Promoter Structure

Antennapedia activates teashirt mesodermal transcription at five sites within an upstream enhancer, 5kb from transcriptional start. Ultrabithorax activates the teashirt enhancer in both epidermis and somatic mesoderm (McCormick, 1995). There are an additional three sites for Antennapedia binding at the proximal promoter and in the first intron of tsh. Far downstream of tshlies an enhancer that is active in epidermis and mesoderm. The downstream enhancer is regulated by Ultrabithorax, Abdominal-A and abdominal-B (Mathes, 1994).

A teashirt gene enhancer is differentially activated by Hox proteins in epidermis and mesoderm. Sites where Antennapedia and Ultrabithorax proteins bind in vitro have been mapped within evolutionarily conserved sequences. Although ANTP and UBX bind to identical sites in vitro, ANTP activates the tsh enhancer only in epidermis while UBX activates the tsh enhancer in both epidermis and in somatic mesoderm. The DNA elements driving tissue-specific transcriptional activation by ANTP and UBX are separable. Next to the homeotic protein-binding sites are extensive conserved sequences likely to control tissue activation through the function of various homeodomain proteins. Local interactions between homeotic proteins and other factors influence activation of targets in proper cell types (McCormick, 1995).

The Drosophila gene teashirt is essential for normal segment identity in the embryonic thorax and abdomen. Null alleles of tsh reduce the size of all trunk segments. During the cellular blastoderm stage, tsh is expressed in a broad central domain that expands into parasegments 3 to 13. A deletion 3' to the tsh transcription unit causes the loss of tsh early expression in the even-numbered parasegments, and the corresponding larval cuticular patterns are disrupted. tsh function in the odd-numbered parasegments in these mutants is normal by both criteria. The in vivo activities of genomic fragments from the deleted region were tested in transgenic embryos. A 2.0 kb enhancer from the 3' region (thousands of base pairs from the tsh 3' terminus) acts mainly in the even-numbered parasegments and is dependent on fushi tarazu (ftz) activity. Ftz protein binds in vitro to four distinct sequences in a 220 bp sub-fragment; these and neighboring sequences are conserved in the equivalent enhancer isolated from Drosophila virilis. Tsh protein produced under the control of the 220 bp enhancer partially rescues a null tsh mutation, with its strongest effect in the even-numbered parasegments. Mutation of the Ftz binding sites partially abrogates the capacity for rescue. These results suggest a composite mechanism for the regulation of tsh, with different activators such as ftz contributing to the overall pattern of expression of this key regulator (Core, 1997).

A cis-acting regulatory element defined in this study is required to drive teashirt (tsh) expression in the regions of the developing adult that give rise to proximal wing and haltere tissues. Loss of this expression results in the fusion of the proximal structures of the wing and halteres to the thoracic cuticle. This represents the first description of a viable adult-specific regulatory allele of tsh with a visible phenotype, and it enlarges our understanding of the expression of tsh and its function during the development of the adult (Soanes, 2001).

The ae mutation is a spontaneous abnormal wing posture mutant. The cause of the abnormal wing posture is the fusion of the proximal ventral radius of the wing hinge to the pleural wing process of the thorax. No other defects are observed and all of the underlying direct and indirect flight muscles are unaffected. The tsh PZ type enhancer trap P element insertion allele, tsh(04319), is located 5' to tsh and exhibits an accurate lacZ representation of the actual wing and haltere disc expression pattern from the tsh locus when compared to Tsh antibody staining. Comparing the fate map of the wing discs and halteres to the tsh-lacZ reporter gene expression, it is apparent that tsh is expressed in tissues fated to give rise to the proximal wing hinge as well as some thoracic structures. The tsh(04319)8.1 derivative allele retains a complete copy of the PZ construct localized to the 5' untranslated leader sequence of tsh, but associated with this construct is a deletion of the immediate 3' flanking genomic sequence, including all of the tsh coding region. The tsh(04319)8.1 allele shows a modified X-gal reporter gene expression pattern in imaginal discs compared to the parental enhancer trap allele tsh(04319). Within tsh(04319)8.1 wing imaginal discs, much of the reporter gene expression regularly observed in the presumptive ventral hinge and the notum is lost or greatly reduced. Some dorsal hinge-specific expression remains but the most distal-specific expression is also lost. When pupal wings are stained, strong expression of the reporter gene is observed in the hinge region of the wing. The most distal X-gal expression from the enhancer trap allele tsh(04319) corresponds to the proximal ventral radius. Only the ventral hinge is affected in the mutant but the reporter gene expression is present in both the dorsal and ventral hinge. This suggests that in the ae background only a subdomain of the wing disc-specific expression from tsh should be affected (Soanes, 2001).

Further, these results indicate that the tsh hinge expression is divisible into, at least, smaller ventral and dorsal-specific components. The imaginal wing disc is formed as an invagination of a single continuous layer of epithelial cells. Within the area of the ventral hinge, the sheet of cells folds around from the wing pouch side to the peripodial membrane side of the disc and histochemical and antibody staining of the third instar wing discs show that tsh is expressed there. Direct analysis of tsh expression within the third instar imaginal wing discs from ae/tsh(04319)8.1 heterozygotes reveals an altered protein expression pattern in the presumptive ventral hinge and to a lesser extent in the dorsal hinge. In the ae/tsh(04319)8.1 mutant, the loss of tsh expression in the presumptive ventral hinge is localized to areas of the disc fated to become the pwp, the pleural wing process, proximal ventral radius and yellow club, and all of these structures are aberrant in the mutant (Soanes, 2001).

The two previously identified cis-acting regulatory elements within the 3' genomic sequence flanking the tsh locus are required for proper expression of tsh within the developing embryo. Until now, no tsh enhancer elements have been identified that drive expression in developing adult tissues. Using the enhancer tester pCasPeR hs 43, the presence of any cis-acting regulatory sequences within the region known to contain the ae insert was sought. The entire wild type 4.8 kb EcoRI (E4.8) sequence was tested and found to be capable of driving reporter lacZ expression within the presumptive wing hinge, in addition to limited expression within the presumptive notum. Spatially, the E4.8 expression pattern only represents a subset of the total wing and haltere disc expression seen using the tsh(04319) reporter line. The enhancer expression described here is confined to the wing and haltere discs only (Soanes, 2001).

An attempt was made to define the minimal sequence(s) required for enhancer function within the ventral hinge. Dissection of the enhancer element(s) revealed that a 915 bp HindIII/BamHI genomic fragment (HB1.0) is fully capable of ventral hinge-specific expression. However, this fragment loses the ability to express the reporter gene in the presumptive dorsal haltere and wing hinge. Overlapping fragments of 481 and 209 bp were not able to activate the reporter gene expression within the hinge region of wing and haltere discs, but were still capable of activating expression within regions of the third instar larval midgut. These observations suggest that the region contains a gut-specific element but not a complete hinge enhancer. The maintenance of hinge-specific expression with loss of the insertional element insert site suggests that the location of the insertion is not the direct cause of the loss of expression from the tsh locus within the ventral hinge. Rather, the insertion is negatively affecting the cis-acting element within the surrounding 915 bp sequence that is still capable of driving reporter gene expression within the relevant tissues (Soanes, 2001).

Transcriptional Regulation

teashirt is independent of Sex combs reduced, Deformed and labial genes. tsh levels are reduced in Antennapedia-null mutants. Therefore, Antp is required to maintain tsh transcription in the posterior prothorax (Röder, 1992).

ANTP activates tsh in anterior midgut mesoderm. In the central midgut mesoderm Ubx, abd-A, dpp, and wg are required for proper tsh expression. The control of tsh by Ubx and abd-A, and probably also by Antp, is mediated by secreted signaling molecules. By responding to signals as well as localized transcription regulators, the TSH transcription factor is produced in a spatial pattern distinct from any of the homeotic genes (Mathies, 1994). A more recent study indicates that ANTP and UBX directly regulate tsh in the midgut mesoderm (McCormick, 1995).

Expression patterns of wingless, teashirt and decapentaplegic are altered in the embryonic midgut of embryos lacking exd, while the expression of their respective regulators (ABD-A, ANTP and UBX) remains normal. (Rauskolb, 1994).

TSH protein acts early and SCR, ANTP and BX-C proteins act late to suppress labial gene expression in different subsets of cells within the trunk region. Salivary gland induction by Sex combs reduced is limited in the trunk by Teashirt protein. Loss of tsh function results in ectopic transcription of Sex combs reduced in parasegment 3. It is not known if this effect is due to direct action of Teashirt protein on the Scr promoter, or to some other mechanism (Andrew, 1994).

The morphological diversification of appendages represents a crucial aspect of animal body plan evolution. The arthropod antenna and leg are homologous appendages, thought to have arisen via duplication and divergence of an ancestral structure. To gain insight into how variations between the antenna and the leg may have arisen, the epistatic relationships among three major proximodistal patterning genes, Distal-less, dachshund and homothorax, have been compared in the antenna and leg of Drosophila. Each segment in the Drosophila leg is considered to be homologous to part or all of a segment in the antenna. The correspondences are based on reproducible homeotic transformations that can occur between parts of the two limbs. Such correlation enables a comparison of the expression domains of Dll, dac and hth between the antenna and the leg. The relative wild-type expression of these three important PD patterning genes of the leg differs in the antenna, indicating that their PD axes are differentially subdivided (Dong, 2001).

The interactions between proximal and medial domains were analyzed. dac is only rarely derepressed in hth-null clones, and ectopic expression of Hth is insufficient to downregulate dac expression in the medial leg. Thus, proximal-to-medial antagonism does not occur via hth. However, ectopic expression of a second proximal leg gene, tsh, can repress dac, and dac expression expands proximally in tsh hypomorphic leg discs. Proximal-to-medial antagonism therefore does occur in the Drosophila leg. Derepression of tsh expression in the dac-null clones has not been observed, but derepression of hth in dac-null clones has been observed. It is therefore concluded that mutually antagonistic interactions between the proximal and medial domains occur via the repression of dac by Tsh and repression of hth by Dac (Dong, 2001).

Appendages are subdivided by mutually antagonistic domains. Gradients of the morphogens Dpp and Wg initiate the PD organization of the Drosophila leg by activating Dll and repressing dac and hth distally, and by allowing the activation of dac while repressing hth medially. This creates three domains, distal, medial and proximal, that are specified respectively by expression of Dll, dac and hth. Further refinement and maintenance of the borders between domains requires mutually antagonistic interactions between proximal and medial domains as well as between medial and distal domains. Specifically, Dll and dac are mutually repressive. Also, mutually repressive interactions between the proximal and medial domains do exist via Tsh repression of dac and Dac repression of hth. Thus, pattern formation in the leg requires mutually antagonistic interactions among all three domains in order to refine and maintain borders that initially were set up by morphogens (Dong, 2001).

A genetic screen designed to isolate regulators of teashirt expression identified one of these regulators as Grunge, a gene that encodes a protein with motifs found in human arginine-glutamic acid dipeptide repeat, Metastasis-associated-like and Atrophin-1 proteins. Grunge is the only Atrophin-like protein in Drosophila, whereas several exist in humans. Evidence exists that Grunge is required for the proper regulation of teashirt but also has multiple activities in fly development. (1) Grunge is crucial for correct segmentation during embryogenesis via a failure in the repression of at least four segmentation genes known to regulate teashirt. (2) Grunge acts positively to regulate teashirt expression in proximoventral parts of the leg. Grunge has other regulatory functions in the leg, including the patterning of ventral parts along the entire proximodistal axis and the proper spacing of bristles in all regions (Erkner, 2002).

Loss of Gug activity also affects the distribution of the Tsh protein. In wild-type embryos at the germ band retraction stage, Tsh is expressed evenly in trunk segments and not the head or tail. In Gug embryos, Tsh is expressed in the trunk but is lost from ventral regions and is expressed in a striped pattern in the dorsal part of the embryo. These results suggest that Gug is a regulator of the tsh gene during embryogenesis (Erkner, 2002).

To analyse the function of the Gug locus in the leg, clones of cells homozygous for Gug35 were induced at different stages of development. Mutant Gug clones were found in all parts of the leg with a frequency similar to that of control Gug+ clones, showing that Gug+ function is not required for cell viability. Mutant and control clones were always restricted to the anterior or posterior compartment, and never changed the overall segmental identity of the legs. Differential behaviour of Gug clones is observed along the dorsoventral axis of the legs. Mutant cells located in dorsal or lateral parts of the leg give rise to essentially wild-type patterns, although they exhibit a slight cell autonomous increase in bristle density, compared with wild type (Erkner, 2002).

By contrast, Gug clones, which occupy any ventral part of the leg, delete specific pattern elements and replace them with patterns that resemble those formed in more lateral distal regions of the leg. Gug clones delete ventral-specific patterns in both the anterior and the posterior compartments. For example, the large ventral bristles of the posterior compartment in the femur of the first leg are not produced. The apical bristle at the distal tip of the anterior tibia, the spur bristles at the tip of each tarsal segment, and the transverse row and sex comb bristles of leg 1 never develop in such clones. Ventrally located Gug clones in posterior or anterior compartments fuse the femur to the tibia, which reflects a defect in the leg-specific morphogenetic process that separates these segments during pupation (Erkner, 2002).

Large Gug clones located in the coxa, trochanter or proximal femur, irrespective of their provenance in the anterior or posterior compartment, lead to fusion of these segments. Pattern elements, which are associated with clones and in neighboring cells, were replaced with those found in more distal parts of the legs. Gug clones in these proximal parts generally bear bracts, as do bristles located more distally. Clones situated in dorsal regions do not affect proximal identity. However, proximal clones, which occupy a large region of both the dorsal and ventral domains, replace all patterns with more distal identities and cause a reversal of the polarity of bristles. These Gug clones have a non-autonomous effect on the polarity of more distally located, ventral bristles. Smaller clones affect patterning if they are located ventrally. Such clones lead to outgrowths forming a partial new axis. Although bristles in these outgrowths show a distal (bracted) identity, they never form a complete new leg. Outgrowths consist of Gug and Gug+ tissue, suggesting that Gug activity is crucial for normal cell communication (Erkner, 2002).

Since Gug+ activity is required for the identity of proximal cells of the leg, a test was made to determine whether the expression of Tsh and Dll is affected in Gug clones. Tsh and Dll are expressed respectively in proximal and distal domains of the wild-type leg. Gug clones were identified by the absence of Myc epitope tag and Tsh expression was simultaneously monitored in third instar leg imaginal discs. In proximoventral positions, Gug activity is required autonomously and non-autonomously for the expression of Tsh in the leg imaginal disc. In dorsal or lateral positions, Tsh expression is not affected by loss of Gug activity in clones. In the peripodial membrane, which corresponds to the future body wall, Tsh does not depend on Gug+ activity, even ventrally. In late third instar discs, Dll is expressed ectopically in such outgrowths, consistent with the observation that lack of Gug+ function replaces proximal with distal cellular identities. Abnormal patterns of Dll expression were not observed in other parts of the legs. These experiments show that tsh and Dll expression depends directly or indirectly on Gug+ activity in the proximoventral leg, confirming the crucial role of Gug in ventroproximal patterning of the leg (Erkner, 2002).

Gug clones lead to outgrowths in the ventroproximal region in a non-autonomous manner. Wg is known to act in the patterning of ventral cells and Dpp acts in the patterning of dorsal positions. Loss of Wg and gain of Dpp signaling in any part of the ventral leg produces outgrowths similar to those described for Gug, specifically in the proximal ventral leg. wg-lacZ and dpp-lacZ expression were examined in Gug clones in the leg discs. When Gug clones produce outgrowths in proximal ventral positions, wg-lacZ expression is diminished and dpp-lacZ is expressed ectopically. In more distal leg parts or in proximal clones that lack outgrowths, Gug clones have no effect on the expression of wg-lacZ or dpp-lacZ. Similarly, no effect of loss of Gug activity is observed on the expression of Wg signaling target genes H15 and Dll or on the expression of the Dpp signaling target gene omb. It is concluded that even though Gug+ activity acts in the patterning of ventral cells of the leg, this effect is not due to a deregulation of the wg or dpp genes, except in a proximoventral position (Erkner, 2002).

Thus Gug determines the global identity of the proximal leg and acts as a positive regulator of tsh specifically in ventroproximal cells. Additionally, Gug activity is required along the entire proximodistal leg axis especially in ventral leg cells. Tsh also acts in the trunk segments of the embryo. Gug activity is required for the normal repression of four segmentation genes known to be required for regulation of tsh during embryogenesis (Erkner, 2002).

Loss of gap gene products and especially the pair rule product ftz affects the normal regulation of tsh. Ftz acts as a positive and probably direct regulator of tsh. Loss of Gug activity does not effect the location of Tsh to the trunk segments of the embryo but Tsh expression is affected (Erkner, 2002).

