/sc expression at the DC site results from activating Pnr-β. Hence, overexpressed pnr-α displays ectopic sensory organs. In contrast, overexpressed pnr-β would repress pnr-α involved together with Ush in repression of ac/sc and would also directly activate proneural expression to promote development of ectopic sensory organs. Both overexpressed pnr-α or pnr-β activates proneural expression, leading to ectopic sensory organs but they act by distinct mechanisms. During activation of proneural expression, overexpressed pnr-β appears to directly stimulate ac/sc through binding to their regulatory sequences whereas overexpressed pnr-α indirectly acts in repressing ush expression (Fromental-Ramain, 2008).

The present data highlight the merit of revisiting pnr function during development since pnr isoforms are expressed in domains that define a novel subdivision of the wing disc. The biological significance of the subdivision is of critical importance since the isoforms exhibit antagonistic activities during regulation of targets genes. A challenging issue will be to understand how the Pnr isoforms molecularly interact with the regulatory sequences of the target genes ac/sc, ush and wg. Sequence analysis revealed that the DC enhancer contains several Pnr binding sites and some of them are involved in regulation of ac/sc expression during neural development (Garcia-Garcia, 1999). These binding sites may correspond to targets for Pnr-β and (Pnr-α)-Ush complexes. Mutagenesis of the Pnr binding sites would be required to understand how the isoforms interact with the regulatory element to antagonistically regulate proneural expression, to clarify the role of Ush during regulation of Pnr target genes, and to resolve the question on how upon dimerization Ush can convert Pnr from an activator to a repressor (Fromental-Ramain, 2008).

XNP/dATRX interacts with DREF in the chromatin to regulate gene expression

The ATRX gene encodes a chromatin remodeling protein that has two important domains, a helicase/ATPase domain and a domain composed of two zinc fingers called the ADD domain. The ADD domain binds to histone tails and has been proposed to mediate their binding to chromatin. The putative ATRX homolog in Drosophila (XNP/dATRX) has a conserved helicase/ATPase domain but lacks the ADD domain. In this study, it is proposed that XNP/dATRX interacts with other proteins with chromatin-binding domains to recognize specific regions of chromatin to regulate gene expression. A novel functional interaction is reported between XNP/dATRX and the cell proliferation factor DREF in the expression of pannier (pnr). DREF binds to DNA-replication elements (DRE) at the pnr promoter to modulate pnr expression. XNP/dATRX interacts with DREF, and the contact between the two factors occurs at the DRE sites, resulting in transcriptional repression of pnr. The occupancy of XNP/dATRX at the DRE, depends on DNA binding of DREF at this site. Interestingly, XNP/dATRX regulates some, but not all of the genes modulated by DREF, suggesting a promoter-specific role of XNP/dATRX in gene regulation. This work establishes that XNP/dATRX directly contacts the transcriptional activator DREF in the chromatin to regulate gene expression (Valadez-Graham, 2012; full text of article).

Response to the BMP gradient requires highly combinatorial inputs from multiple patterning systems in the Drosophila embryo

Pattern formation in the developing embryo relies on key regulatory molecules, many of which are distributed in concentration gradients. For example, a gradient of BMP specifies cell fates along the dorsoventral axis in species ranging from flies to mammals. In Drosophila, a gradient of the BMP molecule Dpp gives rise to nested domains of target gene expression in the dorsal region of the embryo; however, the mechanisms underlying the differential response are not well understood, partly owing to an insufficient number of well-studied targets. This study analyzed how the Dpp gradient regulates expression of pannier (pnr), a candidate low-level Dpp target gene. It was predicted that the pnr enhancer would contain high-affinity binding sites for the Dpp effector Smad transcription factors, which would be occupied in the presence of low-level Dpp. Unexpectedly, the affinity of Smad sites in the pnr enhancer was similar to those in the Race enhancer, a high-level Dpp target gene, suggesting that the affinity threshold mechanism plays a minimal role in the regulation of pnr. The results indicate that a mechanism involving a conserved bipartite motif that is predicted to bind a homeodomain factor in addition to Smads and the Brinker repressor, establishes the pnr expression domain. Furthermore, the pnr enhancer has a highly complex structure that integrates cues not only from the dorsoventral axis, but also from the anteroposterior and terminal patterning systems in the blastoderm embryo (Liang, 2012).

Most blastoderm genes are regulated primarily on either the DV or AP axis. For example, the gap genes are expressed in one or two domains of expression along the AP axis and, although some of them may exhibit regulation along the DV axis, they are nonetheless considered AP genes. pnr represents an interesting case because although it was originally reported as a DV gene, closer inspection of its expression pattern in wild-type and mutant embryos and detailed dissection of its cis-regulatory enhancers revealed that pnr is highly regulated by both AP and DV genes. Its pattern is a composite of two superimposed patterns that each exhibit AP and DV spatial regulation: a dorsal patch and six AP stripes, which are limited to the dorsal 30% of the embryo. The patch domain, but not the stripes, disappeared in dpp mutants, whereas both the patch and stripes expand ventrally in the absence of Brk. The stripes are more sensitive to Brk repression because activation of the patch domain is limited to the region where Dpp is present dorsally, whereas the stripes can be activated along the entire DV axis. Brk in the ventrolateral region and Sna in the ventral-most region repress stripe expression. Since pnr specifies dorsomedial fates, restricting its expression to the dorsal 30% of the circumference is crucial. Ectopic expression of pnr ventrally causes transformation of ventral epidermis into dorsomedial epidermis (Liang, 2012).

Competition between Brk and Smads for binding to overlapping DNA sequences is likely to set the border of the patch domain. Two Smad sites are particularly important for patch expression, and one of these, the M3 site, is a composite site that binds both Brk and Smads, raising the possibility that the patch border is established by competition between activating inputs from Smads in the dorsal region and repressive inputs from Brk emanating from the ventral region. Competition between Brk and Smads for overlapping binding sites has been observed for several Dpp target enhancers (Liang, 2012).

Repression of the AP stripes ventrally requires both Brk sites B1 and B2. The two posterior stripes driven by P3 expand to a lesser degree than the four anterior stripes driven by P4. This can be explained by the fact that P4 lacks Brk site B1, which is a stronger Brk site. Loss of both Brk sites would likely result in expansion to the edge of the mesoderm, as seen in embryos that lack Brk protein. Repression by Sna is likely to involve the Sna binding sites in the pnr enhancer, as genome-wide binding studies have shown that the pnr enhancer is bound by Sna (Liang, 2012).

The positioning of the stripes, as well as of the patch, along the AP axis is regulated by the gap genes. The results suggest that Hb, Gt and Tll set the anterior edge of the pnr domain, whereas Tll sets the posterior, and that direct and indirect interactions among the gap proteins establish the stripe borders relative to one another, as has been observed for eve. For example, the broad central stripe seen in kni^- could be explained by the lack of direct Kni repression. However, owing to the complex cross-regulatory interactions among the gap genes, it is difficult to predict which gap proteins regulate the pnr stripes directly, although genome-wide binding data of the gap factors support their direct binding to the pnr enhancer. Although Bcd does not appear to bind directly to the pnr enhancer, its effects are mediated through its targets Gt and Hb (Liang, 2012).

In depth studies of three genes with different boundary positions in the dorsal region, Race, C15 and pnr, indicate that complex combinatorial mechanisms are employed to establish their expression domains, with each gene having a unique regulatory network of its own. Although they all respond to Dpp signaling, their borders of expression are not set by a simple threshold response to the Dpp gradient that depends on differential binding site affinity (Liang, 2012).

The feature that has been shown to be important for high-level Dpp target expression is the feed-forward motif involving Dpp and Zen. High levels of Dpp/Smads first activate zen expression in the dorsal-most region, the presumptive amnioserosa, and then both Zen and Smads bind and activate the Race enhancer. The intermediate-level target C15 has a different enhancer structure than high-level targets, containing many Smad sites that act in a cumulative manner to drive expression in regions of intermediate Dpp levels. Mutation analysis has shown that the number of intact Smad binding sites, rather than their affinity, is important for the C15 response. Nevertheless, the enhancer structure of C15 might promote high levels of Smad binding in vivo, and this may increase the response to Dpp. Do all intermediate-level Dpp targets have a similar enhancer structure? The enhancer that drives expression of the intermediate-level Dpp target gene tup was examined for putative Smad binding sites (SBEs and GC-rich regions), and observed multiple Smad sites across the enhancer, similar to that seen with C15. Thus, the multiple Smad site signature might be necessary for response to lower than peak levels of Dpp. In addition, intermediate-leveltargets may utilize repression mechanisms to help establish their borders of expression, as was shown for C15 (Liang, 2012).

These studies have revealed that the pnr enhancer resembles that of a high-level target in Smad site organization and Smad binding site affinity. In fact, it was surprisingly easy to convert the low-level target enhancer into a high-level target by mutating a single Smad site. This result could be easily explained if the M3 site had a higher affinity for Smads than those in Race; however, comparison of the binding sites by gel shift showed they have similar affinities. Furthermore, replacing the M3 Smad site with a Race Smad site had little effect on the expression pattern. These results suggest that activation of pnr in its broad domain has little to do with Smad binding affinity. How then does pnr respond to low levels of Dpp? One possible mechanism involves the highly conserved AGCAATTAA site that lies adjacent to the Smad sites. In the absence of this site, the P3 enhancer could not respond to low-level Dpp. It is possible that this site, when bound, leads to greater Smad binding, which would then promote pnr activation (Liang, 2012).

What factor(s) might bind to the AGCAATTAA site? ATTA is the core binding site for Antp class HD proteins. Although Zen binds to the ATTA site in vitro, neither the endogenous pnr pattern nor P3-lacZ expression is significantly affected in zen mutants. To identify candidate factors, the TOMTOM tool at FlyFactor Survey was used, and the best match was to the HD protein Hmx, which binds CAATTAA. However, Drosophila Hmx is expressed only in an anterior region that does not overlap with pnr (see FlyBase). Likewise, although several Antp class HD proteins were predicted to bind to the ATTA core sequence, their timing or domains of expression do not overlap ideally with those of pnr (Liang, 2012).

It has been proposed that the AGCAATTAA site in the Msx2 enhancer might bind a factor in addition to an HD protein via the 5' half of the site, perhaps a transcriptional partner such as FAST1, which was previously shown to function with Smads. Although the search did not reveal any candidates, if this is the case for pnr then the bipartite motif could potentially bind four proteins: Smads, Brk, HD and 'partner X', The combination of these proteins in a given cell along the DV axis would determine pnr transcriptional activity. The fact that the bipartite motif is not present in the enhancers of Race or C15, or in the other pnr enhancers identified, demonstrates the versatility of how Dpp uses different partners to establish multiple target gene domains (Liang, 2012).

Is the structure of the pnr enhancer typical for low-level Dpp targets? This is difficult to address owing to the lack of candidate low-level Dpp targets. Brk is considered a low-level Dpp target in imaginal disc development; however, Dpp represses brk, giving rise to a reciprocal gradient of the Brk repressor. Target gene borders are thus established by competition between Smad and Brk for overlapping binding sites, as mentioned above for pnr. The brk enhancer contains multiple enhancer/silencer modules consisting of activator and repressor (Mad/Medea/Schnurri) binding sites, which contribute to threshold responses to the Dpp gradient, and thus it does not resemble the pnr enhancer. Although good progress has been made in understanding how pnr is expressed in regions with low levels of Dpp, learning the general rules that control broad dorsal patterns will require the analysis of more enhancer elements (Liang, 2012).

What rules do target genes for other morphogens follow? Long before the 'feed-forward' term was it was shown that both the Dl and Bcd morphogens interact with their high-level targets, Twi and Hb, respectively, to activate downstream; thus, combinatorial motifs are generally utilized. Moreover, as more target genes of Dl and Bcd were identified and studied, it became apparent that the affinity threshold model could not explain all cases of differential response to the gradient. For example, analysis of several enhancers that drive Bcd-dependent expression in anterior regions of the embryo revealed a poor correlation between Bcd binding site affinity and the AP limits of the pattern. Also, although Dl targets remain archetypal examples of genes that utilize the affinity threshold mechanism, it was found that genes expressed in the lateral region also require input from the Zelda (Vielfaltig - FlyBase) transcription factor for expression in regions of low-level Dl. Zelda binding sites are present in target enhancers, and it was proposed that Zelda boosts Dl binding to help activate the neuroectodermal genes (Liang, 2012).

Downstream target gene interactions also shape domains of expression, in particular cross-

repression among the targets. In both the Drosophila neuroectoderm and the vertebrate neural tube, morphogen targets are expressed in discrete domains rather than nested overlapping domains due to the repression of one target by another. This mechanism establishes sharp boundaries among the target genes (Liang, 2012).

Thus, it is clear that additional factors help morphogens set threshold responses. Given that the pnr enhancer could potentially interact with four different factors along the DV axis and at least four factors along the AP axis, several combinations of inputs could regulate other Dpp target genes. More generally, depending on the number of different factors that interact with the cis-regulatory regions of target genes, morphogen gradients could elicit multiple threshold responses, as has been seen for morphogens such as Dl in Drosophila, Activin in the Xenopus blastula and Shh in the vertebrate neural tube, where up to seven threshold responses have been described. Only by dissecting enhancers can it be fully understood how target genes integrate diverse inputs (Liang, 2012).

Transcriptional Regulation

pnr lies downstream of decapentaplegic and zerknüllt. In embryos null for dpp, no pannier is expressed (Winick, 1993).

The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGFbeta signaling molecules, Decapentaplegic and Screw. Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation and a newly identified transcriptional repressor, Brinker, which are expressed in neurogenic regions that abut the dorsal ectoderm. The expression of a number of Dpp target genes has been examined in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression. These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos. Brk was found to repress two target genes, tailup/islet (tup) and pannier, that exhibit different limits of expression within the dorsal ectoderm. These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression. Evidence is provided that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase (Ashe, 2000).

The dpp stripe results in an expansion in both the hnt and ush expression patterns. The broadening of these patterns is particularly evident in anterior regions in the vicinity of the eve stripe. Increases in dpp+ gene dose do not expand the pnr expression pattern. For example, four copies of dpp+ result in augmented levels of pnr expression, but the dorsoventral limits of expression are essentially normal. The stripe2-dpp transgene has no obvious effect on the early sog and brk expression patterns (Ashe, 2000).

Previous studies have shown that the pnr expression pattern expands into neurogenic regions in brk- mutant embryos. No such expansion was observed for other Dpp/Scw target genes, including ush. To test the role of the Brk repressor in establishing the different responses of Dpp target genes, the brk-coding sequence was attached to the eve stripe 2 enhancer. Transgenic embryos carrying the stripe2-brk transgene exhibit both the normal pattern (lateral stripes) in the neurogenic ectoderm as well as an ectopic stripe of expression. pnr is normally expressed in a series of 5 stripes in the dorsal ectoderm. The anteriormost stripe is lost in transgenic embryos carrying the stripe2-brk fusion gene. This result suggests that Brk is sufficient to repress pnr in an ectopic location in the embryo (Ashe, 2000).

To examine the relative contributions of the Sog inhibitor and the Brk repressor in establishing different thresholds of Dpp/Scw signaling activity, target genes were analyzed in gastrulation defective (gd) mutants that express either a stripe2-sog or stripe2-brk transgene. Mutant embryos collected from gd-/gd - females lack a Dl nuclear gradient and therefore lack ventral tissues such as the mesoderm and neurogenic ectoderm. All tissues along the dorsoventral axis form derivatives of the dorsal ectoderm, mainly dorsal epidermis. Hereafter, such embryos are referred to as gd-. These mutants lack endogenous sog and brk products, so that the stripe2 transgenes represent the only source of expression. Although the stripe2-sog transgene inhibits Dpp signaling, it does not cause activation of brk. The pnr and tup expression patterns are derepressed in gd- mutants, and exhibit uniform staining in both dorsal and ventral regions. These expanded patterns correlate with the expanded expression of dpp, which is normally repressed in ventral and lateral regions by the Dl gradient. As seen in wild-type embryos, the stripe2-brk transgene represses the anterior portion of the pnr expression pattern. In contrast, the stripe2-sog transgene has virtually no effect on the pattern. These observations suggest that Brk is the key determinant in establishing the lateral limits of the pnr pattern at the boundary between the dorsal ectoderm and neurogenic ectoderm. The failure of stripe2-sog to inhibit pnr expression might be due to redundancy in the action of the Dpp and Scw ligands. Perhaps either Scw alone or just one copy of dpp+ is sufficient to activate pnr. This would be consistent with the observation that the initial pnr expression pattern is essentially normal in dpp-/dpp- and scw-/scw- mutant embryos (Ashe, 2000).

Previous studies have identified mutations in the Drosophila homolog of the mammalian CBP histone acetyltransferase gene, nejire. nej is maternally expressed so that the detection of early patterning defects depends on the analysis of embryos derived from females containing nej germline clones. The complete loss of nej+ activity results in a failure to make mature eggs. However, it is possible to obtain embryos from a strong hypomorphic allele, nej¹. These embryos exhibit dorsoventral patterning defects. Recent studies have shown that CBP interacts with Smad proteins including the Drosophila protein Mad, a transcription factor downstream of Dpp signaling. In nej mutant embryos, there is a loss of the amnioserosa and other derivatives of the dorsal ectoderm. The expression of target genes requiring peak levels of Dpp signaling is essentially abolished. For example, hnt expression is lost in the presumptive amnioserosa, but persists at the posterior pole where it might be separately regulated by the torso signaling pathway (Ashe, 2000).

There is a similar loss of the dorsal rho pattern in mutant embryos. In contrast, the lateral, neurogenic stripes are unaffected, indicating that the nej mutant does not cause defects in the patterning of the neurogenic ectoderm. Moreover, the fact that the rho stripes are excluded from ventral regions, as seen in wild-type embryos, suggests that the patterning of the mesoderm is also normal. Thus, the nej mutation does not appear to cause a general loss of transcriptional activation, but instead results in specific patterning defects in the dorsal ectoderm. Target genes that are activated by lower levels of Dpp signaling such as ush and pnr are also affected by the nej mutation. In the case of ush, there is a loss of staining in central regions of the dorsal ectoderm. Moreover, the residual staining pattern is narrower than the wild-type pattern. This is reminiscent of the ush pattern seen in dpp/+ heterozygotes. However, the nej mutation also causes a narrowing of the pnr pattern, whereas expression is normal in dpp/+ embryos (Ashe, 2000).

A summary is presented of Dpp signaling thresholds in the embryo. The Dpp/Scw activity gradient presumably leads to a broad nuclear gradient of Mad and Medea across the dorsal ectoderm of early embryos. It is conceivable that the early lateral stripes of brk expression lead to the formation of an opposing Brk repressor gradient through the limited diffusion of the protein in the precellular embryo. Peak levels of Dpp and Scw activity lead to the activation of Race and hnt at the dorsal midline. The tup and ush patterns represent another threshold of gene activity. The similar patterns might involve different mechanisms of Dpp signaling since tup is repressed by Brk, whereas ush is not. Finally, the broad pnr pattern represents another threshold of gene activity. It is not inhibited by Sog but is repressed by Brk. It is possible that tup and pnr are differentially repressed by a Brk gradient. Low levels of Brk might be sufficient to direct the lateral limits of the tup pattern, whereas high levels may be required to repress pnr (Ashe, 2000).

The morphogen gradient of Wingless provides positional information to cells in Drosophila imaginal discs. Elucidating the mechanism that precisely restricts the expression domain of wingless is important in understanding the two-dimensional patterning by secreted proteins in imaginal discs. In the pouch region of the wing disc, wingless is induced at the dorsal/ventral compartment boundary by Notch signaling in a compartment-dependent manner. In the notum region of the wing disc, wingless is also expressed across the dorsal/ventral axis, however, regulation of notal wingless expression is not fully understood. Notal wingless expression is established through the function of Pannier, U-shaped and Wingless signaling itself. Initial wingless induction is regulated by two transcription factors, Pannier and U-shaped. At a later stage, wingless expression expands ventrally from the pannier expression domain by a Wingless signaling-dependent mechanism. Interestingly, expression of pannier and u-shaped is regulated by Decapentaplegic signaling that provides the positional information along the anterior/posterior axis, in a concentration-dependent manner. This suggests that the Decapentaplegic morphogen gradient is utilized not only for anterior/posterior patterning but also contributes to dorsal/ventral patterning through the induction of pannier, u-shaped and wingless during Drosophila notum development (Tomoyasu, 2000).

A hierarchy of the activity of these genes during notum development is presented. dpp is initially induced at the dorsal region of the A/P compartment boundary by Hh signaling. Dpp signaling induces two target genes, pnr and ush. Analyses of pnr expression in put-ts and tkv^a12 cells suggest that different thresholds are set for the induction of these genes: low levels for pnr and high levels for ush. wg is induced by Pnr where ush is not expressed. Simultaneously, the Pnr-Ush complex represses wg expression at the dorsal-most region of the presumptive notum. In the later stage, the wg expression domain expands ventrally from the pnr expressing region and wg starts to be expressed in the non-pnr-expressing cells. During this process, Wg signaling plays a crucial role and this separation does not occur in the Wg signaling mutants. The Pnr-Ush complex acts as a repressor for the induction of wg and of DC enhancer-lacZ expression (DC enhancer is an enhancer of the achaete-scute proneural gene complex that activates gene expression in the dorsocentral area). It is interesting that Ush does not simply inhibit Pnr function but switches the activator function of Pnr to a repressor function. Based on the result that the extra doses of Pnr cannot revert the repressor activity of Pnr-Ush, it has been proposed that the activator function of Pnr and the repressor function of the Pnr-Ush complex do not simply compete with each other on the notal wg enhancer element. However, it also seems to be possible that Pnr and the Pnr-Ush complex compete for the binding site at the notal wg enhancer, but the ability of Pnr-Ush complex to bind this site may be greater than that of Pnr. It is also worth noting that FOG-1, a mammalian homolog of Ush, represses the transactivation of alpha-globin and EKLF promoter by GATA-1, but enhances the transactivation of NF-E2 p45 promoter by GATA-1 in a culture cell system. Dorsocentral (DC) bristles are ectopically formed but postvertical bristles on the head are missing in a loss-of-function allelic combination for ush or in pnr^D1 heterozygous flies. These observations suggest that the Pnr-Ush complex acts as a repressor for the DC enhancer, but acts as an activator for the enhancer of postvertical bristles. Only a cis-regulatory element of the DC enhancer has been analyzed at the nucleotide level. Additional studies of the molecular analyses of the cis-regulatory elements of both wg and DC or other enhancers of the achaete-scute complex seem to be necessary in order to reveal the functions of Pnr and Ush (Tomoyasu, 2000).

Generally, at least two different coordinate axes are necessary for positional specification in a two-dimensional field. Morphogen gradients of Dpp and Wg provide this axial information during Drosophila imaginal disc development. In both wing and leg discs, Dpp is induced at the A/P compartment boundary by Hh signaling. In the leg disc, wg is also induced by Hh signaling. Mutual repression between Dpp and Wg signaling separates each expression territory, localizing dpp in the dorsal and wg in the ventral regions abutting the A/P border (a compartment-independent manner). In contrast, wg is induced by Notch signaling only at the D/V compartment boundary in the wing pouch (a compartment-dependent manner). Then, secreted Dpp and Wg proteins provide positional information along the A/P and D/V axes, respectively, to establish Cartesian-like coordinates in the pouch field. Relative positions of dpp and wg expression domains in the notum are more similar to those in the wing pouch (in both cases, the expression domains are orthogonal). However, a D/V compartment boundary does not exist in the notum. The results described here reveal that another compartment-independent mechanism acts to pattern the presumptive notum. Namely, the D/V axis, provided by Pnr, Ush, and Wg, is initially established by the Dpp gradient, which mainly contributes the positional information along the A/P axis. One of the key issues of this patterning model is that Dpp signaling seems to act preferentially along the A/P axis of the notum. This is because two target genes, pnr and ush, are induced farther from the Dpp source along the A/P axis than along the D/V axis. One possible explanation for this phenomenon is that the diffusion of Dpp protein may be positively regulated along the A/P axis. However, such asymmetric induction is not observed on the dad induction; dad is one of the Dpp signaling targets in the wing disc. This suggests that diffusion of Dpp protein is not directionally regulated in the notum region. An alternative explanation would be that an effective range of Dpp morphogen gradient is established in a relatively short range. Cells that respond to Dpp would proliferate or migrate preferentially along the A/P axis. pnr mRNA is detected mainly in the posterior-dorsal region of the presumptive notum. GFP expression of UAS-gfp pnr^md237 is seen along the entire dorsal side of the presumptive notum. This difference between the staining pattern of pnr mRNA and GFP expression of UAS-gfp pnr^md237 in the late third larval stage seems to be caused by a long half-life of gal4 and/or gfp products, suggesting that cells that once have expressed pnr mRNA proliferate preferentially along the A/P axis. However, it seems to be difficult to explain the determination of pnr and ush expression domains only by the Dpp morphogen gradient. The existence of Tkv*-insensitive cells for inducing pnr and ush indicates that some regional subdivision may occur independently of Dpp signaling. Discontinuous expression of dpp in the A/P border of the notum also suggests the existence of a Dpp-independent subdivision. D/V subdivision of the presumptive notum seems to be achieved by several parallel mechanisms, including Dpp signaling (Tomoyasu, 2000).

Because Drosophila is a holometabolous insect, it should destroy larval tissues and replace them with a different population of cells to form the adult structure during the pupal stage. Thus, formation of epidermal structure should occur reiteratively during embryogenesis and metamorphosis. Patterning of larval epidermal structure takes place during embryogenesis; however, patterning of adult structure is mainly performed in larval stage imaginal discs. The Dpp morphogen gradient has been shown here to regulate pnr and ush expression to pattern the presumptive notum, which forms the dorsal structure of the adult, in the wing imaginal disc. pnr and ush are necessary for the formation of amnioserosa, the dorsal structure of the embryo, and both pnr and ush expressions are also positively regulated by Dpp in a concentration-dependent manner during embryogenesis. Furthermore, dorsal closure during embryogenesis and thorax closure in metamorphosis is also analogous, because both processes are regulated by the same signaling cascade, JNK signaling. These similarities between embryogenesis and metamorphosis presumably reflect the evolutionary history of the development in holometabolous insects (Tomoyasu, 2000).

Decapentaplegic (Dpp), a homolog of vertebrate bone morphogenic protein 2/4, is crucial for embryonic patterning and cell fate specification in Drosophila. Dpp signaling triggers nuclear accumulation of the Smads Mad and Medea, which affect gene expression through two distinct mechanisms: direct activation of target genes and relief of repression by the nuclear protein Brinker (Brk). The zinc-finger transcription factor Schnurri (Shn) has been implicated as a co-factor for Mad, based on its DNA-binding ability and evidence of signaling dependent interactions between the two proteins. A key question is whether Shn contributes to both repression of brk as well as to activation of target genes. During embryogenesis, brk expression is derepressed in shn mutants. However, while Mad is essential for Dpp-mediated repression of brk, the requirement for shn is stage specific. Analysis of brk;shn double mutants reveals that upregulation of brk does not account for all aspects of the shn mutant phenotype. Several Dpp target genes are also expressed at intermediate levels in double mutant embryos, demonstrating that shn also provides a brk-independent positive input to gene activation. Shn-mediated relief of brk repression establishes broad domains of gene activation, while the brk-independent input from Shn is crucial for defining the precise limits and levels of Dpp target gene expression in the embryo (Torres-Vazquez, 2001).

Genetic evidence implicates both Shn and Mad in dpp-dependent repression of brk. In the wing disc, cells that lack Mad or shn ectopically express brk and fail to activate the Dpp-responsive genes optomotor-blind, vestigial, spalt and Dad. Abolition of shn or Mad activity results in upregulation of brk in the embryo and in the absence of shn ectopic Dpp cannot suppress brk expression. Since Shn and Mad interact directly, an attractive hypothesis is that a Shn/Mad complex is involved in the Dpp-dependent repression of brk. It has recently been suggested that Dpp signaling bifurcates downstream of Mad/Med into a Shn-dependent pathway, leading to brk repression and a Shn-independent pathway that triggers gene activation. According to this model, Shn acts primarily as a dedicated repressor that switches Mad from a transcriptional activator to a transcriptional repressor on the brk promoter. However several lines of evidence from this study are incompatible with such an interpretation (Torres-Vazquez, 2001).

