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 our 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).

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 elimin