One striking feature of Gug+ function is its role in the formation of proximal specific patterns of the leg. Loss of Gug+ activity in proximal ventral cells changes bristle polarity and replaces proximal with more distal cellular identities. Thus, patterns typical of the coxa, trochanter and proximal femur are replaced with leg tissue that partially resembles that found in more distal femur or tibia. These effects resemble those seen in clones of cells lacking Extradenticle or Homothorax activities. Since Gug+ activity is also crucial for ventral patterning of the leg, the proximal-to-distal change is never complete. Gug mutant clones also affect cell communication in the proximal leg, because they exhibit cellular non autonomy causing neighboring wild-type tissue to differentiate distal patterns in proximal positions (Erkner, 2002).

The role of Gug in patterning the proximal leg is shown at the molecular level, where tsh requires positive input from the Gug gene specifically in ventral proximal parts of the leg imaginal disc. Loss of Gug results in ectopic expression of Dll in this position. Since Gug is ubiquitously produced in the leg, proximodistal specificity of Gug function presumably derives from other proteins located in proximoventral parts. Recently, it has been shown that Dll and possibly Tsh act as mutual repressors only in cells where Wg is signaling. Gug may normally be required for this process (Erkner, 2002).

Gug activity is essential for patterning the ventral parts of the leg along the entire proximodistal leg axis. Loss of Gug in dorsal or lateral parts has no drastic effect on patterning, although the number of bristles is augmented in Gug mutant cells irrespective of dorsoventral position (Erkner, 2002).

Ventrally in the femur-tibia region, loss of ventral Gug activity causes the fusion of these leg segments. During early pupariation, a sack of cells is known to give rise to the femur and tibia. Ventrally situated cells then migrate to meet and separate the femur and tibia. If Gug is missing in these migrating groups of cells, the femur and tibia remain attached. Gug mutant clones also affect the process of segmentation of the tarsus. Similar defects on the morphogenesis of the femur-tibia and tarsus have been observed in clones lacking components of the Notch signaling pathway. The relationship between Gug and Notch signaling activities will be reported elsewhere (Erkner, 2002).

The normal ventral patterning of the legs is specifically under the control of the Wg signaling cascade of molecules; thus, there is a correlation between the domains where Wg signaling occurs and where Gug is active. Furthermore, both Gug and Wg signaling act in domains where wg is transcribed and where Wg is secreted (for example, in the posterior ventral part of the leg) (Erkner, 2002).

Although Wg and Gug act in the same domains of the leg with similar roles, they exhibit distinct functions. Gug seems to act in a fraction of Wg-dependent developmental events. Initially, loss of Wg signaling induces a novel axis in ventral leg parts, irrespective of proximodistal position. Gug, however, induces bifurcated legs only if its activity is removed from proximal ventral parts of the leg. Contrary to the loss of Wg signaling, Gug mosaics do not distalize bifurcated legs properly, presumably because Gug activity is required for this process. Finally, Gug replaces proximal tissue with distal patterns; loss of Wg signaling never produces such homeosis. These observations suggest that Gug functions are related to those controlled by Wg signaling but are more specialized. This specialization may reflect the fact that Gug controls the expression of tsh, which is required to modulate Wg signaling activity (Erkner, 2002).

Repression of Teashirt in the wing disc

The wing imaginal disc comprises the primordia of the adult wing and the dorsal thoracic body wall. During second larval instar, the wing disc is subdivided into distinct domains that correspond to the presumptive wing and body wall. Early activity of the signaling protein Wingless has been implicated in the specification of the wing primordium. Wingless mutants can produce animals in which the wing is replaced by a duplication of thoracic structures. Specification of wing fate has been visualized by expression of the POU-homeodomain protein Nubbin in the presumptive wing territory and by repression of the homeodomain protein Homothorax. Repression of the zinc-finger transcription factor Teashirt (Tsh) is the earliest event in wing specification. Repression of Tsh by the combined action of Wingless and Decapentaplegic is required for wing pouch formation and for subsequent repression of Hth. Thus, repression of Tsh defines the presumptive wing earlier in development than repression of Hth, which must therefore be considered a secondary event (Wu, 2002).

To understand the early specification of the wing field within the imaginal disc the patterns of expression of Wg, Tsh, Hth, Vestigial and Nubbin were examined at very early stages. Vestigial is expressed in every cell of the wing disc primordium in the embryo. In wing discs from early and mid second instar larvae, Vestigial expression has begun to retract from the presumptive notum, but is expressed in both cell layers in the ventral part of the disc. At this stage, Hth is expressed in every cell. By contrast, Tsh is expressed in the presumptive body wall but has already begun to be repressed in the future wing pouch (Wu, 2002).

These early gene expression patterns are best visualized by comparing the horizontal optical sections with optical cross sections that cross the two cell layers. Until mid third instar the epithelium is cuboidal. Later, the peripodial layer becomes a very thin squamous epithelium and the other layer becomes a thick pseudostratified epithelium, which is highly folded. By mid second instar, the patterns have resolved further and the subdivisions become more clear. Tsh is repressed in the presumptive wing territory. Vestigial is expressed in a larger area, foreshadowing its expression along the DV boundary in the body wall, as well as in the wing. Hth continues to be expressed in all cells. These observations indicate that repression of Tsh and restriction of Vestigial expression are the earliest markers of wing specification. These changes occur well before Hth repression is evident. Although Tsh and Vestigial end up in approximately reciprocal patterns by late second instar, the dynamics of their expression does not suggest that they regulate each other's expression. At the earliest stages they overlap considerably. Tsh then begins to be repressed in a small subset of the region where Vestigial is robustly expressed (Wu, 2002).

Wg activity has been implicated in specification of the wing field in the disc. The levels of Wg protein expression are too low to be detected by antibody labeling during the second instar, therefore, use was made of a wg-lacZ reporter gene to visualize wg gene expression. wg-lacZ is expressed in the region of the wing disc where Tsh is repressed, during mid second instar. Repression of Tsh occurs before expression of Nubbin can be detected. By late second instar, signaling between dorsal and ventral compartments induces Wg and Vestigial expression in cells adjacent to the DV boundary. At this stage Nubbin begins to be faintly detectable within the domain of Tsh repression. By early third instar, Vestigial is expressed in a band centered on the DV boundary that extends from the wing primordium into the body wall. Repression of Hth begins in the presumptive wing and Nubbin expression broadens. Hth and Nubbin still overlap at this stage. Nubbin expression becomes stronger in the presumptive wing pouch during mid and late third instar (Wu, 2002).

As Hth expression retracts from the wing pouch, it resolves into three distinct domains in the proximal region. Two of the Hth domains overlap with the proximal rings of Wg expression that are observed in the presumptive wing hinge region. Hth is regulated by Wg at these late stages. Both rings of Wg expression are distal to the Tsh expression domain. The most proximal ring of Hth, which is regulated by secreted Wg, overlaps the edge of the Tsh domain. At this stage, Vestigial and Nubbin expression are centered on the stripe of Wg expression at the DV boundary. Vestigial expression is limited to the distal wing pouch and does not extend as far as the first ring of Hth expression. Nubbin extends more proximally, overlapping the first and second rings of Hth and the first ring of Wg expression. Tsh expression is proximal to the outer ring of Wg expression, which runs through the base of the wing hinge. Thus, the border of Tsh expression coincides with border between wing and the body wall, whereas Hth is expressed in rings in the wing hinge as well as more proximally in the notum (Wu, 2002).

Wing patterning can be subdivided into at least three discrete stages. The earliest observable changes in gene expression patterns that indicate specification of the wing field are repression of Tsh and retraction of Vestigial expression. Wg signaling represses Tsh expression at this stage. Dpp contributes to repression of Tsh. At present, the time at which Dpp acts can only be addressed directly by comparing the effects of clones induced at different stages. Clones induced in late second or early third instar are ineffective, whereas clones induced in early second instar are able to repress Tsh. Interestingly, Wg and Dpp cooperate to repress Hth in the wing pouch, even though this occurs somewhat later that repression of Tsh. These observations support the view that Wg and Dpp act in conjunction to specify the wing field in a manner analogous to the way they cooperate in leg patterning (Wu, 2002).

The combined actions of Wg and Dpp are responsible for both dorsoventral and proximodistal patterning of the leg. The wing makes use of a different strategy from the leg to control DV patterning and outgrowth from the body wall. After the wing field is established, interaction between D and V cells leads to localized activation of Notch signaling, initially in ventral cells. Subsequently Notch is activated in cells on both sides of the DV boundary. Three separate mechanisms have been implicated in limiting Notch activation to cells adjacent to the boundary. Localized Notch activity turns on the vestigial boundary enhancer and Wg expression in cells adjacent to the DV boundary. Subsequently, the long-range Wg morphogen gradient regulates downstream genes, including Achaete-scute, Dll, the vestigial quadrant enhancer, the Wg receptor Fz2 and possibly other genes, to control wing development. Wingless also acts at this stage to regulate growth in the wing hinge and in the wing pouch. This is mediated in part through Vestigial and its co-factor Scalloped, that are both required for cell survival in the wing pouch (Wu, 2002 and references therein).

Evidence has been presented that repression of Tsh in the earliest phase of wing specification appears to be required for subsequent Notch-dependent induction of Wg at the DV boundary. Clones of cells lacking Hth activity cause outgrowth of extra wing tissue along the DV boundary. The results presented here suggest that this is unlikely to be due to an early role of Hth in specification of the size of the wing field because Hth is expressed in the early presumptive wing well after Tsh is repressed. Instead, Hth appears to act in the second stage in conjunction with Tsh to limit the region in which Notch can activate Wg at the DV boundary. Wg expression at the DV boundary extends proximally into the domain of Hth expression in the anterior wing hinge but does not extend into the Tsh domain. In ectopic expression experiments, Hth has a limited ability to repress Notch-dependent activation of Wg on its own, but is able to do so when co-expressed with Tsh. These observations support the view that Hth cooperates with Tsh during later stages to repress Wg activation. This does not exclude a role for Hth as a co-factor in conjunction with Tsh at earlier stages, but if so, the positional information would seem to derive from regulation of Tsh expression (Wu, 2002).

Interestingly, Hth and Tsh can also repress the vestigial quadrant enhancer, which depends on Wg and Dpp signaling in phase 3. Homothorax, Tsh and Vestigial appear to form a loop of mutual repression at this stage, since Vestigial also represses expression of Hth. Together, these observations suggest that Wg and Dpp have a complex regulatory interaction with Hth. Their activities repress it in the pouch, perhaps through activation of Vestigial and Scalloped. At the same time, the outer rings of Wg expression are required for Hth expression in the wing hinge. It is suggested that regulation of Hth may be secondary to regulation of Tsh in specification of the wing field (Wu, 2002).

Homeotic effects

When expressed prior to 8 hours of development, ectopic Teashirt protein induces an almost complete transformation from labial to prothoracic segmental identity. Positive autoregulation of the endogenous teashirt gene and the presence of Sex combs reduced protein in the labium explain this homeosis. Maxillary patterns and a more anterior head segment are partly replaced with trunk patterns. Additional Teashirt protein has no effect on the identity of the trunk segments where the gene is normally expressed; teashirt function is overridden by some homeotic complex acting in the posterior trunk. Strong heat-shock regimes provoke novel defects: ectopic sense organs differentiate in posterior abdominal segments and trunk pattern elements differentiate in the ninth abdominal segment. Teashirt acts in a partially redundant way with certain homeotic complex proteins but co-operates with them for the establishment of specific segment types. It is suggested that Teashirt and HOM-C proteins regulate common sets of downstream target genes (de Zulueta, 1994).

Regulation of Tsh: A mosaic genetic screen reveals distinct roles for trithorax and Polycomb group genes in Drosophila eye development

The wave of differentiation that traverses the Drosophila eye disc requires rapid transitions in gene expression that are controlled by a number of signaling molecules also required in other developmental processes. A mosaic genetic screen has been used to systematically identify autosomal genes required for the normal pattern of photoreceptor differentiation, independent of their requirements for viability. In addition to genes known to be important for eye development and to known and novel components of the Hedgehog, Decapentaplegic, Wingless, Epidermal growth factor receptor, and Notch signaling pathways, several members of the Polycomb and trithorax classes of genes, encoding general transcriptional regulators, were identified. Mutations in these genes disrupt the transitions between zones along the anterior-posterior axis of the eye disc that express different combinations of transcription factors. Different trithorax group genes have very different mutant phenotypes, indicating that target genes differ in their requirements for chromatin remodeling, histone modification, and coactivation factors (Janody, 2004).

Very similar phenotypes were observed in clones mutant for Pc or E(z), which encode components of two distinct complexes implicated in transcriptional repression. Although likely null alleles for both genes were used, the phenotype of E(z) clones appeared slightly stronger, with a greater likelihood of derepressing hth in posterior regions of the eye disc. The E(z) protein has been shown to act as a histone methyltransferase for H3 K27 within a complex that also includes Extra sex combs (Esc), Suppressor of zeste 12 [Su(z)12], and the histone-binding protein NURF-55. esc appears to act only early in embryonic development, while E(z) and Su(z)12 are continuously required to repress inappropriate homeotic gene expression in wing imaginal discs. The core PRC1 complex contains Pc, as well as Ph, Psc, and dRing1, and can prevent SWI/SNF complexes from binding to a chromatin template. Pc, Psc, and ph are all required to prevent homeotic gene misexpression in wing discs; however, Psc and ph act redundantly with closely related adjacent genes. The two complexes are thought to be linked through binding of the Pc chromodomain to K27-methylated H3. The stronger phenotype of E(z) mutations in the eye disc might suggest that methylation of H3 K27 can recruit other proteins in addition to Pc (Janody, 2004).

In the eye disc, loss of E(z) or Pc leads to misexpression of the homeotic gene Ubx, but this does not seem to account for the entire phenotype. Although Ubx is sufficient to turn on tsh ectopically, misexpression of hth and tsh can occur in E(z) or Pc clones in which Ubx is not misexpressed. This suggests that hth and tsh are either direct targets of Pc/E(z)-mediated repression or targets of a downstream gene other than Ubx, possibly one of the homeotic genes not examined. Tsh misexpression would be sufficient to explain the suppression of photoreceptor differentiation in clones close to the morphogenetic furrow, since it is able to maintain expression of Hth and Ey and, in combination with them, to repress eya. Misexpression of Tsh can also account for overgrowth of Pc or E(z) mutant cells at the posterior margin of the eye disc (Janody, 2004).

trithorax group genes were initially identified as suppressors of Polycomb phenotypes and are therefore thought to contribute to the activation of homeotic gene expression. Some members of the group encode components of the Brahma chromatin-remodeling complex, others encode components of the mediator coactivation complex, and still others encode histone methyltransferases. In addition to their distinct biochemical functions, members of the trithorax group act on different sets of target genes during eye development and can also have different effects on the same target genes. Components of the Brahma complex are strongly required for cell growth and/or survival; brm and mor, but not osa, are also absolutely required for photoreceptor differentiation. However, these three genes do not seem to be required for the restricted expression in anterior-posterior domains of the eye disc of the transcription factors examined. In contrast, the mediator complex subunits encoded by skd and kto are not required for cell proliferation, although they are strongly required for photoreceptor differentiation. trx, which encodes a histone methyltransferase, is required primarily for the normal development of marginal regions of the disc. No significant effect on photoreceptor differentiation were seen in clones mutant for kismet1, which encodes chromodomain proteins, or ash21, which encodes a PHD protein. These differences are unlikely to be due to different expression patterns of the trithorax group genes, since Trx, Skd, Kto, and Osa are ubiquitously expressed in the eye disc (Janody, 2004).

The effects of these genes on the rapid transitions between domains of expression of different transcription factors are of particular interest. In the most anterior region of the eye disc, hth expression is enhanced by skd and kto. The domain just posterior to this also expresses tsh and ey, and activation of both of these genes requires trx. However, skd and kto have opposite effects on the two genes, enhancing tsh expression and preventing the maintenance of ey expression in posterior cells. Since Hth and Tsh can positively regulate each other's expression, it is possible that only one of these genes is directly dependent on skd and kto. Next, dac and h are activated transiently and eya is activated and sustained. The establishment of both dac and eya is delayed in trx mutant clones, and h expression is reduced. This delay in establishing the preproneural domain may be due to the failure to activate ey and tsh earlier in development, since Ey and Tsh combine to activate eya. The effect of Pc or E(z) mutations on eya, dac, and h appears very similar to the effect of trx mutations. However, in Pc or E(z) clones, the delay in eya and dac expression is likely to be caused by the failure to repress tsh and hth, since the combination of these two proteins has been shown to repress genes expressed in the preproneural domain. skd and kto clones also show a reduction in h and anterior eya expression, but an inappropriate maintenance of dac and dpp. These mediator complex components may thus contribute both to the activation of genes such as h in the preproneural domain and to the activation of unknown genes that shut off the expression of ey, dac, and dpp. Alternatively, skd and kto could be directly involved in the repression of these genes. Finally, trx is important to prevent misexpression of hth in cells near the posterior and lateral margins. Although Dpp normally represses hth, in trx mutant clones dpp and hth are both inappropriately expressed in marginal cells. This may reflect a role for trx in the process of morphogenetic furrow initiation, perhaps contributing to the ability of dpp to control gene expression (Janody, 2004).