Analysis of Dpp-responsive gene expression in brk; shn double mutants has allowed an assessment the brk-independent input from shn to gene activation at different developmental stages in a range of tissues. Although it has not been demonstrated that each of these markers is a direct target of Dpp signaling, three categories of responses can be distinguished based on these studies. In the first group (class A), exemplified by dpp in the leading edge of the dorsal ectoderm, expression in the double mutant is indistinguishable from that in brk- embryos. Thus, shn contributes to class A gene expression primarily by relief of brk repression. Promoters belonging to class B include Dad and pnr in the dorsal ectoderm during germband extension. Expression of class B genes is downregulated in the double mutant compared with brk- embryos, but is equivalent to wild-type levels. It is inferred from this result that in the absence of Brk and Shn, Mad-mediated activation may be sufficient for expression within the normal domain, but cannot sustain the lateral expansion encountered in brk mutants. A third category of responses (class C) includes dpp and Ubx in the midgut, and sna in the primordia of the wing/haltere imaginal discs. Genes in this class show significantly reduced levels of expression in the double mutant, not only relative to brk- but also compared with wild-type animals. Class C promoters incorporate a brk-independent positive input from shn that is necessary for wild-type levels of expression. The inability of ectopic Dpp to induce sna expression in shn mutants demonstrates the essential nature of the requirement for Shn in activation of class C genes (Torres-Vazquez, 2001).

It is evident that repression of brk is crucial for expression of all three classes of genes described, and as such accounts for a significant part of the positive input from shn to gene activation. In addition, the data suggest that Mad and Shn contribute equally to repression of brk and regulation of class A genes. However, the fact that brk activity is only partially epistatic with respect to class B and C promoters, indicates that the majority of genes examined in this study integrate positive inputs from shn, as well as negative inputs from brk. The near wild-type expression of class B genes in double mutant embryos suggests that the brk-independent input from shn may be crucial at the margins of the expression domains and may be less significant in regions of the embryo that receive moderate to high levels of Dpp signaling. In contrast, the positive input from shn to class C targets appears to be important throughout the domain of expression. The observation that genes such as dCreb-A and Scr, which are repressed by dpp signaling, and which are also sensitive to loss of brk, raises the possibility that Dpp regulates their expression indirectly. In this event, the dpp target genes that mediate repression of dCreb-A and Scr would belong to classes A and C, respectively (Torres-Vazquez, 2001).

The partial restoration of dpp target gene expression in the double mutants relative to shn- embryos provides a basis for interpreting the cuticle phenotype. Homozygous brk;shn animals as well as brk;tkv mutants have an intermediate phenotype in that they show rescue of the dorsal closure defect observed in shn and tkv mutants, but they also display a reduced dorsal epidermis compared with brk-null embryos. Both dpp and pnr have been implicated in dorsal closure, which results from movement of the epidermal cells over the amnioserosa and their suturing at the midline. In light of this, the recovery of their expression in the dorsalmost ectodermal cells in the double mutants correlates well with the restoration of dorsal closure. Likewise, the compromised expression of dorsal ectodermal markers such as Dad and pnr in brk;shn embryos relative to brk null animals, provides molecular correlates for the ventralization observed in the double mutants (Torres-Vazquez, 2001).

Transforming growth factor ß signaling mediated by Decapentaplegic and Screw is known to be involved in defining the border of the ventral neurogenic region in the fruitfly. A second phase of Decapentaplegic signaling occurs in a broad dorsal ectodermal region. The dorsolateral peripheral nervous system forms within the region where this second phase of signaling occurs. Decapentaplegic activity is required for development of many of the dorsal and lateral peripheral nervous system neurons. Double mutant analysis of the Decapentaplegic signaling mediator Schnurri and the inhibitor Brinker indicates that formation of these neurons requires Decapentaplegic signaling, and their absence in the mutant is mediated by a counteracting repression by Brinker. Interestingly, the ventral peripheral neurons that form outside the Decapentaplegic signaling domain depend on Brinker to develop. The role of Decapentaplegic signaling on dorsal and lateral peripheral neurons is strikingly similar to the known role of Transforming growth factor ß signaling in specifying dorsal cell fates of the lateral (later dorsal) nervous system in chordates (Halocythia, zebrafish, Xenopus, chicken and mouse). It points to an evolutionarily conserved mechanism specifying dorsal cell fates in the nervous system of both protostomes and deuterostomes (Rusten, 2002).

The embryonic abdominal (A) PNS of Drosophila consists of three bilateral clusters of neurons (ventral, lateral and dorsal) per segment, which can be most especially appreciated in the serially homologous segments A1-A7. In order to investigate whether the second phase of Dpp signaling is necessary for patterning the PNS, mutant alleles for a gene involved in the Dpp signaling pathway, schnurri (shn), were examined. This gene encodes a zinc-finger transcription factor that is necessary for the transcription of some Dpp target genes and binds directly to the main Dpp mediator Mothers against Dpp (Mad). Unlike the zygotic mutants of dpp, scw, tolloid (tld) or mad, shn mutants have no effect on the initial dpp/scw governed dorsoventral patterning of the blastoderm. They express normally the early Dpp target genes, such as pannier (pnr, stage 7), dpp itself in the dorsal ectoderm (stage 9) and Krüppel (Kr) (which is a marker for the amnioserosa), showing that the dorsal ectoderm is correctly specified. By contrast, several Dpp target genes that are expressed following the second phase of Dpp signaling are affected in shn zygotic mutants: at stage 11, the expression of genes responsive to Dpp signaling, such as dad, pnr, spalt or dpp itself is reduced or lost. Thus, any failures in PNS formation, which are observed in shn mutant embryos, must originate from the second rather than the first phase of Dpp signaling and are likely to be mediated by Shn. PNS malformations were sought in strong shn zygotic mutant embryos using the ubiquitous PNS neuronal marker 22C10. Homozygous shn¹ and shn^k00401 fail to undergo dorsal closure and show severe defects of PNS development. A strong reduction in number of neurons is observed, especially in the dorsal and lateral PNS clusters, although it is difficult to determine exactly which neurons are affected because of the dorsal closure failure. Therefore, another allele, shn^k04412, which does undergo dorsal closure, was also examined. In these embryos, position and identity of PNS neurons could be more clearly assigned. In homozygosity, as well as in transheterozygosity over shn¹, this mutant shows a reduction in the number of dorsal and lateral neurons, similar to the other mutants analyzed. These results are consistent with a role for Shn-mediated Dpp signaling in the formation of the dorsal and lateral PNS (Rusten, 2002).

The Dorsocross T-box genes are key components of the regulatory network controlling early cardiogenesis in Drosophila: The Dorsocross genes act in parallel with tinman to activate the expression of pannier

Cardiac induction in Drosophila relies on combinatorial Dpp and Wg signaling activities that are derived from the ectoderm. Although some of the actions of Dpp during this process have been clarified, the exact roles of Wg, particularly with respect to myocardial cell specification, have not been well defined. The present study identifies the Dorsocross T-box genes as key mediators of combined Dpp and Wg signals during this process. The Dorsocross genes are induced within the segmental areas of the dorsal mesoderm that receive intersecting Dpp and Wg inputs. Dorsocross activity is required for the formation of all myocardial and pericardial cell types, with the exception of the Eve-positive pericardial cells. In an early step, the Dorsocross genes act in parallel with tinman to activate the expression of pannier, a cardiogenic gene encoding a Gata factor. Loss- and gain-of-function studies, as well as the observed genetic interactions among Dorsocross, tinman and pannier, suggest that co-expression of these three genes in the cardiac mesoderm, which also involves cross-regulation, plays a major role in the specification of cardiac progenitors. After cardioblast specification, the Dorsocross genes are re-expressed in a segmental subset of cardioblasts, which in the heart region develop into inflow valves (ostia). The integration of this new information with previous findings has allowed drawing a more complete pathway of regulatory events during cardiac induction and differentiation in Drosophila (Reim, 2005b).

In vertebrate species, genetic studies with loss-of-function alleles have implicated Tbx1, Tbx2, Tbx5 and Tbx20 in the control of heart morphogenesis and the regulation of cardiac differentiation markers. In the case of Tbx5, a small number of cardiac differentiation genes have been identified as direct downstream targets. However, owing to the complexity of the system, the respective positions of these genes within a regulatory network during early cardiogenesis are still poorly understood (Reim, 2005b).

Drosophila offers a simpler system to study regulatory networks in cardiogenesis. The Tbx20-related T-box genes mid and H15 have been shown to play a role in cardiac development downstream of the early function of the NK homeobox gene tin and the Gata gene pannier (pnr). Whereas the role of these genes in the morphogenesis of the cardiac tube is minor, they are involved in processes of cardiac patterning and differentiation during the second half of cardiogenesis, which includes the activation of tin expression in myocardial cells (Reim, 2005a). The present report characterizes the roles of the Tbx6-related Dorsocross T-box genes (which may actually have arisen from a common ancestor of the vertebrate Tbx4, Tbx5 and Tbx6 genes), in Drosophila cardiogenesis. The Doc genes have a fundamental early role; they are required for the specification of all cardiac progenitors that generate pure myocardial and pericardial lineages. They are not required for generating dorsal somatic muscle progenitors and lineages with mixed pericardial/somatic muscle, even though their early expression domains also include cells giving rise to these lineages (Reim, 2005b).

The new information on the regulation and function of Doc fills a major gap in the understanding of early Drosophila cardiogenesis. Previous data have shown that the combinatorial activities of Wg and Dpp are required for the formation of both myocardial and pericardial cells. In addition, the homeobox gene even-skipped (eve) is a direct target of the combined Wg and Dpp signaling inputs in specific pericardial cell/dorsal somatic muscle progenitors. Current data identify the Doc genes as downstream mediators and potential direct targets of combined Wg and Dpp signals during the induction of myocardial and Eve-negative pericardial cell progenitors. The induction of Doc expression by Wg and Dpp occurs concurrently with the induction of tin by Dpp alone, at a time when the mesoderm still consists of a single layer of cells. As a result, tin and Doc are co-expressed in a segmental subset of dorsal mesodermal cells that include the presumptive cardiogenic mesoderm. Conversely, in the intervening subset of dorsal mesodermal cells (the presumptive visceral mesoderm precursors) tin is co-expressed with bagpipe (bap) and biniou (bin), which are both negatively regulated by Wg via the Wg target sloppy paired (slp). Ultimately, these shared responses to Dpp, differential responses to Wg and the specific genetic activities of Doc versus bap and bin lead to the reciprocal arrangement of cardiac versus visceral mesoderm precursors in the dorsal mesoderm (Reim, 2005b and references therein).

Although the Dpp signaling pathway (and likewise, the Wg pathway) is activated in both ectodermal and mesodermal germ layers, tin and bap respond to it only in the mesoderm. The germ layer-specific response of these genes to Dpp relies on two probably interconnected mechanisms. The first of these involves the additional requirement for Tin protein as a mesodermal competence factor for Dpp signals, which is initially produced in the mesoderm downstream of twist. The second involves the specific repression of the responses of tin and bap to Dpp in the ectoderm by yet unidentified factors that bind to the Dpp-responsive enhancers of these two genes. By contrast, the Doc genes are induced by Dpp and Wg with the same spatial and temporal expression patterns in both germ layers. This implies that the (yet unknown) Dpp and Wg-responsive enhancer(s) of the Doc genes are not subject to the ectodermal repressor activities acting on the tin and bap enhancers, and fits with the observation that induction of Doc in the mesoderm does not require Tin as a mesodermal competence factor. However, because of the distinct roles of Doc in the ectoderm and mesoderm, this situation also implies that Doc must act in combination with germ layer-specific co-factors to exert its respective functions. These data suggest that, in the early mesoderm, Doc acts in combination with tin (Reim, 2005b).

A key gene requiring combinatorial Doc and Tin activities for its activation in the cardiac mesoderm is the Gata factor-encoding gene pannier (pnr). pnr expression is activated in the cardiac mesoderm shortly after the induction of Doc and tin, at a time when Doc expression has narrowed to the mesodermal precursors giving rise to pure cardiac lineages. The mechanisms restricting Doc expression to the cardiac mesoderm are currently not known, but as a consequence, pnr expression is also limited to the cardiac mesoderm. It is conceivable that Doc receives continued inputs during this period from the ectoderm through Dpp, whose expression domain narrows towards the dorsal leading edge by then. Together with the observed feedback regulation of pnr on tin and Doc, this situation leads to a prolonged co-expression of Tin, Doc and Pnr in the cardiac mesoderm of stage 11 to stage 12 embryos. Based upon the onset of the expression of early markers such as mid and svp, this is precisely the period when cardiac progenitors become specified (Reim, 2005b).

It is anticipated that the activation of some downstream targets in presumptive cardiac progenitors requires the combination of two, or perhaps all three, of these cardiogenic factors. Potential target genes include mid, svp and hand. However, none of these candidates is essential for generating cardiac progenitors, although mid and svp are known to be required for the normal diversification of cardioblasts within each segment (Reim, 2005b).

The observation that forced expression of Pnr in the absence of any Doc partially rescues cardiogenesis could indicate that the early, combinatorial functions of tin and Doc are primarily mediated by pnr. Alternatively, or in addition, this observation and the fact that a few cardioblasts can be generated without Doc could point to the existence of some degree of functional redundancy among these three factors. In the context of the latter possibility, it is tempting to speculate that the functional redundancy among T-box, Nkx and Gata factors during early cardiogenesis has further increased during the evolution of the vertebrate lineages. This would explain the less dramatic effects of the functional ablation of Tbx5, Nkx2-5 and Gata4/5/6 on vertebrate heart development as compared to the severe effects of Doc, tin or pnr mutations on dorsal vessel formation in Drosophila. Like the related Drosophila genes, these vertebrate genes are co-expressed in the cardiogenic region and developing heart of vertebrate embryos, which at least for Nkx2.5 and Gata6 also involves cross-regulatory interactions that reinforce their mutual expression (Reim, 2005b).

The observed co-expression of Doc, Tin and Pnr allows for the possibility that, in addition to combinatorial binding to target enhancers, protein interactions among these factors play a role in providing synergistic activities during cardiac specification. Physical interactions of Tbx5 with Gata4 and Nkx2-5, as well between Nkx2-5 and Gata4 in vitro as well as synergistic activities cell culture assays have been demonstrated in mammalian systems and may be relevant to human heart disease. In Drosophila, the genetic interactions between Doc, tin and pnr observed both in loss- and gain-of-function experiments reveal similar synergistic activities of the encoded factors during early cardiogenesis. Altogether, these observations make it likely that these Drosophila factors also act through combinatorial DNA binding and mutual protein interactions to turn on target genes required for the specification of cardiac progenitors (Reim, 2005b).

Whereas pnr is expressed only transiently during early cardiogenesis, tin and Doc continue to be expressed in developing myocardial cells, suggesting that they act both in specification and differentiation events. Recently it was shown that the T-box gene mid is required for re-activating tin in cardioblasts (Reim, 2005a). Of note, owing to the action of svp, Doc and tin are expressed in complementary subsets of cardioblasts within each segment. This mutually exclusive expression of tin and Doc implies that they are not acting combinatorially but, instead, act differentially during later stages of myocardial development. Hence, their activities could result in the differential activation of some differentiation genes such as Sulfonylurea receptor (Sur), which is specifically expressed in the four Tin-positive cardioblasts in each hemisegment (Nasonkin, 1999; Lo, 2001), and wingless (wg), which is only turned on in the two Doc-positive cells in each hemisegment of the heart that generate the ostia. Surprisingly, even the activation of some genes that are expressed uniformly in all cardioblasts has turned out to result from differential regulation within the Tin-positive versus Doc-positive cardioblasts. For example, regulatory sequences from the Mef2 gene for the two types of cardioblasts are separable and those active within the four Tin-positive cells are directly targeted by Tin. Likewise, regulatory sequences from a cardioblast-specific enhancer of Toll have been shown to receive differential inputs from Doc and Tin, respectively, in the two types of cardioblasts. In parallel with this differential regulation, it is anticipated that yet unknown differentiation genes are activated uniformly in all cardioblasts downstream of mid/H15 and hand. The integration of the new information on the roles of Doc in cardiogenesis has now provided a basic framework of signaling and gene interactions through all stages of embryonic heart development, which in the future can be further refined upon the identification of new components and additional molecular interactions (Reim, 2005b).

Targets of Activity

Mutants with lesions in the PNR zinc finger domain display an overexpression of achaete and scute and the development of extra neural precursors. Mutant proteins in which the putative zinc finger helices are deleted act as hyperactive repressor molecules, causing a loss of achatae/scute expression and loss of neural precursors (Ramain, 1993). Thus Pannier negatively regulates achaete/scute proneural genes.

The behavior of the hyperactive repressor molecules could be explained one of two ways: either the repressing activity of wild type PNR molecules is negatively regulated by association with other proteins through PNR helical domains, or the helical domains of PNR mask its zinc finger domains (Ramain, 1993).

The consensus GATA sequence defined for Pannier binding is identical to that present in the alpha-globin promoter recognized by the chicken GATA-1 protein. This promoter is active throughout development in erythroid cells and is unlikely to be regulated by stage- and tissue-specific factors. Expression of either GATA-1 or Pannier stimulates a 35-fold increase in activity in a wild type chicken alpha-globin reporter. The effect of Pnr is mediated through a repeat of the GATA motif present in the promoter, since mutation of both GATA sequences abolishes activity. Like GATA-1, Pannier binds as a monomer to the proximal GATA motif to stimulate globin transcription. The consensus binding sequence is GATAAgg. Two dominant alleles of pannier have been described with point mutations resulting in proteins with a single amino acid change in the amino-terminal zinc finger. They are associated with an overexpression of achaete-scute and ectopic dorsocentral bristles on the thorax. Other frameshift deletions that remove the two amphipathic alpha-helices present in the C-terminus of the protein (presumably the activation domain) are associated with decreased ac-sc expression and a loss of dorsocentral bristles. The frameshift mutant proteins do not activate the alpha-globin promoter (Haenlin, 1997).

On each half of the dorsal mesothorax (heminotum), 11 large bristles (macrochaetae) occupy precisely constant positions. The location of each macrochaeta is specified during the third instar larval and early pupal stages by the emergence of its precursor cell (sensory mother cell: SMC) at a precise position in the imaginal wing discs, the precursors of the epidermis of most of the mesothorax and wings. The accurate positioning of SMCs is thought to be the culmination of a multistep process in which positional information is gradually refined. The GATA family transcription factor Pannier and the Wnt secreted protein Wingless are known to be important for the patterning of the notum. Thus, both proteins are necessary for the development of the dorsocentral mechanosensory bristles. Pannier has been shown to directly activate the proneural genes achaete and scute by binding to the enhancer responsible for the expression of these genes in the dorsocentral proneural cluster. Moreover, the boundary of the expression domain of Pannier appears to delimit the proneural cluster laterally, while antagonism of Pannier function by U-shaped, a Zn-finger protein, sets its limit dorsally. Therefore, Pannier and U-shaped provide positional information for the patterning of the dorsocentral cluster. In contrast and contrary to previous suggestions, Wingless does not play a similar role, since the levels and vectorial orientation of its concentration gradient in the dorsocentral area can be greatly modified without affecting the position of the dorsocentral cluster. Thus, Wingless has only a permissive role on dorsocentral achaete-scute expression. Evidence is provided indicating that Pannier and U-shaped are main effectors of the regulation of wingless expression in the presumptive notum (Garcia-Garcia, 1999).

An enhancer that directs expression specifically at the DC proneural cluster is present within a 5.7 kb fragment of AS-C DNA. Different subfragments were assayed for enhancer activity in vivo. A 1.4 kb subfragment (AS1.4DC) directs lacZ transcription from a minimal hsp70 promoter in the DC proneural cluster: beta-galactosidase and Scute endogenous accumulations precisely colocalized at this cluster. This fragment and the corresponding region of the AS-C from D. virilis were sequenced. Stretches of conserved DNA were present throughout the fragment, although they appeared to cluster within three regions. Subfragments containing each one of these regions were assayed for DC enhancer activity. Only the most 3' subfragment (PB0.5DC) shows such an activity, but to a much lesser extent than AS1.4DC. Interestingly, the activity is usually limited to only one cell, which is the posterior DC SMC. However, when assayed with the sc promoter, the PB0.5DC fragment directs lacZ activity in most cells of the DC cluster. Consequently, the sequences essential for specifying transcription in the DC cluster are contained within the PB0.5DC subfragment, although additional sequences that reinforce this expression are present in the larger AS1.4DC fragment. The AS1.4DC fragment was used to study DC enhancer activity (Garcia-Garcia, 1999).

The Pnr protein, which is a GATA-1 transcription factor, is known to regulate ac-sc expression at the DC cluster by acting directly or indirectly through the DC enhancer. The sequence of AS1.4DC was examined: within it, seven putative GATA-1 factor binding sites were found. Three of them fit the vertebrate consensus sequence (WGATAR: sites 1, 2 and 4); three comply with the consensus obtained in a random oligonucleotide selection experiment performed with Pnr protein (GATAAG: sites 3, 5 and 6), and one fits both consensus sequences (site 7). Interestingly, sites 5, 6 and 7 are within the PBO.5DC subfragment and two of them are conserved in D. virilis. Site-directed mutagenesis of site 7 strongly decreases enhancer activity of the AS1.4DC-lacZ construct (abbreviated DC-lacZ). Additional mutagenesis of other sites displaying the vertebrate consensus does not further reduce the residual activity. However, mutagenesis of all seven sites completely abolishes activity. These data suggest that Pnr interacts with some of these sites and that this interaction is essential for DC-lacZ activity. The capacity of Pnr to activate transcription of an AdhCAT reporter gene linked to either the complete AS1.4DC enhancer fragment or to each of the three subfragments was tested in transfection assays performed in chicken embryonic fibroblast (CEF) cells. Pnr stimulates transcription to similar levels from the complete enhancer and from subfragment PB0.5DC. In contrast, no stimulation was detected with the other subfragments. Notably, PB0.5DC displays DC enhancer activity in flies and contains three putative Pnr-binding GATA sites. Mutagenesis of only one of these (site 7) does not affect AdhCAT activity. But simultaneous removal of two sites (either sites 5 and 7, or 6 and 7) strongly impairs activity and mutagenesis of all three sites essentially abolished it. This suggests that a minimum of two GATA sites are necessary for transcriptional activation. Further evidence for a direct interaction of Pnr with the GATA sites of the enhancer was obtained in electrophoretic mobility-shift assays (EMSA) conducted with two different GST-Pnr fusion proteins that included the DNA binding domain of Pnr. Additonally, it has been shown that although relatively high levels of Wg protein are necessary for full DC-lacZ activity, the precise levels of this protein and the orientation of its gradient do not convey information for the position and the shape of the DC cluster (Garcia-Garcia, 1999).

In the prospective notum, the stripe of diffusible Wg protein straddles the lateral border of the domain of expression of pnr. This is compatible with the location of the Wg source being on the border of, but still within, the pnr domain. In accordance with this location, pnr appears to activate wg, since it has been found that a wg-lacZ construct, which reproduces the notal band of Wg accumulation, is not expressed in pnr mutant discs and is ectopically expressed in the dorsalmost area of the disc in a pnr dominant gain-of-function combination. In contrast, other data suggest that Pnr represses wg. Thus, the notal wg stripe is expanded dorsally in strong hypomorphic pnr combinations. Moreover, in flies in which pnr is overexpressed there was no expansion of the domain of WG mRNA, which in fact accumulates in a stripe that is even narrower than that seen in the wild type. The repressing effects appeared to be restricted to the domain of accumulation of Ush, which suggests the participation of Pnr/Ush heterodimers in the repression. Consistent with this assumption, the Pnr^D1 mutant protein, which is incapable of interacting with Ush, promotes wg expression within the entire dorsalmost area of the disc in pnr mutants animals. Interestingly, Pnr D1 can not induce the expansion of the wg expression domain in the presence of wild-type Pnr, suggesting that Pnr+/Ush heterodimers interfere with the Ush-resistant function of Pnr^D1. Such interference may also account for the repression of the Pnr^D1-mediated dorsal expansion of DC-lacZ expression by Pnr+. Taken together, these results suggest that during development of the wing disc, Pnr is necessary both for activation of wg and (together with Ush) for its repression in the dorsalmost region of the presumptive notum. This dorsal repression probably takes place from the start of wg expression, since the earliest detectable accumulation of WG mRNA is already restricted to the presumptive mid notal region. A wg-lacZ enhancer trap line, which shows expression throughout the dorsalmost part of the early third instar wing discs and posterior refinement to the notal stripe, might have a reduced sensitivity to the repression by Pnr/Ush (Garcia-Garcia, 1999).

A model is provided for the dorsal-lateral patterning of the DC area by Pnr and Ush. In the third instar wing disc and in the dorsalmost part of the prospective notum, Ush is present at high concentrations and the Pnr/Ush heterodimers are relatively abundant. These heterodimers would act as repressors and prevent activation of downstream genes. In the DC area, defined along the dorso-lateral axis by lower concentrations of Ush and the presence of Pnr, there is sufficient free Pnr to activate genes like ac-sc, DC-lacZ and wg. ac-sc is transcribed in the more dorsal part of the area because its activation requires relatively high concentrations of Pnr. wg is only transcribed at the edge of the Pnr domain because its expression is very sensitive to both Pnr and Pnr/Ush, and consequently low concentrations of the former are sufficient for activation and low concentrations of the latter, even in the presence of high concentrations of free Pnr, impose repression. The inability of extra doses of the activator Pnr to revert the repression by Pnr/Ush in the dorsalmost region of the notum suggests that activator and repressor do not compete for overlapping sites at the DC as-sc and notal wg enhancers. The presence of Pnr/Ush at their site(s) would block the activating effect of bound Pnr. Additional inputs, notably decapentaplegic, are known to act on the DC enhancer (Garcia-Garcia, 1999).

Embryonic target gene activation in the absence of brinker is independent of SMAD activity. Thus brk acts either parallel to or downstream of SMADs as a specific repressor of low and intermediate level Dpp target genes. brk is expressed like another dpp antagonist, short gastrulation (sog), in ventrolateral regions of the embryo abutting the dorsal dpp domain, and in brk mutants dpp expression expands to cover the entire ectoderm. In this situation sog is largely responsible for Dpp gradient formation, since brk;sog double mutant embryos have almost no polarity information in the ectoderm. The double mutants consist mainly of mesoderm and unstructured dorsal epidermis. Thus, brk and sog together specify the neuroectoderm of Drosophila embryos (Jazwinska, 1999).

The cuticle of brk mutant embryos has an enlarged region carrying dorsal hairs and a smaller region carrying ventral denticles. The number of sna-expressing neuroblasts in the ventral neurogenic region is reduced. This indicates that brk mutations lead to an expansion of dorsolateral fates and a reduction of ventrolateral fates. However, despite these lateral fate shifts, the number of Kr-expressing amnioserosa cells is not different from wild type. Thus, brk specifically affects cell fates depending on low or intermediate levels of Dpp signaling, while those that require peak levels are not altered. To identify the underlying causes of the visible changes in cell fate, the effect of brk was examined on the expression of two groups of dorsal/ventral (DV) patterning genes. The first group consists of dpp, zerknullt (zen) and tolloid (tld), whose expression is initiated very early in syncytial blastoderm stages. Since they are ventrally repressed by Dorsal (Dl) protein, their expression domains are confined to the dorsal 40% of the egg's circumference. In brk mutant embryos dpp, zen and tld expression is initiated normally. However, in contrast to wild type their expression domains expand ventrally during mid-cellularization. These data demonstrate that brk is not required for the early ventrolateral repression of these genes, but is essential to prevent their lateral expansion during cellularization. The second group of DV patterning genes includes rhomboid (rho), u-shaped (ush) and pannier (pnr), which are not direct targets of repression by maternal Dl. The initiation of their expression during cellularization requires prior formation of the Dpp activity gradient. Therefore, they are candidates for being direct targets of Dpp signaling in the embryo. They are expressed in domains straddling the dorsal midline that are 12 (rho), 14 (ush) and 32 (pnr) cells wide at cellular blastoderm (cell counts at approx. 50% egg length). The two narrowly expressed genes rho and ush are not changed in brk mutant embryos. This is also true for late zen expression, which in brk mutant embryos, as in wild type, refines to a narrow 5- to 6- cell-wide stripe along the dorsal midline despite the prior expansion. However, pnr expression expands in brk mutant embryos and low ectopic pnr levels can be seen in a broad lateral domain that stops about five cells short of mesodermal sna expression. Thus, brk does not affect the Dpp target genes that are expressed in dorsalmost regions and supposedly depend on highest Dpp levels. However, a target gene that is expressed in a wider domain, and is therefore presumably activated by intermediate levels of Dpp, is expanded. In summary, brk mutations affect the Dpp activity gradient in the embryo by expanding the domains of expression of dpp and one of its activators (tld) into ventrolateral regions. Despite the uniform expression of dpp in the entire ectoderm, Dpp activity levels appear to be only mildly increased in the ventrolateral region since only low-level (zen) or intermediate-level (pnr) target genes are ectopically expressed, causing a reduction in the size of the nervous system and ventral epidermis accompanied by an expansion of dorsal epidermis. Peak levels of Dpp in dorsalmost positions appear to be normal, judging from both target gene expression and cell type differentiation (Jazwinska, 1999).