Further study will be needed to determine which genes are direct targets of each trithorax group protein. However, the results point to a strong specificity of these general transcriptional regulators, suggesting that they may be specialized to mediate the effects of particular signaling pathways or to control specific subsets of downstream genes (Janody, 2004).

Differing strategies for the establishment and maintenance of teashirt and homothorax repression in the Drosophila wing

Secreted signaling molecules such as Wingless (Wg) and Decapentaplegic (Dpp) organize positional information along the proximodistal (PD) axis of the Drosophila wing imaginal disc. Responding cells activate different downstream targets depending on the combination and level of these signals and other factors present at the time of signal transduction. Two such factors, teashirt (tsh) and homothorax (hth), are initially co-expressed throughout the entire wing disc, but are later repressed in distal cells, permitting the subsequent elaboration of distal fates. Control of tsh and hth repression is, therefore, crucial for wing development, and plays a role in shaping and sizing the adult appendage. Although both Wg and Dpp participate in this control, their specific contributions remain unclear. In this report, tsh and hth regulation were analyzed in the wing disc; Wg and Dpp act independently as the primary signals for the repression of tsh and hth, respectively. In cells that receive low levels of Dpp, hth repression also requires Vestigial (Vg). Furthermore, although Dpp is required continuously for hth repression throughout development, Wg is only required for the initiation of tsh repression. Instead, the maintenance of tsh repression requires Polycomb group (PcG) mediated gene silencing, which is dispensable for hth repression. Thus, despite their overall similar expression patterns, tsh and hth repression in the wing disc is controlled by two very different mechanisms (Zirin, 2004).

In the course of a screen for mutations affecting the PD axis of the wing, an allele of the Drosophila Smad4 homolog Med was isolated. Like other Dpp pathway mutations, Medadro clones located in the wing pouch cell autonomously de-repress hth. This is evident even in late-induced clones, demonstrating the continuous role of Dpp signaling in shaping the wing blade/hinge subdivision during larval development. By contrast, no de-repression of tsh was detected resulting from any manipulations of the Dpp pathway (Zirin, 2004).

The ability of ectopic Dpp activity to repress tsh in early-induced proximal clones was interpreted to suggest that Wg and Dpp cooperate to repress tsh in the early pouch. However, because Dpp is dispensable for tsh repression, this model must be an over-simplification. It is concluded that Wg, not Dpp, must be considered the primary repressor of tsh in the wing. The lack of synergy between the two pathways is reminiscent of the regulation of Dll, which is activated in the leg by the combined activities of Wg and Dpp, but requires only Wg for its expression in the wing pouch (Zirin, 2004).

In the absence of Dpp signaling, wing pouch cells co-express hth, nub and Dll, but not tsh. This combination of factors is normally found only in the distal hinge (DH), suggesting a transformation from pouch to DH when the Dpp pathway is compromised. The expression of the Iro-C genes, normally restricted to the notum, extends to the distal limit of the tsh domain in dpp mutant discs, leading to the hypothesis that Dpp signaling is essential for the separation of wing and body wall. However, because loss of Dpp signaling transforms wing pouch to DH, an alternative view is proposed in which Dpp further divides an already extant appendage/trunk subdivision by repression of hth in the pouch and Iro-C in the proximal hinge (PH). According to this proposal, the distal limit of tsh expression, initiated by early Wg expression and maintained by PcG silencing, denotes the boundary between the appendage and the body (Zirin, 2004).

The results suggest that repression of hth in the wing disc occurs only in cells with a history of vg expression and continuous Dpp input. Consistent with this, ectopic vg expression in the medial DH and loss of brk in the lateral DH both result in hth repression. The requirement for vg can be separated into two distinct stages. The first stage occurs in the second instar, when vg expressed at the DV compartment boundary determines which cells are competent to repress hth in response to Dpp signaling. Thus, both vg or Dpp-pathway mutant clones induced at this early stage fail to repress hth (Zirin, 2004).

In the third instar, vg expression is required for hth repression only at the lateral edges of the wing pouch, whereas Dpp signaling is required at all positions along the AP axis. Accordingly, the boundary between the lateral hinge and pouch is dictated by the threshold of Dpp activity that permits the Vg-dependent repression of hth. Wg signal transduction is also required to repress hth in pouch cells far from the AP boundary. However, the requirement for Wg signaling in this part of the wing pouch could be due to its role in vg activation. Alternatively, it is possible that Wg and Vg are independently required to repress hth in these cells (Zirin, 2004).

A model that encompasses these observations is that Vg and Dpp activate another factor that directly represses hth. This factor would be activated in Vg-positive cells by Dpp signaling beginning in the late second instar. By the third instar, high levels of Dpp signaling would be sufficient to maintain its activation, with additional input by Vg and Wg required only at the lateral regions of the pouch. Even further from the source of Dpp, in the lateral hinge, high levels of Brk would prevent expresssion of this factor, thus allowing hth expression despite the presence of Vg. This model is consistent with the idea that Brk is a transcriptional repressor and Vg is a transcriptional activator. There is also precedent for the idea that early vg expression predisposes cells to a particular Dpp response, which was also proposed for the activation of the vgQE (Zirin, 2004).

The above model does not apply to PH cells, which have a distinct response to Dpp signaling. For example, Medadro clones located near the AP boundary of the PH ectopically expressed vg. tsh is an attractive candidate for mediating this switch in response to Dpp signaling, since it is expressed in the PH but not the DH, and is reported to bind Brk in vitro. However, the absence of reagents to readily examine tsh loss-of-function clones prevents this idea from being tested (Zirin, 2004).

If tsh repression marks a fundamental subdivision along the PD axis, then the maintenance of tsh repression is crucial for the maintenance of this subdivision. Although Wg signaling is clearly required for the initiation of tsh repression, it is dispensable by the time the DV margin is established. The elbow-no ocelli (el-noc) gene complex has been identified as a target of both Dpp and Wg that is necessary for tsh repression in the wing. However, tsh de-repression is observed in el-noc loss-of-function clones induced only in first or early second instar larvae. tsh repression must therefore be maintained by a wg- and el-noc-independent mechanism. The possibility of redundant Wg- and Dpp-mediated tsh repression was ruled out by making clones doubly mutant for both signaling pathways. Such clones upregulate hth, and lose Dll expression, but show no ectopic tsh expression. Thus, neither of the two major long-range signaling systems of the wing pouch is involved in the maintenance of tsh repression (Zirin, 2004).

Instead, analysis of Su(z)12daed and Pc mutant clones indicates that the maintenance of tsh repression is mediated by a heritable silencing mechanism. By inducing Pc mutant clones in third instar discs, it was demonstrated that this ectopic tsh expression represents a failure to maintain rather than a failure to establish repression. The weak hth levels observed in some PcG mutant clones may be due to the ability of tsh to upregulate hth. This interpretation is supported by the fact that hth expression is seen only in large Pc mutant clones, and only in cells expressing the highest levels of tsh. The general absence of hth expression in PcG mutant clones, together with the ectopic hth expression resulting from late Dpp pathway disruption, points to the need for continuous signaling input to maintain hth repression. By contrast, tsh requires PcG gene activity, but not continuous Wg or Dpp input, to maintain its repression during the third instar (Zirin, 2004).

At this stage the possibility that the affects of PcG mutant clones on tsh repression described here are indirectly due to the de-repression of another factor cannot be ruled out. It is suggested that this is unlikely, however, in part because the spatial distribution of tsh de-repression in PcG mutant clones differs significantly from reports of Hox gene de-repression. Additionally, the ectopic tsh expression in Pc mutant clones is repressible by Nrt-Wg, indicating that tsh is still subject to regulation by Wg signaling (Zirin, 2004).

In the embryo, Hox genes are repressed in some segments by the transient presence of the gap genes. This initial repression is then maintained by the PcG proteins through a heritable silencing mechanism. A model of tsh repression follows this general outline, whereby Wg signaling is required transiently to establish the limits of the tsh expression domain. PcG proteins subsequently maintain the tsh silenced state, while the appendage is further subdivided along the PD axis. Similar mechanisms may be important for tsh regulation in other tissues, as is suggested by tsh de-repression in PcG mutant clones in the eye disc (Zirin, 2004).

tsh and hth repression are distinct events during the development of the wing imaginal disc. The requirement for PcG activity in tsh, but not hth, repression points to the primacy of tsh repression in determining appendage versus trunk fate. PcG regulation ensures a strict and inflexible pattern of gene expression, ideal for defining the fundamental divisions of the disc. Within the specified appendage domain, Wg and Dpp signaling can then modify the shape and size of the hinge and wing blade through continuous input into transcription factors that control patterning and growth. In the absence of Tsh, Hth is an essential mediator of this process, since it promotes hinge development at the expense of wing pouch growth. The complexity of hth relative to tsh regulation may, therefore, reflect the greater need for plasticity in the response of hth to the Wg and Dpp morphogen gradients (Zirin, 2004).

The Drosophila gap gene giant has an anterior segment identity function mediated through disconnected and teashirt

The C2H2 zinc-finger-containing transcription factors encoded by the disconnected (disco) and teashirt (tsh) genes contribute to the regionalization of the Drosophila embryo by establishing fields in which specific Homeotic complex (Hom-C) proteins can function. In Drosophila embryos, disco and the paralogous disco-related (disco-r) are expressed throughout most of the epidermis of the head segments, but only in small patches in the trunk segments. Conversely, tsh is expressed extensively in the trunk segments, with little or no accumulation in the head segments. Little is known about the regulation of these genes; for example, what limits their expression to these domains? This study reports the regulatory effects of gap genes on the spatial expression of disco, disco-r, and tsh during Drosophila embryogenesis. The data shed new light on how mutations in giant (gt) affect patterning within the anterior gt domain, demonstrating homeotic function in this domain. However, the homeosis does not occur through altered expression of the Hom-C genes but through changes in the regulation of disco and tsh (Sanders, 2008).

disco and disco-r, referred to together as the disco genes, and teashirt (tsh) are differentially expressed in the embryonic head and trunk segments and are therefore markers for head and trunk segment types. In the head segments the disco genes are required for the proper development of the larval mouthpart structures, while in the trunk segments, these genes are necessary for development of the Keilin's organs, small thoracic sensory structures and some peripheral neurons. By contrast, tsh is necessary for proper development of most of the ventral trunk epidermis. Both the disco genes and tsh are also members of the proximal-distal patterning network. The disco and tsh genes encode C2H2 zinc-finger transcription factors that are expressed early in embryonic development with precise, nearly reciprocal expression patterns in the trunk and head segments, but not much is known as to how these patterns are established. What is known is that ectopically expressing tsh in the head segments converts the expression of the disco genes to a trunk-like pattern. The Spalt major (Salm) protein represses tsh expression in the posterior labial segment, but otherwise little is known regarding the regulation of disco and tsh—in particular, what factors distinguish the head and trunk modes of expression. The gap genes are logical candidates for this role (Sanders, 2008).

Patterning the Drosophila embryo involves initial establishment of the axes, regionalization of the embryo, definition of the segments and their polarity, and the specification of unique identities to each segment. The early acting components of this genetic cascade include both maternally and zygotically expressed genes that set in motion the segmentation and segment identity processes. The gap genes are among the earliest zygotic factors involved in these processes. Regulated by maternal morphogens in the blastoderm, and by one another, these genes act via overlapping gradients to divide the embryo into broad regions and to regulate the expression of downstream segmentation genes (Sanders, 2008).

Comparative studies in other insects have revealed significant conservation in the function of many segmentation genes, but less clear is the functional conservation of the gap genes between insect species. Earlier studies -- one in Tribolium castaneum, examining a giant (gt) homolog (Tc'giant), and one in Oncopeltus fasciatus, examining a hunchback (hb) homolog (Of'hb) -- conclude that the function of these gap genes is one of segmentation and segment identity, differing somewhat from the segmentation function characterized in Drosophila. This difference is likely due to the differential patterning of long vs. short germ-band insect embryos. The results presented in this study indicate that Drosophila gt, in fact, has an embryonic segment identity role similar to that observed in Tc'giant. Surprisingly, this identity function arises not only through changes in homeotic (Hom-C) gene expression, but also from the regulation of disco and tsh. This, in conjunction with other gap genes, defines the position of the embryonic head and trunk segment types (Sanders, 2008).

To explore the regulation of the disco gene during embryogenesis, disco mRNA accumulation was studied in homozygous gap mutant embryos. disco is normally expressed in the clypeolabrum, the optic lobe region, the antennal segment, the gnathal segments (mandibular, maxillary, and labial), the embryonic leg primordia, and transiently in similar positions in the abdominal segments and the proctodeum. Five homozygous gap gene mutants exhibited altered disco mRNA distribution -- hunchback (hb), Krüppel (Kr), giant (gt), tailless (tll), and caudal (cad). Of these, hb, Kr, and gt affected the gnathal/thoracic disco expression domains. disco-r mRNA accumulation was examined in hb12, Kr2, gtQ292, and gtX11 mutant embryos. Alterations in disco-r expression mirrored those of disco. Because the effects on disco and disco-r mRNA accumulation appeared to be identical, the remaining studies focused on the regulation of disco. It is noted that gt mutations had a particularly interesting effect, indicating a central role for gt in disco regulation and possibly head-trunk boundary formation. Therefore, this study concentrated on gt. Indeed, the effects of hb and Kr could be interpreted through their known cross-regulation of gt (Sanders, 2008).

Two significant conclusions are drawn from this study: (1) In its anterior expression domain, gt acts in both segment identity and segmentation roles, and these two roles are functionally separable; and (2) the distinction between the gnathal and trunk segment types is determined by the gap genes and is reflected by the head and trunk expression patterns of disco and tsh, which appear to be regulated by a series of repressive interactions (Sanders, 2008).

The assertion that gt acts in both segment identity and segmentation is based upon the following observations:

  1. The dorsal cuticle of gt embryos displayed ectopic dorsal hair development anterior to the first thoracic segment, and the ventral cuticle of some gtQ292 individuals displayed first thoracic-like denticle belts in the labial segment. This ventral transformation was masked in some gt mutant alleles (i.e., gtX11).
  2. In many gt mutant embryos, the reduction or loss of En protein from the posterior labial segment likely prevented the formation of the labial/first thoracic segmental groove, resulting in the fusion of the two segments and, consequently, the presence of only one organizer for denticle development in the fused segment.
  3. In the labial segment of gt mutant embryos, the expression of the homeotic genes Scr, Antp, disco, and tsh recapitulated their expression in the first thoracic segment, indicating a transformation to a first thoracic identity.
  4. In gt mutant embryos, expression of the gnathal-specific Hom-C gene pb is significantly reduced in the labial segment, indicating a loss of gnathal identity. Such a separation of segmentation and segment identity functions as this study describes for the anterior domain of different gt alleles has been observed in the posterior gt domain.

Initial characterizations of the gt mutant phenotype, based on SEM studies, described a fusion between the labial, first thoracic, and second thoracic segment, which, later in development, resolved such that the first and second thoracic segments separated, but the labial segment remained fused with the thoracic segment. The loss of the third (labial) En stripe has been described as indicating the deletion of the labial posterior compartment, and it was suggested that this may be the extent of the 'gap' phenotype in the anterior gt domain. The current examination of gtQ292 embryos revealed clear indications of a homeotic transformation of the labial segment to a first thoracic identity. The presence of ectopic hairs in the dorsal cuticle of gtX11 mutants has been noted, this observation was not related to a change in segment identity (Sanders, 2008).

En-expressing cells are the first to regress during the formation of the segment groove. There is an absolute requirement for En expression in cells adjacent to the developing groove. Thus, in gt mutant embryos, the amount of En accumulation retained in the posterior labial compartment likely determines the extent of segmentation that will occur between the labial and first thoracic segments. Embryos hemizygous for gtX11 almost completely lose the third En stripe, coinciding with the virtually complete fusion between the labial and first thoracic segments in these individuals. Consequently, a duplication of the first thoracic segment was never observed in embryos of this genotype. This lack of ventral denticle duplication in gtX11 embryos follows when considering the loss of En in the posterior labial segment. Since more of the labial En stripe remains in gtQ292 embryos, a segment border can form. Interestingly, both gtX11 and gtQ292 develop ectopic dorsal hairs anterior to the first thoracic segment. En staining revealed that at least a portion of the dorsal ridge fuses (or never properly separates) from the dorsal labial segment, creating a segment that resembles the first thoracic segment. It is likely that the ectopic dorsal hairs arise from the dorsal ridge, which has been transformed toward dorsal first thoracic identity (Sanders, 2008).

The case for a gt segment identity function is strengthened by the alterations in the homeotic genes expressed in the gnathal and thoracic regions. In all gtQ292mutants examined, the labial segment expressed Scr, Antp, and tsh. This combination of segment identity factors is normally found in the first thoracic segment. Further, the labial segment shows significant reduction or alteration in pb and disco expression, both markers of gnathal identity (Sanders, 2008).