In developing Drosophila notum, wingless expression is regulated by positive and negative Decapentaplegic signaling so that only notal cells receiving optimal levels of Decapentaplegic signal express wingless. This Decapentaplegic-dependent regulation of notal wingless expression includes multiple mechanisms involving pannier and u-shaped. In the medial notum, Pannier and U-shaped form a complex. The expression of pannier and u-shaped is positively regulated by Decapentaplegic signals emanating from the dorsal-most region. The Pannier/U-shaped complex serves as a repressor and a transcriptional activator, respectively, for wingless and u-shaped expression. In the more lateral region, wingless expression is up-regulated by U-shaped-unbound Pannier. wingless expression is also weakly regulated by its own signaling (Sato, 2000).

To further clarify the notion that notal wg expression occurs only in cells receiving optimal levels of Dpp signal, wg expression was examined in Mothers against dpp (Mad) and thickveins (tkv) mutant clones generated at two different stages. Mad encodes a transactivator acting downstream of Dpp signals, while tkv encodes a type I receptor for Dpp. Early and late clones were generated at late first instar and late second instar, respectively; resultant clones were observed in late third instar. A dorsal shift of wg expression was detected in both early and late clones homozygous for Mad^1-2, a hypomorphic mutant allele of Mad. In contrast, in the case of tkv^a12 (a strong hypomorphic mutant allele of tkv), a dorsal shift of wg expression was observed only in late clones. Little or no wg expression could be detected in any of the early clones. These results suggest that Dpp-signaling activity in cells within early tkv^a12 clones is much lower than that in Mad^1-2 and late tkv^a12 clones, and hence, medial-notal cells in Mad^1-2 and late tkv^a12 clones but not early tkv^a12 clones possess residual levels of Dpp-signaling activity, sufficient to induce wg expression. Consistent with this, twin spot analysis in the wing pouch where Dpp signals are autonomously required for cell proliferation has showen that Mad^1-2 mutant clones are recovered much more frequently than tkv^a12 clones. The absence of wg misexpression in late medial tkv^a12 clones situated along the anterior notal edge is possibly due to Bar-dependent repression of wg expression in the future anterior notum (Sato, 2000).

achaete-scute (ac-sc) expression in the notum is affected in several allelic combinations of pnr, whose function is prevented by ush gene product (Ush) as a result of direct binding to Pnr. Since pnr appears involved in notal wg expression, and pnr and ush are expressed in the future medial notum in a graded fashion with peaks within the dpp expressing dorsal-most region, the late third instar notum was examined by staining for wg protein and pnr or USH RNA. ush and wg expression areas were found to abut on each other except for the future scutellum, while almost all wg expressing cells were situated in a ventral-most region of the pnr expression domain. Although somewhat ambiguous, a similar relationship among wg, pnr and ush expression areas was detected in small discs at an early third instar stage, the earliest stage of notal wg expression. It may thus follow that wg expression occurs in lateral-notal cells expressing pnr but not ush throughout third instar larval notal development. wg-LacZ expression occurs in a region much broader than the authentic wg expression domain; wg-LacZ expression was always observed to be expanded medially or dorsally, suggesting that the authentic wg expression domain shifts ventrally as a disc grows (Sato, 2000).

To determine the role of pnr in wg expression, examination was made of wg expression on various pnr mutant backgrounds. Strong wg misexpression occurs in medial pnr-null-mutant (pnr^VX6) clones. wg-LacZ or Wg signals are detected in the entire medial notum transheterozygous for pnr^VX6 and pnr^VI, from which most, if not all, pnr activity is absent. In contrast, a significant reduction of wg expression occurs in pnr-null-mutant (pnr^VX6) clones generated within the authentic wg expression domain. These findings indicate that pnr is involved in both negative and positive regulation of notal wg expression; Pnr serves as a positive regulator of wg expression in the future lateral notum including the authentic wg expression domain, while it is a negative factor of wg expression in the medial notum. Consistent with this, ubiquitous or clonal expression of wild-type pnr induces wg misexpression in the notum ventral to the authentic wg expression domain, while no or little wg misexpression occurs in the future medial notum (Sato, 2000).

As in the case of ac-sc expression, ush appears to serve as a negative factor for notal wg expression, since (1) wg is misexpressed in ush-null-mutant (ush¹) clones in the future medial notum and (2) virtually all endogenous wg expression is abolished when wild-type ush is overexpressed throughout the notal region. In contrast to medial ush¹clones, no appreciable change in wg expression is detected in ush¹clones generated within the authentic wg expression domain. That the authentic wg expression domain is demarcated by medial ush expression may indicate that medial ush expression is involved in the establishment of the dorsal boundary of the authentic wg expression domain. Based on the fact that Pnr mutants such as Pnr^D1 and Pnr^D4, lacking ability to bind to Ush, are still capable of activating ac-sc in the presence of ush activity, wild-type Pnr has been proposed to be inactivated by ush through direct interactions of Ush with Pnr. However, the results presented here show that this may not be the case in notal wg expression and ac-sc expression for the DC macrochaetae formation. If Ush serves only as the inhibitor of Pnr as predicted, a wild-type copy of pnr added in trans to Pnr^D1 would not decrease the area of wg expression, since wild-type Pnr is considered to either activate wg expression or neutralize the negative function of Ush or both. The results presented here are apparently at variance with this consideration. Both wg and ac-sc misexpression found in the future medial notum of Pnr¹⁴/Pnr^VI discs are abolished in Pnr^D1/+ discs with no loss of wg and ac-sc expression in the authentic wg expression domain. This negative effect of wild-type pnr is reversed by halving the copy number of endogenous ush. It is concluded that, in the medial notum, Pnr forms a complex with Ush and the resultant Pnr/Ush complex represses wg and ac-sc expression directly or indirectly to establish the dorsal boundaries of the authentic wg expression domain and the ac-sc expression area for the DC macrochaetae formation (Sato, 2000).

Notal wg expression is regulated not only by dpp signaling but also by Pnr and Ush. Thus, pnr and ush expression may be under the control of Dpp signaling or conversely, Dpp signaling is regulated by pnr and ush. The second possibility, however, seems to be unlikely, since neither pnr nor ush mutant clones exhibit any appreciable change in brinker (brk)-LacZ expression. brk is a general Dpp target gene whose expression is negatively regulated by Dpp signaling. Loss of Dpp signaling causes cell-autonomous brk misexpression in the wing pouch and notum of wing imaginal discs. To determine the feasibility of the first possibility, pnr and ush expression was examined in tkv^a12, Mad^1-2 or tkv^Q253D(tkv^QD) clones; tkv^QD is a constitutively active form of tkv. pnr and ush are misexpressed in lateral UAS-tkv^QD clones generated in late second instar, an observation indicating that pnr and ush expression is under the control of Dpp signaling. Unlike wg expression, pnr and ush expression are abolished not only in early tkv^a12 clones but also in late tkv^a12 and early Mad^1-2 clones, both expressing wg, suggesting that pnr and ush expression requires higher levels of Dpp-signaling activity than those required for wg expression. Loss of ush expression in tkv^a12 and Mad^1-2 clones might be a secondary effect due to the loss of pnr expression, since the maintenance of ush expression requires both pnr and ush activities. pnr and ush expression may be independently initiated by Dpp signaling, since pnr expression normally occurs in ush mutant clones and no ush misexpression is induced by ubiquitous pnr expression. It is concluded that the graded expression of pnr and ush is determined by Dpp signaling and hence, Pnr and Ush act downstream of Dpp (Sato, 2000).

In the larval notal region, dpp expression is not continuous but is broken by the authentic wg expression domain, thus suggesting that notal development could be regulated by Dpp signals emanating separately from dorsal and ventral sources up to the wg expression domain. As anticipated, the expression of dad (dad-LacZ), a downstream component of Dpp signaling whose expression is positively regulated by Dpp signaling, is detected not only in medial but also in lateral notum. However, double-staining of dad-LacZ and either PNR or USH RNA expression shows that unlike dad-LacZ, pnr and ush are not induced in the postero-lateral notum in spite of the presence of active Dpp signals. In addition, ectopic wg expression induced by tkv^QD is restricted to the antero-lateral notum. It may thus follow that an unidentified factor represses the expression of a fraction of Dpp target genes, which include pnr, ush and wg but not dad, in the postero-lateral notum (Sato, 2000).

Wg signaling represses wg transcription for refinement of its own expression domain in the wing margin. Thus, an examination has been made of notal wg expression on Wg-signaling mutant backgrounds. In contrast to wing-margin, wg expression in the notum is activated by its own signaling though much less effectively. armadillo (arm) and disheveled (dsh) encode Wg signal transducers and wg^ts is a temperature-sensitive Wg secretion mutant. Weak partial wg misexpression is noted in about 50% of lateral clones (19 of 40 clones) expressing Deltaarm, which constitutively activates Wg signaling. Ectopic wg expression was also detected in a cell non-autonomous fashion when wg misexpressing clones were induced in the lateral notum. In contrast, wg transcription is considerably reduced in dsh null mutant clones. When wg^ts mutant discs are incubated at a non-permissive temperature, for 48 h, an appreciable reduction of wg expression is detected in the authentic wg domain without any significant change in pnr and ush expression. Taken together, these results indicate that Wg signaling weakly activates wg transcription in the future lateral notum. The failure of induction of wg misexpression in Deltaarm and wg clones in future medial notum may indicate that wg expression due to auto-activation is repressed by Pnr/Ush complexes in the medial notum. One unexpected finding is that, in the hinge region, strong wg misexpression occurred only in cells surrounding wg expressing cells, suggesting possibly a new type of Wg-dependent wg expression (Sato, 2000).

The entire notal ush expression area is included in the notal pnr expression domain and hence, notal ush expression might be positively regulated by pnr. This possibility using a pnr hypomorphic mutant and a significant reduction of notal ush expression was in pnr hypomorphic mutant flies. Thus, it is concluded that Pnr is involved in the up-regulation of notal ush expression. In the case of wg expression, Ush free of Pnr serves as an activator, while a Pnr/Ush complex serves as a repressor. To determine which forms of Pnr are involved in ush expression, examination was made of USH RNA expression in the notum expressing pnr ubiquitously and the notum transheterozygous for pnr^D1 and pnr^Vl. Neither wild-type Pnr free of Ush nor Pnr^D1, incapable of binding to Ush but capable of activating wg expression, could induce ush expression. It may thus follow that a Pnr/Ush complex (but not Pnr free of Ush) is required for ush expression as a positive transcriptional regulator (Sato, 2000).

A summary is presented of wg regulation in the notum. In both future medial and lateral notal regions, dpp is expressed and Dpp signaling is active. However, ventral Dpp signals are neutralized by an unknown mechanism as far as pnr, ush and wg expression is concerned. Notal wg expression, except for that in the scutellum, is regulated through four different pathways, three under the control of Dpp signals emanating from the dorsal-most region. pnr and ush expression is up-regulated by Dpp signaling, but ush expression is much narrower than that of pnr, possibly because of the requirement of higher Dpp-signaling activity for ush expression than that for pnr expression. In the future medial notum, Pnr and Ush form a complex repressing wg expression, while Ush-unbound Pnr activates lateral wg expression. The authentic wg domain and the medial notum abut one another. Unlike wg expression, ush expression in the future medial notum is positively regulated by the Pnr/Ush complex. This regulation appears required for the maintenance of medial ush expression. Dpp signaling is also capable of activating notal wg expression through an unidentified factor X. This route includes neither Pnr nor Ush. In addition, wg expression is weakly up-regulated by its own signaling in the lateral notum (Sato, 2000).

The D-mef2 gene is a direct transcriptional target of Tin and Pnr in cardioblasts. A defined heart enhancer for the gene contains a pair of essential Tin binding sites and a required GATA element located in close proximity to one of the Tin recognition sequences. Coexpression of the two factors in CNS midline cells results in the ectopic activation of the D-mef2 enhancer normally expressed only in cardial cells. This result is compatible with the nuclear colocalization and physical interaction of Tin and Pnr in cultured cells and provides an embryological assay for identifying regions of the proteins that are essential for their functional synergism. Nine deleted or point mutant versions of Tin were tested in the synergism assay. Tin(N351Q) has a single amino acid change in the homeodomain and is unable to bind DNA. Coexpression of this mutant with wild-type Pnr fails to activate the D-mef2 enhancer. While a competent homeodomain must be present in Tin for synergism with Pnr, this region by itself is not sufficient as it fails in the coactivation assay. The TN domain is a highly conserved 12 amino acid region found in Tin and most other NK-2 class proteins. A 10-amino acid deletion was made within this domain to generate the Tin(1-35, 46-416) mutant, but this altered protein is still able to function combinatorially with Pnr. Thus, the TN domain is dispensable in the synergism assay (Gajewski, 2001).

A transcriptional activation domain has been mapped to the N-terminal 114 amino acids of Tin by using a cell transfection strategy. To determine whether this region is required for functional interaction with Pnr, the Tin(111-416) deletion was generated and tested. This truncated protein remained competent to synergize with Pnr in the activation of the D-mef2 enhancer, showing that the Tin transactivation domain is not required. However, larger N-terminal deletions result in Tin proteins that are functionally inactive. Specifically, removal of an additional 41 amino acids in Tin(152-416) has identified residues 111 to 151 as essential for Tin synergism with Pnr. The Tin(1-109, 192-416) variant that contains the transactivation domain and homeodomain, but lacks internal sequences including the required 41-amino acid region, is likewise nonfunctional in the D-mef2 enhancer coactivation assay. Therefore, these studies identify two distinct regions of Tin needed for its combinatorial function with Pnr, an internal segment of 41 amino acids adjacent to the transactivation domain and the conserved homeodomain (Gajewski, 2001).

The ectopic activation assay was used to determine those regions of Pnr that are essential for its functional synergism with Tin. Six deleted or point mutant forms were tested for enhancer activation in CNS midline cells. Pnr(1-457) represents a C-terminal truncation of the GATA factor that maintains zinc fingers 1 and 2, but deletes two putative amphipathic a helices. This C-terminal region has been shown to contain a transcriptional activation domain, and the inability of the truncated protein to synergize with Tin demonstrates an essential requirement of this Pnr sequence. Pnr(E168K) and Pnr(C190S) contain single amino acid changes in the N-terminal zinc finger that correspond to mutations found in dominant alleles pnr. These mutations may affect the formation of the first zinc finger and result in proteins that heterodimerize poorly with the Ush antagonist. However, two different dominant mutant Pnr proteins are able to synergize with Tin and direct D-mef2 expression in the CNS. In contrast, the mutation of a conserved cysteine residue in zinc finger 2 in Pnr(C247S) inactivates the protein in the synergism assay. This amino acid change is likely to influence the formation of the C-terminal zinc finger and identifies this region as an essential functional domain of Pnr in the coactivation of D-mef2. It is important to note that, although Pnr(1-457) and Pnr(C247S) fail to synergize with Tin, they are competent to bind the homeodomain protein in the GST pull-down assay. In combination, these results substantiate that intrinsic functional properties of Pannier are perturbed in the two mutant forms of the GATA factor (Gajewski, 2001).

An unexpected finding of this work is that, while the C-terminal transactivation domain of Pnr is required in the combinatorial assay, the N-terminal transactivation domain of Tin is not. One could envision a mechanism wherein the presence of the single domain provided by Pnr is sufficient for the activation properties of the heterodimeric complex. Additionally, it can not be ruled out that a second transactivation domain exists in Tin that was not revealed previously in cell transfection studies. Also of note is the nonrequirement of a proposed cardiogenic domain of Tin that maps to the N-terminus of the protein. Specifically, Tin(111- 416) is competent to work with Pnr in the cooperative activation of the D-mef2 heart enhancer, despite the absence of residues 1 through 42. Instead, an internal 41-amino acid region between the Tin transactivation domain and homeodomain has emerged as a vital sequence for functional interaction with Pnr. A repressor activity of Tin has been ascribed to residues 111 through 188, and it is plausible that, based on the biological assay being used, multiple functional characteristics may be uncovered within this region (Gajewski, 2001).

In the context of Tin's synergistic interaction with Pnr in regulating a defined cardiac enhancer, association of the two through this domain may prevent Pnr from interacting with other proteins such as Ush. At the same time, because Tin has the potential to act as a transcriptional repressor that recruits Groucho via this domain, the interaction of Tin and Pnr through the essential 111 to 151 subregion may be beneficial to Tin in its role as a transcriptional activator by eliminating its possible association with inhibitory cofactors. Preliminary results suggest the molecular interaction of Tin and Pnr may be due in part to the presence of this domain (Gajewski, 2001).

In Drosophila, muscles attach to epidermal tendon cells are specified by the gene stripe (sr). Flight muscle attachment sites are prefigured on the wing imaginal disc by sr expression in discrete domains. The mechanisms underlying the specification of these domains of sr expression have been examined. The concerted activities of the wingless (wg), decapentaplegic (dpp) and Notch (N) signaling pathways, and the prepattern genes pannier (pnr) and u-shaped (ush) establish domains of sr expression. N is required for initiation of sr expression. pnr is a positive regulator of sr, and is inhibited by ush in this function. The Wg signal differentially influences the formation of different sr domains. These results identify the multiple regulatory elements involved in the positioning of Drosophila flight muscle attachment sites (Ghazi, 2003).

Pnr, a GATA-binding protein normally functions as a transcriptional activator and is antagonized by Ush in its function. Loss of function pnr mutants show no sr expression in the domain covered by pnr. This, along with sr expansion in mutants of ush, would suggest that pnr activates sr in the notum, and is inhibited by ush. However, there is also loss of sr expression in pnr `gain of function' mutants. The reason for this is not completely clear. One possibility is that since the mutation causes an increase in wg activity in the region this may cause a down-regulation of sr. This is supported by a similar effect seen on misexpression of activated armadillo in the pnr domain. Results with both pnr and ush have been taken into account to suggest that pnr positively regulates sr and is antagonized by ush (Ghazi, 2003).

These results indicate that each sr domain is regulated by a combination of prepattern genes and signaling molecules. But, a precise description of the 'combinatorial code' for regulation of each sr domain is beyond the scope of this work and can be achieved by generation of domain specific markers for sr. Based on expression pattern data, and existing literature, it is suggested that high levels of Pnr, low (or absence of) Ush and moderate levels of Wg determine the initial induction of domain a. The distinction between medial (a) and lateral (b-d) domains is established by the presence of very high levels of Wg (the cells where the Wg gradient originates). Lateral expression domains are probably induced in domains controlled by the lateral prepattern gene iro. The differences between different lateral domains arise as a result of expression of different genes in the region. For instance, the lateral-most domain d appears to be regulated by ush and does not encounter Wg at all. Whereas, all cells of b receive uniformly moderate levels of Wg, only cells at the borders of c receive high Wg levels, and these differences result in the distinct identities of the two domains. Dpp, either through its effects on these regulatory genes and/or through direct effects on sr influences the process (Ghazi, 2003).

The eyegone (eyg) gene is involved in the development of the eye structures of Drosophila. eyg and its related gene, twin of eyegone (toe), are also expressed in part of the anterior compartment of the adult mesothorax (notum). The anterior compartment is termed the scutum and consists of the part of the notum from the anterior border to the suture with the scutellum. In the absence of eyg function the anterior-central region of the notum does not develop, whereas ectopic activity of either eyg or toe induces the formation of the anterior-central pattern in the posterior or lateral region of the notum. These results demonstrate that eyg and toe play a role in the genetic subdivision of the notum, although the experiments indicate that eyg exerts the principal function. However, by itself the Eyg product cannot induce the formation of notum patterns; its thoracic function requires co-expression with the Iroquois (Iro) genes. The restriction of eyg activity to the anterior-central region of the wing disc is achieved by the antagonistic regulatory activities of the Iro and pnr genes, which promote eyg expression, and those of the Hh and Dpp pathways, which act as repressors. It is argued that eyg is a subordinate gene of the Iro genes, and that pnr mediates their thoracic patterning function. The activity of eyg gives rise to a new notum subdivision that acts upon the pre-extant one generated by the Iro genes and pnr. As a result the notum becomes subdivided into four distinct genetic domains (Aldaz, 2003).

A significant functional feature of eyg/toe is that it is unable to induce notum structures by itself, but requires co-expression of its activator the Iro gene, and probably pnr. For example, whereas ectopic eyg/toe activity induces scutum-like structures in the scutellum (which is also part of the notum and which expresses pnr), it fails to do so in most of the wing. Interestingly, it only induces notal structures in the middle of the wing, precisely the place where there is Iro gene activity in normal development. This mode of action is unlike that of selector or selector-like genes, such as the Hox genes, en, Dll, pnr or the Iro genes, which are able to induce, out of context, the formation of the patterns they specify. This indicates that eyg/toe is not of the same rank, but that it is developmentally downstream of the Iro genes and pnr, and appears to mediate their 'thoracic' function. The restriction of eyg/toe activity to the thorax, unlike the Iro genes and pnr, which are also expressed in the abdomen, is fully consistent with this role. eyg/toe is also expressed in a similar domain in the metathorax, suggesting that it may perform a parallel role in this segment (Aldaz, 2003).

Localized expression of eyg/toe is achieved by the activity of two antagonistic factors: the promoting activity induced by the Iro and pnr genes, and the repressing activities exerted by the Hh and the Dpp pathways. The latter are probably mediated by Hh and Dpp target genes that are yet to be identified (Aldaz, 2003).

Both Iro genes and pnr act as activators of eyg/toe expression. In Iro gene-mutant clones eyg is abolished, and ectopic Iro gene activity results in ectopic eyg expression. Although pnr^- clones do not lose eyg activity, the probable explanation is that they show Iro gene activity, which upregulates eyg. However, ectopic pnr expression induces eyg activity. Because the Iro gene and pnr expression domains cover the entire notum, in the absence of any other regulation they would induce eyg activity in the whole structure (Aldaz, 2003).

The result of the antagonistic activities of the Iro genes and pnr in one case and of the Hh and Dpp signalling pathways in the other, subdivides the notum into an eyg/toe expressing domain and a non-expressing domain. The localized expression of eyg/toe contributes to the morphological diversity of the thorax, as it distinguishes between an anterior-central region and a posterior-lateral one. It provides another example of a genetic subdivision of the body that is not based on lineage. It also provides an example of a patterning gene acting downstream of the combinatorial code of selector and selector-like genes. Its mode of action supports a model in which the genetic specification of complex patterns, such as the notum, is achieved by a stepwise process involving the activation of a cascade of regulatory genes (Aldaz, 2003).

The precise regulation of wingless (wg) expression in the Drosophila eye disc is key to control the anteroposterior and dorsoventral patterning of this disc. This study identifies an eye disc-specific wg cis-regulatory element that functions as a regulatory rheostat. Pannier (Pnr), a transcription factor previously proposed to act as an upstream activator of wg, is sufficient to activate the eye disc enhancer but required for wg expression only in the peripodial epithelium of the disc. It is proposed that this regulation of wg by Pnr appeared associated to the development of the peripodial epithelium in higher dipterans and was added to an existing mechanism regulating the deployment of wingless in the dorsal region of the eye primordium. In addition, this analysis identifies a separate ventral disc enhancer that lies adjacent to the eye-specific one, and thus altogether, they define a 1-kb genomic region where disc-specific enhancers of the wg gene are located (Pereira, 2006).

The T-box genes midline and H15 are conserved regulators of heart development: The expression of midline and H15 is dependent on Wingless signaling and tinman and pannier

The Drosophila melanogaster genes midline and H15 encode predicted T-box transcription factors homologous to vertebrate Tbx20 genes. All identified vertebrate Tbx20 genes are expressed in the embryonic heart and both midline and H15 are expressed in the cardioblasts of the dorsal vessel, the insect organ equivalent to the vertebrate heart. The midline mRNA is first detected in dorsal mesoderm at embryonic stage 12 in the two progenitors per hemisegment that will divide to give rise to all six cardioblasts. Expression of H15 mRNA in the dorsal mesoderm is detected first in four to six cells per hemisegment at stage 13. The expression of midline and H15 in the dorsal vessel is dependent on Wingless signaling and the transcription factors tinman and pannier. The selection of two midline-expressing cells from a pool of competent progenitors is dependent on Notch signaling. Embryos deleted for both midline and H15 have defects in the alignment of the cardioblasts and associated pericardial cells. Embryos null for midline have weaker and less penetrant phenotypes while embryos deficient for H15 have morphologically normal hearts, suggesting that the two genes are partially redundant in heart development. Despite the dorsal vessel defects, embryos mutant for both midline and H15 have normal numbers of cardioblasts, suggesting that cardiac cell fate specification is not disrupted. However, ectopic expression of midline in the dorsal mesoderm can lead to dramatic increases in the expression of cardiac markers, suggesting that midline and H15 participate in cardiac fate specification and may normally act redundantly with other cardiogenic factors. Conservation of Tbx20 expression and function in cardiac development lends further support for a common ancestral origin of the insect dorsal vessel and the vertebrate heart (Miskolczi-McCallum, 2005).

In order to determine where mid and H15 fit in the genetic hierarchy controlling heart development, their expression was examined in several mutant backgrounds. The initiation of mid expression in the dorsal mesoderm in early stage 12 occurs after the expression of tin and pnr, as well as after the period of Wg signaling in the dorsal mesoderm, suggesting that mid and H15 are regulated downstream of the factors that confer cardiac fate. Indeed, the dorsal vessel expression of mid and H15 is completely lost in both wg^cx4 and tin^ec40 mutant embryos, which fail to specify dorsal mesoderm. Embryos mutant for pnr have greatly decreased numbers of cardioblasts. Accordingly, mid and H15 expression is variably lost in pnr^vx6 null mutant embryos, with most embryos completely lacking mid expression in the dorsal mesoderm. Ectopic expression of pnr throughout the mesoderm using the GAL4/UAS system is able to induce ectopic expression of mid and H15. These results indicate that the initiation of mid and H15 in the dorsal mesoderm is downstream of factors required for the specification of cardiac fate (Miskolczi-McCallum, 2005).

A second study by Qian (2005) provides more detailed information on a cellular fate switch accompanying loss- and gain-of-function of this gene pair. Referring to midline and H15 by the alternative name neuromancer (nmr) the Qian study shows that gene function causes a switch in cell fates in the cardiogenic region, in that the progenitors expressing the homeobox gene even skipped (eve) are expanded, accompanied by a corresponding reduction of the progenitors expressing the homeobox gene ladybird (lbe). As a result, the number of differentiating myocardial cells is severely reduced whereas pericardial cell populations are expanded. Conversely, pan-mesodermal expression of nmr represses eve, while causing an expansion of cardiac lbe expression, as well as ectopic mesodermal expression of the homeobox gene tinman. In addition, nmr mutants with less severe penetrance exhibit cell alignment defects of the myocardium at the dorsal midline, suggesting nmr is also required for cell polarity acquisition of the heart tube. In exploring the regulation of nmr, it was found that the GATA factor Pannier is essential for cardiac expression, and acts synergistically with Tinman in promoting nmr expression. Moreover, reducing nmr function in the absence of pannier further aggravates the deficit in cardiac mesoderm specification. Taken together, the data suggest that nmr acts both in concert with and subsequent to pannier and tinman in cardiac specification and differentiation. It is proposed that nmr is another determinant of cardiogenesis, along with tinman and pannier (Qian, 2005).

Pannier has been shown to be required, along with Tinman, for specification of the heart primordium. pannier null mutant embryos exhibit a reduction of both myocardial and pericardial cell populations, but eve expression is less affected than that of lbe. Thus, the functional relationship between pannier and nmr in cardiac cell type specification was examined. Double-labeling for pannier RNA and nmr^H15-LacZ shows that reporter gene expression overlaps with that of pannier at the dorsal edge of the mesoderm. In pannier null mutants, mesodermal nmr expression is completely missing, which is in contrast to the presence of residual tinman and other cardiac marker genes. In addition, pan-mesodermal expression of pannier is sufficient to initiate nmr expression ectopically. In contrast, misexpression of nmr throughout the mesoderm is unable to induce pnr ectopically, and in nmr1⁻,nmr2^meso− embryos no detectable change in pannier expression is observed. These findings are consistent with the idea that nmr acts downstream of pannier (Qian, 2005).

Pannier function in the eye disc

The dorsoventral midline of the Drosophila eye disc is a source of signals that stimulate growth of the eye disc, define the point at which differentiation initiates, and direct ommatidial rotation in opposite directions in the two halves of the eye disc. This boundary region seems to be established by the genes of the iroquois complex, which are expressed in the dorsal half of the disc and inhibit fringe expression there. Fringe controls the activation of Notch and the expression of its ligands, with the result that Notch is activated only at the fringe expression boundary at the midline. The secreted protein Wingless activates the dorsal expression of the iroquois genes. Pannier, which encodes a GATA family transcription factor expressed at the dorsal margin of the eye disc from embryonic stages on, acts upstream of wingless to control mirror and fringe expression and establish the dorsoventral boundary. Loss of pannier function leads to the formation of an ectopic eye field and the reorganization of ommatidial polarity; ubiquitous pannier expression can abolish the eye field. Pannier is thus the most upstream element yet described in dorsoventral patterning of the eye disc (Maurel-Zaffran, 2000).