There are two potential explanations for the differential effects on En accumulation and the ventral cuticle phenotype in the gt alleles that were examined. First, the available gtQ292 stock may, over time, have acquired second site suppressors responsible for the occasional persistence of En accumulation in the posterior labial segment. However, when this allele was crossed into a different genetic background, the presence of En accumulation and ventral cuticle transformation was still observed. If it is a second site suppressor of the gt mutation, then it must lie on the X chromosome carrying the gtQ292 allele. A second possibility is that the gtQ292 allele is a strong hypomorph, rather than an amorphic allele, and the residual Gt function is sufficient in some individuals to allow the labial En segmentation process to proceed, although the segment identity process remains faulty. Regardless of which explanation proves to be true, it appears that the anterior gt domain regulates embryonic patterning at two different levels -- segmentation and segment identity -- and that these two processes are functionally separable from one another (Sanders, 2008).

This conclusion is not without precedent. Early reports suggested a possible homeotic function in addition to the segmentation function of Gt in its posterior expression domain. Homeotic transformations and segmentation defects are observed in the mutant phenotypes of other gap genes. For example, in hb mutants, the loss of mid-abdominal segmentation is accompanied by mirror image duplications. The current results are significant, as they are the first to definitively demonstrate a segment identity role of the anterior gt domain (Sanders, 2008).

A recent study characterized the expression and function of a gt homolog in Tribolium (Tc'gt). As in Drosophila, Tc'gt mRNA is expressed in two primary domains -- one in the anterior of the embryo, overlying the gnathal region, and a second in the region of the third thoracic segment to the second abdominal segment. Although the anterior Tc'gt domain is similarly placed as compared to Drosophila, the posterior domain is shifted forward approximately five segments. RNA interference and morpholinos were used to knock down the expression of Tc'gt to explore its function. Interestingly, it was found that Tc'gt has a role in the identity specification of the maxillary and labial segments, but did not have a role in segmentation. The maxillary and labial segments were transformed to a first and second thoracic identity, respectively, while all three thoracic segments exhibit a third thoracic identity. There was no loss of the gnathal Tc'engrailed (Tc'En) stripes, although thoracic and abdominal Tc'En accumulation was affected to varying degrees in different embryos. Although the region affected by the loss of Tc'gt function is broader than the transformed region observed in Drosophila gtQ292 mutants, the nature of the homeotic change is quite similar. A gnathal segment(s) is transformed to a thoracic identity, and this identity change is separate from the segmentation process. The segment identity function for gt may have been present in the last common ancestor of the holometabolous insects, and the segmentation role of the anterior gt might have been acquired separately to accommodate the long germ-band mode of development (Sanders, 2008).

In the head, disco is expressed in most cells of the segmental epidermis, while there is little or no expression of tsh. By contrast, tsh is expressed throughout most of the trunk segmental epidermis, while disco is limited to the limb primordia. The genetic studies presented in this study demonstrate that the difference between head and trunk expression patterns, and therefore segment types, is dependent upon the gap genes, and particularly, on gt (Sanders, 2008).

In gt mutant embryos, both disco and tsh expression are altered reciprocally. disco expression is severely reduced in the labial segment and in fact is altered such that the remaining disco mRNA resembled the embryonic limb primordia expression observed in the thoracic segments. There is a concomitant expansion of tsh expression into the labial segment. It has been demonstrated that UAS-driven ectopic tsh expression in the gnathal segments reduces and alters disco expression such that it mimics the expression pattern of the thoracic segments (Robertson, 2004). Similarly, in gt mutants, it is the expansion of tsh expression into the labial segment that is responsible for the changes in disco expression. When both gt and tsh are absent, disco expression in the labial lobe recovers significantly, and the overall morphology of the labial segment and adjoining dorsal ridge is notably improved (Sanders, 2008).

The results may support a direct role for gt in the regulation of tsh. Although previous work demonstrated the requirement of Antp for appropriate tsh expression in the thoracic segments, and the loss of gt results in ectopic Antp protein in the labial segment, Antp is not required for ectopic tsh activation in the labial and maxillary segments of gt mutants. It is likely that gt directly limits the anterior expression of both tsh and Antp. Gt functions as a short-range repressor and has been shown to bind with high affinity to the CD1 sequence (TAT GAC GCA AGA) derived from the Kr regulatory region. There is a sequence ~0.5 kb upstream of the transcription start site of tsh that is similar to the CD1 sequence (TAT GAA GGA AGG), differing by only three bases. Although it remains to be investigated as to whether the Gt protein can bind to this sequence, the similarity in sequence to a known in vivo Gt-binding site supports direct repression of tsh by Gt (Sanders, 2008).

The results outline a model for the positioning of the gnathal/trunk boundary in the Drosophila embryo, involving a network of repressive factors. gt is a key player in this model. The anterior domain of gt is limited by its interactions with the zygotic gap genes hb and Kr, both of which act as repressors of gt expression. Gt in turn limits tsh expression, preventing expression in the labial segment. tsh expression is further limited by the expression of salm in the anlagen of the maxillary and labial segments. However, the results demonstrate that salm alone is insufficient for repressing tsh in the posterior labial segment in embryos lacking gt function. In the trunk segments, Tsh limits disco expression to only the embryonic appendage primordial, so that, lacking Tsh, disco expression is expanded through much of the gnathal segments (Sanders, 2008).

Questions remain regarding the activation of tsh and disco. disco mRNA accumulates in the cellular blastoderm prior to gastrulation, implying the involvement of maternal factors or early acting gap genes. However, none of the gap mutants that were tested affected the initiation of the initial anterior disco domain. disco was significantly affected by the loss of maternal bcd. This suggests that bcd and/or maternal hb may play a role in the initial activation of the anterior disco domain, after which Tsh acts to limit the disco to the gnathal region. tsh expression initiates prior to gastrulation, first with a central stripe that resolves to form a striped pattern reminiscent of the pair-rule genes. Again, none of the gap genes that were examined eliminated tsh expression. Although Kr is expressed in the central region of the embryo, where tsh is first transcribed, it is not the activator of tsh. Early tsh may respond to maternal factors and/or a combination of gap gene products in a concentration-specific manner, which would account for the inability to detect a single activator in the gap mutant studies. Finally, although several instances were found where Tsh represses disco, there is no evidence that the reverse is true. What leads to the repression of tsh and concomitant maintenance of disco in the maxillary segment of gt mutant embryos is unclear at this time (Sanders, 2008).

Org-1 is required for the diversification of circular visceral muscle founder cells and normal midgut morphogenesis

The T-Box family of transcription factors plays fundamental roles in the generation of appropriate spatial and temporal gene expression profiles during cellular differentiation and organogenesis in animals. This study reports that the Drosophila Tbx1 orthologue optomotor-blind-related-gene-1 (org-1) exerts a pivotal function in the diversification of circular visceral muscle founder cell identities in Drosophila. In embryos mutant for org-1, the specification of the midgut musculature per se is not affected, but the differentiating midgut fails to form the anterior and central midgut constrictions and lacks the gastric caeca. It was demonstrate that this phenotype results from the nearly complete loss of the founder cell specific expression domains of several genes known to regulate midgut morphogenesis, including odd-paired (opa), teashirt (tsh), Ultrabithorax (Ubx), decapentaplegic (dpp) and wingless (wg). To address the mechanisms that mediate the regulatory inputs from org-1 towards Ubx, dpp, and wg in these founder cells, known visceral mesoderm specific cis-regulatory-modules (CRMs) of these genes were dissected. The analyses revealed that the activities of the dpp and wg CRMs depend on org-1, the CRMs are bound by Org-1 in vivo and their T-Box binding sites are essential for their activation in the visceral muscle founder cells. It is concluded that Org-1 acts within a well-defined signaling and transcriptional network of the trunk visceral mesoderm as a crucial founder cell-specific competence factor, in concert with the general visceral mesodermal factor Biniou. As such, it directly regulates several key genes involved in the establishment of morphogenetic centers along the anteroposterior axis of the visceral mesoderm, which subsequently organize the formation of midgut constrictions and gastric caeca and thereby determine the morphology of the midgut (Schaub, 2013).

The analysis of org-1 expression and function during visceral mesoderm development defined this gene as a new and essential lineage specific regulator of circular visceral muscle founder cell identities and midgut patterning in Drosophila. The data add new insights into the developmental regulatory mechanisms responsible for the diversification of the circular visceral muscle founder cell lineage and midgut morphogenesis (Schaub, 2013).

The initial expression of org-1 occurs in the segmented trunk visceral mesoderm (TVM), where it is coexpressed with tin, bap, bin and Alk. It has been documented that the induction of tin and bap in the dorsal mesoderm involves the combined binding of Smad proteins (Medea and Mad) and Tin to Dpp-responsive enhancers of the tin and bap genes, whereas the segmental repression of bap is mediated by binding of the sloppy paired (slp) gene product. Genetic analysis of org-1 has shown that org-1 is activated downstream of tin but independently of bap and bin, and that dpp provides the key signals for its induction. This suggests a regulatory mechanism analogous to that of bap, in which the combined binding of Smads and Tin activates a Dpp-responsive org-1 enhancer, whereas Wg activated Slp is required for its mutual segmental repression (Schaub, 2013).

The similarities in the early expression patterns of bap, bin, Alk and org-1 in the trunk visceral mesoderm primordia raise the question of the contribution of org-1 to the early development of the TVM as such. Whereas bap and bin are crucially required for the specification of the trunk visceral mesoderm and visceral musculature, loss of org-1 function, like the loss of Alk, has no obvious impact on the specification of the early TVM. Therefore, it is notable that during the subdivision of the visceral mesoderm primordia into founder and fusion-competent myoblasts (cFCs and FCMs), org-1 expression is extinguished in the FCMs and only sustained in the cFC lineage of the circular visceral musculature. This lineage-specific restriction and maintenance of org-1 expression crucially depends on Jeb mediated Alk/Ras/MAPK signaling and points toward a possible cFC lineage specific function of org-1. The genetic analysis demonstrates that org-1 is not required for cFC specification, but plays a decisive role in the induction of the visceral mesoderm specific expression of patterning genes in the founder cells of the circular musculature. Thus, org-1 is critical for the processes of cell fate diversification that provide individual fields of cells along the anteroposterior axis of the visceral mesoderm with their specific identities (Schaub, 2013).

Proper anteroposterior patterning of the trunk visceral mesoderm and the formation of localized organizer fields are prerequisites for eliciting the morphogenetic events that shape the midgut. The formation of these organizer fields depends on the appropriate spatial expression domains of the homeotic selectors Scr, Antp, Ubx and abd-A, the secreted factors dpp and wg, as well as the zinc finger proteins opa and tsh, which are required for the formation of the midgut constrictions as well as the gastric caeca. The regulatory mechanisms responsible for the establishment of the spatial, temporal and tissue-specific expression patterns of these genes in the TVM are only partially understood. Genetic and molecular analyses with the FoxF gene bin, which is expressed in all trunk visceral mesoderm precursors and their descendents, have demonstrated that bin is a direct upstream regulator of dpp in PS7 and is also required for the expression of wg in PS8 of the TVM. Thus, Bin serves as an essential TVM-specific competence factor in conjunction with the dpp/wg signaling feedback loop. The current findings have defined Org-1 as an additional tissue-specific regulator with an even broader range of downstream patterning genes in the TVM, but with a narrower spatial range of action. org-1 acts specifically within the visceral muscle founder cell lineage as a positive regulator upstream of opa, tsh, Ubx, dpp as well as wg (Schaub, 2013).

This combination of genetic data and functional enhancer analyses provides convincing evidence that both dpp and wg are direct transcriptional targets of Org-1 in the cFCs. Prior dissections of the dpp visceral mesoderm (VM) enhancer had shown that it is also regulated by the direct binding of Ubx, Exd, dTCF (a Wg effector) and Bin, and that minimal synthetic variants that contain only the binding motifs for Ubx, Exd, Bin, and dTCF within conserved sequence contexts (which happen to include the Org-1 motif) are active as VM enhancers. Likewise, the wgXC enhancer fragment integrates Org-1 with the direct regulatory inputs of Abd-A as well as CREB and Smad (Mad/Medea) proteins mediating Dpp signaling (Schaub, 2013).

Org-1 is the first transcription factor known to be required for Ubx expression in PS7 of the visceral musculature. Extensive work on an Ubx visceral mesoderm CRM (UbxRP) indicated that dpp and wg regulate Ubx through indirect autoregulation. Of note, in bin embryos, which also lack visceral mesodermal dpp and wg expression, Ubx is still expressed. Genetic data show that the UbxRP element, while requiring org-1, is not directly regulated by Org-1, since mutation of its four predicted T-Box binding sites did not have any effects. Taking into account that no UbxRP reporter activity was detected in the cFCs at pre-fusion stages, it is suggested that UbxRP represents a late enhancer element and responds to dpp and wg only after they are activated by Org-1 in the founder cells. To clarify whether the regulation of Ubx by Org-1 is direct or indirect, the identification and dissection of a founder cell specific CRM will be required (Schaub, 2013).

tsh and opa were described as homeotic target genes of Antp in PS4-6 (tsh) and PS4-5 (opa) as well as of abd-A in PS8 (tsh) and PS9-12 (opa) of the visceral musculature. The current data show that tsh and opa expression is already activated in the respective cFCs of the visceral parasegments where it requires org-1. The later activation of tsh in PS8 during muscle fusion follows the org-1 dependent founder cell specific initiation of wg in PS8, which acts upstream of tsh. Thus it was conceivable that the regulation of tsh by org-1 is indirect. However, ectopic activation of wg in an org-1 loss of function background is not able to rescue tsh expression and Antp and abd-A expression is not altered upon loss of org-1. These observations suggest that Org-1 acts directly on tsh and opa, e.g., via functional cooperation with Antp and Abd-A, respectively, during the early activation of tsh and opa in the founder cells (Schaub, 2013).

It was reported that the absence of Jeb/Alk signaling causes loss of dpp expression in the founder cells in PS7 of the visceral mesoderm. In light of the current findings that org-1 loss-of-function produces a similar phenotype, and of the previous demonstration that org-1 expression is downstream of Jeb/Alk, this observation could simply be explained by the action of a linear regulatory cascade from Jeb/Alk via org-1 towards dpp. Alternatively, Jeb/Alk may provide additional inputs towards dpp (and other patterning genes) in parallel to org-1, which could explain the slightly stronger phenotype of Alk as compared to org-1 mutations with respect to dpp. A possible candidate for an additional effector of Jeb/Alk signals in this pathway is extradenticle (exd), which is known to be required for normal dpp expression in PS7 of the visceral mesoderm, presumably through direct binding of Exd in a complex with Hox proteins and Homothorax (Hth) to a PS7-specific enhancer element (a derivative of which was used in this study). Like org-1, exd is also needed for the expression of tsh and wg in the visceral mesoderm (Additionally, it represses dpp in PS4-6 through sequences not contained in the minimal PS7 enhancer). It is thought that Exd complexed with Hox proteins and Hth increases the binding preference of these Hox complexes for specific binding sites within visceral mesodermal enhancers of their target genes (Schaub, 2013).

Since exd is expressed in both founder and fusion-competent cells in the visceral mesoderm, it is unlikely that it fulfills its roles in the regulation of dpp, wg, and tsh in the founder cells as a downstream gene of org-1. However, it is known that Exd requires nucleocytoplasmic translocation for it to be functiona and, interestingly, it has been shown that Jeb/Alk signals trigger nuclear localization of Exd specifically in the cFCs of the visceral mesoderm. Because nuclear Exd appears to be hyperphosphorylated as compared to cytoplasmic Exd, nuclear translocation of Exd may be triggered by Alk-mediated phosphorylation. Alternatively, Jeb/Alk signals may induce the expression of hth in the cFCs and Hth could then translocate Exd to the nuclei, as has been shown in other contexts. This would be compatible with the observation that Hth is upregulated in the founder cells in an org-1-independent manner (Schaub, 2013).

The combined data show that Jeb/Alk signals exert at least two parallel inputs towards patterning genes in the cFCs, which are the induction of org-1 and the nuclear translocation of Exd. Taken altogether, a model is suggested in which combinatorial binding of Org-1, nuclear Exd/Hth and the homeotic selector proteins to the corresponding visceral mesoderm specific CRMs is required for the initiation of lineage specific expression of opa, tsh, dpp, Ubx and wg in the founder cells of the respective parasegments. As shown in the examples of dpp (PS7) and wg (PS8), accessory Bin is required for the activation as a general visceral mesodermal competence factor, whereas Dpp and Wg effectors mediate autoregulatory stabilization of their expression (Schaub, 2013).

Extensive work has shown that during somatic muscle development individual founder myoblasts acquire distinct identities, which are adopted by the newly incorporated nuclei upon myoblast fusion, thus leading to the morphological and physiological diversification of the differentiating muscles. It is proposed that the same principle is active during visceral muscle development. In this view, Org-1 acts as a muscle identity factor in both the somatic and visceral mesoderm. In the visceral mesoderm, Org-1 helps diversifying founder cell identities and, after myoblast fusion, their differential identities are transmitted to the respective differentiating circular gut muscles. The activation of downstream targets of this identity factor in the developing muscles leads to the observed morphogenetic differentiation events of the midgut and the establishment of the signaling center in PS7/8 that is also required for Dpp and Wg mediated induction of labial in the endodermal germ layer. As is the case for identity factors in the somatic muscle founders, Org-1 in the visceral mesoderm acts in concert with other, spatially restricted activities such as Hox factors and signaling effectors to achieve region-specific outputs. The main difference is that, in the trunk visceral mesoderm, Org-1 is present in all founder cells whereas in the somatic mesoderm this identity factor (like others) is expressed in a particular subset of founder myoblasts. Thus, in contrast to the somatic mesoderm, the spatial expression of Org-1 does not contribute to its function in visceral muscle diversification and instead, it solely relies on spatially-restricted co-regulators during this process (Schaub, 2013).

The pool of trunk visceral mesodermal fusion-competent cells contributes to the formation of both circular and longitudinal midgut muscles, depending on whether they fuse with resident founder cells of the trunk visceral mesoderm or with founders that migrated in from the caudal visceral mesoderm. The restricted expression of the identity factor Org-1 in the founder myoblasts in the trunk visceral mesoderm and its exclusion from the FCMs represents an elegant mechanism to ensure that the respective patterning events only occur in the developing circular musculature but not in the longitudinal muscle fibers, which extend as multinucleate syncytia throughout the length of the midgut (Schaub, 2013).

Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation

Paired box 6 (Pax6) is considered to be the master control gene for eye development in all seeing animals studied so far. In vertebrates, it is required not only for lens/retina formation but also for the development of the CNS, olfactory system, and pancreas. Although Pax6 plays important roles in cell differentiation, proliferation, and patterning during the development of these systems, the underlying mechanism remains poorly understood. In the fruit fly, Drosophila melanogaster, Pax6 also functions in a range of tissues, including the eye and brain. This report describes the function of Pax6 in Drosophila eye-antennal disc development. Previous studies have suggested that the two fly Pax6 genes, eyeless (ey) and twin of eyeless (toy), initiate eye specification, whereas eyegone (eyg) and the Notch (N) pathway independently regulate cell proliferation. This study shows that Pax6 controls eye progenitor cell survival and proliferation through the activation of teashirt (tsh) and eyg, thereby indicating that Pax6 initiates both eye specification and proliferation. Although simultaneous loss of ey and toy during early eye-antennal disc development disrupts the development of all head structures derived from the eye-antennal disc, overexpression of N or tsh in the absence of Pax6 rescues only antennal and head epidermis development. Furthermore, overexpression of tsh induces a homeotic transformation of the fly head into thoracic structures. Taking these data together, this study demonstrates that Pax6 promotes development of the entire eye-antennal disc and that the retinal determination network works to repress alternative tissue fates, which ensures proper development of adult head structures (Zhu, 2017).

In contrast to vertebrates that have a single Pax6 gene, the Drosophila genome contains two Pax6 homologs, ey and toy. Both genes are expressed broadly throughout the entire eye-antennal disc but are later limited to a far more restricted domain within the undifferentiated cells of the eye field. Whereas most studies on Pax6 in the eye-antennal disc have focused on the developing compound eye, several reports have hinted at a role for both genes outside of the eye. However, the underlying mechanism of how Ey/Toy promote eye-antennal disc development has been elusive. This is, in part, because of the use of single Pax6 mutants to study development. The phenotypes associated with individual mutants are variable and often restricted to the eye. Several studies have suggested that Ey and Toy function redundantly to each other. This finding most likely explains the variability of phenotype severity and penetrance. Thus, the combined loss of both Ey/Toy may be a more accurate reflection of the effect that Pax6 loss has on Drosophila development. Indeed, this appears to be the case as it is reported that the combined loss of both ey and toy leads to the complete loss of all head structures that are derived from the eye antennal disc. This study attempted to determine the mechanism by which Ey/Toy support eye-antennal disc development (Zhu, 2017).

Previous studies in the fly eye proposed that Pax6 is concerned solely with eye specification, whereas Notch signaling and other retinal determination proteins, such as Eyg, Tsh, and Hth, control cell proliferation and tissue growth. This study proposes an alternate model in which Ey/Toy are in fact required for cell survival and proliferation in addition to eye specification. The data indicate that Ey/Toy regulate growth of the eye-antennal disc through Tsh, N/Eyg, and additional N-dependent proliferation promoting genes. It is proposed that on simultaneous removal of Ey and Toy the eye-antennal disc fails to develop, in part, because the expression of eyg and tsh is lost in complete absence of Pax6. Expression of tsh and activation of the N pathway are sufficient to restore tissue growth to the eye-antennal disc. Support for this model linking Ey/Toy to cell proliferation via Eyg and Tsh comes from studies showing that eyg loss-of-function mutants display a headless phenotype identical to that seen in the ey/toy double knockdowns, that cells lacking eyg do not survive in the eye disc, and overexpression of Tsh causes overproliferation (Zhu, 2017).

The results also show that the combined loss of Ey and Toy affects the number of cells that are in S and M phases of the cell cycle. This observation directly supports the model that Ey/Toy control growth of the eye-antennal disc and is consistent with studies in vertebrates that demonstrate roles for Pax6 in the proliferation of neural progenitors within the brain. Earlier studies observed cells undergoing apoptosis in Pax6 single-mutant eye-antennal discs and showed that blocking cell death alone can partially rescue the head defects of the eyD and toyhdl mutants. Although this study shows that retinal progenitor cells lacking both Pax6 proteins undergo even greater levels of apoptosis, blocking cell death does not restore the eye-antennal disc. What accounts for the differences in the two experiments? In the eyD and toyhdl rescue experiments, each genotype contained wild-type copies of the other Pax6 paralog, but this study has knocked down both Pax6 genes simultaneously. Another possible difference is that Pax6 levels are being reduced while the eyD and toyhdl mutants are likely functioning as dominant negatives. It is concluded from these results that a reduction in cell proliferation but not elevated apoptosis levels is the proximate cause for the complete loss of the eye-antennal disc (Zhu, 2017).

Although the activation of Tsh and the Notch pathway can restore antennal and head epidermal development, neither factor is capable of restoring eye development to the ey/toy double-knockdown discs. This is most likely because both Pax6 genes are also required for the specification of the eye. In particular, Ey/Toy are required for the activation of several other retinal determination genes, including so, eya, and dac. Thus, the results suggest that Notch signaling, Eyg, and Tsh can restore nonocular tissue growth to the eye field but cannot compensate for the Pax6 requirement in eye specification (Zhu, 2017).

Finally, the results using the double knockdown of ey/toy are consistent with the dosage effects that are seen in mammalian Pax6 mutants. Although mutations in ey have just eye defects, the combined loss of ey/toy lacks all head structures. Mice that are heterozygous for Pax6 mutations have small eyes, whereas those that are homozygous completely lack eyes, have severe CNS defects, and die prematurely. Similarly, human patients carrying a single mutant copy of Pax6 suffer from aniridia, whereas newborns that are homozygous for the mutant Pax6 allele have anophthalmia, microcephaly, and die very early as well. As a master control gene of eye development, Pax6 appears to initiate both retinal specification and proliferation. These data demonstrate that the functions of Ey and Toy in the eye-antennal disc are redundant and dependent upon gene dosage, thereby making the roles of Pax6 in the Drosophila similar to what is observed in vertebrates where Pax6 controls both specification and proliferation of the brain and retina in a dosage-sensitive manner (Zhu, 2017).

Targets of Activity

The homeotic gene teashirt (tsh) is known to regulate segmental identity of the trunk region in the Drosophila embryo. There is also a requirement for tsh function in the development of adult head structures. Animals homozygous for a viable tsh allele or heterozygous for various embryonic recessive lethal alleles display miniaturized maxillary palps, a phenotype characteristically induced by dominant gain-of-function mutations of the Antennapedia (Antp) homeotic gene. Animals transheterozygous for tsh and Antp mutations display an enhanced antenna-to-leg and a striking reduced-eye phenotype, suggesting aggravated Antp misexpression in eye-antennal discs of these animals. In agreement with this, in the developing eye-antennal discs of the tsh mutant animals, a significant amount of Antp protein is detected overlapping the domains where tsh is normally expressed. teashirt is expressed in the presumptive palpus of the antennal disc and in the primordia of the sensillae, in the rostral membrane. In pharate adults the antennal segments display expression beyond the proximal domain and extending up to the second and third antennal segments Apparently, during later stages of development, tsh expression is not restricted to the proximal domains of the antenna. These results suggest that tsh specifies adult head segments by repressing Antp expression. No obvious antenna-to-leg transformation is seen in any tsh mutants (Bhojwani, 1997).

A candidate tsh target that is shared with some HOM-C genes has been identified: the modifier of variegation gene modulo (mod). mod is strongly expressed in parasegment 2, where it constitutes an early marker for the salivary placodes, the primordia of the salivary glands. In a Sexcombs reduced mutant, no mod expression is detected in PS2, indicating that the high level of mod expression in the parasegment is under the positive control of the Scr protein. Tsh represses mod in PS3 and this repression is performed independent of Scr. In vitro, Tsh recognizes two specific sites within a 5' control element of the mod gene, which, in vivo, respond to tsh activity. Tsh is therefore a DNA binding protein and might directly control mod expression (Alexandre, 1996).

Expression of Creb-A in salivary glands depends on Sex combs reduced, since Scr mutants do not express CrebA in salivary glands and embryos expressing Scr in new places also express CrebA in new places. Activation is blocked by the trunk gene, teashirt and the posterior homeotic gene Abdominal-B. As with two other salivary gland genes, forkhead and trachealess, activation of CrebA in the salivary gland by Scr is blocked by dpp (Andrew, 1997).

Based on its pattern of expression, eyegone is thought to play a role in salivary gland organogenesis. Salivary gland primordium (SGP) development responds to positional information. On the anteroposterior axis, Sex combs reduced (Scr) specifies parasegment 2. In Scr minus embryos, no salivary glands are formed and eyg expression is lost, except for a small patch of cells present at the PS1/PS2 border. In a teashirt minus mutation, Scr is expanded to both PS2 and PS3 and results in enlarged SGPs. The SGP expression of eyg is duplicated in PS3, although its appearance and fading are delayed slightly. Along the dorsoventral axis, the SGP is restricted dorsally by decapentaplegic (dpp), and ventrally by the spitz group of genes. In dpp minus embryos, eyg expression expands dorsally to form a ring that is interrupted ventrally. In several spitz-group mutant embryos, such as single minded (sim), the SGPs from each side move ventrally, and eyg expression expands ventrally. Expression in the trunk is also disordered, which may be a secondary effect of the disruption of the mesoderm (Jun, 1998).

The Drosophila wing imaginal disc gives rise to three body parts along the proximo-distal (P-D) axis: the wing blade, the wing hinge and the mesonotum. The more distal portion of the hinge is continuous with the wing blade, but contains three identifiable structures: the costa (Co), the radius (Ra) and the allula (Al). A second, more proximal part of the hinge (or axillary region), is morphologically demarcated from the rest of the wing and consists of several sclerites (Scl), which are mostly devoid of trichomes, and the axillary cord (aCrd). The tegula (Te), although positioned just anterior to the sclerites, fate maps in the wing disc to a distinct and more dorso-proximal region than these hinge structures, and therefore is not considered a part of the hinge. Correspondingly, the distalmost portion of third instar wing discs is referred to as the wing pouch, which will give rise to the wing blade. Surrounding the wing pouch is a region that will give rise to the hinge and, more proximally, there is a large dorsal territory that will give rise to the mesonotum (mnt) and a thin ventral region that gives rise to the pleura (pl) (Casares, 2000).

Several genes are known to be expressed in the wing pouch including vestigial (vg), scalloped (sd), nubbin (nub) and Distal-less (Dll), which encode transcription factors, and four-jointed (fj), which encodes a putative secreted factor. Development of the wing blade initiates along part of the dorsal/ventral (D/V) compartment boundary and requires input from both the Notch and wingless (wg) signal transduction pathways. wg is expressed along the D/V compartment boundary within the wing blade and in two concentric rings that surround the wing blade region. The rings of wg expression have been fate mapped to the adult hinge and, using a wg-lacZ reporter gene, they map within the hinge as follows: the outer wg ring (OR) maps to the proximal hinge, and the inner wg ring (IR) stains structures in the distal hinge, including the medial costa (mCo), distal radius (dRa) and part of the allula (Al). hth is also highly expressed in the wing hinge region of third instar wing discs, straddling both wg rings. Using a hth-lacZ reporter gene, hth expression maps to the same structures in the adult hinge as does wg. In late third instar wing discs, teashirt (tsh), which encodes a Zn-finger transcription factor, is strongly expressed in cells that are more proximal than hth-expressing cells, although low levels of tsh and hth overlap in the proximal hinge region. Consistent with this expression pattern, tsh-expressing cells fate map in the adult to the axillary sclerites and pleura (Casares, 2000).

In the wing blade, wg activates the gene vestigial (vg), which is required for the wing blade to grow. wg is also required for hinge development, but wg does not activate vg in the hinge, raising the question of what target genes are activated by wg to generate hinge structures. wg is shown to activate the gene homothorax (hth) in the hinge and hth is shown to be necessary for hinge development. Further, hth also limits where along the D/V compartment boundary wing blade development can initiate, thus helping to define the size and position of the wing blade within the disc epithelium. teashirt (tsh), which is coexpressed with hth throughout most of wing disc development, collaborates with hth to repress vg and block wing blade development. These results suggest that tsh and hth block wing blade development by repressing some of the activities of the Notch pathway at the D/V compartment boundary (Casares, 2000).

The overlap between wg and high levels of hth in the hinge region suggested that wg might play a role in activating hth in this region of the wing disc. Four experiments tested this idea. (1) hth expression was examined in discs in which the wg signaling cascade was compromised due to the expression of a dominant negative form of dTCF, a downstream transcription factor in the wg signal transduction pathway. Expression of dominant negative dTCF (dTCFDN) using ptc-Gal4 results in the repression of hth in the hinge. Similar results are obtained when flip-out clones expressing dTCFDN are generated in the hinge between 72 and 96 hours of development. (2) A second piece of evidence supporting a role for wg in the upregulation of hth is the finding that in wgspd-fg mutant discs, in which wg expression is specifically reduced in the IR of third instar discs, hth expression is no longer upregulated. (3) Clones of cells doubly mutant for frizzled1 and frizzled2 (fz1-;fz2-), which are required for the reception of the Wg signal, were generated. fz1-;fz2- clones show a cell-autonomous loss of hth expression. These findings suggest that wg signaling, mediated by the Frizzled family of receptors, is required for high levels of hth expression in the hinge region of wing discs. (4) A test was performed to see if ectopic expression of wg could trigger the expression of hth in more proximal regions of the wing disc, where hth levels are usually low and tsh levels are high. Based on the expression patterns of tsh and hth in third instar discs, it was predicted that, if induced by wg, hth would also repress tsh. Flip-out clones of wild-type (secreted) Wg or of a membrane-tethered, and therefore non-diffusable, form of Wg, Nrt-Wg were generated, and the expression of wg, hth and tsh were monitored. Expression of either Wg or Nrt-Wg in clones in the notum (just dorsal to the hinge region) non-autonomously induces high levels of hth and repressed tsh, recapitulating the situation found in the wild-type hinge. In contrast to clones induced in the notum portion of the disc, Nrt-Wg was never observed to activate hth in the wing pouch. Instead, activation of the Wg pathway in the wing pouch induces higher levels of vg expression. These data suggest that hth is a wg target gene in the wing hinge. In addition, they suggest that, in response to wg, cells in the wing pouch are biased in favor of activating vg, whereas in more proximal positions, induction of hth is favored (Casares, 2000).

The gene homothorax is required for the nuclear import of Extradenticle, The functions of exd/hth and of the Hh/Wg/Dpp pathway are mutually antagonistic: exd blocks the response of Hh/Wg/Dpp target genes such as optomotor-blind and dachshund; high levels of Wg and Dpp eliminate exd function by repressing hth. This repression is mediated by the activity of Dll and dac. One prerequisite for appendage development is the inactivation of the exd/hth genes (Azpiazu, 2000 and references therein).

The negative regulation of hth is a modification of the original uniform expression found in the early wing disc. Since the wing disc derives from the second thoracic embryonic segment, which shows high and uniform levels of hth expression, the initial levels of hth in the thoracic region and the wing hinge are likely to be inherited from the progenitor cells. The mechanism of hth activation during embryogenesis is not known, although its expression is modulated by the activity of the BX-C genes. At least during the larval period, hth expression in the wing disc is positively regulated by tsh. This is based on two findings. (1) An increase of Tsh levels in the hth domain results in increased levels of Hth product; possibly, one of the normal functions of tsh in the wing hinge is to maintain high hth levels. (2) Ectopic tsh expression in the wing pouch causes ectopic hth activity, at least in the clones located close to the hinge (or far from the DV border). The results indicate that tsh is not the only factor involved: hth and tsh are normally co-expressed only in the proximal ring of hth expression, therefore there should be other factor(s) maintaining hth expression in the distal ring. Since dsh mutant clones show a reduction in hth expression in the hinge, a wg response gene is likely to be involved. In addition, the fact that tsh cannot activate hth near the DV border in the wing pouch may suggest that other factors are required (Aspiazu, 2000).