The pnr gene is expressed in the dorsalmost embryonic cells, in a domain of the notum surrounding the dorsal midline, and at the dorsal anterior margin of the eye disc. The FLP-FRT system was used to generate clones of cells mutant for pnr^VX6 , a null allele. Mutant clones were produced in the eye disc using the yeast FLP recombinase expressed under the control of the eye-specific enhancer of eyeless. Only clones produced at the dorsal margin of the eye disc give rise to a phenotype. In such discs an ectopic field of differentiating photoreceptors appears anterior to the main eye field. In adult flies this results in the formation of an ectopic eye field in the dorsal head cuticle, which is either separate from or fused with the normal eye. Interestingly, these ectopic eye fields do not arise exclusively from the pnr mutant cells within the clone itself, but also contained a domain of wild-type cells. These observations suggested that the new boundary of pnr expression present at the edge of the clone could be responsible for the induction of this new eye field. Frequently a duplication of the antenna is also observed, probably reflecting the function of pnr expressed dorsally in the antennal disc (Maurel-Zaffran, 2000).

To test the hypothesis that the boundary of pnr expression, rather than the absence of pnr, could be important for promoting eye growth, all pnr function in the eye was removed. Adult eyes and eye discs were examined from flies containing very large pnr mutant clones. In some cases, a dramatic loss of the eye and an absence of differentiating photoreceptors in the eye disc, resulting from the loss of all pnr function, were observed. In other cases eye overgrowth was observed; probably these eye discs retain some pnr-expressing cells, allowing the establishment of an ectopic pnr expression boundary. Only a small percentage of adults with large pnr clones were recovered; most animals died as late pupae and their heads were sometimes entirely missing, probably due to loss of all tissues deriving from the eye-antennal disc. A similar phenotype has been reported for some hypomorphic combinations of pnr alleles (Maurel-Zaffran, 2000 and references therein).

In the notum, the activity of pnr as a transcriptional activator is inhibited by binding to the zinc finger protein U-shaped (Ush), which is expressed in an adjacent domain. Ush does not appear to be required in the eye disc, since clones mutant for ush develop normally even when they are very large. However, ectopically expressed ush is able to inhibit the function of pnr in the eye disc; expression of ush with a pnr-GAL4 driver results in phenotypes similar to those induced by pnr mutant clones. Thus pnr is likely to act by activating the transcription of target genes in the eye as well as in the notum (Maurel-Zaffran, 2000).

The pnr boundary was eliminated by inducing ubiquitous pnr expression using the UAS/GAL4 system. A form of pnr was used that is resistant to inhibition by Ush, but appears to behave like wild-type pnr in the absence of Ush function. As a consequence, a complete loss of the eye is observed, confirming the importance of the border of pnr expression. From these experiments, it is concluded that the dorsally restricted expression of pnr is critical for eye development. A boundary between pnr-expressing cells and pnr-non-expressing cells appears to be necessary to induce growth and differentiation of the eye field (Maurel-Zaffran, 2000).

Recently, several studies have established that N activation along the dorsoventral midline of the eye disc is critical for eye growth as well as for positioning the equator. This local activation is the consequence of the ventrally restricted expression of fng, which is negatively controlled by the iro-C homeobox genes expressed in the dorsal half of the eye disc. Either loss of fng function, or ubiquitous expression of fng, caup or mirr, abolishes eye growth. The iro-C genes appear to act redundantly, as both ara and caup must be removed from clones of cells to promote the formation of ectopic dorsal eyes similar to those reported for pnr. The similar effects observed for gain or loss of pnr function suggest strongly that pnr might act in the same pathway as the iro-C and fng. To confirm this and to order pnr with respect to these genes, expression of mirr and fng was examined in eye discs mutant for pnr or misexpressing pnr. In eye discs in which pnr function had been removed, mirr expression is greatly reduced, whereas fng is derepressed dorsally. In eye discs expressing constitutively active pnr, mirr expression is expanded ventrally, shifting the point of morphogenetic furrow initiation to the ventral side. fng expression is dramatically reduced in discs overexpressing pnr D4. It thus appears that pnr acts upstream of the iro-C genes, activating their expression dorsally. Consistent with this, it has been found that ubiquitous expression of ara abolishes photoreceptor differentiation, and that removal of pnr function does not restore photoreceptor formation. If pnr were downstream of ara, blocking its function should have induced ectopic eye development even in the presence of ara (Maurel-Zaffran, 2000).

The results above show that pnr acts upstream of the iro-C genes to regulate dorsal eye development. Another molecule that has been shown to act upstream of the iro-C in this context is Wg. wg is required to inhibit the initiation of the morphogenetic furrow at the lateral margins of the eye disc, preventing ectopic eye differentiation there. The dorsal ectopic eyes induced by removing pnr function thus suggest that the functions of pnr and wg may be related. Consistent with this idea, the block in morphogenetic furrow initiation caused by expressing wg throughout the eye disc, like the block caused by expressing pnr D4 , can be rescued by co-expressing an activated form of N. pnr and wg may thus act in the same cascade to prevent eye differentiation (Maurel-Zaffran, 2000).

In situ hybridization has been used to show that pnr mRNA is restricted to the dorsal margin of the eye disc, anterior to and overlapping the morphogenetic furrow. wg is expressed at the dorsal and ventral edges of the eye disc with stronger expression dorsally, and its dorsal domain of expression resembles that of pnr. To test the epistatic relationship between wg and pnr, PNR mRNA expression was examined in eye discs from which wg function had been removed. Adult flies carrying wg minus clones show a transformation of the dorsal head cuticle into ectopic eye tissue, as well as missing antennae. Eye-antennal discs carrying such clones were identified by a severe reduction in the size of the antennal disc. In these eye discs PNR mRNA expression is wild type, showing that wg is not required for pnr expression. Overexpression of either Wg or an activated form of Armadillo (Arm), a downstream component of the Wg pathway, has no effect on pnr expression. Thus, wg is neither necessary nor sufficient for pnr expression. When pnr mutant clones are produced, dorsal wg expression is lost. Conversely, when a form of Pnr that is resistant to inhibition by U-shaped is overexpressed, although the size of the eye disc is dramatically reduced, and a derepression of wg expression is observed in both the eye and the antennal discs. It is concluded that pnr indeed activates wg expression at the dorsal margin (Maurel-Zaffran, 2000).

The role of wg in directing dorsal development is unexpected because wg is also expressed at the ventral anterior margin of the eye disc, although at a lower level than at the dorsal margin; this expression must have an upstream regulator other than pnr. However, the effects of loss of wg are more robust on the dorsal than the ventral side of the eye disc, and misexpression of wg symmetrically at both lateral margins dorsalizes the eye disc. These observations may be explained by the finding that at early stages wg is limited to the dorsal side of the eye disc and may exert its dorsalizing effect at this time (Maurel-Zaffran, 2000).

Dorsoventral (DV) patterning is crucial for eye development in invertebrates and higher animals. DV lineage restriction is the primary event in undifferentiated early eye primordia of Drosophila. In Drosophila eye disc, a dorsal-specific GATA family transcription factor pannier (pnr) controls Iroquois-Complex (Iro-C) genes to establish the dorsal eye fate whereas Lobe (L), which is involved in controlling a Notch ligand Serrate (Ser), is specifically required for ventral growth. However, fate of eye disc cells before the onset of dorsal expression of pnr and Iro-C is not known. L/Ser have been shown to be expressed in entire early eye disc before the expression of pnr and Iro-C is initiated in late first instar dorsal eye margin cells. The evidence suggests that during embryogenesis pnr activity is not essential for eye development. Evidence that loss of L or Ser function prior to initiation of pnr expression results in elimination of the entire eye, whereas after the onset of pnr expression it results only in preferential loss of the ventral half of the eye. Dorsal eye disc cells also become L or Ser dependent when they are ventralized by removal of pnr or Iro-C gene function. Therefore, it is proposed that early state of the eye prior to DV lineage restriction is equivalent to the ventral half and requires L and Ser gene function (Singh, 2003).

Previously, L/Ser were thought to be required for ventral eye growth after the DV lineage restriction boundary was established, which corresponds to the onset of expression of dorsal eye selectors. The results, however, clearly suggest that L/Ser are required much earlier for the growth of the entire early eye disc, even before the DV patterning is established. In contrast to the function of dorsal selector genes in eye patterning, L and Ser have been shown to play a distinct role in controlling ventral-specific growth of eye disc (Singh, 2003).

Loss-of-function phenotypes of L or Ser are restricted to the ventral eye. The spatial as well as temporal requirement of these genes in the ventral eye pattern formation were examined. Extent of loss of ventral eye pattern in loss-of-function clones of L/Ser varies along the temporal scale. During early eye disc development, prior to onset of pnr expression in dorsal eye, removal of L or Ser function results in complete elimination of the eye field, whereas later when dorsal eye selector genes starts expressing the eye suppression phenotype becomes restricted only to the ventral eye. Therefore, DV lineage border in the eye can also be interpreted as the border between the cells sensitive and insensitive to the L/Ser gene function (Singh, 2003).

The eye antennal disc has the most complex origin in the embryo. The eye disc is initiated from a small group of ~70 precursor cells on each side contributed by six different head segments of the embryo. These embryonic precursors do not physically separate from the surrounding larval primordia and are therefore difficult to discern morphologically (Singh, 2003 and references therein).

Once the cells for the eye-antennal disc are committed, these discs proliferate and undergo differentiation into an adult eye, which requires generation of DV lineage restriction in eye. There are possibly three different ways by which genesis of DV lineage in the eye can be explained. Early first instar larval eye disc may initiate either from only dorsal, only ventral or from both DV lineages. Based on results from studies of expression patterns and analysis of mutant phenotypes, it is proposed that larval eye primordium initially comprises only the ventral-equivalent state rather than well-defined DV or dorsal states alone. The initial state of eye is referred to as ventral equivalent state because, at this stage, dorsal and ventral identity is not yet generated. DV lineage restriction is established later after the onset of pnr expression. The cells of the initial ventral-equivalent state are similar to the ventral eye cells that are generated after DV specification. The similarity is in terms of their requirement of L/Ser for growth and maintenance, and the absence of the dorsal selector expression. How dorsal lineage is initiated in the early eye disc is not yet clear. Once the DV lineage restriction is established, N signaling is initiated at the equator, a border between dorsal and ventral compartments. Activation of N signaling promotes proliferation, which is followed by differentiation of eye disc into adult compound eye (Singh, 2003).

The ventral-equivalent state model is supported by two observations: 1) presence of Ser and L expression in the dorsal and ventral eye disc of the early first instar larva and 2) change of dorsal eye fate to ventral upon removal of dorsal selectors. The mutants, which affect ventral eye development, show two major phenotypes in eye: either there is no or very small eye, or there is a preferential loss of ventral eye based on the time they affect their function but none of the mutants for dorsal eye selectors show phenotypes of loss of only dorsal eye. Conversely, loss-of-function clones of pnr or Iro-C causes dorsal eye enlargement or ectopic eye formation rather than loss of only dorsal eye clonal tissue. This phenotype is probably due to generation of ectopic boundary of pnr-expressing and non-expressing cells (rather than absence of pnr), which could be important for promoting eye growth. Overexpression of Ush or Fog proteins in eye discs results in loss of pnr activity, causing complete elimination of eye development. By removing pnr activity at different time points it was found that pnr activity in embryo and early first instar is not essential for eye disc development. Later, pnr becomes essential for DV patterning consistent with its strong expression in dorsal margin of eye disc after the early first instar stage (Singh, 2003).

In contrast to enlargements or ectopic eyes induced by loss-of-function clones of dorsal selectors, the loss-of-function clones of L or Ser always resulted in the elimination of the eye fate. L/Ser are primarily required for the maintenance and development of ventral or ventral-equivalent state of the eye, whereas dorsal genes establish the DV border. This suggests that dorsal genes and L/Ser, although involved in a common goal of generation of DV lineage in eye, probably affect eye development at two different tiers (Singh, 2003).

Fng, another essential component of DV patterning in eye, is expressed preferentially in the ventral domain of early eye disc and is required for restriction of N signaling to the DV border. Although fng is known to act upstream of Ser in the wing and eye discs, there is also an apparent difference between the two genes. Unlike L/Ser, the main function of fng seems to affect DV ommatidial polarity but not the growth. This suggests that fng may be selectively required for DV patterning after dorsal selectors initiate domain specification. This may be the reason why phenotypes of loss-of-function clones of fng are different from those of L and Ser in the eye. It has been observed that the pattern of fng expression is not altered in L mutants, and vice versa, supporting the independent functions of these two genes in controlling DV border formation and growth of ventral domain (Singh, 2003).

The function of Pnr in organizing the DV pattern from an initial ventral-equivalent state raises an interesting question of whether similar patterning processes occur in other developing tissues and organs. Interestingly, Pnr is expressed in a broad dorsal domain in early embryos, but later refined in a longitudinal dorsal domain extending along the thoracic and abdominal segments. During this stage, Pnr has an instructive and selector-like function, determining the identity of the medial dorsal structures. It has been shown that loss of pnr eliminates the dorsomedial pattern in the larval cuticle whereas the dorsolateral pattern extends dorsally without cell loss. This suggests that DV pattern in the larval cuticle is established with the onset of Pnr expression in the dorsomedial domain, and ventral may be the initial fate of epidermal cells (Singh, 2003).

Hand is a direct target of Tinman and GATA factors during Drosophila cardiogenesis and hematopoiesis

The Hand gene family encodes highly conserved basic helix-loop-helix (bHLH) transcription factors that play crucial roles in cardiac and vascular development in vertebrates. In Drosophila, a single Hand gene is expressed in the three major cell types that comprise the circulatory system: cardioblasts, pericardial nephrocytes and lymph gland hematopoietic progenitors. Drosophila Hand functions as a potent transcriptional activator, and converting it into a repressor blocks heart and lymph gland formation. Disruption of Hand function by homologous recombination also results in profound cardiac defects that include hypoplastic myocardium and a deficiency of pericardial and lymph gland hematopoietic cells, accompanied by cardiac apoptosis. Targeted expression of Hand in the heart completely rescues the lethality of Hand mutants, and cardiac expression of a human HAND gene, or the caspase inhibitor P35, partially rescues the cardiac and lymph gland phenotypes. These findings demonstrate evolutionarily conserved functions of HAND transcription factors in Drosophila and mammalian cardiogenesis, and reveal a previously unrecognized requirement of Hand genes in hematopoiesis (Han, 2006).

The existence of hemangioblasts, which serve as common progenitors for hematopoietic cells and cardioblasts, has suggested a molecular link between cardiogenesis and hematopoiesis in Drosophila. However, the molecular mediators that might link hematopoiesis and cardiogenesis remain unknown. This study shows that the highly conserved bHLH transcription factor Hand is expressed in cardioblasts, pericardial nephrocytes and hematopoietic progenitors. The homeodomain protein Tinman and the GATA factors Pannier and Serpent directly activate Hand in these cell types through a minimal enhancer, which is necessary and sufficient to drive Hand expression in these different cell types. Hand is activated by Tinman and Pannier in cardioblasts and pericardial nephrocytes, and by Serpent in hematopoietic progenitors in the lymph gland. These findings place Hand at a nexus of the transcriptional networks that govern cardiogenesis and hematopoiesis, and indicate that the transcriptional pathways involved in development of the cardiovascular, excretory and hematopoietic systems may be more closely related than previously appreciated (Han, 2005).

To search for cis-regulatory elements capable of conferring the specific expression pattern of Hand in cardioblasts, pericardial nephrocytes and lymph gland hematopoietic progenitors, a series of reporter genes were generated containing lacZ and the hsp70 basal promoter linked to genomic fragments within a 13 kb genomic region encompassing the gene, and reporter gene expression was examined in transgenic embryos. A 513 bp minimal enhancer was identified referred to as Hand cardiac and hematopoietic (HCH) enhancer, between exons 3 and 4 of the Hand gene. HCH is both necessary and sufficient to direct lacZ expression in the entire embryonic heart and lymph gland in a pattern identical to that of the endogenous Hand gene. Further deletions of this enhancer caused either a partial or complete loss of activity. The 513 bp HCH enhancer showed the same expression pattern in the heart and lymph gland as larger genomic fragments that were positive for enhancer activity. It is concluded that this enhancer fully recapitulates the temporal and spatial expression pattern of Hand transcription in the distinct cell types derived from the cardiogenic region (Han, 2005).

The homeobox protein Tinman is essential for the formation of the cardiac mesoderm, from which the heart and blood progenitors arise. However, its potential late functions remain unknown. It is believed that Tinman is not required for the entirety of heart development in flies, because it is not maintained in all the cardiac cells at late stages. The data reveal at least one function for the late-embryonic Tinman expression, which is to maintain Hand expression. The fact that ectopic Tinman can turn on Hand expression dramatically in the somatic muscles is striking and suggests the existence of a Tinman-co-factor in muscle cells that can cooperate with Tinman to activate Hand expression; this co-factor would not be expected to be expressed in pericardial cells or the lymph gland. This co-factor should also be expressed in Drosophila S2 cells, since transfected Tinman can increase activity of the HCH enhancer in S2 cells by more than 100-fold. The generally reduced activity of the HCH enhancer that results from mutation of the Tinman-binding sites also suggests that Tinman activity is required to fully activate the Hand enhancer (Han, 2005).

Although Pannier and Serpent bind to the same consensus sites, these GATA factors produce distinct phenotypes when overexpressed in the mesoderm. Ectopic Pannier induces cardiogenesis, shown by the extra number of cardioblasts and pericardial nephrocytes, but does not affect the lymph gland hematopoietic progenitors. Ectopic Serpent, however, induces ectopic lymph gland hematopoietic progenitors, but reduces the number of cardioblasts and pericardial cells. Interestingly, pericardial cells with ectopic Serpent expression have a tendency to form cell clusters such as the lymph gland progenitors, suggesting a partial cell fate transformation. These results suggest that Pannier functions as a cardiogenic factor, whereas Serpent functions as a hematopoietic factor. Although both can activate Hand expression, Pannier and Serpent activate the HCH enhancer in different cell types. This assumption is also supported by the specific expression pattern of Serpent and Pannier in late embryos. Serpent is detected specifically in the lymph gland hematopoietic progenitors but not in any cardiac cells. Pannier expression in the cardiogenic region of late embryos is not clear because of the interference by the high level Pannier expression from the overlaying ectoderm. However, the lymph gland was examined in late stage embryos and no Pannier expression was detected in these cells. Together with the evidence from loss-of-function and gain-of-function experiments with Serpent, it is concluded that the HCH-5G-GFP transgene is not expressed in the lymph gland because Serpent could not bind to the mutant enhancer in the lymph gland cells; whereas the lack of HCH-5G-GFP expression in cardiac cells is due to the inability of Pannier to bind the mutant enhancer in these cardiac cells (Han, 2005).

Since tin and pnr are not expressed in all the cardiac cells of late stage embryos but the Hand-GFP transgene is expressed in these cells, it is likely that additional factors control Hand expression in the heart. One group of candidates is the T-box family. Since Doc1, Doc2 and Doc3 genes (Drosophila orthologs to vertebrate Tbx5) are expressed in the Svp-positive cardioblasts where tin is not expressed, but H15 and midline (Drosophila orthologs to vertebrate Tbx-11) are expressed in most of the cardiac cells in late embryos, it is likely that the T-box genes activate Hand expression in cells that do not express tin and pannier. However, the enhancer lacking GATA and Tinman sites has no activity, indicating that the additional factors that may activate Hand expression in the heart and lymph gland also requires these crucial Tinman and GATA sites, probably through protein interaction between Tinman and the GATA factors (Han, 2005).

In mammals, the adult hematopoietic system originates from the yolk sac and the intra-embryonic aorta-gonad-mesonephros (AGM) region. The AGM region is derived from the mesodermal germ layer of the embryo in close association with the vasculature. Indeed, the idea of the hemangioblast, a common mesodermal precursor cell for the hematopoietic and endothelial lineages, was proposed nearly 100 years ago without clear in vivo evidence. Recently, this idea was substantiated by the identification of a single progenitor cell that can divide into a hematopoietic progenitor cell in the lymph gland and a cardioblast cell in the dorsal vessel in Drosophila (Mandal, 2004). In addition to providing the first evidence for the existence of the hemangioblast, this finding also suggested a close relationship between the Drosophila cardiac mesoderm, which gives rise to cardioblasts, pericardial nephrocytes and pre-hemocytes, and the mammalian cardiogenic and AGM region, which gives rise to the vasculature (including cardiomyocytes), the excretory systems (including nephrocytes) as well as adult hematopoietic stem cells. In fact, in both Drosophila and mammals, the specification of the cardiogenic and AGM region requires the input of Bmp, Wnt and Fgf signaling. In addition to the conserved role of the NK and GATA factors, GATA co-factors (U-shaped in Drosophila and Fog in mice) also play important roles in cardiogenesis and hematopoiesis in both Drosophila and mammals. Recent studies have shown that the Notch pathway is required for both cardiogenic and hematopoietic progenitor specification in Drosophila. It is likely that Notch also plays an important role in mammalian hematopoiesis (Han, 2005).

This study found that Drosophila Hand is expressed in cardioblasts, pericardial nephrocytes and pre-hemocytes, and is directly regulated by conserved transcription factors (NK and GATA factors) that control both cardiogenesis and hematopoiesis. The bHLH transcription factor Hand is highly conserved in both protein sequence and expression pattern in almost all organisms that have a cardiovascular system. In mammals, Hand1 is expressed at high levels in the lateral plate mesoderm, from which the cardiogenic region and the AGM region arise, in E9.5 mouse embryos. Functional studies of Hand1 and Hand2 using knockout mice have demonstrated the essential role of Hand genes during cardiogenesis, whereas the functional analysis of Hand genes during vertebrate hematopoiesis has not yet been explored. It will be interesting to determine whether mammalian Hand genes are also regulated in the AGM region by GATA1, GATA2 and GAT3 (vertebrate orthologs to Drosophila Serpent), and whether they play a role in mammalian hematopoiesis (Han, 2005).

In summary, this study places Hand at a pivotal point to link the transcriptional networks that govern cardiogenesis and hematopoiesis. Since the Hand gene family encodes highly conserved bHLH transcription factors expressed in the cardiogenic region of widely divergent vertebrates and probably in the AGM region in mouse, these findings open an avenue for further exploration of the conserved transcriptional networks that govern both cardiogenesis and hematopoiesis, by studying the regulation and functions of Hand genes in vertebrate model systems (Han, 2005).

The ATP-sensitive potassium (K_ATP) channel-encoded dSUR gene is required for Drosophila heart function and is regulated by Tinman and Pannier

The homeobox transcription factor Tinman plays an important role in the initiation of heart development. Later functions of Tinman, including the target genes involved in cardiac physiology, are less well studied. Focus was placed on the dSUR gene, which encodes an ATP-binding cassette transmembrane protein that is expressed in the heart. Mammalian SUR genes are associated with K_ATP (ATP-sensitive potassium) channels, which are involved in metabolic homeostasis. Experimental evidence is provided that Tinman directly regulates dSUR expression in the developing heart. A cis-regulatory element was identified in the first intron of dSUR that contains Tinman consensus binding sites and is sufficient for faithful dSUR expression in the fly’s myocardium. Site-directed mutagenesis of this element shows that these Tinman sites are critical to dSUR expression, and further genetic manipulations suggest that the GATA transcription factor Pannier is synergistically involved in cardiac-restricted dSUR expression in vivo. Physiological analysis of dSUR knock-down flies supports the idea that dSUR plays a protective role against hypoxic stress and pacing-induced heart failure. Because dSUR expression dramatically decreases with age, it is likely to be a factor involved in the cardiac aging phenotype of Drosophila. dSUR provides a model for addressing how embryonic regulators of myocardial cell commitment can contribute to the establishment and maintenance of cardiac performance (Akasaka, 2006; full test of article).

Because cardiac dSUR expression depends on Tin, 40 kb of the dSUR locus was scanned for Tin consensus binding sites (TNAAGTG). Three large genomic fragments were chosen based on the high density of potential Tin-binding sites (En1, 4,095 bp; En2, 2,151 bp; and En3, 2,291 bp). The enhancer activity of these En fragments was then examined in transgenic flies. Two fragments located upstream of the ATG start (En1 and En2) do not show any reporter activity in the embryonic heart. In contrast, En3 exhibits a pattern of reporter gene expression identical to the endogenous dSUR pattern. This En3 fragment is downstream of the ATG start and contains six Tin sites. To determine whether these Tin sites are required for cardiac expression, they were mutated. Of the mutated Tin sites, only a mutated T3 site reduced the enhancer’s transcriptional activity. Mutations in both T2 and T3 (241 bp apart) abolished reporter gene expression in the cardiac progenitor cells, suggesting that Tin is capable of directly activating dSUR expression in the appropriate myocardial cells. Shorter fragments (S, 890 bp; SS, 359 bp; and SSS, 297 bp) containing both T2- and T3-binding sites were tested for enhancer activity. These three fragments mimicked the cardiac dSUR expression and showed a similar expression level as seen with En3. Within the context of the short SSS fragment, the T3-binding site is absolutely essential for reporter gene activation. The En3 fragment was also scanned for Mad/Media (Dpp pathway)-binding sites (GCCGCGACG). No Mad/Media sites were found with appreciable conservation within this enhancer, which is consistent with Dpp signaling only indirectly regulating dSUR expression, possibly by means of tin. However, a direct regulation by Dpp by means of degenerate or not well conserved sites cannot be excluded (Akasaka, 2006).

An EMSA was performed to test whether Tin can directly bind to the T3 site. A DNA template (28 bp) composed of dSUR genomic sequence containing the T3-binding site produced a specific Tin-binding complex. Thus, Tin can directly associate with the T3 element in dSUR, which is consistent with the possibility that dSUR expression is directly controlled by Tin (Akasaka, 2006).

The Tin expression pattern varies by developmental stage, and, likewise, its downstream target genes may also change during development. In vertebrates, GATA-4 provides the binding efficiency to Nkx2.5 in cardiomyocytes; therefore, these two transcription factors can act cooperatively to activate cardiac genes. Similarly, the Drosophila counterparts Pnr and Tin physically interact and synergistically control cardiac gene expression of genes such as Dmef2. To further characterize the role of Pnr in dSUR activation, Pnr was expressed panmesodermally and the expression of dSUR was compared to that of dHand, which marks all cardiac lineages. Panmesodermally expressed Pnr activates both ectopic dHand and dSUR expression but only to a moderate extent. In contrast, a dominant-negative Pnr (Pnr-EnR) did not induce, and instead reduced, dSUR and dHand expression. Moreover, both dSUR and dHand were strongly activated when tin and pnr were coexpressed, suggesting that, like dHand, dSUR activation depends on genetic synergy between Tin and Pnr (Akasaka, 2006).

Next, whether a reduction of Pnr activity could be compensated for by overexpression of tin was examined. tin and pnr-EnR were co-expressed throughout the mesoderm and it was found that the reduced dSUR and dHand expression, which was due to Pnr-EnR, was not rescued by forced panmesodermal tin expression. This finding suggests that dSUR activation requires not only Tin but also Pnr activity (Akasaka, 2006).

Furthermore, Pnr consensus binding site (WGATAR) was sought within the En3 fragment to explain synergistic activation by Tin and Pnr. There are two well conserved Pnr sites in the SS fragment. However, when the enhancer activity was examined when both of these Pnr consensus sites were mutated [SS(P2P3)], it was found that this enhancer was equivalent to the WT SS fragment. This finding implies that Pnr could bind to Tin directly or to other nonconsensus Pnr-binding sites, such as TGATA (which exists in the SSS fragment), to activate dSUR expression in the embryonic heart (Akasaka, 2006).

To address the possibility that Tin and Pnr may be acting in a complex in regulating cardiac dSUR transcription, an in vitro reporter assay with a luciferase plasmid was used, in which expression was driven by six concurrent T3. Cotransfection of the T3 reporter plasmid with Tin but not the Pnr expression vector into Drosophila Schneider cells resulted in a 3-fold activation of luciferase activity compared with the reporter construct alone. In contrast, when Tin and Pnr were cotransfected, the luciferase activity was elevated 9-fold compared with the reporter construct alone (or with a mutant T3-binding site), suggesting that Pnr acts as a cofactor with Tin to synergistically activate dSUR transcription (Akasaka, 2006).

It has been shown that in corpora cardiaca (CC) cells of Drosophila, dSUR controls glucose homeostasis by increasing secretion of adipokinetic hormone (AKH) in response to low glucose concentration in the hemolymph. Evidence indicates that AKH release likely is increased by the SUR inhibitor sulfonylurea and is decreased by ectopic expression of constitutively active (and thus ATP-independent) ion channel Kir2.1, suggesting striking parallels between endocrine cells in Drosophila and mammals in controlling blood glucose. Therefore, the role of dSUR was examined in cardiac physiology and heart homeostasis in adults. The findings suggest that there are striking functional similarities between Drosophila and mammalian SUR in heart function. In the mammalian heart, there are two types of K_ATP channels, sarcK_ATP and mitochondrial K_ATP, which are candidate regulators of acute hypoxia and IPC. Impairing sarcK_ATP channel activity by genetic manipulation of mouse Kir6.2 results in compromised recovery of contractile function after hypoxia. The data are consistent, with dSUR in Drosophila providing a similar protective mechanism against hypoxia. Moreover, a recent study in goldfish K_ATP channel function revealed that the involvement of K_ATP in IPC is widely conserved, including in highly hypoxia-tolerant species (Akasaka, 2006).

To further address dSUR function, external electrical pacing of the heart was performed in dSUR knock-down mutants. Rapid electrical pacing per se is a nonhypoxic stimulus that may induce an IPC effect in mammals by activating K_ATP channels. Indeed, Kir6.2 mutant hearts exhibit diminished electrical tolerance against catecholamine-induced ventricular arrhythmia because of a failure to achieve action potential shortening and by causing early after-depolarization. Thus, the elevated heart failure rate in dSUR knock-down hearts may be due to K_ATP channel insufficiency. Interestingly, IPC is no longer observed in older human patients, and in female guinea pigs, SUR2A expression is reduced in old ventricular tissue compared with young ventricular tissue. Moreover, human SUR2 mutations found in two independent families were recently shown to cause dilated cardiomyopathy, with an onset around middle age. These mutations result in the structural abnormalities of the K_ATP channel and impair the ATP-dependent channel gating. Patients carrying these mutations showed ventricular tachycardia with normal coronary angiography, suggesting that human cardiomyocyte K_ATP channels play a role in maintaining membrane electrical stability and that the reduction of the K_ATP channel activity causes electrical disturbance, especially in older hearts. In this study it was observed that pacing-induced heart failure steeply increases in aging flies, which can be reversed by exposure to the K_ATP channel activator pinacidil. These observations, together with the drastically reduced dSUR expression in old flies, suggest that dSUR serves as an indicator of cardiac aging. Given the experimental advantages of Drosophila, such as a small genome size and short life span, dSUR and cardiac aging provide a unique model not only for assessing the control of physiological heart functions, such as the response to hypoxia, but also for the analysis of age-related human diseases (Akasaka, 2006).

Two or four bristles: Functional evolution of an Enhancer of scute in Drosophilidae - upstream regulation of the scute enhancer by Pannier has been evolutionarily conserved

Changes in cis-regulatory sequences are proposed to underlie much of morphological evolution. Yet, little is known about how such modifications translate into phenotypic differences. To address this problem, focus was placed on the dorsocentral bristles of Drosophilidae. In Drosophila melanogaster, development of these bristles depends on a cis-regulatory element, the dorsocentral enhancer, to activate scute in a cluster of cells from which two bristles on the posterior scutum arise. A few species however, such as D. quadrilineata, bear anterior dorsocentral bristles as well as posterior ones, a derived feature. This correlates with an anterior expansion of the scute expression domain. This study shows that the D. quadrilineata enhancer has evolved, and is now active in more anterior regions. When used to rescue scute expression in transgenic D. melanogaster, the D. quadrilineata enhancer is able to induce anterior bristles. Importantly, these properties are not displayed by homologous enhancers from control species bearing only two posterior bristles. Evidence is provied that upstream regulation of the enhancer, by the GATA transcription factor Pannier, has been evolutionarily conserved. This work illustrates how, in the context of a conserved trans-regulatory landscape, evolutionary tinkering of pre-existing enhancers can modify gene expression patterns and contribute to morphological diversification (Marcellini, 2006).

Unfortunately, to date, the mechanism responsible for restricting the activity of the DC enhancer in the anterior direction has not been discovered. However, the direct input of Pnr and U-shaped (Ush), essential for the correct activity of the Dm-DCE along the dorso-lateral axis, has been extensively analyzed. In order to shed light on the ancestry and the functional conservation of the regulation by Pnr and Ush, the sequences of orthologous DCEs, as well as their relative activities in various mutant backgrounds was tested (Marcellini, 2006).

Sequence alignments reveal that the DCEs are greatly variable in size and have undergone considerable turnover. Only the extremities display significant levels of similarity between all species examined. The central region is poorly conserved. The elements from D. melanogaster (1.5 kb) and D. eugracilis (2 kb) are more similar to each other than to the others, in accordance with their closer phylogenetic relationship. The enhancers from D. virilis and D. quadrilineata share a relatively large size (4.1 and 3.3 kb, respectively) and a conserved stretch of about 300 nucleotides that is absent from the D. melanogaster and D. eugracilis sequences. Putative binding sites for Pnr are present in all species. Mutation of a specific Pnr binding site severely reduces activity of the Dm-DCE. This site is embedded within a stretch of 16 nucleotides perfectly conserved between the four species. Interestingly, two other neighbouring GATA sequences can be recognised as homologous between all species. Conservation overall, however, is low, and the number, spacing, and orientation of the remaining putative Pnr binding sites are extremely variable (Marcellini, 2006).

In D. melanogaster, pnr is expressed in a broad medial domain, but activates sc in discrete proneural clusters. Expression of sc mediated by the Dm-DCE is a direct consequence of Pnr binding. DCE function is restricted dorsally through the repressor activity of Ush, which forms heterodimers with Pnr and prevents activation of sc. It was found that the activity of the Dv-DCE and the Dq-DCE in D. melanogaster is restricted to a lateral cluster of cells completely included within the expression domain of pnr. This suggests that, despite significant sequence turnover, the divergent DCEs require Pnr and are efficiently repressed dorsally by Ush. Behaviour of the DCEs was examined in the context of various mutant alleles of pnr. pnr^VX4, a strong loss of function allele, pnr^V1, a hypomorphic allele and pnr^D1, a gain of function allele with a missense mutation that disrupts the interaction of Pnr with Ush were used. Activity of the Dm-DCE was compared with that of the Dv-DCE and that of the Dq-DCE. It was observed that the enhancers react in a similar fashion to four different mutant backgrounds. The expression domains are reduced in loss of function genotypes and expanded in gain of function genotypes (Marcellini, 2006).

This study has presented evidence that the activity of Pnr is conserved and positively regulates the DC enhancers from distantly related Drosophilidae. When assayed in D. melanogaster, the Dv-DCE and Dq-DCE are active in groups of cells completely included within the expression domain of Dm-pnr. It is significant that an essential, high-affinity Pnr binding site in the Dm-DCE is conserved in the DCEs of the other species. Note that the three conserved Pnr binding sites are clustered in a region of the DCE that is required for activity and is sufficient in D. melanogaster to direct weak expression by itself. Expression of sc mediated by the Dm-DCE is restricted dorsally through the repressor activity of Ush that associates with Pnr to prevent activation. In gain-of-function pnr alleles that are insensitive to Ush, activity of the Dv-DCE and the Dq-DCE, like the Dm-DCE, expands dorsally. Most of the open reading frame of pnr was cloned from D. quadrilineata, and it was found that, as in D. virilis, the two zinc fingers are perfectly conserved, suggesting that Dq-Pnr and Dv-Pnr may also bind Ush within their respective species. Hence, it is most likely that Pnr and Ush are direct, evolutionarily conserved regulators of the DCE within Drosophilidae. Indeed the expression domain of pnr, as well as other upstream regulators, has been found to be conserved in other families of flies. Even Pnr from the mosquito Anopheles gambiae is able to regulate ac-sc in transgenic D. melanogaster, suggesting conservation of pnr function throughout the Diptera (Marcellini, 2006).

D. quadrilineata is phylogenetically distant from D. melanogaster and displays four instead of two DC bristles. The results demonstrate that this secondary gain is partly due to evolution of the cis-regulatory sequence that drives sc expression at the DC site. A Dq-DCE-sc minigene, present in transgenic mutant D. melanogaster devoid of the endogenous DC proneural cluster of ac-sc expression, is not only able to rescue posterior bristles, but also allows development of more anterior bristles. It thus mimics the DC phenotype of D. quadrilineata itself. Expression driven by the Dq-DCE in D. melanogaster extends anteriorly in a domain that is longer and thinner. Although the Dq-DCE was not tested in D. quadrilineata itself, it is active in D. melanogaster in a domain that is similar to the DC domain of sc expression in D. quadrilineata visualized by in situ hybridisation. This suggests that the Dq-DCE autonomously reproduces an expression pattern similar to the endogenous one in D. quadrilineata. Expression of sc mediated by the Dm-DCE is restricted laterally through lack of Pnr, dorsally through the repressor activity of Ush and posteriorly through the antagonistic activity of Islet, but it is not yet known what restricts expression in an anterior direction. The anterior expansion seen with the Dq-DCE indicates that this sequence may be at least partially insensitive to whatever factors limit anterior expression driven by the Dm-DCE. Alternatively it may contain new information not present in the other species (Marcellini, 2006).

These observations demonstrate an altered response of the D. quadrilineata sequence to the upstream regulators of D. melanogaster. This response should reside in the sequence of the Dq-DCE itself that is sufficient to modify the phenotype of D. melanogaster when used to drive sc. Thus the exchange of a single, well-defined enhancer is sufficient, not only to reproduce an expression pattern, but also to partially transform a morphological trait of one species into that of another. It is proposed that a change in cis, within a pre-existing regulatory element of sc, contributed to the evolution of the bristle pattern observed in D. quadrilineata by altering the region where it is expressed (Marcellini, 2006).

The Dv-DCE, in D. melanogaster, drives expression in a larger cluster that expands predominantly in a dorsal direction. A Dv-DCE-sc minigene, however, allows the development of only two bristles positioned at the correct locations. The most likely explanation for the fact that the expanded expression driven by Dq-DCE-sc leads to additional bristles, whereas that of the Dv-DCE-sc does not, is probably linked to the different locations of the cells expressing sc. It seems that, in D. melanogaster, the region anterior to the two DC bristles is competent to produce bristles. This region is situated between the domains of expression of sr, a repressor of macrochaete development, and overlaps a band of expression of wingless (wg), a gene encoding a secreted factor that is required to maintain sc expression and to repress sr. It is possible to select for additional anterior DC bristles, but not for macrochaetes on either side of the DC row where sr is expressed but wg is not. Notably, anterior DC bristles were present in the ancestor common to D. melanogaster and D. virilis. The curved shape of the Dq-DCE-driven expression domain means that it avoids overlap with the domains of expression of sr and shows significant overlap with that of wg. Therefore only the Dq-DCE drives expression in an anterior location that is competent to produce bristles (Marcellini, 2006).

Nevertheless transgenic D. melanogaster expressing Dq-DCE-sc do not perfectly reproduce the bristle pattern of D. quadrilineata. The anterior-most DC bristle, the scapular bristle, is absent. This bristle is situated in the prescutum, anterior to the transverse suture. It may be that this difference is attributable to changes in factors that negatively or positively regulate the enhancer in trans. It is also possible that full enhancer activity requires sequences on either side of the fragment tested. Additionally, the modification of cis-regulatory elements lying elsewhere within the D. quadrilineata ac-sc complex could also have contributed to the emergence of the additional bristles. However, it is equally possible that other extraneous factors are responsible that cannot be controlled for in these experiments. For instance, it has been shown that differences in the timing of bristle precursor formation between species can influence the development of macrochaetes (Marcellini, 2006).

The two DC bristles resulting from the activity of Dv-DCE-sc are situated at exactly the correct positions despite the fact that the Dv-DCE drives expression in a cluster of cells that is larger and displaced dorsally when compared with that of D. melanogaster. Thus the fly can compensate for this degree of imprecision in sc expression at the DC site. The explanation for this probably lies in the manner in which the bristle precursors are selected from the proneural cluster. Notch-mediated lateral signalling allows the selection of only two cells destined to become precursors with the appropriate spacing. However, the choice of these cells is not random, but biased by external factors such as the repressors emc and sr, whose activity causes the precursors to arise at similar positions within the DC cluster of all individuals. Their site of origin is in fact located within the region of overlap of expression driven by the Dm-DCE and the Dv-DCE. Positioning of bristle precursors thus results from restricted expression of sc in the proneural clusters as well as other cues that constrain the choice of precursor cell. Together, these two inputs lead to a robust patterning mechanism that is resistant to mild perturbations such as the shifting of the proneural cluster observed for Dv-DCE activity (Marcellini, 2006).

The ability of poorly conserved enhancers to drive expression of reporter genes in homologous tissues when transferred between species of similar morphology has been widely documented in the literature. Where a detailed comparison of enhancer activity allowed a rigorous assessment of the degree of conservation, two different outcomes have been observed. On the one hand, transferring enhancers between related species of Drosophila (e.g., even-skipped), or of nematodes (e.g., lin-48) revealed a perfect conservation of activity, a phenomenon attributed to stabilizing selection. On the other hand, the regulatory regions exchanged between species of sea urchins (e.g., endo-16) or ascidians (e.g., Otx) did not perfectly recapitulate the endogenous expression pattern. The DCEs from D. eugracilis and D. virilis behave like the latter: they drive reporter gene expression in a cluster of cells that is not perfectly co-incident with that of the endogenous DC cluster. The slightly different expression patterns could be due to the divergent sequences, or could result from co-evolution between the enhancer and its regulatory environment. Indeed earlier experiments have hinted that co-evolution between Pnr and its target sequences may be occurring (Marcellini, 2006).

The role of the sensory macrochaetes in behaviour is not known. Many species of Acalyptrata have ancient stereotyped patterns in which the number and precise position of each bristle is invariant. The bristle patterns of the Drosophilidae are remarkably conserved, and the majority of the nearly 4,000 species have two DC bristles. The evolutionary stability of the many bristle patterns suggests a role for selective forces to maintain them. D. quadrilineata is unusual among Drosophilidae in having four or five DC bristles. The anterior-most DC bristles would allow additional positional sensory input, and it is possible that they confer a selective advantage. However, it is important to note that not all morphological change needs be driven by selection. Kimura proposed a neutral theory of molecular evolution in which mutations with null or negligible effect can become passively fixed in populations. Similarly, natural selection alone may not explain the infinite number of subtle morphological variations displayed by the many species of Drosophila described. Exploratory behaviour is an intrinsic property of biological systems, and one may therefore also speculate that evolution can proceed through a series of viable, seemingly useless, phenotypes (Marcellini, 2006).

Antagonistic and cooperative actions of the EGFR and Dpp pathways on the iroquois genes regulate Drosophila mesothorax specification and patterning; a role for Pnr/Ush

In Drosophila, restricted expression of the Iroquois complex (Iro-C) genes in the proximal region of the wing imaginal disc contributes to its territorial subdivision, specifying first the development of the notum versus the wing hinge, and subsequently, that of the lateral versus medial notum. Iro-C expression is under the control of the EGFR and Dpp signalling pathways. To analyze how both pathways cooperate in the regulation of Iro-C, several wing disc-specific cis-regulatory elements of the complex were isolated. One of these (IroRE²) integrates competing inputs of the EGFR and Dpp pathways, mediated by the transcription factors Pointed (downstream of EGFR pathway) and Pannier/U-shaped and Mothers against Dpp (Mad), in the case of Dpp. By contrast, a second element (IroRE¹) mediates activation by both the EGFR and Dpp pathways, thus promoting expression of Iro-C in a region of elevated levels of Dpp signalling, the prospective lateral notum near the anterior-posterior compartment boundary. These results help define the molecular mechanisms of the interplay between the EGFR and Dpp pathways in the specification and patterning of the notum (Letizia, 2007).

The Iro-C genes ara and caup show similar patterns of expression in the wing disc. In early second instar larvae, they are expressed in the whole prospective mesothorax region. Later, in the third instar, their expression is restricted to the lateral notum. In addition, at this developmental stage, novel domains of expression appear in the prospective regions of the L1, L3 and L5 veins, tegula, dorsal radius, dorsal and ventral pleura and alula. The expression of mirr is slightly different, being absent from the L3, L5 and tegula domains but present at the other domains. The Iro-C harbours two additional transcription units, lincoyan (linc), whose pattern of expression at the notum is identical to that of ara and/or caup and quilapan (quil), which is ubiquitously expressed. Previous genetic analysis suggested the existence of enhancer-like REs that would drive the coincident expression of ara and caup in the wing disc. Thus, In(3L)iro^DFM2, associated with a breakpoint within the ara transcription unit, removes ara expression in the wing disc except in the L3 vein domain, in contrast to caup expression which is only lost from that domain. This suggests the existence of vein L3-specific RE(s) distal to the In(3L)iro^DFM2 breakpoint and other RE(s), specific for the remaining domains of Iro-C expression, located proximal to such breakpoint. To identify notum-specific REs, the regulatory potential of 31 different genomic fragments, spanning approximately 110 kb of genomic Iro-C DNA was analyzed (Letizia, 2007).

Only five of those fragments drove lacZ expression at specific regions of the imaginal wing disc. One of them, 3.3 kb in length and named Iro regulatory element². The IroRE² was reduced to a 1.6 kb subfragment (sequence of the IroRE2-B fragment), which maintained enhancer activity in the notum and was activated by EGFR and repressed by Dpp signalling. Thus, IroRE²-lacZ was expressed in the proximal region of early third instar wing discs (the presumptive notum region) and at the presumptive lateral notum in third instar wing discs. Note, however, that the pattern of IroRE²-mediated lacZ expression does not exactly coincide with that of ara/caup. Thus, ß-gal was not detected in a triangular area, located near the notum/hinge border and centred around the AP compartment boundary, where expression of ara/caup is enhanced. This is precisely the region where expression of lacZ was driven by another Iro-C genomic fragment of 3.9 kb, IroRE¹. Accordingly, an IroRE¹-IroRE² composite RE was found to drive lacZ expression in a pattern very similar, albeit not identical, to that of the endogenous ara/caup genes (Letizia, 2007).

Two other genomic fragments, IroRE³ and IroRE⁴ (3.4 and 3.7 kb), adjacent to each other, drove lacZ expression in a stripe of cells located at the proximal region of the presumptive lateral notum, which partially overlapped with the caup expression domain. Finally, IroRE⁵ (2.8 kb) drove expression mainly in the prospective alula and peripodial membrane (Letizia, 2007).

A common theme in development is the convergence of different signalling pathways to implement a given developmental program. For instance during embryonic development, the antagonistic activity of the EGFR and Dpp pathways sets the limits between the neuroectoderm and the dorsal ectoderm. A similar situation applies to the specification of prospective body regions within the wing imaginal disc. During the early second instar, EGFR and Dpp pathways act antagonistically on the regulation of the Iro-C restricting its expression to the prospective notum region where it specifies notum development rather than hinge. Later, at the early third instar, again the concomitant activity of EGFR and Dpp signals (the latter now also emanating form the most proximal region of the wing disc) partition the prospective notum into two different subdomains, the medial and the lateral notum, the latter being specified by ara/caup expression. Thus, to understand how regionalization of the adult fly body is achieved it is important to elucidate the mechanisms responsible for the joint interpretation of both signalling pathways (Letizia, 2007).

This study shows that the opposing effects of the EGFR and Dpp pathways on Iro-C expression result from the convergence of both pathways on at least two distinct Iro-C regulatory elements, IroRE¹ and IroRE². These two REs drive gene expression in two complementary domains of the prospective notum region of the wing disc, and appear to mediate most of the regulation of the Iro-C genes by the Dpp and EGFR pathways in this region of the wing disc. Furthermore, IroRE¹ provides a regulatory mechanism for the coexistence at the prospective lateral notum of Iro-C expression and Dpp pathway activity, notwithstanding the negative regulation of Iro-C by such pathway (Letizia, 2007).

The transcriptional regulation of the Iro-C genes is modular. Thus, the non-coding Iro-C genomic DNA contains a series of five separate enhancers that control the expression of a reporter gene in sub-domains within the realm of Iro-C expression in the prospective notum region of the wing disc. None of the identified fragments reproduces on its own the entire pattern of expression of Iro-C in the prospective notum. However, IroRE¹ and IroRE² promote expression in complementary domains that entirely cover the territory of the presumptive lateral notum. Furthermore, IroRE²-mediated transcription recapitulates expression of Iro-C at the whole prospective notum at the second larval instar. It is hypothesized that the combined activity of both REs would be responsible for a great part of the regulation of Iro-C expression in the notum territory. Moreover, although IroRE³, IroRE⁴ and IroRE⁵ mediate lacZ expression in patterns only partly related to that of the Iro-C genes, these REs probably contribute to the complex regulation of the Iro-C. In addition, the possibility cannot be excluded of other RE(s) located outside the tested region that would help to establish the final pattern of Iro-C expression. Indeed, Iro^DFM3, a deficiency obtained by imprecise excision of the iro^rF209 P-lacZ element that extends up to the mirr promoter, maintains some lacZ expression in part of the central notum (Letizia, 2007).

The identified REs might act simultaneously on ara and caup expression to give rise to their almost coincident patterns of expression. Such coincidence cannot be attributed to cross-regulation between ara and caup since in iro^rF209 mutant discs (iro^rF209 is an ara null allele expression of caup is unmodified. Regulation of ara/caup would be, accordingly, similar to that of the achaete-scute genes of the AS-C, which show identical patterns of expression due to the use of shared enhancers. Expression of the vertebrate Iroquois (Irx) genes appears to be similarly regulated. Thus, the analysis of the regulatory potential of highly and ultra conserved non-coding regions present in the intergenic regions of the Irx clusters suggests these genes to be regulated by partially redundant enhancers shared by the components of each cluster (Letizia, 2007).

Expression of mirr in the notum region of the wing disc largely coincides with that of ara/caup and most likely is under the control of the same REs. Thus, activity of the IroRE² may account for the unmodified expression of mirr in iro¹ imaginal discs (associated with an inversion breakpoint located within the caup transcription unit). In addition, differences in the expression of ara/caup and mirr might be due to the presence of repressor RE(s) or insulator sequences that would prevent the action of the RE(s) controlling ara/caup on the mirr promoter. This is consistent with the previous observation of ectopic expression of mirr in Mob1 mutants, a regulatory mutation mapped within the Iro-C (Letizia, 2007).

The identification of REs present in the Iro-C has allowed unveiling of some of the molecular mechanisms of its transcriptional regulation at the level of DNA-protein interaction and analysis of the interplay of positive and negative inputs from convergent signalling pathways (Letizia, 2007).

EGFR activation in the proximal region of the wing disc leads to expression of Iro-C. This study demonstrates that both IroRE¹ and IroRE² mediate positive regulation by the EGFR pathway. It is shown that Pnt mediates activation of IroRE²-lacZ by the EGFR pathway. Furthermore, EGFR-dependent activation is cell context dependent. This suggests the existence, in the cells receiving EGFR signalling, of presently unknown factors that would contribute to ara/caup activation and/or the presence of counteracting repressing mechanisms, which should prevent their activation. Clearly, the Dpp pathway is so far the best candidate, since it has been shown that it can repress Iro-C and the IroRE²-lacZ transgene (Letizia, 2007).

The molecular mechanism of Dpp-dependent regulation of Iro-C expression appears to be more complex. The Dpp pathway can repress or activate Iro-C through different REs and different effector proteins. IroRE² appears to mediate Dpp-dependent repression at the medial notum (most probably through direct binding of the heterodimer Pnr/Ush and Mad) and at the hinge and lateral notum (independently of Pnr, Ush and the GATA factor Grn in these domains). Dpp-dependent repression of Iro-C may be mediated, in addition, through a different RE, namely, through a brk silencer element (brkSE), shown to mediate Dpp-dependent repression of brk by binding of a Medea/Mad/Schnurri repressor complex, which is present at the Iro-C within IroRE⁵ (Letizia, 2007).

Despite the Dpp-mediated repression through IroRE², a high level of Iro-C proteins accumulates in the lateral region of the notum, near the strong source of Dpp at the AP border. Furthermore, in this region of the wing disc Iro-C expression is refractory to Dpp-dependent repression. It is noteworthy that, IroRE¹ mediates lacZ expression exclusively in that region of the wing disc and it appears to provide a regulatory mechanism for the co-existence of Iro-C expression and Dpp pathway activity, since the Dpp pathway does not repress but, on the contrary, activates IroRE¹-mediated lacZ expression. Activation is restricted to the lateral notum, most likely because of the presence, in the hinge and medial notum territories, of repressors [Muscle segment homeobox, Msh; also known as Drop and Pnr/Ush, respectively] that would counteract activation. Putative binding sites for both Msh (consensus sequence G/C TTAATTG) and GATA proteins are indeed present in IroRE¹. Thus, IroRE¹ and IroRE² represent two different REs in the same gene that respond in opposite ways to the same positional information, i.e. Dpp signalling. In addition a Dpp-independent mechanism based in the mutual repression between Iro-C and the homeoprotein Msh helps to maintain the distal border of Iro-C expression. This repression could be mediated by direct binding of Msh to one putative Msh binding site present in the Iro-RE^2-B sequence (Letizia, 2007).

U-shaped protein domains required for repression of cardiac gene expression in Drosophila: Antagonism by Pannier

U-shaped is a zinc finger protein that functions predominantly as a negative transcriptional regulator of cell fate determination during Drosophila development. In the early stages of dorsal vessel formation, the protein acts to control cardioblast specification, working as a negative attenuator of the cardiogenic GATA factor Pannier. Pannier and the homeodomain protein Tinman normally work together to specify heart cells and activate cardioblast gene expression. One target of this positive regulation is a heart enhancer of the Drosophila mef2 gene and U-shaped has been shown to antagonize enhancer activation by Pannier and Tinman. Protein domains of U-shaped required for its repression of cardioblast gene expression were mapped. Such studies showed GATA factor interacting zinc fingers of U-shaped are required for enhancer repression, as well as three small motifs that are likely needed for co-factor binding and/or protein modification. These analyses have also allowed for the definition of a 253 amino acid interval of U-shaped that is essential for its nuclear localization. Together, these findings provide molecular insights into the function of U-shaped as a negative regulator of heart development in Drosophila (Tokusumi, 2007).

Through the use of an established assay to monitor Pannier-dependent cardioblast gene activity, and the generation and analysis of 20 different versions of the U-shaped protein, six U-shaped domains required for its repression of mef2 gene expression were identified. Three previously identified GATA-interacting zinc fingers of U-shaped are critical for this inhibitory property, which likely reflects the necessity of multiple zinc fingers forming a strong and stable interaction with the Pannier GATA factor. Whether Pannier-U-shaped complex formation interferes with the physical interaction of Pannier and Tinman in the synergistic activation of D-mef2 target sequences remains to be determined (Tokusumi, 2007).

U-shaped may also directly antagonize Pannier function as has been shown in the process of sensory bristle formation. Heterodimerization of U-shaped with Pannier converts the GATA transcriptional activator into a transcriptional repressor, an event that leads to the non-activation of target genes such as ac, sc, and wg in the dorsal notum of the wing disc. It is noteworthy that the results demonstrated the requirement of a binding site for the CtBP transcriptional co-repressor protein. In the context of the cardiogenic mesoderm, the combination of Pannier, U-shaped, and CtBP may prevent mesodermal cells from initiating gene expression programs needed for the specification of the cardioblast fate. In contrast, the combination of Pannier, Dorsocross, and Tinman is known to activate a regulatory network programming heart cell specification and cardioblast differentiation. Additional studies will be needed to elucidate the potential role of CtBP as an antagonist of cardiac gene expression and heart development. If U-shaped-CtBP interaction plays a crucial inhibitory role, then one would predict comparable dorsal vessel phenotypes for CtBP and U-shaped in loss- and gain-of-function genetic backgrounds (Tokusumi, 2007).

Finally, these studies have defined a 253 amino acid region required for nuclear localization of U-shaped. Within this interval, two highly basic amino acid sequences have been defined as being essential for U-shaped ability to inhibit Pannier-mediated cardiac gene expression. Perhaps, these motifs are required to facilitate the binding and stable interaction of co-repressor proteins with U-shaped. Another possibility is that these sequences serve as sites for post-translational modification, such as acetylation and/or methylation. Selective protein modification(s) may be a requisite for U-shaped to act as a negative modulator of Pannier transcription factor function during cardiogenesis in Drosophila (Tokusumi, 2007).