Altogether, the results presented here suggest the subdivision of the non-thoracic part of the wing disc into two major domains: the wing hinge, where hth is expressed and Exd is functional (nuclear), and the wing pouch where hth is not expressed, and Exd is cytoplasmic and therefore inactive. By homology with the leg disc, the latter would be the genuine appendage part of the disc. These two regions are formed by two antagonistic genetic systems: in the hinge, the high levels of hth, inherited from the embryo and probably maintained by wg, tsh and maybe other regulators, prevent wg response to Notch signaling, which is necessary for the development of the wing pouch. In the wing pouch, the activities of the Wg and Dpp pathways suppress hth so that Notch may induce wg activity and the appendage is formed. In addition to its role in preventing excessive proliferation, hth may also contribute, together with tsh, wg and nub, to the partition of the wing hinge into two regions that correspond to the outer and inner rings of hth expression. The outer ring domain expresses tsh, wg and hth; has nuclear Exd and does not express vg and nub. The inner domain expresses wg, nub and hth, has nuclear Exd and does not express tsh. The individual role of these genes is not yet established, but it is possible that they function in some combinatorial manner (Aspiazu, 2000).

It is also not clear what is the role of hth in the specification of the wing hinge, where it is expressed at high levels. Its ectopic expression in the pouch does not produce any specific transformation towards hinge structures, but rather a general proximalization of the whole pattern. This is in contrast with the effect of ectopic tsh that induces sclerites and very proximal hinge structures. Since tsh activates hth in the hinge, it suggests that the formation of proximal hinge requires the activity of the two gene products (Aspiazu, 2000).

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. These genes can be roughly split into three categories based on their time of action during development. (1) Prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. (2) The initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). cnc and tsh act as as negative regulators of pb expression in the mandible and first thoracic segments, respectively. (3) Maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct (Ruscha, 2000).

The early phase reflects a requirement for gap gene function for normal expression of pb to occur during later stages. Specifically, btd, gt, and hb have been identified as being required for proper gnathal expression of pb. The function of the head gap gene btd has been shown to be required only during early stages of embryogenesis. The expression patterns of gt and hb are such that they are no longer expressed in the labial segment at the time when pb expression begins. This is taken as a strong indication that the gap genes influence pb indirectly. Consistent with this hypothesis, no gt or hb binding sites could be detected in the regulatory elements of the pb reporter. In the case of hb, the role that various trans-acting factors might play in mediating loss of pb expression in the labial segment was investigated. Expression of Scr, the positive regulator of pb in the labial segment, is not eliminated. Further, repression of pb is not attributable to expansion of tsh expression. One possibility is that another negative regulator is being expressed such that Scr can no longer activate pb. Given the negative regulatory interactions that occur between the gap genes, it is possible that one of the other gap genes might be misexpressed and downregulate pb. However, it may be misexpression of cnc or some other gene that has yet to be identified. Alternatively, the 'hit-and-run' hypothesis, proposed to explain the long-term repression of Ultrabithorax (Ubx) by hb, may describe how transient expression of the gap genes is required very early in development to permit later expression of pb. In this hypothesis, heritable changes in chromatin structure, mediated by the PcG genes, are invoked to explain how hb regulates Ubx long after hb expression has ceased. In the case of pb regulation, one or more of these gap genes may be required to alter chromatin structure in and around the pb locus, thereby allowing the various trans-acting factors access to the pb cis-acting regulatory elements (Rusch, 2000).

During the middle phase, the initial expression pattern of pb is set by a variety of trans-acting factors. Focus was placed on the identification of those factors that determine the ectodermal pattern of pb expression along the A/P axis of the embryo. The Hox genes Dfd and Scr act as positive regulators of pb and Dfd can bind to pb regulatory elements in vitro. It is thought likely that Scr also regulates pb directly based on the similarity with which mutations in Dfd and Scr affect expression of pb and the pb reporter. In addition to the Hox genes, the region-specific homeotics cnc and tsh have been identified as negative regulators of pb and serve to restrict pb expression to the gnathos. It is not clear whether these genes regulate pb directly, though in the case of tsh the sequence TGGAAAAGT has been identified in the 500-bp regulatory fragment used in the pb reporter; this sequence is very similar to the identified tsh binding site. While this regulatory paradigm does not completely describe the regulation of the endogenous gene, based on the presence of pb residual expression, it is sufficient to explain the behavior of the 500-bp pb reporter. This mechanism of regulation places pb downstream of the Hox genes and is the first instance in Drosophila where one Hox gene is positively and directly regulated by another, a distinction previously accorded only to vertebrate Hox genes. Studies by others have suggested that wg may be mediating the nonautonomous residual expression of pb that is uncovered by mutations in Dfd and/or Scr. With the exception that wg has the strongest effect on pb expression of the segment polarity genes tested, the results shed little light on the mechanism that underlies this phenomenon. However, non-cell autonomous signaling has been implicated to explain regulation of ectodermal pb function by mesodermal expression of Scr; perhaps the residual expression in the embryo is an example of this pathway. Further experiments, including identification of an enhancer that mediates this residual expression, are needed (Rusch, 2000).

The manner in which Hh molecules regulate a target cell remains poorly understood. In the Drosophila embryo, Hh is produced in identical stripes of cells in the posterior compartment of each segment. From these cells a Hh signal acts in both anterior and posterior directions. In the anterior cells, the target genes wingless and patched are activated whereas posterior cells respond to Hh by expressing rhomboid and patched. This study examines the role of the transcription factor Cubitus interruptus (Ci) in this process. So far, Ci has been thought to be the most downstream component of the Hh pathway, capable of activating all Hh functions. However, the study of a null ci allele indicates that it is actually not required for all Hh functions. Whereas Hh and Ci are both required for patched expression, the target genes wingless and rhomboid have unequal requirements for Hh and Ci activity. Hh is required for the maintenance of wingless expression before embryonic stage 11 whereas Ci is necessary only later during stage 11. For rhomboid expression Hh is required positively whereas Ci exhibits negative input. These results indicate that factors other than Ci are necessary for Hh target gene regulation. Evidence is presented that the zinc-finger protein Teashirt is one candidate for this activity. It is required positively for rhomboid expression and Teashirt and Ci act in a partially redundant manner before stage 11 to maintain wingless expression in the trunk (Gallet, 2000).

Before stage 11, either Tsh or Ci is sufficient for wg regulation because only the loss of both gene activities results in a downregulation of wg, a situation comparable with that observed in hh mutants. It is interesting to note that Ci seems to display differential requirements for wg maintenance and naked cuticle differentiation in the abdomen versus the thorax. While Ci is dispensable until stage 11 for wg expression and naked cuticle differentiation in the abdomen, its presence in the thorax is required. This specific Ci function in the thorax is currently being studied (Gallet, 2000).

Both Ci and Tsh transcription factors, when overexpressed can induce ectopic wg expression. The two factors do not display the same features: Tsh has three atypical, widely spaced, zinc-finger motifs, whereas Ci has conserved spacer regions between its five zinc fingers; the binding sites identified so far for these two proteins are different. It would be interesting to know whether Tsh can bind directly to the wg promoter and to identify its binding sites. It is also noteworthy that between stage 8 and 10 wg requires, in parallel to Hh, its own activity for the maintenance of its transcription. It has previously been shown that Tsh is a modulator of Wg signaling. Tsh becomes phosphorylated and accumulates at a higher level in the nucleus in Wg-receiving cells compared with cells lacking Wg signal. Hence, in the trunk Tsh could be employed both by Wg and Hh signaling in order to maintain wg transcription (Gallet, 2000).

The redundancy exhibited between Tsh and Ci for wg regulation changes after stage 10, since loss of either Ci or Tsh results in the downregulation of wg transcripts. It is not known if this observation is the result of a cooperation between Tsh and Ci. At least one other gene, gooseberry, is required for the maintenance of wg transcription at this stage, indicating that multiple inputs for the maintenance of wg expression are necessary for normal embryonic development (Gallet, 2000).

Studies on the developing wing blade show that Ci transduces all Hh-delivered information. However, this study and others on the Hh pathway support the idea that Ci is not always involved in Hh signaling, showing that branchpoints are common for distinct Hh signaling steps for the following five reasons. (1) It has been shown that neither Ci nor Fused (Fu) are involved in the Hh-dependent formation of Bolwig's organ in Drosophila. (2) A Hh-responsive wg reporter gene with no Ci-binding sites does not require Ci activity for its regulation until stage 11. (3) Studies on the talpid 3 gene in chicken suggest that Gli proteins, the vertebrate homologues of Ci, regulate only a subset of Hh target genes, the others being regulated by an unidentified transcription factor. (4) A Sonic hedgehog response element on the COUP-TFII promoter binds to a factor distinct from Gli. (5) Hh signaling does not require Ci activity to regulate rho. Although the authors favor the idea that Tsh regulates rho expression directly in response to Hh signal the hypothesis that Tsh plays a more permissive role allowing Hh to regulate rho via another factor apart from Ci cannot be excluded (Gallet, 2000 and references therein).

In conclusion, Hh requires at least two different transcription factors during Drosophila embryogenesis to regulate its multiple target genes and to instruct cells with precise behaviors. The transcription factors may act independently (e.g. Ci for ptc; Tsh for rho), cooperatively (e.g. Ci and Tsh for wg maintenance during the cell specification phase) or redundantly (e.g. Ci and Tsh for wg maintenance earlier during the stabilization phase). The possibility that other transcription factors like gooseberry might be recruited for Hh signaling cannot be excluded, especially since denticle density is weaker in tsh;ci double mutants as compared with hh single mutants. Furthermore the dorsal phenotypes of the tsh;ci double mutants are weaker than those of hh. (1) wg transcripts are still present in dorsal patches in tsh;ci mutations whereas they are not present in hh embryos. (2) Dorsal cuticle is not as severely perturbed in tsh;ci larvae as compared with hh null ones (Gallet, 2000).

Teashirt regulates homothorax in the eye disc

teashirt (tsh) encodes a Drosophila zinc-finger protein. Misexpression of tsh has been shown to induce ectopic eye formation in the antenna. tsh can also suppress eye development. This novel function of tsh is due to the induction of homothorax (hth), a known repressor of eye development, and requires Wingless (Wg) signaling. Interestingly, tsh has different functions in the dorsal and ventral eye, suppressing eye development close to the ventral margin, while promoting eye development near the dorsal margin. It affects both growth of eye disc and retinal cell differentiation (Singh, 2002).

Since ectopic tsh can regulate hth expression, the endogenous expression of these genes in imaginal discs was compared. Expression of tsh was examined using tsh-GAL4-driven UAS-GFP (tsh>GFP). In the eye disc, tsh expression can be detected as early as first larval instar in the entire disc proper, overlapping with hth and the pro-eye gene eyeless (ey. In the late second instar eye disc, tsh>GFP expression retracts anteriorly and occupied nearly three quarters of the disc. hth expression also retracts anteriorly. Ey is also expressed in the same region. In early third instar eye discs, tsh expression regresses to the anterior two-thirds of the disc. hth expression is restricted to the anterior margin in a 10- to 15-cell wide domain. tsh and hth expression overlaps in a 3- to 4-cell wide stripe. Ey expression largely overlaps tsh. In late third instar eye disc, tsh>GFP expression is anterior to the MF and is similar to the expression pattern determined by tsh-lacZ, anti-Tsh antibody and in situ hybridization. The co-expression of tsh and hth during the early phase of eye disc development is consistent with the finding that tsh induces hth expression (Singh, 2002).

Interestingly, although tsh is expressed symmetrically in the dorsal and ventral halves of the eye disc, overexpressing tsh in these regions suppresses eye development in the ventral region, while it promotes eye development in the dorsal region. Why would overexpressing tsh in a region where it is normally expressed cause phenotype reciprocal to the loss-of-function tsh mutant phenotype? It is possibly a dose effect, since the ectopic expression of two copies of tsh transgene causes a stronger effect. The normal level of Tsh may be balanced with some opposing forces for proper development, thus too little and too much of Tsh will cause reciprocal effects. A similar case is Wg, which is normally expressed in both dorsal and ventral margins. Reducing Wg level causes ectopic MF formation, while raising Wg level blocks MF initiation (Singh, 2002).

The eye-suppression function of tsh is accompanied by the induction of hth at the transcriptional level. Eye suppression is reduced when the hth dose is reduced, suggesting that Hth is the major mediator of tsh-induced eye suppression. This is consistent with the known role of hth as a repressor of eye development. In the wing disc, tsh also induces Hth, but tsh has additional effects (e.g. protecting wg from suppression by Hth and splitting the wing pouch). Whether tsh has additional effects in the eye disc awaits further study (Singh, 2002).

The eye-suppression function of tsh requires Wg signaling, since blocking Wg signaling by co-expressing dTCFDeltaN or sgg with tsh, or overexpressing tsh in a wgts mutant at the non-permissive temperature blocks the suppression effect. The critical time for wg involvement is 48-72 hours AEL, corresponding to the second instar larval stage. At this stage, the expression patterns of tsh, hth and wg in the eye disc overlap considerably, consistent with their functional interaction (Singh, 2002).

Tsh can induce Hth and suppress eye development only in the ventral margin of the eye disc. Internal clonal induction of tsh expression (Act>tsh) clones has no eye-suppression effects. The restriction of eye suppression to the eye disc margin, where wg is expressed, suggests that tsh does not induce wg but requires high level Wg signaling. Indeed, clonal expression of tsh internal in the eye disc does not induce Wg expression. When Tsh is co-expressed with Wg or an activated Arm, eye suppression can occur away from the margin, possibly because higher levels of Wg signaling are provided by the ectopic expression. Tsh also requires a high level of Wg to repress Ubx transcription in the embryonic midgut (Singh, 2002).

Ectopic expression of Wg in the region ahead of MF induces Hth, while blocking Wg signaling (by clonal expression of dTCFDeltaN) reduced Hth in the presumptive head region of the eye disc. These locations correspond to tsh expression domain, consistent with the Tsh-Wg collaboration. Act>hth clones can block MF initiation without inducing ectopic wg expression, also suggesting that hth acts downstream of Wg. Thus, these results suggest that Tsh collaborates with Wg signaling to induce Hth to suppress eye development (Singh, 2002).

Tsh and Wg signaling also collaborate during embryonic development. Tsh acts in the late phase of Wg signaling to promote the naked cuticle cell fate of larvae. Tsh phosphorylation and nuclear accumulation is partially promoted by Wg signaling. Hypophosphorylated Tsh can bind directly to the intracellular Arm. The effect of Tsh overexpression on embryo development is dependent on the interaction with Arm. Tsh can also associate with Sgg, an inhibitory component of Wg signaling that promotes Arm degradation and acts downstream of Sgg. Whether the same molecular interaction operates in the eye disc awaits further study (Singh, 2002).

Based on the loss-of-function phenotype and overexpression phenotype, tsh suppresses eye development only in the ventral eye, while promoting eye development in the dorsal eye. The DV difference in Tsh function is not likely to be due to wg, since wg is expressed in both dorsal and ventral margins, with even higher levels in dorsal parts. In a wg temperature-sensitive mutant, an ectopic MF initiates more on the dorsal side. Wg signaling upregulates hth in both dorsal and ventral regions of the eye disc. Thus, wg can induce hth and suppress eye development in both ventral and dorsal margins, but through different mechanisms. Tsh collaborates with Wg signaling for eye suppression only in the ventral margin, but not in the dorsal margin. Whether Wg requires other co-factors in the dorsal margin is not known (Singh, 2002).

The critical period for eye suppression by tsh is in the second instar larval stage, based on tsh mutant clones and on misexpression of tsh in wgts background. At this time, the morphogenetic furrow has not initiated and photoreceptor differentiation has not begun. tsh mutant clones induced in second instar caused enlargement in the ventral eye field and reduction of eye cells in the dorsal eye field. In the ventral overgrowth, not all cells have differentiated into photoreceptors. These results suggest that the primary effect of tsh function is on growth in the early eye disc. When the relative frequency and size of Act>GFP and Act>tsh+GFP clones are compared, the results show that tsh promotes growth in the dorsal and suppressed growth in the ventral region. A dorsal clone anterior to the MF shows overgrowth, suggesting that the effect can be a general growth promotion and is not limited to differentiating retinal cells (Singh, 2002).

However, tsh8 mutant clones in the dorsal eye cause a transformation of eye cells into cuticle fate, suggesting that tsh also plays a role in promoting eye fate (in the dorsal part of the disc). This role is consistent with the finding that tsh could induce ectopic eye formation in antenna. In the ventral eye disc, a role in directly suppressing photoreceptor fate is also supported by the finding of an isolated ventral eye field in the eye disc with tsh8 clone induction. This direct role is consistent with the ventral activation of hth, which can directly suppress photoreceptor differentiation. Thus, tsh can affect both the growth of the eye disc and the differentiation of photoreceptors (Singh, 2002).