Drosophila dLMO-PA isoform acts as an early activator of achaete/scute proneural expression

The Drosophila bHLH proneural factors Achaete (Ac) and Scute (Sc) are expressed in clusters of cells (proneural clusters), providing the cells with the potential to develop a neural fate. Mediodorsal proneural patterning is mediated through the GATA transcription factor Pannier (Pnr) that activates ac/sc directly through binding to the dorsocentral (DC) enhancer of ac/sc. Besides, the Gfi transcription factor Senseless (Sens), a target of Ac/Sc, synergizes with ac/sc in the presumptive sensory organ precursors (SOPs). This study investigated, through new genetic tools, the function of dLMO, the Drosophila LIM only transcription factor that was already known to control wing development. dLMO gene encodes two isoforms, dLMO-RA and dLMO-RB. dLMO null and dLMO-RA⁻ deletions have similar phenotypes, lacking thoracic and wing margin sensory organs (SO), while dLMO-RB⁻ deletion has normal SOs. At early stages, dLMO-RA is expressed in proneural clusters, however later it is excluded from the SOPs. dLMO functions as a Pnr coactivator to promote ac/sc expression. In the late SOPs, where dLMO-PA is not expressed, Pnr participates to the Sens-dependent regulation of ac/sc. Taken together these results suggest that dLMO-PA is the major isoform that is required for early activation of ac/sc expression (Asmar, 2008).

The lack of dLMO protein leads to very distinctive phenotypes. The mutant animals are not able to fly, they have a short life span and show an abnormal gait behaviour. In addition, they show a discreet bristle phenotype. In Drosophila, there are two paralogous LMO factors, dLMO and CG5708. These genes are expressed in the CNS where redundancy is not excluded. However CG5708 is not expressed in the wing discs and presumptive SOPs. Therefore it is concluded that the mild phenotype observed for the adult PNS in dLMO mutants, is not attributed to gene redundancy. dLMO encodes two distinct isoforms, dLMO-PA and -PB, which only differ from their N-terminus. Only dLMO-RA is broadly expressed in the notum, and contributes to the PNS phenotype. dLMO function is also critical in the developing central nervous system for the activity of the ventral lateral neurons, LN_vs. It is highly probable that dLMO-RB has some subtle biological activities in the brain, where it has a specific pattern (Asmar, 2008).

In vertebrate, multiproteic complexes composed by GATA-1, LMO2, Ldb-1 and the bHLHs E47 and SCL, are required for normal differentiation of haematopoietic cells. The current results highlight several evidences in favour of dLMO as a GATA coactivator in Drosophila . (1) A genetic synergism exists between pnr⁻ and dLMO⁻ null alleles. (2) dLMO modulates the activity of a DC:ac-lacZ reporter, the model target of Pnr, in vivo. Loss of function dLMO mutants show reduced level of the DC:ac-lacZ expression, whereas in gain-of-function dLMO mutants the DC:ac-lacZ expression is increased. (3) dLMO-PA isoform directly interact with Pnr in GST pull down assay. Therefore it is concluded that dLMO might enhance the proneural activity of Pnr through direct interaction with the GATA factor. Consistently, dLMO expression overlaps with the dorsal-most domain of Pnr during third instar larval stages. Though Pnr controls the development of both DC and SC bristles, dLMO null alleles affect only DC bristles. dLMO expression, that overlaps both SC and DC proneural clusters in the notum, is significantly weaker in the SC region, suggesting that regulation of proneural ac/sc expression is differentially sensitive and responds to local combinations of transcription factors. These data support previously published studies demonstrating that the proneural activity of Pnr is prominently repressed in the SC region by the LIM-HD transcription factor Isl (Asmar, 2008).

At later stages, dLMO expression is excluded from the corresponding SOP and its derivative cells. In contrast, the proneural factor Sens, that plays an important role for sensory organ specification, is first broadly expressed in proneural clusters at low levels where it functions as a repressor of ac/sc, and then later, is expressed at high levels in the presumptive SOPs, where it acts as a transcriptional activator that directly interacts and synergizes with the proneural proteins, Ac and Sc. It has been shown that both Gfi-1 and GATA-1, the mammalian ortholog of Sens and Pnr respectively, are essential for development of the closed related erythroid and megakaryocytic lineages. The Sens/Pnr interaction is evolutionary conserved in Drosophila neurogenesis. It is suggestd that Pnr could participate to the Sens-dependent positive autoregulation of Ac/Sc in late SOPs where dLMO is not expressed. The synergism between Pnr and Sens would need more detailed investigations. Taken together, these studies have shown dLMO-PA as a co-activator for Pnr during the establishment of proneural fields and revealed another level of proneural ac/sc regulation during late neurogenesis in the Drosophila PNS (Asmar, 2008).

Transcriptional regulation by CHIP/LDB complexes

It is increasingly clear that transcription factors play versatile roles in turning genes 'on' or 'off' depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. This study used the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, 189 down-stream targets of CHIP/LDB were identified; these genes are enriched for the binding sites of Apterous (AP) and Pannier (PNR), two well studied transcription factors associated with CHIP/LDB complexes. Extensive genetic screens were performed and target genes were identified that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing, novel roles were uncovered for two of the target genes, Gs-alpha, in early development of these structures. Taken together, these results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required for development (Bronstein, 2011).

Drosophila SSDP was identified on the basis of its ability to bind the nuclear adaptor protein CHIP/LDB (van Meyel, 2003; Chen, 2002). Both nuclear localization of SSDP and its ability to modulate the transcription activity of the CHIP-AP complex during wing development depend on its interaction with CHIP/LDB. This study implemented a combination of molecular, bioinformatic and genetic approaches that allowed has led to insight into the effect of SSDP on the transcriptional activity of CHIP/LDB complexes and their role in development. A genome wide screen was conducted for SSDP target genes in Drosophila using expression microarrays with mRNA isolated from larvae bearing hypomorphic alleles of ssdp. Analysis of transcription factor binding site enrichment served as an orthogonal assay that validates and extends the microarray results and thus contributes to understanding of the relation between the CHIP-AP and CHIP-PNR transcription complexes in specific tissues (e.g. wing and thorax) (Bronstein, 2011).

SSDP proteins directly bind DNA and mouse SSDP1 activates the expression of a reporter gene in both yeast and mammalian cells indicating that it is capable of regulating transcription activity. Enrichment was found for SSDP binding sites upstream of the genes identified in the microarray experiments on flies lacking SSDP. Moreover, in agreement with the positive transcriptional role of SSDP, enrichment for SSDP binding sites was restricted to the genes showing decreased expression in mutants. This strongly suggests that a significant number of these genes are bona fide SSDP target genes (Bronstein, 2011).

Consistent with the involvement of SSDP with the CHIP-AP complex, it was found that upstream regulatory regions of the SSDP putative target genes are also enriched for the AP binding site and the SSDP binding site. These sites are likely to be functionally significant, since loss of ssdp enhances the wing notching phenotype of a dominant allele of ap. Additionally, over-expression of Dlmo, whose product negatively regulates the CHIP-AP complex, also interacts with mutants of SSDP target genes, demonstrating that SSDP target genes are involved in the CHIP-AP pathway. The efficiency of finding genetic interactions among the genes differentially expressed in the microarray experiments, demonstrated the power of this approach. Specifically, 72% of the loci tested with Dlmo^Bx2 is more than an order of magnitude higher than an EP insertion screen (1.3% interacting) in a Dlmo^Bx1 sensitized background. Combined microarray and genetic loss of function screen allowed the identification of a similar number of Dlmo-interacting genes by screening a much smaller group of putative target genes (Bejarano, 2008). Of the 35 genes identified by Bejarano only CG1943 was found in the 189 genes identified in the current microarray screen. This study specifically identified down-stream targets of SSDP, while Bejarano searched for any modifiers of the Dlmo wing notching phenotype and thus uncovered genes that function in other regulatory pathways or genes that are upstream of the CHIP-AP complexes. This may explain the limited overlap between the current results and those of Bejarano (Bronstein, 2011).

In contrast to the enrichment of SSDP binding sites in the genes down-regulated in ssdp mutants, the PNR binding site was enriched specifically in the genes up-regulated in the ssdp mutants. A model is therefore presented in which loss of SSDP disrupts the balance between the CHIP-AP and CHIP-PNR complexes. Mammalian SSDP proteins protect LDB, LHX and LMO proteins from ubiquitination and subsequent proteasome-mediated degradation by interfering with the interaction between LDB and the E3 ubiquitin ligase, RLIM. It is therefore possible that in the absence of SSDP proteins, CHIP/LDB and LMO can escape degradation by interacting with GATA and beta-HLH proteins that are not subjected to proteasome-mediated regulation. The N-terminus of CHIP/LDB proteins is responsible for interaction with both PNR and RLIM. Thus, PNR/GATA proteins may partially interfere with the interaction between CHIP/LDB and RLIM making the CHIP/LDB-PNR/GATA complex more resistant to proteasome regulation and less dependant on the levels of SSDP proteins then the CHIP/LDB-LHX/AP complex (Bronstein, 2011).

According to the current model, in cells where both the CHIP-AP and CHIP-PNR complexes are active, loss of SSDP should result in the same phenotype as over-expression of PNR. Indeed, it was found that ssdp^L7/+ flies display duplications of scutellar sensory bristles, similar to gain of function mutations in pnr. In addition, lowered levels of pnr in ssdp^L7/+; pnr^VX6/+ flies suppresses scutellar bristle duplication. This indicates that the duplicated scutellar bristle phenotype of ssdp^L7/+ flies depends on the presence of PNR. As predicted by the model, since both AP and PNR regulate bristle formation, the functional interactions between SSDP target genes and ssdp^L7 and/or Chip^e5.5 resulted in either suppression or enhancement of the duplicated scutellar bristle phenotype (Bronstein, 2011).

These results in flies indicate that SSDP contributes differentially to CHIP/LDB complexes containing AP versus PNR. By contrast, mouse SSDP proteins positively contribute to the transcription activity and assembly of both LDB-GATA and LDB-LHX complexes, but the relative contribution of mammalian SSDP proteins to LDB complexes containing LHX proteins versus GATA proteins has not been specifically examined. It is possible that SSDP alters the balance of LIM-based CHIP/LDB complexes and GATA-containing CHIP/LDB complexes in the development of mice, as occurs in flies (Bronstein, 2011).

The search for enrichment of transcription factor binding sites upstream of the putative SSDP target genes identified additional transcription factors that may warrant future study. Some of these factors are associated with SSDP and CHIP/LDB complexes. For example, the binding sites for PNR and ZESTE (Z) were both enriched in the up-regulated putative SSDP target genes. This is in agreement with previous studies showing that Z can recruit the BRAHMA (BRM, the Drosophila homolog of the yeast SWI2/SNF2 gene) complex via its member OSA, which together negatively regulate the CHIP-PNR complex during sensory bristle formation through direct and simultaneous binding of OSA to both CHIP and PNR (Bronstein, 2011).

Some of the additional regulatory inputs at SSDP target genes may be evolutionarily conserved. For example, enrichment of STAT92E and SSDP binding sites was found in the down-regulated SSDP target genes. This may be significant, as a known role of ssdp is regulation of the JAK/STAT pathway during Drosophila eye development. Interestingly, mammalian STAT1 confers an anti-proliferative response to IFN-γ signaling by inhibition of c-myc expression. Similarly, expression of mammalian SSDP2 in human acute myelogenous leukemia cells and prostate cancer cells leads to cell cycle arrest and inhibits proliferation accompanied by down-regulation of C-MYC. These findings indicate that both in Drosophila and in mammals SSDP and STAT proteins have similar functions and may share common target genes (Bronstein, 2011).

While the transcription factor binding site analysis utilized all of the 189 putative SSDP target genes, genetic screens were conducted on a subset of them due to the availability of mutants. This suggests that more genetic interactions will be found among the untested genes. Even among this more limited subset, there are interesting new stories that suggest future experimental directions. For example, an insertion mutation in the Xbp1 gene suppressed the duplicated scutellar bristle phenotype characteristic of ssdp^L7/+ and Chip^e5.5/+ flies, indicating that XBP1 contributes positively to bristle formation. In contrast, when Xbp1 was silenced in ap-expressing cells both the wings and the scutum displayed a marked excess of sensory bristles while the scutellum was not affected. These results suggest that in the wing and scutum XBP1 acts as a negative regulator of bristle formation. Silencing of Xbp1 in pnr-expressing cells caused a similar excess of bristle on the scutum, accompanied by a reduced number of scutellar bristles, further emphasizing the opposing effects of XBP1 in these two distinct parts of the thorax. Such contrasting phenotypes have been previously documented for several pnr mutants as well. In flies and mammals XBP1 regulates the ER stress response, also termed the unfolded protein response (UPR). Since one of the functions of the ER is the production of secreted proteins, UPR-related pathways are widely utilized during the normal differentiation of many specialized secretory cells. In this respect it would be interesting to examine whether SSDP and CHIP/LDB complexes affect the production of secreted morphogens, such as Wingless (WG), the secreted ligands of the EGFR receptor, Spitz (SPI) and Argos (AOS), or the secreted Notch binding protein Scabrous (SCA) via XBP1 during wing and sensory bristle formation. Alternatively, the transcription factor XBP1 may directly regulate the expression of genes required for differentiation of the wing and sensory bristles. Indeed, carbohydrate ingestion induces XBP1 in the liver of mice, which in turn directly regulates the expression of genes involved in fatty acid synthesis. This role of XBP1 is independent of UPR activation and is not due to altered protein secretory function. Curiously, the two GO function categories 'cellular carbohydrate metabolism' and 'cellular lipid metabolism' which are enriched among Xbp1 target genes in mouse skeletal muscle and secretory cells were also enriched in the list of putative SSDP target genes. Whether this reflects a secondary effect due to the down-regulation of Xbp1 in ssdp mutants or a direct regulation of these processes by SSDP is yet to be determined (Bronstein, 2011).

Additional novel functions for CHIP/LDB complexes are implied by the results regarding the Gs-alpha60A (a.k.a. CG2835) gene. G protein coupled receptors are important regulators of development by for example, signaling via the protein kinase A (PKA) pathway. Activation or inhibition of PKA signaling during pupal wing maturation perturb proper adhesion of dorso-ventral wing surfaces resulting in wing blistering. This phenotype may be due to miss-regulation of wing epithelial cell death in ap-expressing cells. Interestingly, similar wing blisters occur in the wing of Dlmo^Bx2 flies. Moreover, it was found that mutant alleles of Gs-alpha60A enhanced the wing blistering phenotype of Dlmo^Bx2. Silencing of G-salpha60A in ap-expressing cells caused a curled wing phenotype. Such a phenotype can result from differences in the size of the dorsal and ventral wing blade surfaces. In addition, silencing of this gene in pnr-expressing cells caused the posterior pair of scutellar bristles to form in reversed orientation. Bristle orientation have been proposed to be regulated by planar cell polarity genes. Taken together these results point to novel aspects of regulation of wing and sensory bristle development by SSDP and CHIP/LDB complexes mediated by G-alpha proteins (Bronstein, 2011).

This genome-wide expression profiling and bioinformatics analysis of ssdp mutant larvae, combined with genetic screens resulted in gained insight into the intricate context-dependent transcriptional regulation by CHIP/LDB complexes. It was possible to identify 28 putative SSDP target genes that are involved in wing development and 23 putative SSDP target genes that play a role in scutellar bristle formation. Examination of two of these, xbp1 and Gs-alpha60A, suggests novel aspects of developmental regulation such as the involvement of SSDP and CHIP/LDB complexes in ER function and PKA signaling. Furthermore, it was shown that SSDP proteins contribute differentially to transcription activity, and probably to the balance in formation of CHIP-AP and CHIP-PNR complexes. Furthermore potential novel partners of SSDP in regulating transcription of downstream genes during fly development were. It stands to reason that an extension of the genetic analysis to mammals and other vertebrates will reveal a host of additional functions of SSDP and CHIP/LDB during the multifaceted process of transcriptional regulation that underlies the development of multicellular organisms (Bronstein, 2011).

A transcription factor collective defines cardiac cell fate and reflects lineage history

Cell fate decisions are driven through the integration of inductive signals and tissue-specific transcription factors (TFs), although the details on how this information converges in cis remain unclear. This study demonstrates that the five genetic components essential for cardiac specification in Drosophila, including the effectors of Wg and Dpp signaling, act as a collective unit to cooperatively regulate heart enhancer activity, both in vivo and in vitro. Their combinatorial binding does not require any specific motif orientation or spacing, suggesting an alternative mode of enhancer function whereby cooperative activity occurs with extensive motif flexibility. A fraction of enhancers co-occupied by cardiogenic TFs had unexpected activity in the neighboring visceral mesoderm but could be rendered active in heart through single-site mutations. Given that cardiac and visceral cells are both derived from the dorsal mesoderm, this 'dormant' TF binding signature may represent a molecular footprint of these cells' developmental lineage (Junion, 2012).

Dissecting transcriptional networks in the context of embryonic development is inherently difficult due to the multicellularity of the system and the fact that most essential developmental regulators have pleiotropic effects, acting in separate and sometimes interconnected networks. This study presents a comprehensive systematic dissection of the cis-regulatory properties leading to cardiac specification within the context of a developing embryo. The resulting compendium of TF binding signatures, in addition to extensive in vivo and in vitro analysis of enhancer activity, revealed a number of insights into the regulatory complexity of developmental programs (Junion, 2012).

Nkx (Tinman in Drosophila), GATA (Pannier in Drosophila), and T box factors (Doc in Drosophila) regulate each other’s expression in both flies and mice, where they form a recursively wired transcriptional circuit that acts cooperatively at a genetic level to regulate heart development across a broad range of organisms. The data demonstrate that this cooperative regulation extends beyond the ability of these TFs to regulate each other’s expression. All five cardiogenic TFs (including dTCF and pMad) converge as a collective unit on a very extensive set of mesodermal enhancer elements in vivo (Tin-bound regions) and also in vitro (in DmD8 cells). Importantly, this TF co-occupancy occurs in cis, rather than being mediated via crosslinking of DNA-looping interactions bringing together distant sites. Examining enhancer activity out of context, for example, in transgenic experiments and luciferase assays, revealed that the TF collective activity is preserved in situations in which these regions are removed from their native genomic 'looping' context (Junion, 2012).

In keeping with the conserved essential role of these factors for heart development, the integration of their activity at shared enhancer elements may also be conserved. Recent analyses of the mouse homologs of these TFs (with the exception of the inductive signals from Wg and Dpp signaling) in a cardiomyocyte cell line support this, revealing a signifcant overlap in their binding signatures (He, 2011; Schlesinger, 2011), although interestingly not in the collective 'all-or-none' fashion observed in Drosophila embryos. This difference may result from the partial overlap of the TFs examined, interspecies differences, or the inherent differences between the in vivo versus in vitro models. Examining enhancer output for a large number of regions indicates that this collective TF occupancy signature is generally predictive of enhancer activity in cardiac mesoderm or its neighboring cell population, the visceral mesoderm—expression patterns that cannot be obtained from any one of these TFs alone (Junion, 2012).

There are currently two prevailing models of how enhancers function. The enhanceosome model suggests that TFs bind to enhancers in a cooperative manner directed by a specific arrangement of motifs, often having a very rigid motif grammar. An alternative, the billboard model, suggests that each TF (or submodule) is recruited independently via its own sequence motif, and therefore the motif spacing and relative orientation have little importance. The results of this study indicate that cardiogenic TFs are corecruited and activate enhancers in a cooperative manner, but this cooperativity occurs with little or no apparent motif grammar to such an extent that the motifs for some factors do not always need to be present. This is at odds with either the enhanceosome (cooperative binding; rigid grammar) or billboard (independent binding; little grammar) models and represents an alternative mode of enhancer activity, which was termed a 'TF collective' (cooperative binding; no grammar), and likely constitutes a common principle in other systems (Junion, 2012).

The data suggest that the TF collective operates via the cooperative recruitment of a large number of TFs (in this case, at least five), which is mediated by the presence of high-affinity TF motifs for a subset of factors initiating the recruitment of all TFs. The occupancy of any remaining factor(s) is most likely facilitated via protein-protein interactions or cooperativity at a higher level such as, for example, via the chromatin activators CBP/ p300, which interact with mammalian GATA and Mad homologs. This model allows for extensive motif turnover without any obvious effect on enhancer activity, consistent with what has been observed in vivo for the Drosophila spa enhancer and mouse heart enhancers (Junion, 2012).

Integrating the TF occupancy data for all seven major TFs involved in dorsal mesoderm specification (the five cardiogenic factors together with Biniou and Slp) revealed a very striking observation: the developmental history of cardiac cells is reflected in their TF occupancy patterns. Visceral mesoderm (VM) and cardiac mesoderm (CM) are both derived from precursor cells within the dorsal mesoderm. Once specified, these cell types express divergent sets of TFs: Slp, activated dTCF, Doc, and Pnr function in cardiac cells, whereas Biniou and Bagpipe are active in the VM. Despite these mutually exclusive expression patterns, the cardiogenic TFs are recruited to the same enhancers as VM TFs in the juxtaposed cardiac mesoderm. Moreover, dependent on the removal of a transcriptional repressor, these combined binding signatures have the capacity to drive expression in either cell type. This finding provides the exciting possibility that dormant TF occupancy could be used to trace the developmental origins of a cell lineage. It also explains why active repression in cis is required for correct lineage specification, which is a frequent observation from genetic studies. At the molecular level, it remains an open question why the VM-specific enhancers are occupied by the cardiac TF collective. It is hypothesized that this may occur through chromatin remodeling in the precursor cell population. An 'open' (accessible) chromatin state at these loci in dorsal mesoderm cells, which is most likely mediated or maintained by Tin binding prior to specification, could facilitate the occupancy of cell type-specific TFs in both CM and VM cells. Such early 'chromatin priming' of regulatory regions active at later stages has been observed during ES cell differentiation. The current data provide evidence that this also holds true for TF occupancy and not just chromatin marks. On a more speculative level, this developmental footprint of TF occupancy may reflect the evolutionary ancestry of these two organs. Visceral and cardiogenic tissues are derived from the splanchnic mesoderm in both flies and vertebrates. These complex VM-heart enhancers may represent evolutionary relics containing functional binding sites that reflect enhancer activity in an ancestral cell type (Junion, 2012).

Taken together, the collective TF occupancy on enhancers during dorsal mesoderm specification illustrates how the regulatory input of cooperative TFs is integrated in cis, in the absence of any strict motif grammar. This more flexible mode of cooperative cis regulation is expected to be present in many other complex developmental systems (Junion, 2012).

The Iroquois Complex Is Required in the Dorsal Mesoderm to Ensure Normal Heart Development in Drosophila

Drosophila heart development is an invaluable system to study the orchestrated action of numerous factors that govern cardiogenesis. Cardiac progenitors arise within specific dorsal mesodermal regions that are under the influence of temporally coordinated actions of multiple signaling pathways. The Drosophila Iroquois complex (Iro-C) consists of the three homeobox transcription factors araucan (ara), caupolican (caup) and mirror (mirr). The Iro-C has been shown to be involved in tissue patterning leading to the differentiation of specific structures, such as the lateral notum and dorsal head structures and in establishing the dorsal-ventral border of the eye. A function for Iro-C in cardiogenesis has not been investigated yet. Loss of the whole Iroquois complex, as well as loss of either ara/caup or mirr only, affect heart development in Drosophila. The data indicate that the GATA factor Pannier requires the presence of Iro-C to function in cardiogenesis. A detailed expression pattern analysis of the members of the Iro-C revealed the presence of a possibly novel subpopulation of Even-skipped expressing pericardial cells and seven pairs of heart-associated cells that have not been described before. Taken together, this work introduces Iro-C as a new set of transcription factors that are required for normal development of the heart. As the members of the Iro-C may function, at least partly, as competence factors in the dorsal mesoderm, these results are fundamental for future studies aiming to decipher the regulatory interactions between factors that determine different cell fates in the dorsal mesoderm (Mirzoyan, 2013).

Tissue patterning requires the spatial and temporal coordinated action of signals providing instructive or permissive cues that result in the specification of different cell types and their subsequent differentiation into different lineages. This analyses of Iro-C deficient embryos demonstrate that ara/caup and mirr are required in the dorsal mesoderm for normal heart development. The heart phenotypes could be caused by alterations of the fine balance of the interactions between factors of the cardiac signaling network. In early stage Drosophila embryos the mesoderm is patterned along the anterior-posterior (AP) axis with cardiac and somatic mesodermal domains alternating with visceral mesodermal domains. The tin-positive mesoderm is specified as cardiac and somatic mesoderm under the influence of combined Dpp and Wg signaling. Subsequently, the cardiac and somatic mesodermal domains are further subdivided by the action of the Notch pathway and MAPK signaling activated by EGFR and FGFR. The Eve-expressing cell clusters that give rise to pericardial and DA1 somatic muscle cells, as well as the Doc expression pattern, distinguish the cardiac and somatic mesodermal domain from the visceral mesodermal domain. The early expression pattern of Ara/Caup and Mirr at stages 10/11 suggests a role for Iro-C in patterning the dorsal mesoderm along the AP axis. Consistent with their previously described functions in other developmental contexts, members of the Iro-C may integrate signaling inputs and interact with other transcription factors to specify different dorsal mesodermal derivatives. Activation of the Iro-C by the EGFR pathway is required for the specification of the notum. Mirr was shown to interpret EGFR signaling by eliciting a specific cellular response required for patterning the follicular epithelium. During Drosophila eye development, mirr expression can be regulated by Unpaired, a ligand that activates JAK/Stat signaling. In fact, the JAK/Stat signaling pathway has only recently been added to the signaling pathways that function in Drosophila cardiogenesis. In chromatin immunoprecipitation experiments caup was identified as a target of Stat92E, which is the sole transcriptional effector of the JAK/Stat signaling pathway in Drosophila. Interestingly, the increase of Odd-pericardial cells and the additional Tin-expressing cells that were the characteristic phenotypes in ara/caup (iro^DFM1) and in mirr (mirr^e48) mutants are highly similar to the phenotypes in stat92E mutants described by Johnson (Johnson, 2011). Also, as described for stat92E mutants, cell adhesion defects were noticed in a number of embryos as determined by the distant location of some Tin-expressing cells from the forming heart tube. As for establishing a possible link between JAK/Stat and Iro-C in the dorsal mesoderm and specifically in cardiogenesis, it would be necessary to determine for example whether caup and mirr can rescue the heart phenotype of stat92E mutants. Also, it would be interesting to compare the expression of the other crucial heart marker genes, Tup, Doc and Pnr, in stat92E mutants at early stages to determine to what extent the phenotypes of embryos mutant for Iro-C and for JAK/Stat signaling are similar (Mirzoyan, 2013).

Members of the Iro-C were shown to be positively or negatively regulated by signaling pathways that play crucial roles in heart development. Conversely, the Iro-C factors can also regulate the activity of at least one of these pathways. Specifically, Ara/Caup, as well as Mirr were shown to regulate the expression of the glycosyltransferase fringe and as a consequence modulate Notch signaling activity in the eye. In the dorsal mesoderm, the lateral inhibitory function of Notch signaling establishes the proper number of heart and muscle progenitors. Given the fact that Iro-C can regulate Notch activity it may be that the loss of Iro-C leads to an imbalance of progenitor cell specification resulting in an abnormal number of heart cells. Further studies are required to decipher the molecular mechanism by which Iro-C could integrate diverse signaling inputs and thereby function in the specification and differentiation of the different dorsal mesodermal derivatives (Mirzoyan, 2013).

To determine whether Iro-C can be positioned into the early transcriptional network that determines a cardiac lineage, this study investigated the interdependency between crucial cardiac factors and Iro-C during cardiogenesis. Analyses of the expression of Ara/Caup and Mirr in tin³⁴⁶, Df(3L)DocA, pnr^VX6 and tup^isl-1 embryos demonstrated the dependency of Ara/Caup and Mirr on all four factors. The strongest loss of Ara/Caup and Mirr expression was observed in tin³⁴⁶ and Df(3L)DocA mutants, which clearly places tin and Doc upstream of Ara/Caup and Mirr. In tup^isl-1 and in pnr^VX6 mutant embryos, Ara/Caup and Mirr were strongly downregulated, however regarding Ara/Caup, some expression remained in segmental patches suggesting a different level of regulation. The currently available data indicates a positive and a negative regulatory effect of pnr on Iro-C. Whereas pnr restricts Iro-C expression in the dorsal ectoderm and in the wing disc, there is also evidence that pnr can positively regulate Iro-C in the wing disc. Whether Pnr activates or represses Iro-C appears to depend on the presence of U-shaped (Ush), a protein that modulates the transcriptional activity of Pnr. In the wing disc it was shown that an Iro-C-lacZ (IroRE²-lacZ) construct was activated in cells that contained Pnr but were devoid of Ush. The data demonstrate that in the dorsal mesoderm, the expression of Ara/Caup and Mirr depends on pnr. Additionally this analyses show that pnr expression is independent of Iro-C. This finding is intriguing with respect to the downregulation of Tup and Doc in Iro-C mutants. Pnr is required for the maintenance of Doc and for the initiation and/or maintenance of Tup. Since Iro-C mutants exhibit a reduction in Doc-positive cells despite the presence of pnr, members of the Iro-C appear to be required independently or in addition to pnr to maintain expression of Doc. This could be investigated by expressing ara, caup and/or mirr in the mesoderm of pnr mutants to determine whether these factors are able to restore Doc expression. Alternatively, it may be that Iro-C is required indirectly meaning that its main function is to provide a molecular context in which Pnr can be active. For example, it is known that Ush can bind to Pnr thereby inactivating Pnr function. It is conceivable that the absence of Iro-C affects the spatial expression of Ush. If, in the absence of Iro-C, the expression domain of Ush shifts into the Pnr expression domain, Ush could bind to Pnr and inactivate it in the region where Pnr is required to maintain the expression of Tup and Doc. Adding to the complexity of the interpretation of the observed phenotypes is the finding that the majority of embryos that are mutant for ara/caup or for mirr were characterized by supernumerary Tin-positive cells in the cardiac region by stage 11/12. This phenotype could still be observed at later stages when the heart tube forms. The additional Tin-positive cells are pericardial cells as determined by the expression of Prc around the Tin-expressing cells. Also, no increase was observed of Dmef2-positive myocardial cells. Hence, the data suggests a different level of regulation of Tin by the Iro-C. Similar to the findings of Johnson (2011), it may be that Iro-C is normally required to restrict Tin expression at an early stage. The regulation of Tin expression can be divided into four phases. The phenotype this study observed occurs when Tin expression becomes restricted to the myo- and pericardial cells in the cardiac region. In summary, the data adds Iro-C to tin, pnr, Doc and tup whose concerted actions establish the cardiac domains in the dorsal mesoderm. Further studies are required to re-evaluate the current understanding of the interactions between factors of the cardiac transcriptional network (Mirzoyan, 2013).

According to the expression pattern of Ara/Caup and Mirr it was possible to distinguish between an early and late role for these factors, the latter being a role in the differentiation of heart cells (Mirzoyan, 2013).

This analyses of the expression of Ara/Caup and Mirr during embryogenesis led to the identification of hitherto unknown heart-associated cells. Seven pairs of Ara/Caup and Mirr expressing cells and seven pairs of Mirr only expressing cells were detected that were located along the dorsal vessel. No co-expression was detected with any of the known pericardial cell markers. Because there are seven pairs of these cells segmentally arranged, it was tempting to speculate that these cells may function, for example, as additional attachment sites for the seven pairs of alary muscles. The alary muscles attach the heart to the dorsal epidermis and their extensions can be visualized by Prc. Due to the lack of markers little is known about the development of the alary muscles. Previous work demonstrated that the alary muscles attach to the dorsal vessel in the vicinity of the Svp pericardial cells and, in addition, more laterally to one of two distinct locations on the body wall. Maybe it is the Mirr-positive cells that identify the more lateral locations. Clearly, a detailed analysis is needed to identify the function of the Ara/Caup- and Mirr- as well as Mirr-expressing cells that are positioned along the heart tube and whose existence has now been revealed. Additionally, on each side at the anterior end of the dorsal vessel four pericardial cells were identified that co-express Ara/Caup and Eve. Their location at the anterior tip of the heart is intriguing. Further analysis is required to unambiguously determine whether these cells are, for example, the wing heart progenitor cells or the newly identified heart anchoring cells. It is also possible that they represent a yet undefined, novel subpopulation of pericardial cells. In any case, this finding suggests that Ara/Caup plays a role in the diversification of pericardial heart cell types. Future experiments aim to determine the developmental fate of these cells (Mirzoyan, 2013).

Taken together, this investigation of a role for Iro-C in heart development introduces ara/caup and mirr as additional components of the transcriptional network that acts in the dorsal mesoderm and as novel factors that function in the diversification of heart cell types (Mirzoyan, 2013).

The results show that the role of the Iro-C and its individual members, respectively, appears to be rather complex and awaits in-depth analyses. Nevertheless, this work raises important questions regarding the current understanding of interactions between the well-characterized transcription factors that will be addressed in future studies (Mirzoyan, 2013).

Protein Interactions

The genes pannier and u-shaped (ush) are required for the regulation of achaete-scute during establishment of the bristle pattern in Drosophila. pnr encodes a protein belonging to the GATA family of transcription factors, whereas ush encodes a novel zinc finger protein. Genetic interactions between dominant pnr mutants bearing lesions situated in the amino-terminal zinc finger of the GATA domain and ush mutants have been described. The number of ectopic bristles in pannierD/+ flies increases in flies bearing only a single copy of u-shaped+ but decreases when three copies are present. Activation of a chicken alpha-globin promoter sequence by Pannier in cultured cells is inhibited by Ush. When both Ush and wild-type Pnr are expressed simultaneously, activation is abolished. Stimulation by Pnr is lost progressively in a concentration-dependent manner. Similarly, activation by chicken GATA-1 is also lost after cotransfection with the Ush expression vector. Because Pnr and GATA-1 have no homology outside their GATA DNA-binding domain, and since Ush alone has no effect on globin promoter activity, these observations suggest that the function of Ush is mediated through the GATA DNA-binding domain (Haenlin, 1997).

The GATA factor Pannier activates the achaete-scute (ASC) proneural complex through enhancer binding and provides positional information for sensory bristle patterning in Drosophila. Chip acts as a cofactor of the dorsal selector Apterous, and both Apterous and Chip also regulate ASC expression. Chip cooperates with Pannier in bridging the GATA factor with the HLH Ac/Sc and Daughterless proteins to allow enhancer-promoter interactions, leading to activation of the proneural genes, whereas Apterous antagonizes Pannier function. Within the Pannier domain of expression, Pannier and Apterous may compete for binding to their common Chip cofactor, and the accurate stoichiometry between these three proteins is essential for both proneural prepattern and compartmentalization of the thorax (Ramain, 2000).

Pnr is a member of the GATA-1 family of transcription factors and activates proneural function by binding to the dorsocentral (DC) enhancer located 4 kb and 30 kb upstream of ac and sc, respectively. Reported in this study is the characterization of Chip^E, a viable allele of Chip, that interacts with pnr genetically. Chip^E mutants show reduced ac-sc expression in the DC, associated with loss of DC bristles, and produce a phenotype similar to that of loss-of-function pnr alleles. This genetic interaction correlates with a physical interaction between Chip, Pnr, and the bHLH heterodimers (Ac/Sc-Da). Pnr interacts with the N terminus of Chip through its COOH terminus encompassing two helices that are conserved between D. melanogaster and D. virilis and that are probably both involved in protein-protein interactions. Chip dimerizes with the bHLH heterodimers through its C-terminal LID, known to mediate heterodimerization with LIM-containing proteins (Ramain, 2000).

In vertebrates, the Ldb1/NLI protein associates with GATA-1, Lmo2, and the bHLH E47, Tal1/SCL in an erythroid complex whose function is poorly understood. A similar Drosophila complex functions in vivo to regulate the ac/sc genes directly during establishment of the proneural prepattern.

Accurate coexpression of ac/sc is mediated by Pnr binding to the DC. The ac and sc promoters include E boxes that are targets for the Ac/Sc-Da heterodimers and support autoregulation during development. In cultured chicken embryonic fibroblast (CEF) cells, Pnr and the Ac/Sc-Da heterodimer activate expression of a CAT reporter linked respectively to the DC enhancer and to the ac promoter. Physical interactions between Pnr and the bHLHs lead to synergistic activation of the reporter when the regulatory sequences are associated. Pnr and the Ac/Sc-Da heterodimers are both required in flies for expression of a LacZ reporter linked to the promoter associated with the enhancer, but the analysis of Chip^E shows that Chip is also required for full activation in vivo. The interactions between Pnr and the bHLH mediated by Chip suggest that Pnr might also be involved in autoregulation. Interestingly, Chip interacts with Ac/Sc through the Ac/Sc bHLH domains, and it has been shown that the overexpression of a homologous bHLH domain is sufficient to mediate the proneural function of Ac/Sc (Ramain, 2000).

Chip has been identified in a genetic screen for mutations that reduce activity of the wing margin enhancer of the cut locus, and it has been proposed that Chip may act as a bridge allowing enhancer-promoter communications. Thus, if the flies have a unique functional Chip allele, they display a cut margin phenotype, and this effect is observed exclusively when they carry a gypsy insertion between the enhancer and the promoter on one chromosome. It has been proposed that binding of the Su(Hw) insulator protein to the gypsy insertion blocks communication on the mutant chromosome, thereby interfering with the functioning of the wild-type homolog. The interchromosomal insulation is detectable when Chip activity is reduced, and Chip and Su(Hw) are antagonistic to each other, suggesting that Chip may be a facilitator target of Su(Hw) (Ramain, 2000).

The Chip^E mutation specifically disrupts interactions with the bHLH and strongly affects the expression of a LacZ reporter linked to the ac promoter/DC enhancer in flies, suggesting that Chip also mediates enhancer-promoter communication in the ASC. Further evidence is provided by the Hw¹ mutant. Hw¹ carries a gypsy insertion within ac such that sc, which is located further downstream from the DC enhancer, is no longer expressed. In addition, the removal of the gypsy insulator largely restores sc expression in the DC proneural cluster (Ramain, 2000).

Thus, a complex containing Pnr, Chip, and the Ac/Sc-Da heterodimer activates ac-sc expression, and its function is antagonized by Ush, Ap, and Emc. Ush and Emc dimerize respectively with Pnr and the HLH Ac/Sc. The repressing effect of Ap may reflect its ability to interact with Chip, thereby depriving Pnr of its essential cofactor. Alternatively, Ap may weaken the enhancer activity of Pnr. Thus, Ap may compete directly with Chip for binding to Pnr (Ramain, 2000).

Within the domain of Pnr expression, Ap and Pnr compete for binding to their common Chip cofactor. Ap activity is mediated by a Chip dimer, whereas activation of ac-sc by Pnr requires a Chip monomer. Chip acts as a bridge between the Ac-Da heterodimer bound to the E boxes of the ac promoter and Pnr bound to the GATA sites of the DC enhancer. The activity of the resulting complex is antagonized by Ush and Emc, which negatively regulate Pnr and Ac/Sc functions during development. The repressing effect of Ap is mediated either by dimerization of Ap with Chip and/or Pnr or by Chip-assisted binding of Ap to sites located between the DC enhancer and the ac promoter. Additional cofactors, such as dLMO, may participate in this complex (Ramain, 2000).

Chip is required in flies for ASC activation, whereas it appears dispensable in CEF cells. This observation may reflect the nature of the reporter used in the transfection experiments where the DC enhancer is close to the ac promoter and poorly mimics the genomic organization of the ASC, where the DC enhancer has to regulate ac and sc simultaneously. Furthermore, the chromatin structure and its modifications associated with gene expression are probably not reproduced in the transient expression assay. Thus, ASC expression in flies may require additional coactivators recruited by Chip, including chromatin remodeling factors. Moreover, the activation of ac/sc probably requires the assembly of a higher-order nucleoprotein complex containing multiple transcription factors (enhanceosome), and Chip may allow the correct positioning of Pnr and the Ac/Sc-Da heterodimer in this structure (Ramain, 2000).

Chip^E mutants affect the scutellar and dorsocentral bristles in opposite fashions. It will be of interest to compare the regulation of the activity of the corresponding enhancers by Chip and Pnr (Ramain, 2000).

It has been proposed that appropriate combinations of proteins represent the positional cues that activate a given enhancer of the ASC complex. The disc is divided in large territories, but almost nothing is known concerning how these territories are further subdivided or how the positional information revealed by the accurate ac-sc expression is created. The present study provides a link between the spatial regulation of the proneural genes and the compartmentalization of the disc. ac-sc expression is stimulated by a complex containing the prepattern factor Pnr, Chip, and the bHLH proteins Ac/Sc and Da. Chip is an essential cofactor of the dorsal selector Ap, and these interactions coordinate the spatial transcription of the proneural genes. Ap is expressed specifically in the dorsal compartment of the wing pouch, and the juxtaposition of Ap-expressing with Ap-nonexpressing cells defines the dorsal/ventral organizing boundary where wingless (wg) expression is induced. On the thorax, ap and Chip are ubiquitously expressed, whereas wg expression occurs in a stripe straddling the lateral border of the domain of pnr expression. Moreover, Pnr activates wg. Pnr associates with Chip, and the domain of pnr expression appears devoid of Ap activity. As a consequence, this domain may define a boundary between a region devoid of Ap activity and a region where Ap is active. Alternatively, Pnr may associate with Ap, and the resulting heterodimer may regulate wg. Further studies will help to resolve this issue (Ramain, 2000).

The coexpression of Pnr and Tinman in the embryo results in a synergistic activation of cardiac gene expression and the ectopic induction of heart-like cells. To investigate a possible physical interaction of the two proteins, mammalian CV-1 cells were cotransfected with Pnr and Tin expression vectors and the cellular distribution of the factors was monitored by confocal microscopy and coimmunoprecipitation analysis. Cells expressing GFP-tagged Tin and Myc-tagged Pnr exhibit colocalization of the proteins in the nucleus. Furthermore, analysis of nuclear extracts by immunoprecipitation followed by Western blot reveals that Tin is coimmunoprecipitated with Pnr. Conversely, Pnr is also detected in the Tin immunoprecipitate. These results indicate that the two proteins form a physical complex in the cultured cells, consistent with their ability to work combinatorially in the regulation of cardiac gene expression (Gajewski, 2001).

In support of this in vivo data, regions of Tin that are able to bind to the Pnr protein were delineated. Eight GST-Tin fusion proteins containing all or part of the cardiogenic factor were used in in vitro pull-down assays with 35S-labeled full-length Pnr. The strongest interactions were observed with either wild-type Tin or truncated versions that contained the homeodomain region (Tin A1, A2, A23, and A31). Additionally, weaker but discernible binding of Tin and Pnr was observed with truncated proteins that contained the functionally defined 111 to 151 region (Tin A4, A5, and A34). These molecular results are consistent with the requirement of this internal domain for the functional synergism of Tin with Pnr in the activation of the D-mef2 cardiac enhancer in the CNS (Gajewski, 2001).

Enhancer-promoter communication mediated by Chip during Pannier-driven proneural patterning is regulated by Osa

The GATA factor Pannier activates proneural achaete/scute (ac/sc) expression during development of the sensory organs of Drosophila through enhancer binding. Chip bridges Pannier with the (Ac/Sc)-Daughterless heterodimers bound to the promoter and facilitates the enhancer-promoter communication required for proneural development. This communication is regulated by Osa, which is recruited by Pannier and Chip. Osa belongs to Brahma chromatin remodeling complexes, and this study shows that Osa negatively regulates ac/sc. Consequently, Pannier and Chip also play an essential role during repression of proneural gene expression. This study suggests that altering chromatin structure is essential for regulation of enhancer-promoter communication (Heitzler, 2003).

Chip^E is a viable allele of Chip that is associated with a point mutation in the LIM-interacting domain (LID), which specifically reduces interaction with the bHLH proteins Ac, Sc, and Da. As a consequence, the Chip^E mutation disrupts the functioning of the proneural complex encompassing Chip, Pnr, Ac/Sc, and Da. A homozygous Chip^E mutant shows thoracic cleft and loss of the DC bristles, similar to loss of function pnr alleles (Heitzler, 2003).

To identify new factors that regulate this proneural complex, a screen was performed for second-site modifiers of the Chip^E phenotypes. One allele of osa (osa^E17) was found among the putative mutants. Osa^E17 corresponds to a loss-of-function allele, and homozygous embryos die with normal cuticle patterning. Both osa^E17 and null alleles of osa (osa⁶¹⁶ or osa¹⁴⁰⁶⁰) enhance the cleft but suppress the loss of DC bristle phenotypes of Chip^E flies. Indeed, Chip^E flies with only one copy of osa⁺ (Chip^E;osa⁶¹⁶/+) are weak and sterile but show wild-type DC bristle pattern (Heitzler, 2003).

These genetic interactions suggest that Osa can antagonize the function of Pnr. Moreover, overexpressed Osa (+/UAS-osa;Gal4-pnr^MD237/+) induces a thoracic cleft and the loss of DC bristles similar to the loss-of-function pnr alleles. In contrast, loss-of-function osa alleles display an excess of DC bristles similar to overexpressed Pnr. For example, (osa¹⁴⁰⁶⁰/+), (osa⁶¹⁶/+), and (osa^E17/+) flies exhibit respectively 2.35 ± 0.12, 2.38 ± 0.12, and 2.43 ± 0.17 DC bristles per heminotum (Oregon wild-type flies have 2.00 DC bristles/heminotum). Furthermore, transallelic combination of osa¹⁴⁰⁶⁰ with the hypomorphic osa^4H (osa^4H/osa¹⁴⁰⁶⁰) accentuates the excess of DC bristles compared with (osa¹⁴⁰⁶⁰/+). (osa^4H/osa¹⁴⁰⁶⁰) flies display 4.17 ± 0.19 DC bristles per heminotum. In contrast, (osa^4H/osa^4H) flies display 2.50 ± 0.11 DC bristles per hemithorax. The development of the extra DC bristles revealed by phenotypic analysis was compared with the positions of the DC bristle precursors detected with a LacZ insert, A101, in the neuralized gene that exhibits staining in all sensory organs. In (osa¹⁴⁰⁶⁰/osa^4H) discs, additional DC precursors are observed that lead to the excess of DC bristles. The pnr^D alleles encode Pnr proteins carrying a single amino acid substitution in the DNA binding domain that disrupts interaction with the U-shaped (Ush) antagonist. Consequently, Pnr^D constitutively activates ac/sc, leading to an excess of DC bristles. This excess is accentuated when osa function is simultaneously reduced (pnr^D1/osa⁶¹⁶) (Heitzler, 2003).

Since osa shows genetic interactions with trithorax group genes encoding components of the Brm complex like moira (mor) and brm, whether mutations in mor and brm suppress the Chip^E phenotype was investigated. Loss of one copy of brm⁺ in (Chip^E; brm²/+) flies suppresses the lack of DC bristles observed in Chip^E flies, similar to loss of one copy of osa⁺. This shows that brm and osa both act during Pnr-dependent patterning, in agreement with the fact that they have been shown to be associated in the Brm complex. In contrast, reducing the amount of Mor by half [(Chip^E;mor¹/+) flies] is not sufficient to modify the Chip^E phenotype. This does not definitely exclude the possibility that mor is directly or indirectly involved, via the Brm complex, in Pnr-dependent patterning (Heitzler, 2003).

The complete osa open reading frame of 2715 amino acids and the intronic splicing signals were PCR amplified from genomic DNA prepared from homozygous embryos (osa^E17 and osa¹⁴⁰⁶⁰) and homozygous first instar larvae (osa^4H). For osa¹⁴⁰⁶⁰ and osa^4H, the sequence analysis revealed a single mutation in the N terminus that causes a glutamine to stop codon substitution. The conceptual translation of osa¹⁴⁰⁶⁰ leads to a truncated Osa protein lacking both functional domains, whereas Osa^4H retains the ARID domain but lacks the C-terminal EHD. Wild-type osa function is necessary for patterning of the DC bristles. Although osa^E17 behaves as a stronger allele than osa¹⁴⁰⁶⁰ and osa^4H, molecular identity of the mutation is unknown. Hence, the osa^E17 phenotype may result from a mutation in regulatory sequences that affects osa expression (Heitzler, 2003).

It has been shown that a complex containing Pnr, Chip, and the (Ac/Sc)-Da heterodimer activates proneural expression in the DC proneural cluster and promotes development of the DC macrochaetae. Osa and Pnr/Chip have antagonistic activities during development because loss of osa function (osa^4H and osa¹⁴⁰⁶⁰) displays additional DC bristles. However, since the current study reveals that osa genetically interacts with pnr and Chip, it was asked whether Osa physically interacts with the Pnr and Chip proteins. Immunoprecipitations of protein extracts made from Cos cells cotransfected with expression vectors for tagged Osa and either Pnr or tagged Chip were immunoprecipitated. Because Osa is a large protein, several expression vectors encoding contiguous domains of Osa were used. Osa coimmunoprecipitates with Pnr and Chip and can be detected on Western blots with appropriate antibodies. The interactions appear to require the overlapping domains Osa E (His1733/Glu2550) and Osa F (Ala2339/Ala2715) corresponding to the EHD. Enhancer-promoter communication during proneural activation and development of the DC bristles requires regulatory sequences scattered over large distances and appears to be negatively regulated by interaction of Pnr and Chip with Osa through the EHD. Interestingly, the EHD is not conserved in yeast. In yeast, the UAS sequences are generally close to the promoter and there is no requirement for long-distance interactions. This observation could support the idea that the EHD is essential for long-distance enhancer-promoter communication. Alternatively, yeast may just lack proteins like Chip or Pnr (Heitzler, 2003).

The DNA-binding domain and the C-terminal region are essential for the function of Pnr during development of the DC sensory organs. The pnr^VX1 and pnr^VX4 alleles (collectively pnr^VX1/4) are characterized by frameshift deletions that remove two C-terminal alpha-helices and result in reduced proneural expression and loss of DC bristles (Heitzler, 2003).

The molecular interactions between Osa and Pnr^D1 and between Osa and Pnr^VX1 were investigated. Pnr^D1 protein interacts with the EHD as efficiently as wild-type Pnr. In contrast, the physical interaction is disrupted when the C terminus of Pnr encompassing the alpha-helices is removed. Because the C terminus of Pnr is required for the Pnr-Osa interaction in transfected cells extracts, the abilities of in vitro translated 35S-labeled Osa domains to bind to GST-C^TPnr attached to glutathione-bearing beads were investigated. Only Osa E and Osa F interact with the C terminus of Pnr. The interaction between Chip and Osa and it was found that Osa associates with the N-terminal homodimerization domain of Chip, also required for the interaction between Chip and Pnr, was investigated. Furthermore, Osa E and Osa F also bind to immobilized GST-Chip. Deletion of the alpha helix H1 disrupts the interactions between Pnr and Osa. Interestingly, the same deletion also disrupts the interaction with Chip. Therefore, the functional antagonism between Chip and Osa during neural development may result from a competition between these proteins for association with Pnr. Alternatively, the deletion of H1 may affect the overall structure of the C terminus of Pnr and disrupt the physical interactions with Chip and Osa. To discriminate between these hypotheses, immunoprecipitations of protein extracts containing a constant amount of Pnr, a constant amount of the tagged Osa E domain, and increasing concentrations of Chip were performed. Pnr immunoprecipitates with immunoprecipitated tagged Osa E and the amount of Pnr immunoprecipitated increases in the presence of increasing concentrations of Chip. The presence of increasing amounts of Chip does not inhibit the Osa-Pnr interaction as would be expected if Osa and Chip were to compete for binding to Pnr. In contrast, it suggests that Chip and Pnr act together to recruit Osa and to target its activity and possibly the activity of the Brm complex to the ac/sc promoter sequences (Heitzler, 2003).

Using expression vectors encoding contiguous domains of Osa, it was shown that the EHD of Osa mediates interactions with Pnr and Chip. Because the EHD is lacking in the truncated Osa¹⁴⁰⁶⁰ and Osa^4H, it is hypothesized that the loss of interaction with Pnr and Chip are responsible for the excess of DC bristles observed in osa^4H and osa¹⁴⁰⁶⁰ (Heitzler, 2003).

To investigate whether these interactions between Osa, Pnr, and Chip function in vivo during DC bristle development, the effects of both loss of function and overexpression of osa were examined on the activity of a LacZ reporter whose expression is driven by a minimal promoter sequence of ac fused to the DC enhancer (transgenic line DC:ac-LacZ). It was found that expression of the LacZ transgene is increased in osa¹⁴⁰⁶⁰/osa^4H wing discs when compared with the wild-type control. For overexpression experiments, the UAS/GAL4 system was used, using as a driver the pnr^MD237 strain that carries a GAL4-containing transposon inserted in the pnr locus (driver: pnr-Gal4). This insert gives an expression pattern of Gal4 indistinguishable from that of pnr. It was found that overexpressed Osa leads to a strong reduction of LacZ staining in the DC area, consistent with the lack of DC bristles. Thus, overexpressed Osa represses activity of the ac promoter sequences required for DC ac/sc expression and development of the DC bristles. It has been previously reported that wingless expression is also required for patterning of the DC bristles. However, the repressing effect of Osa on development of the DC bristles is unlikely to be the result of an effect of Osa on wingless expression because overexpressed Osa driven by pnr^MD237 has no effect on the expression of a LacZ reporter inserted into the wingless locus. Thus, Osa acts through the DC enhancer of the ac/sc promoter sequences to repress ac/sc and neural development (Heitzler, 2003).

Chip^E disrupts the enhancer-promoter communication and strongly affects expression of the LacZ reporter driven by the ac promoter linked to the DC enhancer. Because null alleles of osa suppress the loss of DC bristles displayed by Chip^E, the consequences of reducing the dosage of osa was examined in Chip^E flies. The expression of the LacZ reporter is not affected in Chip^E flies when Osa concentration is simultaneously reduced (Heitzler, 2003).

In conclusion, Pnr function during proneural patterning is regulated by interaction with several transcription factors. Pnr function is negatively regulated by Ush, which interacts with its DNA-binding domain. Chip associates with the C terminus of Pnr, bridging Pnr at the DC enhancer with the AC/Sc-Da heterodimers bound at the proneural promoters, thus activating proneural gene expression. The current study reveals that Pnr function can also be regulated by interaction with Osa. Thus, Osa activity is specifically targeted to ac/sc promoter sequences and the binding of Osa therefore has a negative effect on Pnr function, leading to reduced expression of the proneural ac/sc genes. Osa belongs to Brm complexes, which are believed to play an essential role during chromatin remodeling necessary for gene expression. For example, in vitro transcription experiments with nucleosome assembled human beta-globin promoters have shown that the BRG1 and BAF155 subunits of the mammalian SWI/SNF homolog are essential to target chromatin remodeling and promote transcription initiation mediated by GATA-1. In contrast to what was observed in vitro, the current results suggest that in vivo the SWI/SNF complexes can also act to remodel chromatin in a way that represses transcription. Alternatively, the observed repression of proneural genes may simply define a novel function of Osa, independent of chromatin remodeling (Heitzler, 2003).

The Drosophila LIM-homeodomain protein Islet antagonizes proneural cell specification in the peripheral nervous system: Isl antagonizes Pnr activity both by dimerization with the DNA-binding domain of Pnr and via competitive inhibition of the Chip-bHLH interaction

The pattern of the external sensory organs (SO) in Drosophila depends on the activity of the basic helix-loop-helix (bHLH) transcriptional activators Achaete/Scute (Ac/Sc) that are expressed in clusters of cells (proneural clusters) and provide the cells with the potential to develop a neural fate. In the mesothorax, the GATA1 transcription factor Pannier (Pnr), together with its cofactor Chip, activates ac/sc genes directly through binding to the dorsocentral enhancer (DC) of ac/sc. The LIM-homeodomain (LIM-HD) transcription factor Islet (Isl) was identified by genetic screening and its role in the thoracic prepatterning was investigated. isl loss-of-function mutations result in expanded Ac expression in DC and scutellar (SC) proneural clusters and formation of ectopic sensory organs. Overexpression of Isl decreases proneural expression and suppresses bristle development. Moreover, Isl is coexpressed with Pnr in the posterior region of the mesothorax. In the DC proneural cluster, Isl antagonizes Pnr activity both by dimerization with the DNA-binding domain of Pnr and via competitive inhibition of the Chip-bHLH interaction. It is proposed that sensory organ prepatterning relies on the antagonistic activity of individual Chip-binding factors. The differential affinities of these binding-factors and their precise stoichiometry are crucial in specifying prepatterns within the different proneural clusters (Biryukova, 2005).

During Drosophila development, the expression of transcription factors divides the dorsal thorax into three domains -- one median and two lateral domains. The lateral domains are specified by the homeobox-containing proteins of the iroquois-complex (iro), whereas the GATA factor Pnr is required to establish the median domain. Within the mesothorax, Pnr together with U-shaped (Ush) and Chip plays a key role in dorsal closure. This report presents evidence that Isl is an essential regulator of the dorso-median patterning of the thorax. isl⁻ clones generated adjacent to the thoracic midline, induce a strong cleft, suggesting that Isl is required for proper dorsal closure during metamorphosis. Ectopic expression of Pnr leads to wing-to-thorax transformations, consistent with its role as medio-dorsal patterning factor. Ectopic Isl expression does not exhibit this phenotype, excluding the LIM-HD factor from a direct function as a prothoracic selector. Pnr is also known to activate wingless (wg) in dorsal thorax. isl loss-of-function has no significant effect on wg expression. However, overexpressed Isl strongly reduces the size of the wg thoracic stripe. This result is consistent with a repressive activity of Isl on Pnr (Biryukova, 2005).

Iro proteins and Pnr are direct activators of the proneural genes in their respective domains. Pnr binds directly to the DC enhancer of ac/sc, providing therefore region-specific control of the proneural prepatterning. Flies with reduced or lack of Pnr function fail to activate ac/sc and to develop DC and SC sensory organs. The proneural activity of Pnr is antagonized by Ush, the vertebrate homologue of the FOG (friend of GATA). Ush is expressed only in the dorsal-most cells of the medial region. As a consequence, the segregation of the sensory organ precursors occurs along two stripes at the border of the medial domain of Pnr expression, where Ush is absent or insufficient to repress Pnr (Biryukova, 2005).