Increased avidity for Dpp/BMP2 maintains the proliferation of progenitors-like cells in the Drosophila eye

During organ development, the progenitor state is transient, and depends on specific combinations of transcription factors and extracellular signals. Not surprisingly, abnormal maintenance of progenitor transcription factors may lead to tissue overgrowth, and the concurrence of signals from the local environment is often critical to trigger this overgrowth. Therefore, identifying specific combinations of transcription factors/signals promoting (or opposing) proliferation in progenitors is essential to understand normal development and disease. This study used the Drosophila eye as a model where the transcription factors hth and tsh are transiently expressed in eye progenitors causing the expansion of the progenitor pool. However, if their co-expression is maintained experimentally, cell proliferation continues and differentiation is halted. Hth+Tsh-induced tissue overgrowth was shown to require the BMP2 Dpp and the abnormal hyperactivation of its pathway. Rather than using autocrine Dpp expression, Hth+Tsh cells increase their avidity for Dpp, produced locally, by upregulating extracellular matrix components. During normal development, Dpp represses hth and tsh ensuring that the progenitor state is transient. However, cells in which Hth+Tsh expression is forcibly maintained use Dpp to enhance their proliferation (Neto, 2016).

Abnormal maintenance of transcription factors that promote an undifferentiated, proliferative state is often an initiating event in tumors. However, abnormal growth is dependent on specific non-autonomous signals provided by the microenvironment. This study used an experimental system that results in continuous growth to identify these signals and the mechanism of action. In this system, the GAL4-driven maintenance during eye development of hth and tsh, two transcription factors normally transiently co-expressed in eye progenitors, cause cells to increase their avidity for Dpp. This, in turn, leads to a hyper-activation of the pathway, which is necessary to maintain the proliferative/undifferentiated phenotype. The increased avidity for Dpp was shown to be mediated, at least partly, through increased expression of the proteoglycans components encoded by dally and dlp, functionally modified by slf (Neto, 2016).

Progenitor cells, forced to maintain Hth and Tsh (hth+tsh progenitor-like cells) trap Dpp produced at local sources, which then causes an increased in intracellular signaling. The mechanism responsible of this trapping seems to be the increase of extracellular matrix (ECM) components. First, a cell-autonomous increase was found in dally transcription and Dlp membrane levels, the two glypican moieties of heparane sulphate proteoglycans. Second, the RNAi-mediated attenuation of sfl function, a gene encoding an enzyme required for the biosynthesis of these proteoglycans, is required for the overgrowth/eye-suppression phenotype induced by hth+tsh maintenance. A third line of support comes from examination of the effects of hth+tsh or hth+tsh+slf RNAi on the pMad profiles. Considering that the Dpp production remains unaltered, hth+tsh tissue shows an increase in both pMad signal amplitude and range, which is consistent with the increase in proteoglycans simultaneously augmenting Dpp diffusion and stability. On the contrary, reducing proteoglycan biosynthesis in hth+tsh+slf RNAi cells results in the retraction of the pMad signaling range back towards control values, which again is expected if Dpp's diffusion depends on proteoglycans (Neto, 2016).

By forcing the expression of hth and tsh in eye precursors, these cells are exposed to signaling levels higher than they would normally encounter. This is because during normal eye development Dpp, produced at the furrow, represses first hth and then, closer to the furrow, also tsh, so that the cells approaching the furrow and receiving the highest Dpp levels no longer co-express hth and tsh. The loss of hth marks the transition between proliferation/undifferentiation and cell quiescence/commitment. This transition coincides with a transient proliferative wave (the so-called 'first mitotic wave') that precedes entry into G1. This transition zone corresponds to a region where low, but not null, levels of Hth and pMad signals overlap. If the interaction between hth+tsh and the Dpp pathway described in this study were to hold also in the zone of hth/Dpp signal overlap during normal eye development (remember that hth-positive cells co-express normally tsh too), one prediction would be that the mitotic wave would be lost if either hth or dpp-signaling were removed. Indeed this has been shown to be the case: RNAi-mediated attenuation of hth or abrogation of Dpp signaling result in the loss of the first mitotic wave. However, it is not thought that the mechanisms driving Dpp-mediated proliferation of optix> hth+tsh cells are necessarily the same as those operating normally in hth+tsh-expressing progenitors during eye development, because of the following experiment. Discs were generated expressing in their dorsal domain an RNAi targeting Hth's partner, the Pbx gene extradenticle (exd). In the absence of Exd, Hth is degraded. Therefore, a depletion of Exd causes an effective loss of Hth. Knowing that in optix>hth+tsh the stability and diffusion of Dpp were increased, the prediction would be that the loss of hth (in exd-depleted cells) should cause a decrease in both the stability and diffusion of Dpp. However, when the dorsal ('exd-') with the ventral ('exd+') pMad profiles of D>exdRNAi discs was quantified, it was found that both the stability and diffusion of Dpp increased by the loss of hth. This result suggests that during normal eye development hth (perhaps together with tsh) influences Dpp signaling, but the mechanisms described in this study as triggered by forced hth+tsh expression are likely different (Neto, 2016).

The upregulation of dally and dlp by hth+tsh is likely the consequence of the transcriptional activity of Hth+Tsh in partnership with the YAP/TAZ homologue, Yki, as previous work showed that loss of the protocadherin genes fat (ft) and dachsous (ds) , which causes the activation of Yki, results in an upregulation of dally and dlp in the wing primordium. In fact, previous studies have found, in imaginal tissues, binding of Yki and Hth to nearby sites on the dlp locus, suggesting that some of this regulation might be direct. All these data make Yki a necessary component of the molecular machinery responsible for the increased avidity of hth+tsh cells for Dpp. However, in the eye primordium, the overexpression of Yki induces a different phenotype than hth+tsh. More importantly, in the eye primordium, yki+ clones do not cause the autonomous upregulation of pMad signal that hth+tsh clones do. Therefore, a specific stoichiometry among Hth, Tsh and Yki is likely necessary to induce the Dpp signaling-dependent properties of hth+tsh cells, at least in the developing eye. Alternatively, Hth and Tsh may activate Yki-independent targets that would be required for the full expression of the phenotype. Recently, another study has found that Yki and the Dpp pathway synergize in stimulating tissue overgrowth, both in eye and wing primordia, through the physical association between Yki and Mad. The current results suggest that hth+tsh progenitor-like cells establish a positive feedback, in which the growth promoting activity of the Hth:Tsh:Yki complex would be enhanced by increasing levels of pMad activated by Dpp. This feedback would be region-specific, as it depends on sources of Dpp that are localized within the eye primordium. Further work is needed to investigate the molecular mechanisms behind this feedback. Finally, it has been shown recently that tissue growth promoted by the PI3K/PTEN and TSC/TOR nutrient-sensing pathways also requires Dally, which, in turn, increases the avidity of the growing tissue for Dpp. Therefore, increasing the avidity for Dpp by augmenting proteoglycan levels may be a common strategy of tissues to sustain their growth (Neto, 2016).

Protein Interactions

Wnt signaling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts inside cells to stabilize Armadillo where Arm binds to at least two DNA-binding factors that regulate specific target genes. One Armadillo-binding protein in Drosophila is the zinc finger protein Teashirt. A 23 amino acid domain (between aa 692 and 715) in Arm is necessary for the interaction with Tsh. This domain lies in the most conserved part of the C-terminal domain of Arm. Wingless signaling promotes the phosphorylation and the nuclear accumulation of Teashirt. This process requires the binding of Teashirt to the C-terminal end of Armadillo. Evidence is presented that the serine/threonine kinase Shaggy is associated with Teashirt in a complex (Gallet, 1999).

To investigate the effects of Wg signaling on Tsh phosphorylation, Western blots were performed on proteins extracted from stage 9-11 embryos mutant for different components of the Wg pathway. Mutant embryos that constitutively transduce Wg and those lacking signal transmission were selected. In wild-type embryos, different hyperphosphorylated forms of Tsh are present. In constitutive Wg signaling mutant embryos, the most hyperphosphorylated forms are predominant. Conversely, in Wg signaling loss-of-function mutants, the upper band is fainter and the 116 kDa form is more apparent. By probing the same blot with an anti-tubulin antibody and by densitometric analysis, the relative amount of Tsh in the different mutants can be correlated: there are equal amounts of Tsh in wild-type and in embryos with gain of Wg signaling function, but there is less Tsh in mutants lacking Wg signalling function. This is consistent with a decreased level of nuclear Tsh observed in the absence of Wg function. Taken together, the results indicate that the Tsh phosphorylation and the increasing nuclear level of Tsh is in part dependent on the Wg pathway. Nevertheless, even in mutants lacking signal transmission, Tsh is still phosphorylated and localized in the nucleus, indicating that other factors are acting on Tsh independently of Wg (Gallet, 1999).

Wg signal acts by inhibiting the activity of Sgg, which would otherwise promote the degradation of Arm inside the cell. Thus Arm accumulates inside the cell and can interact with its partners. Loss of Sgg activity causes the stabilization of intracellular Arm everywhere in the segment promoting the production of naked cuticle in the trunk. When Wg does not signal, Sgg is thought to promote phosphorylation of Arm on an N-terminal motif, leading to Arm degradation via the ubiquitination pathway. In order to test the interaction between Tsh and Sgg, germ-line clones of sgg were induced. The distribution of Tsh was examined in such embryos. As expected, nuclear Tsh level is high, as in embryos constitutively expressing the Wg pathway. In order to analyse the epistasis between tsh and sgg, sgg cuticles were examined with or without tsh activity. Whereas sgg germ-line clones give naked cuticle, absence of tsh gives larvae with reduced naked cuticle and a lawn of denticles. Therefore Tsh acts downstream of Sgg. Finally, mmunoprecipitations were performed to test whether Sgg and Tsh are in a complex. Using affinity-purified anti-Tsh, Sgg co-immunoprecipitates with Tsh. Together, these results show that Tsh is epistatic to Sgg; that the nuclear Tsh level is also Sgg-dependent, and that in vivo Tsh is in a protein complex with Sgg (Gallet, 1999).

How could the C-terminal domain of Arm act as transcripitional transactivator? Two models are presented for Arm function. In the first model, Arm/beta-catenin could be considered as a bridge between a general DNA-binding factor (e.g. TCF) and a specific transcription factor like Tsh, which modulates a specific function for signaling. In this model the transactivating domain of Arm could bind several different and localized factors allowing tissue specific output. In the shuttle model, on Wg signaling, Arm could interact with general (dTCF) as well as specific factors (Tsh) and translocate them into the nucleus to activate Wg target genes. Arm/beta-catenin shares homology with the importins/karyopherins, which are cytoplasmic receptors for proteins containing nuclear localization sequences (NLS) allowing their docking to the nuclear pore. In agreement with this, it has been shown that Arm is able to interact with the nuclear pore machinery. In this model Arm/beta-catenin is detected in the nucleus in cells where Wg is signaling. Furthermore, Tsh is able to produce naked cuticle in the absence of zygotic dTCF, suggesting that Tsh and dTCF act independently for Wg signaling. Further experiments are required to establish whether Tsh acts directly with dTCF and especially to determine whether or not Arm/beta-catenin is part of a transcription complex or acts simply as a nuclear shuttle. These two models can be considered together where, upon an extracellular signal (e.g. Wg/Wnt), a cytoplasmic protein (e.g. Arm/beta-catenin) recruits specific factors (e.g. Tsh) and stimulates their entry into the nucleus, where this bipartite complex could collaborate with general DNA-binding factors (e.g. TCF) to regulate specific target genes of the pathway. There are several results in favour of this model: (1) TCF is localized in the nucleus independent of Arm; (2) TCF is able to bind other specific factors, including Groucho or dCBP, to repress Wg/Wnt target genes; (3) Tsh does not contain canonical NLS signal and requires an unknown mechanism for entry into the nucleus. Nevertheless, Tsh is the first example of a protein binding Arm in its C-terminal domain and potentially is able to participate in the transactivating process (Gallet, 1999 and references).

One function of the Wingless signaling pathway is to determine the naked, cuticle cell fate choice in the trunk epidermis of Drosophila larvae. The zinc finger transcripton factor Teashirt (Tsh) binds to the transactivator domain of Armadillo to modulate Wingless signaling output in the embryonic trunk and contributes to the naked cell fate choice. The Hedgehog pathway is also necessary for the correct specification of larval epidermal cell fate, which signals via the zinc finger protein, Cubitus interruptus (Ci). Ci also has a Wingless-independent function, which is required for the specification of the naked cell fate; previously, it had been assumed that Ci induces naked cuticle exclusively by regulation of wg. Wg and Hh signaling pathways may be acting combinatorially in the same, or individually in different, cells for this process, by regulating common sets of target genes. (1) The loss of the naked cuticular phenotype in embryos lacking ci activity is very similar to that induced by a late loss of Wg function. (2) Overexpression of Ci causes the suppression of denticles (as Wg does) in absence of Wg activity in the anterior trunk. Using epistasis experiments, it has been concluded that different combinations of the three proteins Tsh, Ci, and Arm are employed for the specification of naked cuticle at distinct positions both along the antero-posterior axis and within individual trunk segments. Finally, biochemical approaches suggest the existence of protein complexes consisting of Tsh, Ci, and Arm (Angelats, 2001).

The cuticles of ci null embryos resemble those that lack wg function specifically during the cell fate specification phase. In both genotypes, the bands of naked cells are reduced though they are not totally lost and the number of denticles is increased, suggesting that both Wg and Ci are required for the patterning of the naked regions. Closer comparison of the denticle identities from these embryos reveals that the expansion of denticles belts correspond, in both cases, to an increase in the number of denticles of types 2, 3, and 4. Since the EGF pathway, and particularly rhomboid (rho), is required to specify these denticle identities, rho expression was examined in wgts embryos. When wgts embryos are shifted to the restrictive temperature at stage 10-11, rho expression is expanded posteriorly in a similar way as that observed in ci94 embryos. These observations support the idea that Ci has a function related to the late activity of Wg signaling (Angelats, 2001).

Thus examination of rhomboid expression and cuticle patterns shows the close similarity of phenotype between a late loss of wg function and the loss of function of ci. Following ectopic expression, Ci is able to promote the specification of naked cuticle in the absence of Wg signaling, showing that Ci is acting downstream or in parallel to Wg during the specification phase. This capacity of Ci to induce the naked cell fate was previously explained by ectopic expression of wg, but the experiments described here show that Ci can also act directly for the specification of the naked cell fate choice especially in the anterior trunk segments. It is believed that UASCi produces high levels of full-length Ci, resulting in the saturation of its normal negative regulation, producing naked cuticle (Angelats, 2001).

Ci, Arm, and Pangolin act in a combinatorial fashion to regulate the expression of dpp in the wing disc. Thus, in addition to the well-known regulatory effects of Hh on wg, it is proposed that downstream components of these signaling pathways may interact directly for gene regulation. Similar arguments may apply in vertebrates where Wnt signaling has been shown to be critical for the regulation of the Ci orthologs Gli2 and Gli3. These considerations and the current results support the hypothesis that Wg and Hh signaling components (Arm and Ci, respectively) have overlapping and thus common functions for patterning, at least in some cells (Angelats, 2001).

The capacity of Ci to mimic Wg activity seems to be position-specific since Ci never suppresses the denticles in the most posterior part of the abdomen (from A5 to the tail) in the absence of Wg activity. In this region of the body, the presence of another unidentified factor may modify the activity of Ci (Angelats, 2001).

Morphological examination of the wild-type trunk segments shows that a typical thoracic segment has fewer and smaller denticle belts compared to those in any abdominal segment. Consequently, thoracic segments generally possess more naked cuticle than abdominal ones. Ci and Arm exhibit differences in their ability to induce naked cuticle in different parts of the trunk. The activity of Arm is crucial for the transduction of the Wg signaling pathway and plays a pivotal role in the trunk for naked cuticular identity. Despite this, ectopic production of stabilized Arm or Wg does not replace denticles of the prothoracic beard with naked cuticle. Loss of Ci activity affects this process of patterning, suggesting that Ci activity acts with Arm signaling for the patterning of the beard. In accord with this hypothesis, ectopic Ci, with or without Wg/Arm signaling, suppresses denticles in the beard. In this context, it is interesting to note that loss of the Wg signaling component sgg induces naked cuticle in the trunk, as expected for constitutive Wg signaling, but in the prothorax no beard develops, contrary to the effects of ectopic Wg signaling (Angelats, 2001).

Tsh activity is also critical for the identity of the prothoracic segment, raising the possibility that Tsh cooperates with the Hh and Wg signaling pathways for patterning of the beard. In conclusion, it is thought that different combinations of dTcf/Arm, Ci, and Tsh proteins are acting to specify the naked cuticular choice, both in different A/P positions along the body and at distinct positions within segments, presumably by acting on common and overlapping sets of downstream target genes (Angelats, 2001).

The differential effects of ectopic Ci or ArmS10c along the A/P axis for the induction of naked cuticle may depend on the Hox proteins, which are known to act in distinct parts of the trunk for segmental identity in combination with Tsh. For example, tsh cooperates with the Sex combs reduced Hox gene for patterning of the prothorax. These results are consistent with the idea that combinations of signaling effectors, Tsh and Hox proteins determine epidermal patterning, since their binding sites are often clustered on the enhancers of target genes (Angelats, 2001).

Consistent with the idea that signaling effectors and Tsh act together during epidermal patterning, Ci, Arm, and Tsh form protein complexes in vivo. Tsh is a phosphoprotein whose phosphorylation is induced in part by Wg signaling. Additionally, hyper-phosphorylated forms of Tsh are found in the nucleus whereas hypophosphorylated forms are predominantly in the cytoplasm. By coimmunoprecipitation, only a hyperphosphorylated form of Tsh coimmunoprecipitates with Ci, suggesting that the interaction between the two proteins takes place in the nucleus. However, only hypo-phosphorylated Tsh interacts with Arm (Angelats, 2001).

These results favour the existence of bipartite complexes (Arm-Tsh and Tsh-Ci) rather than tripartite complexes in vivo. However, the existence of a complex containing these three molecules cannot be excluded (Angelats, 2001).

In Drosophila, the development of the compound eye depends on the movement of a morphogenetic furrow (MF) from the posterior (P) to the anterior (A) of the eye imaginal disc. Several subdomains along the A-P axis of the eye disc have been described that express distinct combinations of transcription factors. One subdomain, anterior to the MF, expresses two homeobox genes, eyeless (ey) and homothorax (hth), and the zinc-finger gene teashirt (tsh). Evidence suggests that this combination of transcription factors may function as a complex and that their combination plays at least two roles in eye development: it blocks the expression of later-acting transcription factors in the eye development cascade, and it promotes cell proliferation. A key step in the transition from an immature proliferative state to a committed state in eye development is the repression of hth by the BMP-4 homolog Dpp (Bessa, 2002).

Anterior to the MF, at least three cell types can be distinguished by the patterns of Hth, Ey, and Tsh expression. The most anterior domain in the eye field, which is next to the antennal portion of the eye-antennal imaginal disc, expresses Hth, but not Tsh or Ey. In a slightly more posterior domain, all three of these factors are coexpressed (region II). In a more posterior domain, Tsh and Ey, but not Hth, are coexpressed. This domain, which also expresses hairy, is equivalent to the pre-proneural (PPN) domain. The MF, marked by the expression of Dpp, is immediately posterior to the PPN domain, and therefore abuts Tsh + Ey-expressing cells (Bessa, 2002).

Domain II is the only region of the eye-antennal imaginal disc that strongly expresses all three of these transcription factors. Posterior to the MF, Hth, but not Tsh or Ey, is expressed in cells committed to become pigment cells. Hth and Ey, but not Tsh, are coexpressed in a narrow row of margin cells that frame the eye field and separate the main epithelium of the eye disc from the peripodial membrane. Finally, Hth is also strongly expressed in peripodial cells, whereas Ey and Tsh are weakly expressed in a subset of these cells (Bessa, 2002).

The expression patterns of So, Dac, and Eya were also examined in wild-type eye discs. All three of these transcription factors are expressed in the PPN domain but not in domain II. Their expression domains have the same anterior limit but different posterior limits. Furthermore, the anterior limits of their expression domains are not sharp, but instead decrease gradually as Hth levels increase. Thus, cells in the PPN domain express So, Dac, and Eya as well as Tsh, Ey, and Hairy. Anterior to the PPN domain there is a gradual transition into domain II, where cells express Hth, Ey, and Tsh, but not So, Eya, Dac, or Hairy (Bessa, 2002).

In late second/early-third-instar eye discs, before or just as the MF is initiated, most eye disc cells express tsh, hth, and ey, although the levels of Hth are lower close to the posterior margin. Therefore, at this stage of development most eye disc cells express the same combination of transcription factors as domain II of third-instar discs. In both cases, these cells are uncommitted and dividing asynchronously (Bessa, 2002).

The overlapping expression patterns of ey, hth, and tsh in domain II raised the possibility that their gene products could be functioning together. As a first test of this idea, it was determined whether their protein products could interact with each other in vitro and in vivo. Histidine (his)-tagged Hth, alone or together with its partner protein Extradenticle (Exd), specifically binds to 35S-labeled Ey and Tsh in vitro. In vivo, it was found that both Exd and Tsh could be coimmunoprecipitated (IP) from wild-type embryos with Hth. Ey and Hth could not be IPed from wild-type embryos, perhaps because the number of cells coexpressing these transcription factors is too few. These results suggest that Hth, Exd, Tsh, and Ey have the potential to interact with each other in vivo. However, additional experiments are required to definitively test this idea (Bessa, 2002).

Tests were made of the ability of these factors to regulate each other's expression in the eye disc. Clones of cells were generated that express the yeast transcription factor Gal4 in flies containing UAS-Ey, UAS-Hth, or UAS-Tsh transgenes. These clones were generated during the second instar, when all three of these genes are coexpressed throughout the eye disc, and they were analyzed during the third instar, when the Hth expression pattern is distinct from the Tsh and Ey expression patterns. Tsh or Ey overexpressing clones in the PPN domain up-regulate Hth. The ability to maintain Hth expression was limited to the PPN domain; Ey- or Tsh-expressing clones within or posterior to the MF did not alter Hth expression. Hth can maintain Ey and Tsh expression posterior to its normal expression domain. Although this effect was not limited to the PPN domain, it was only observed in ~50% of the clones generated during the second instar, suggesting that other factors or the timing of clone induction limit this response. In addition, ectopic Tsh can also induce Ey expression in a subset of the eye imaginal disc. Together with the protein interaction experiments, the ability of these transcription factors to maintain or induce each other's expression suggests that these proteins may function together in eye development (Bessa, 2002).

Expression of Tsh or Ey maintains Hth expression in the PPN domain, where Tsh and Ey are already expressed. This result is interpreted as suggesting that hth is under two competing controls: maintenance by Tsh and Ey and repression by other factors, in particular the Dpp pathway, and that expressing higher levels of Tsh or Ey can shift this balance in favor of maintenance (Bessa, 2002).

Because Hth is coexpressed and can interact in vitro with Tsh and Ey, the possibility was considered that combinations of these transcription factors might be required to repress eya and dac. Consistent with this idea, it was found that the simultaneous expression of Tsh and Hth efficiently represses eya and dac expression. Importantly, the dual expression of Tsh and Hth is maintained by Ey expression; consequently, these clones expressed all three of these transcription factors. Other pairs of these transcription factors (Hth + Ey and Tsh + Ey) were also tested, and it was found that they can also partially repress eya (Bessa, 2002).

The above results suggest that the combination of Hth + Ey + Tsh, which is normally present in domain II, is able to repress the expression of eya. To test if hth normally plays a role in the repression of these genes, hth- clones were examined. Although hth- clones anterior to the MF are rare, it was found that both dac and eya are de-repressed in anterior hth- clones (Bessa, 2002).

In summary, these data suggest that the combination of the factors expressed in domain II is necessary and sufficient to repress eya and dac. In contrast, Hth is sufficient to repress the pre-proneural gene hairy. Conversely, eya and dac, together with Dpp, repress hth as the MF advances. It is suggested that one function for this reciprocal antagonism may be to prevent premature and uncoordinated differentiation anterior to the MF. However, as the MF advances, hth must be repressed to allow differentiation to occur (Bessa, 2002).

These experiments have shed new light on the nature and function of the cells anterior to the MF. Two domains anterior to the hairy-expressing PPN domain have been defined. One of these domains (II) expresses three transcription factors: Ey, which was already known to play a central role in eye development; Hth, which also plays a role in suppressing eye development in the ventral head, and Tsh, which, because of its ability to induce ectopic eyes elsewhere in the head, has also been implicated in eye development. The results suggest that, although these cells have not committed to become a particular cell type, they are predisposed to become eye tissue. Furthermore, it is suggested that the combination of Hth, Ey, and Tsh performs at least two functions during eye development: it represses the expression of later-acting transcription factors in the eye development cascade, and it promotes cell proliferation. Each of these points is discussed and these findings are integrated with the current view of eye development (Bessa, 2002).

These experiments suggest that one of the functions mediated by Ey-Hth-Tsh is to repress eya and dac. This proposal stems from both ectopic expression experiments, showing that the coexpression of Ey, Hth, and Tsh represses these genes, and from loss-of-function experiments, showing that hth- clones anterior to the MF de-repress these genes. Similarly, hth is de-repressed in both eya- and dac- clones, suggesting that this antagonism exists in both directions. Interestingly, the antagonism between these two sets of genes is analogous to that observed in other appendages. In the leg, hth and tsh are required for the development of proximal fates, and have been shown to be mutually antagonistic with dac and Distal-less (Dll), two genes required for intermediate and distal leg fates, respectively. Similarly, in the wing, hth and tsh are required for proximal wing fates, and oppose the activity of vestigial (vg), which is required for more distal wing fates (Bessa, 2002).

It is proposed that the putative Ey-Hth-Tsh complex promotes cell proliferation in early eye discs and in cells anterior to the PPN domain in third-instar discs. This suggestion is based on three observations. (1) In young discs, when most of the growth of the eye disc occurs and before the MF initiates, all eye disc cells express all three of these transcription factors. (2) hth- clones are only rarely observed anterior to the MF. The lack of hth- clones observed in this region of the eye disc suggests that hth is playing an important role in either the survival or proliferation of these cells (Bessa, 2002).

(3) Linking this combination of transcription factors with the growth of the eye disc stems from the observation that, when coexpressed, these factors can induce cell proliferation. This was most readily observed in clones that include cells at the edge of the eye disc. These cells may be unique in the eye disc because they express wg. Interestingly, activation of the wg pathway by generating axin- clones in the eye disc also induces proliferation and the maintenance of ey, hth, and tsh expression. Thus, proliferating eye disc cells express hth, ey, and tsh and are in a state in which the wg pathway is activated. It is speculated that this state, which can be induced by the expression of Tsh, Ey, and Hth at the edge of the eye disc, mimics the normal state of eye disc cells during the second instar, when the disc is growing most rapidly. Consistent with this idea, anterior hth expression in the eye disc is autonomously lost in dishevelled- (dsh-) clones, showing that these cells require wg signaling to maintain their anterior identity (Bessa, 2002).

The proliferation-inducing ability of Ey, Tsh, and Hth is interesting in light of the fact that the mammalian homologs of hth, the meis genes, are proto-oncogenes. The proliferation observed in Drosophila may require wg signaling and the coexpression of tsh, which has been implicated in modulating wg signaling during Drosophila development. Given these findings, it will be of interest to determine if the oncogenic potential of the meis genes also depends on the activities of wg and/or tsh homologs (Bessa, 2002).

Transcription factors often act in unique combinations to elicit distinct biological outputs. The combination examined here is Ey-Hth-Tsh. Because Hth and Tsh are also required for leg and wing development, Ey must make this combination specific for eye development. It is suggested that this combination of factors is used transiently during eye development to promote the proliferation of eye disc cells and to prevent the premature expression of later-acting transcription factors that are required for eye development. Consistent with this second role, ectopic expression of Hth blocks eye development. Similarly, forcing the expression of Ey can also interfere with eye development. The ability of these factors to repress eye development may in part be due to the ability of the Ey-Hth-Tsh combination to repress eya and dac (Bessa, 2002).

The progression of the MF across the eye is an elegant mechanism for gradually changing the combination of transcription factors as development proceeds. So, Eya, and Dac also have the ability to positively activate each other's expression, as is the case with Hth, Ey, and Tsh. Thus, both ahead of and behind the MF, eye disc cells are in different, but relatively stable states, in part because the factors expressed within these regions -- Hth, Tsh, and Ey in domain II and Eya, So, and Dac posterior to the MF -- can reinforce each other's expression. These two states are important for promoting proliferation and differentiation, respectively. Signals coming from the MF convert one state into another, and a key to flipping this switch is the repression of hth. Remarkably, in the vertebrate retina, Sonic hedgehog, a homolog of Drosophila Hh, is expressed in a wave-like fashion as retina cells differentiate. Furthermore, Pax6, the vertebrate ey homolog, is required to keep retinal cells multipotent: this is reminiscent of the uncommitted state of anterior cells in the fly eye disc. Given these intriguing parallels, it will be very interesting to determine if homologs of hth and tsh play analogous roles in the vertebrate retina before the initiation of differentiation (Bessa, 2002).

In the embryonic midgut of Drosophila, Wingless (Wg) signaling elicits threshold-specific transcriptional response, that is, low-signaling levels activate target genes, whereas high-signaling levels repress them. Wg-mediated repression of the HOX gene Ultrabithorax (Ubx) is conferred by a response sequence within the Ubx B midgut enhancer, called WRS-R. It further depends on the Teashirt (Tsh) repressor, which acts through the WRS-R without binding to it. Wg-mediated repression of Ubx B depends on Brinker, which binds to the WRS-R. Brinker binds to a site distinct from that occupied by the Wg effector, the Pangolin/Armadillo activator complex. Brinker thus acts at short range to block the activity of this complex. Furthermore, Brinker blocks transcriptional activation by ubiquitous Wg signaling. Brinker binds to Tsh in vitro, recruits Tsh to the WRS-R, and mutual physical interactions are found between Brinker, Tsh, and the corepressor dCtBP. This suggests that the three proteins may form a ternary repressor complex at the WRS-R to quench the activity of the nearby-bound Pangolin/Armadillo transcription complex. Finally, brinker and tsh produce similar mutant phenotypes in the ventral epidermis, and double mutants mimic overactive Wg signaling in this tissue. This suggests that Brinker, which was initially discovered as an antagonist of Dpp signaling, may have a widespread function in antagonizing Wg signaling (Saller, 2002).

Brinker can bind to the corepressor dCtBP, so Brinker may recruit dCtBP instead of, or in addition to, Groucho. Recall that Tsh plays a critical role in the Wg-mediated repression in the midgut. Moreover, Tsh can bind to Brinker as well as to dCtBP, so it seems plausible that Tsh plays a pivotal role in assisting Brinker in the recruitment of dCtBP. Like Groucho, dCtBP is a corepressor with quenching activity. In addition, Tsh may itself be involved in the quenching process. It has been suggested that quenching may be based on obstruction of the interaction between the activation domain of a transcriptional activator and the general transcription machinery -- intriguingly, hypophosphorylated Tsh binds to the carboxy-terminal activation domain of Armadillo to modulate Wg signaling (Saller, 2002 and references therein).

The Drosophila midgut has provided a model system in which Wg signaling regulates gene transcription in a concentration-dependent manner; low signaling levels activate Wg target genes, whereas high levels repress the same genes. The discovery that Brinker confers transcriptional repression by Wg completes the picture of the DNA-binding proteins that interpret these different signaling thresholds. Pangolin confers Wg-induced stimulation of target genes, but its activity can be blocked by Brinker, which confers Wg-mediated repression of the same genes. Pangolin depends on Armadillo for its activity, whereas Brinker depends on Tsh to block the activity of the Pangolin/Armadillo complex. In turn, the availability of Armadillo depends directly on Wg signaling, which promotes its stabilization and nuclear translocation, whereas the availability of Tsh depends on transcription of its gene (which itself depends on wg). In other words, high Wg signaling induces locally the expression of the Tsh corepressor, which then cooperates with Brinker to repress Wg target genes in the same cells. One of these targets is wg itself, so Brinker and Tsh take part in the negative feedback loop of Wg signaling in the middle midgut (Saller, 2002).

Ubx B is not only a Wg-responsive enhancer, but it is also stimulated by Dpp signaling. Furthermore, Dpp signaling antagonizes Wg-mediated repression. This can be explained in two ways: (1) high levels of Dpp-activated Mad are expected to compete with Brinker for binding to the WRS-R; (2) the brinker gene itself may be down-regulated by Dpp signaling, since this is the case in other tissues, so Brinker may only be present at very low levels in cells within the Dpp-signaling domain. brinker expression cannot be detected in this domain, whereas low levels of expression are detectable in the neighboring Wg-signaling domain. In contrast, in the latter domain, in which the levels of activated Mad are expected to be low, Brinker successfully competes with Mad for binding to the WRS-R and, together with Tsh, which is present at high levels in this domain, blocks the activity of Pangolin/Armadillo. Note that Dpp signaling promotes this repression indirectly, by contributing to the stimulation of Tsh expression in ps8 (Saller, 2002).

Dpp and Wg signaling cooperate in multiple developmental contexts. In some contexts they synergize, whereas in other contexts, they antagonize each other. Given that most, if not all, Dpp target genes, and multiple Wg target genes, are repressible by Brinker, this suggests that Brinker may have a universal key role in this decision between synergy and antagonism: absence of Brinker allows synergy between Dpp and Wg, whereas presence of Brinker (and Tsh) mediates antagonism (Saller, 2002).

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

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