Several lines of evidence indicate that Isl interferes with the proneural activity of Pnr as a repressor. (1) isl loss-of-function mutants show an opposite phenotype with regard to Pnr or Chip loss-of-function mutants: an excess of DC and SC sensory organs. (2) A genetic synergism exists between Pnr^D and isl⁻ alleles. This genetic interaction is less sensitive than that between Pnr^D and ush⁻, implying an alternative route for Isl to modulate the Pnr proneural activity. (3) Isl is coexpressed with Pnr within the posterior mesothorax. (4) Isl modulates the activity of a DC:ac-lacZ reporter. Loss-of-function isl mutants expand the DC:ac-lacZ expression as in ush⁻ or Pnr^D constitutive mutants, whereas overexpressed Isl reduces the DC:ac-lacZ expression (Biryukova, 2005).

In the DC region, the regulation of Pnr concentration is critical for the proper position and shape of the DC proneural cluster. Isl expression overlaps with the dorsal-most domain of Pnr and DC proneural activity coincides with the posterior border of Isl expression. Therefore, it proposed that both Isl and Ush restrict Pnr activity in the mesothorax. Interestingly, the regulation of the concentration of the mammalian Pnr ortholog, GATA-1, is similarly critical for proper erythroid, megakaryocytic, eosinophilic and mast cell lineages (Biryukova, 2005).

Ush behaves as either an activator or a repressor of Pnr, depending on developmental context. No evidence was found for a direct Isl-Ush interaction by GST pull down assay: Ush, Pnr and Isl could be co-immunoprecipitated from transient transfected S2 cells. Both Ush and Isl may behave as positive cofactors of Pnr for nonneural activities, such as cardiac development, embryonic dorsal closure and metamorphosis. Several reports emphasize the role of the Pnr homolog, GATA-1 and Isl1 in human blood disorders. It seems likely that GATA:Islet interactions represent a conserved mechanism to specify different cell fates in humans and other organisms (Biryukova, 2005).

Isl proteins are known as positive regulators of transcription in vertebrates. In flies, Isl mediates repression of Pnr-driven proneural activity via binding to the DNA-binding domain of Pnr. Interestingly, these interactions are less specific than for the Pnr-Ush interaction, where the amino-terminal zinc finger of Pnr is specifically involved (Biryukova, 2005).

Genetic analyses of mutants reveal that the DC and the SC proneural clusters show differential sensitivities during neurogenesis. Ush mutants display ectopic DC bristles and a few additional SC bristles. This phenotype is similar to Pnr^D constitutive mutants, in which Pnr-Ush interactions are greatly reduced. In contrast, isl mutants show the opposite phenotype, with a large excess of SC bristles and a few additional DC bristles. The Chip^E mutant exhibits antagonistic phenotypes: lack of DC bristles, reflecting Pnr loss-of-function and an excess of SC bristles, reflecting Isl loss-of-function. The differential topography of DC and SC enhancer binding sites presumably underlies differential transcription-complex binding affinities (Biryukova, 2005).

Chip is the ortholog of Ldb factors that are ubiquitous multiadaptor proteins in vertebrates. Each Ldb-dependent developmental event is specified by modification of the transcriptional complex and is dependent on the stoichiometry of the region-specific Ldb partners. During normal development of the thorax, different partners of Chip (i.e., Isl, Ap and Pnr) are expressed in the same region. The Chip^E mutant is highly sensitive to the dosage of these factors. In Chip^E flies, removing one copy of either Pnr or Isl causes pupal lethality associated with extreme morphogenetic phenotypes. Removing one copy of Ap, however, rescues the Pnr-dependent phenotypes of Chip^E flies. Taken together, these results indicate selective competition between the different partners of Chip, suggesting that hierarchical protein interactions depending on differential affinities and the strict stoichiometry of Chip and its partners, are critical to establish proper transcriptional codes within different proneural fields (Biryukova, 2005).

isl mutants were isolated in genetic screens for dominant enhancers of the Chip^E phenotype. This study demonstrates that the LIM-HD transcription factor Isl can bind to the LID of Chip. The binding of the LID domain of Chip with LIM domains has been conserved throughout evolution as has Chip binding with bHLHs proteins. LID contains two subdomains: a small N-terminal hydrophobic β patch (VMVV) followed by a large α helix. Chip^E mutation has a single substitution that changes an Arg to Trp (R504W) in the middle of the α helix. This residue is highly conserved among species and mediates high-affinity contact with the LIM domains. Interestingly, the R504W substitution in Chip abolishes, or strongly reduces, both interactions with the bHLHs and also interactions with Isl. This result implies that bHLHs and Isl recognize the same site within the LID domain of Chip. The data argue that competition between bHLHs and Isl for the LID domain of Chip may be critical for modulating the activity of transcription complexes during development. In vertebrates, the NLI homolog of Chip mediates direct coupling of the proneural bHLH factors Ngn2, NeuroM and the LIM-HD transcription factors (Isl1 and Lhx3). This interaction leads to transcriptional synergism and the synchronization of motor neuron subtype specification with neurogenesis in the embryonic spinal cord of chicken. This work demonstrates that Isl is able to interfere with proneural activity of Chip-Pnr-bHLH transcription complex and therefore, Isl is thought to be able to antagonize proneural specification (Biryukova, 2005).

Interestingly, the Chip^E mutation has little or no effect on interactions with other LIM-containing factors, such as Ap and dLMO, suggesting that different factors have different affinities with the Chip LID domain. Therefore, the Chip^E mutation changes the hierarchy of the affinities among the different partners of Chip in the mesothorax (Biryukova, 2005).

A transcription-complex 'cassette' model is proposed for the specification of region-specific patterns of specialized cell types. In this model, the presence of one of a number of alternative binding factors modifies the specificity of a core transcription complex. This model makes the prediction that, while the core components of the transcription complex will be strongly conserved in evolution, the specificity cassette components will vary significantly between species showing divergent morphogenetic patterns. Comparison of these variable components in related species should provide insights into the fundamental mechanisms of encoding the pattern of differentiated cell types within morphogenetic fields (Biryukova, 2005).

Toutatis, a TIP5-related protein, positively regulates Pannier function during Drosophila neural development

The GATA factor Pannier (Pnr) activates proneural expression through binding to a remote enhancer of the achaete-scute (ac-sc) complex. Chip associates both with Pnr and with the (Ac-Sc)-Daughterless heterodimer bound to the ac-sc promoters to give a proneural complex that facilitates enhancer-promoter communication during development. Using a yeast two-hybrid screening, Toutatis (Tou; see Teutates the supposed deified spirit of male tribal unity in ancient Celtic polytheism, best known under the name Toutatis, through the Gaulish catchphrase "By Toutatis!", invented for the Asterix comics by Goscinni and Uderzo), which physically interacts with both Pnr and Chip, was identified. Loss-of-function and gain-of-function experiments indicate that Tou cooperates with Pnr and Chip during neural development. Tou shares functional domains with chromatin remodelling proteins, including TIP5 (termination factor TTFI-interacting protein 5) of NoRC (nucleolar remodelling complex), which mediates repression of RNA polymerase 1 transcription. In contrast, Tou acts positively to activate proneural gene expression. Moreover, Iswi associates with Tou, Pnr and Chip, and is also required during Pnr-driven neural development. The results suggest that Tou and Iswi may belong to a complex that directly regulates the activity of Pnr and Chip during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).

Transcriptional activation of many developmentally regulated genes is mediated by proteins binding to enhancers scattered over the genome, raising the question on how long-range activation is restricted to the relevant target promoter. Numerous studies have highlighted the essential role of boundaries, which maintain domains independent of their surrounding (Vanolst, 2005).

The patterning of the large sensory bristles (macrochaetae) on the thorax of Drosophila melanogaster is a powerful model to study how enhancers communicate with promoters during regulation of gene expression. Each macrochaeta derives from a precursor cell selected from a group of equivalent ac-sc-expressing cells, the proneural cluster. ac and sc encode basic helix-loop-helix proteins (bHLH) that heterodimerize with Daughterless (Da) to activate expression of downstream genes required for neural fate. Transcription of ac and sc in the different sites of the imaginal disc is initiated by enhancers of the ac-sc complex and the expression is maintained throughout development by autoregulation mediated by the (Ac-Sc)-Da heterodimers binding to E boxes within the ac-sc promoters. Each enhancer interacts with specific transcription factors that are expressed in broader domains than the proneural clusters and define the bristle prepattern. Thus, the GATA factor Pannier (Pnr) binds to the dorsocentral (DC) enhancer and activates proneural expression to promote development of DC sensory organs. The Drosophila LIM-domain-binding protein 1 (Ldb1), Chip physically interacts both with Pnr and the (Ac-Sc)-Da heterodimer to give a multiprotein proneural complex which facilitates the enhancer-promoter communication (Vanolst, 2005 and references therein).

Chromatin plays a crucial role in control of eukaryotic gene expression and is a highly dynamic structure at promoters. In Drosophila, the polycomb (Pc) group and the trithorax (Trx) group proteins are chromatin components that maintain stable states of gene expression and are involved in various complexes. The Pc group proteins are required to maintain repression of homeotic genes such as Ultrabithorax, presumably by inducing a repressive chromatin structure. Members of the Trx group were identified by their ability to suppress dominant Polycomb phenotypes. Evidence has been provided that enhancer-promoter communication during Pnr-driven proneural development is negatively regulated by the Brahma (Brm) chromatin remodelling complex, homologous to the yeast SWI/SNF complex (Vanolst, 2005).

Evidence is presented that Toutatis (Tou), a protein that associates both with Pnr and Chip and that positively regulates activity of the proneural complex encompassing Pnr and Chip during enhancer-promoter communication. Tou has been previously identified in a genetic screen for dominant modifiers of the extra-sex-combs phenotype displayed by mutant of polyhomeotic (ph), a member of the Pc group in Drosophila. Tou shares functional domains with Acf1, a subunit of both the human and Drosophila ACF (ATP-utilizing chromatin assembly and remodelling factor) and CHRAC (chromatin accessibility complex), and with TIP5 of NoRC (nucleolar remodelling complex). Hence, Tou regulates activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling. Moreover, Iswi, a highly conserved member of the SWI2/SNF2 family of ATPases, is also necessary for activation of ac-sc and neural development. Since Iswi is shown to physically interact with Tou, Pnr and Chip, it is suggested that a complex encompassing Tou and Iswi directly regulates activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).

In Drosophila, Chip has been postulated to be a facilitator required both for activity of the DC enhancer of the ac-sc complex. Enhancer-promoter communication at the ac-sc complex is negatively regulated by the Brm complex whose activity is targeted to the ac-sc promoter sequences through dimerization of the Osa subunit with both Pnr and Chip. The Brm complex is thought to remodel chromatin in a way that represses transcription (Vanolst, 2005).

Tou and Iswi appear to act together as subunits of a multiprotein complex to positively regulate activity of Pnr and Chip during enhancer-promoter communication. Tou and Iswi therefore display opposite activity to that of the Brm complex, raising questions about their molecular function during neural development. Tou shares essential functional domains with members of the WAL family of chromatin remodelling proteins, including Acf1 of ACF and CHRAC. Importantly, Acf1 and TIP5 associate in vivo with Iswi, showing that Iswi can mediate both activation and repression of gene expression. Tou positively regulates Pnr/Chip function during the period of ac-sc expression in neural development, and it associates with Iswi. Since Iswi also positively regulates Pnr/Chip function, it is hypothesized that a complex encompassing Tou and Iswi acts during long-range activation of proneural expression, possibly through chromatin remodelling. Further studies will help to resolve this issue (Vanolst, 2005).

Interestingly, Chip and Pnr seem to play similar roles both during recruitment of the Brm complex and recruitment of Tou and Iswi, since they dimerize with Osa, Tou and Iswi. In addition, Pnr and Chip apparently cooperate to strengthen the physical association with Osa and Tou. However, Osa, on the one hand, and Tou and Iswi, on the other, display antagonistic activities during neural development. Since they are ubiquitously expressed, accurate regulation of ac-sc expression would require a strict control of the stoichiometry between Osa, Tou and Iswi. It remains to be investigated whether the functional antagonism between Osa and Tou/Iswi relies on a molecular competition for association with Pnr and Chip. Determination of this would require a complete molecular definition of the putative complex encompassing Tou and Iswi, together with a full understanding of how this complex and the Brm complex molecularly interact with the proneural complex to regulate enhancer-promoter communication during development (Vanolst, 2005).

Biochemical analysis of Iswi and Iswi-containing complexes, together with genetic studies of Iswi and associated proteins in flies and in budding yeast, has revealed roles for Iswi in a wide variety of nuclear processes, including transcriptional regulation, chromosome organization and DNA replication. Accordingly, Iswi was found to be a subunit of various complexes, including NURF (nucleosome remodelling factor), ACF and CHRAC. Iswi-containing complexes were primarily recognized as factors that facilitate in vitro transcription from chromatin templates. However, genetic analysis in Drosophila and in Saccharomyces cerevisiae have provided evidence that Iswi-containing complexes are involved in both transcriptional activation and repression in vivo. For example, immunostaining of Drosophila polytene chromosomes of salivary glands showed that Iswi is associated with hundreds of euchromatic sites in a pattern that is non-overlapping with RNA polymerase II. It suggests that Iswi may play a general role in transcriptional repression. In contrast, it was also demonstrated that expression of engrailed and Ultrabithorax are severely compromised in Iswi-mutant Drosophila larvae. Recent studies have also shown that a mouse Iswi-containing complex, NoRC, plays an essential role during repression of transcription of the rDNA locus by RNA polymerase I. Tou, a protein that is structurally related to the TIP5 subunit of NoRC. Tou positively regulates enhancer-promoter communication during Pnr-driven proneural development and its activity is targeted to the ac-sc promoter sequences through dimerization with Pnr and Chip. Evidence is provided that Iswi is required during neural development. Overexpression of Iswi^K159R in the precursor cells of the sensory organs using the sca^Gal4 driver leads to flies lacking multiple bristles, suggesting that Iswi functions late during neural development, essential for either cell viability or division of the precursor cell. Using the Iswi¹/Iswi² transheterozygous combination and individuals overexpressing Iswi^K159R in earlier stages of development and in less restricted patterns, it has been shown that Iswi also regulates ac-sc expression. Interestingly, the regulation is probably direct since Iswi associates with the transcription factors Pnr and Chip, known to promote ac-sc expression at the DC site. Since Iswi interacts with Tou, it is proposed that Tou and Iswi may positively regulate activity of Pnr and Chip during enhancer-promoter communication, possibly as subunits of a multiprotein complex involved in chromatin remodelling (Vanolst, 2005).

Drosophila LIM-only is a positive regulator of transcription during thoracic bristle development

The Drosophila LIM-only (Lmo) protein DLMO functions as a negative regulator of transcription during development of the fly wing. This study reports a novel role of Dlmo as a positive regulator of transcription during the development of thoracic sensory bristles. New dlmo mutants, which lack some thoracic dorsocentral (DC) bristles, were isolated. This phenotype is typical of malfunction of a thoracic multiprotein transcription complex, composed of Chip, Pannier (Pnr), Achaete (Ac), and Daughterless (Da). Genetic interactions reveal that dlmo synergizes with pnr and ac to promote the development of thoracic DC bristles. Moreover, loss-of-function of dlmo reduces the expression of a reporter target gene of this complex in vivo. Using the GAL4-UAS system it was also shown that dlmo is spatially expressed where this complex is known to be active. Glutathione-S-transferase (GST)-pulldown assays showed that Dlmo can physically bind Chip and Pnr through either of the two LIM domains of Dlmo, suggesting that Dlmo might function as part of this transcription complex in vivo. It is proposed that Dlmo exerts its positive effect on DC bristle development by serving as a bridging molecule between components of the thoracic transcription complex (Zenvirt, 2008).

The results presented in this study uncover a novel role of Dlmo in regulation of the development of the thoracic DC bristles. Homozygous, or hemizygous, loss-of-function (dlmo^hdp) mutants lack the anterior pair of the DC bristles. Moreover, these dlmo mutants displayed genetic interactions with mutants in genes known to regulate DC bristle development, such as pnr and ac, to reduce the number of DC bristles. Consistently, overexpression alleles of dlmo (dlmo^Bx) also exhibited genetic interactions with these pnr and ac mutants, resulting in an increased number of bristles. In addition, the finding that overexpression of pnr under the regulation of dlmo-GAL4 affects DC bristle development suggests that dlmo is expressed in the region of the wing disc that gives rise to these bristles (Zenvirt, 2008).

These results suggest a role of Dlmo in positive regulation of transcription. The negative role of Dlmo in modulation of transcription during Drosophila wing development has been well documented. The findings indicate that in another context, namely in regulation of DC bristle development by the Chip, Pnr, Ac and Da (CPAD) complex, Dlmo has another role, as a positive regulator of transcription. Lowering the level of Dlmo (in dlmo^hdp mutants) results in a reduction in the expression of a reporter driven by regulatory sequences of a bona fide target gene of the CPAD transcription complex, suggesting that Dlmo is a positive regulator of CPAD-dependent transcription. While the mechanism by which Dlmo positively regulates transcription in the context of the CPAD complex remains to be elucidated, a first clue to this mechanism may lie in the finding that Dlmo can bind constituent proteins of this complex, including Pnr and Chip, in vitro. Should these interactions also take place in vivo, Dlmo may exert its positive role in transcriptional regulation as a component of the CPAD complex (Zenvirt, 2008).

Insights into the mechanism of positive transcriptional regulation by Dlmo can be gleaned from LMO2, one of the mammalian homologs of Dlmo. LMO2 was demonstrated to participate in a multiprotein transcription complex that contains Ldb1, a GATA factor (GATA-1 or GATA-2), and the bHLH transcription factors TAL1 and E2A, which are homologous, respectively, to the fly components of the CPAD complex, Chip, Pannier, Achaete, and Daughterless. Various lines of evidence indicate that in mammals LMO2 serves as a bridge between components of the complex, and silencing of LMO2 causes disruption of the complex and decreases in the activation of transcription of its target genes, just as does silencing of Ldb1 or Tal1. Similarly to LMO2, Dlmo might serve as a bridge between components of the CPAD complex. LIM domains are protein-interaction modules and could serve Dlmo to bind components of the CPAD complex. This suggestion is supported by the finding that each single LIM domain of Dlmo is capable of binding components of the CPAD complex in vitro, and it agrees with similar reports on other LIM-containing proteins. Notably, a single LIM domain from LMO2 and LMO4 is sufficient to interact with Ldb1 or the related protein CLIM-1a. However, both LIM domains are required for the highest-affinity interactions (Zenvirt, 2008).

This proposed mode of action of Dlmo, as a bridging molecule, which binds a different protein through each one of its LIM domains, predicts that a Dlmo molecule with one defective LIM domain and one intact LIM domain would bind only one protein at a time and not be able to bridge between molecules. Indeed, in the new dlmo mutants it was found that deletions that span the second zinc finger of the second LIM domain of Dlmo, namely dlmo^hdp48-1 and dlmo^hdp185-1, resulted in dlmo loss-of-function mutations. These mutants display partial loss of thoracic DC bristles along with reduced expression of a target gene of the thoracic transcription complex. Interestingly, the wing size of these mutants is normal, unlike the small wings of mutants with lesions in the 5'-UTR of Dlmo, such as dlmo^hdp58-1, dlmo^hdp67-2, and dlmo^hdpR590. This may suggest that the defective Dlmo protein, which has only a single intact LIM domain, is sufficient for its function in the context of the wing, where Dlmo acts as a negative regulator that binds only one protein (CHIP), but is not sufficient when Dlmo acts as a bridging molecule in the thoracic CPAD transcription complex. Finally, the finding that Dlmo can bind other Dlmo molecules to generate homodimers or multimers might provide Dlmo with a greater flexibility of bridging between distant components of the complex. This possibility remains to be examined (Zenvirt, 2008).

In conclusion, Dlmo appears to have a dual role in regulation of transcription, depending on the context. Such a phenomenon has been documented for other transcription cofactors, whose dual function in transcription regulation varies according to their binding partners, the specific tissue, or the developmental stage. Likewise, these results indicate Dlmo has such a dual role, being a negative regulator with respect to the Ap-Chip complex and a positive regulator in the context of the CPAD complex (Zenvirt, 2008).

The kinase Sgg modulates temporal development of macrochaetes in Drosophila by phosphorylation of Scute and Pannier

Evolution of novel structures is often made possible by changes in the timing or spatial expression of genes regulating development. Macrochaetes, large sensory bristles arranged into species-specific stereotypical patterns, are an evolutionary novelty of cyclorraphous flies (see The development and evolution of bristle patterns in Diptera) and are associated with changes in both the temporal and spatial expression of the proneural genes achaete (ac) and scute (sc). Changes in spatial expression are associated with the evolution of cis-regulatory sequences, but it is not known how temporal regulation is achieved. One factor required for ac-sc expression, the expression of which coincides temporally with that of ac-sc in the notum, is Wingless (Wg). Wingless downregulates the activity of the serine/threonine kinase Shaggy (Sgg; also known as GSK-3). This study demonstrates that Scute is phosphorylated by Sgg on a serine residue and that mutation of this residue results in a form of Sc with heightened proneural activity that can rescue the loss of bristles characteristic of wg mutants. It is suggested that the phosphorylated form of Sc has reduced transcriptional activity such that sc is unable to autoregulate, an essential function for the segregation of bristle precursors. Sgg also phosphorylates Pannier, a transcriptional activator of ac-sc, the activity of which is similarly dampened when in the phosphorylated state. Furthermore, it was shown that Wg signalling does not act directly via a cis-regulatory element of the ac-sc genes. It is suggested that temporal control of ac-sc activity in cyclorraphous flies is likely to be regulated by permissive factors and might therefore not be encoded at the level of ac-sc gene sequences (Yang, 2012).

achaete-scute products become detectable in wing discs only at mid third larval instar. The known upstream regulators, Pnr and the Iro-C genes, are selector genes that pattern the medial and lateral halves of the notum, respectively. Therefore their activity is not restricted to ac-sc activation and bristle patterning and they are expressed for a considerable period before ac-sc gene products are detected. Furthermore, although activation of ac-sc in proneural clusters by Pnr and Iro-C dramatically increases transcription at these sites, the ac-sc genes are also expressed at low levels over the entire disc epithelium, presumably through activity of the basal promoters. Indeed maintenance of proneural genes in an active state of basal transcription is a general feature of neuroepithelia. So what prevents accumulation of Ac-Sc at earlier stages in disc development (Yang, 2012)?

This study has shown that Sc is phosphorylated by Sgg, an enzyme that is expressed constitutively. Furthermore a mutated form of Sc that is resistant to phosphorylation has significantly greater bristle-forming activity than the wild-type protein. This suggests reduced transcriptional activity of phospho-Sc. One possibility is that the turnover of phospho-Sc is rapid, owing to phosphorylation-dependent ubiquitination and degradation. It has been reported that mutations in the GSK-3β consensus motif in β-catenin abolishes ubiquitination and leads to protein stability. GSK-3β also induces ubiquitination and degradation of Drosophila myc protein through the proteasome pathway and mutation of residues in the phosphorylation domain affects stability of this protein. Indeed it has been shown that mutation of the phosphorylation site SPTS to APAA stabilizes the Sc protein. This suggests that before expression of wg at the mid third larval instar, the stability and transcriptional activity of any Sc present, whether derived from transcription mediated by the basal promoter or enhanced by Pnr and the Iro-C proteins, would be reduced through phosphorylation by Sgg (Yang, 2012).

Development of neural precursors requires high levels of Sc, which are needed for the process of lateral inhibition and singling out of precursors as well as for autoregulation. During this process in Drosophila, Sc binds its own promoter, through a specific regulatory sequence, the sensory organ precursor enhancer (SOPE), to further activate transcription in presumptive precursors (Culi, 1998). Therefore, any factors that diminish the activity of Sc itself have the potential to prevent sufficient accumulation to allow selection of precursors and maintenance of precursor cell fate. Expression of wg at mid third larval instar would lead to inactivation of Sgg. The consequent accumulation of a more active nonphosphorylated form of Sc might allow levels of Sc to accumulate sufficiently for precursor cell development. Achaete does not appear to be a target for Sgg. However, this protein has been shown to be dispensable for bristle development (Yang, 2012).

Pnr is also a target for phosphorylation by Sgg and, like Sc, a mutated phosphorylation-resistant form of Pnr is hyperactive. So phosphorylation of Pnr might also result in ubiquitination and increased degradation, a situation that would be modified by Wg signalling at mid third larval instar. The effects of phosphorylation on Pnr and Sc appear to be quantitative, rather than all or nothing. Pannier has other targets before Wg signalling and activation of ac-sc (the iro genes and wg itself) and if the sole function of Wg were to be the inactivation of Sgg then one would expect loss of sgg function to have no bristle phenotype. So de-phosphorylation might just give an extra little boost to the system. Interestingly it has been shown that the Drosophila transcription factor Mad is also a target of Sgg and that phosphorylation-resistant Mad proteins are hyperactive (Eivers, 2009). Mad is activated by Dpp/TGFβ signalling, which in turn regulates expression of both pnr and the Iro-C genes in the thorax. Thus, it appears that inactivation of Sgg by the Wg signal can stimulate the levels of Sc via multiple routes: by increasing the levels of expression of pnr and the Iro-C genes as well as the activity of Pnr and Sc themselves. Thus, expression of wg at mid third larval instar might result in levels of Sc sufficient for macrochaete development. It is not known how the second phase of ac-sc expression for microchaetes is regulated (Yang, 2012).

Wingless is unlikely to be the only factor regulating temporal ac-sc expression. Indeed, although loss of sgg function can affect bristles over the entire notum, the effects of wg appear to be restricted to the medial notum. Other factors must be involved on the lateral notum. One possibility is NFkappa-B/Rel, a factor that is required for functioning of the the sensory organ precursor enhancer (SOPE) and singling out of precursors, and that also indirectly affects the stability of sc transcripts (Culi, 1998; Ayyar, 2007). Another event that coincides with the accumulation of ac-sc products at mid third larval instar is a small peak of 20H-ecdysteroid (not associated with a moult). Indeed ecdysone has been implicated in temporal regulation of expression of the proneural gene atonal and the development of atonal-dependent sense organs (Yang, 2012).

Wingless signalling has important functions in the thorax, likely to be ancient, that are linked to the development and patterning of flight muscles. So wg was probably already expressed on the notum of the ancestor of the Cyclorrapha, before the evolution of macrochaetes. The rapid development of the notum and short pupal period in many Nematocera leaves little requirement for any temporal control of expression. By contrast, the prolonged period of growth and patterning during the larval and pupal life of Drosophila allows time for two discrete phases of proneural gene expression. Wingless might then have been co-opted for the regulation of ac-sc and the evolution of macrochaetes in the lineage leading to the Cyclorrapha. The current results suggest that the Wg signal does not involve transcriptional regulation of target genes but instead is mediated simply through inactivation of Sgg. The phosphorylation sites are strongly conserved in the sc genes of C. vicina and C. capitata, two other species of Schizophora, suggesting a conserved mechanism of regulation by Wg and Sgg. By contrast, the same sites are not conserved in the other genes of the Drosophila AS-C, or in the ac-sc homologues of A. gambiae, although other potential Sgg phosphorylation sites can be detected in these proteins. Phosphorylation of Sc by Sgg could have been recently acquired in the Cyclorrapha. The ac and sc genes themselves have arisen from duplication events thought to have taken place during evolution of the Cyclorrapha (see Negre, 2009). Phosphorylation of Pnr by Sgg might also have been acquired in the lineage leading to the Schizophora, as one of the sites is conserved in the pnr protein of C. vicina, but not that of Megaselia abdita or A. gambiae (Yang, 2012).

Uniform proneural gene expression, together with Notch-mediated lateral inhibition, is sufficient to generate a pattern of evenly spaced, but randomly positioned, bristles such as that seen in Nematocera and for the microchaetes of the Cyclorrapha. For this process, the SOPE, a very ancient regulatory element that predates the Diptera (Ayyar, 2010), is the only cis-regulatory element of ac-sc that would be required. Factors that act through the SOPE could be co-opted to modulate the temporal activity of ac-sc. This includes factors regulating activity of Sc, which itself binds the SOPE (Culi, 1998). Control at this level could be superimposed on the ancestral state without the need to acquire new regulatory sequences for the binding of novel transcriptional repressors and activators. By contrast, the spatially restricted expression underlying the macrochaete pattern is linked to changes at the AS-C complex and the acquisition of novel cis-regulatory elements that possibly arose in association with gene duplication events. This illustrates the power of evolution to make use of factors acting both in cis and in trans to effect morphological change (Yang, 2012).

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