Targets of activity

During the evolution of insects from a millipede-like ancestor, the Hox genes are thought to have promoted the diversification of originally identical body structures. In Drosophila, antennae and legs are homologous structures that differ from each other as a result of the Hox gene Antennapedia (Antp), which promotes leg identities by repressing unknown antennal-determining genes. Four lines of evidence are presented that identify extradenticle (exd) and homothorax (hth) as antennal-determining genes. (1) Removing the function of eitherexd or hth (which is required for the nuclear localization of Exd protein), transforms the antenna into leg; such transformations occur without activation of Antp. (2) In most antennal cells, hth is expressed and Exd is nuclear, whereas both are restricted to proximal cells of the leg. (3) Antp is a repressor of hth. (4) Ectopic expression of Meis1, a murine hth homolog, can trigger antennal development elsewhere in the fly. Taken together, these data indicate that hth is an antennal selector gene, and that Antp promotes leg development by repressing hth, consequently preventing the nuclear transport of Exd (Casares, 1998).

Now that it is clear that hth determines antennal fate, it is worthwhile reconsidering the transformation to leg that is produced by hth or exd mutant cells in the antenna. This is the same phenotype seen with dominant Antp mutants, but the leg develops without the activity of Antp, Scr or Ubx. It follows that a leg can be generated without Hox activity, suggesting that the leg pathway is the ground state for ventral appendages. Thus the ground pattern for both larvae and adults is thoracic. Nor does Antp "select" for a specific leg pathway -- it simply represses hth in the leg primordia, thereby blocking antennal development and allowing the development of legs by default. This supports the idea that Antp promotes a ground (mesothoracic) pattern by repressing cephalic genes. This basal pattern is modified by Scr toward prothoracic (first leg) or by Ubx toward metathoracic (third leg) in their respective primordia. The downregulation of hth by Antp explains the phenotype of the dominant Antp mutants is due to homothorax repression. It also explains the ability of other Hox genes such as Ubx, abdominal-A, and Abdominal-B to induce the transformation of antennae into legs. These genes prevent the nuclear translocation of Exd (most likely through hth repression), so the antennal to leg transformations are probable nonspecific and caused by a property that is common to Antp and other Hox proteins (Morata, 1998 and Casares, 1998).

During embryogenesis, in contrast with leg development, Antp selects for a specific developmental pathway. Loss-of-function mutations and experiments to induce ectopic expression show that Antp determines the larval mesothoracic pattern -- a function that is clearly distinct from the other Hox genes. Why legs should be different is not clear, but different Hox genes have similar effects on appendages, possibly because these appendages have no hth activity, without which the Hox genes lack specificity (Morata, 1998 and references).

One possible mode of Hth/Exd action in suppressing eye development is by activating Wingless signaling, which suppresses the initiation of the morphogenetic furrow (MF) in the eye disc or by suppressing Dpp signaling, which is required for MF initiation. Hth is present in the periphery of the eye disc, weaker in the posterior margin, and stronger in the anterior half of the lateral margins. The distribution is consistent with a role in suppressing dpp expression (expressed in the posterior and lateral margins but not in the most anterior of the lateral margins) and activating wg expression (expressed in the anterior half of the lateral margins). hth mutant clones located in the ventral region of the head can cause ectopic eye formation, whereas clones in the dorsal region have no effect. This correlates with the stronger expression and inhibitory effect of wg in the dorsal margin. hth-expressing clones can block the propagation and possibly the initiation of the MF. These effects are consistent with either a loss of Dpp function or an enhanced Wg function. Another possible mode of Hth/Exd action is by interacting negatively with a nuclear protein, which is required for eye development. A similar mode of action has been proposed for proboscipedia (pb). Ubiquitous expression of a mutant Pb protein can suppress eye development. The effect is independent of DNA binding by Pb and is attributable to perturbed interaction with other proteins. The many nuclear factors required for eye development (eyeless, dachshund, sine oculis, and eyes absent) are candidates for this suppressive interaction with Exd or Hth (Pai, 1998).

The cap'n'collar locus encodes three transcript and protein isoforms. The cncB transcript is expressed in an embryonic pattern that includes the labral, intercalary and mandibular segments, while cncA and cncC are expressed ubiquitously. CncB suppresses the segmental identity function of the Hox gene Deformed (Dfd) in the mandibular segment of Drosophila embryos. Evidence has been provided that the CncB-mediated suppression of Dfd requires the Drosophila homolog of the mammalian small Maf proteins, Maf-S, and that the suppression occurs even in the presence of high amounts of Dfd protein. Interestingly, the CncB/Maf-S suppressive effect can be partially reversed by overexpression of Homothorax (Hth), suggesting that Hth and Extradenticle proteins antagonize the effects of CncB/Maf-S on Dfd function in the mandibular segment (Veraksa, 2000).

Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development

Homothorax is shown to limit Dpp and Wg expression in the leg disc. Expression of the Dpp and Wg targets omb and H15 is restricted to those cells that do not express Hth. To determine if hth inhibits target gene activation by Dpp and Wg, hth was either removed from its endogenous domain or either a GFP-Hth fusion protein or the murine hth homolog MEIS-1B was misexpressed in the distal portion of the leg disc. Removing hth function results in the expansion of wg and dpp target gene expression. Dorsally situated hth- clones result in the expansion of omb expression, as marked by the omb-lacZ reporter gene. Does hth repress Distal-less and dachshund? Similar to removing exd function, when hth loss-of-function clones were examined, dac was found to be only partially derepressed, and derepression was found to be more likely to occur in clones that arise near endogenous dac expression. hth- clones have no effect on Dll expression, regardless of where they are situated. However, when clones of GFP-Hth- or MYC-MEIS-expressing cells are generated, both Dll and dac can be repressed. These results suggest that the expression of Dll and dac requires two conditions: (1) the absence of Hth and (2) sufficient activity in the Dpp and Wg pathways. High levels of Wg and Dpp signaling are shown to repress the nuclear localization of Exd by repressing hth transcription. The direct action of both the Wg- and Dpp-signaling pathways is required to specify cell fates along the P/D axis. High levels of Wg and Dpp signaling are required to activate Dll, a determinant of distal cell fates, and to repress expression of dac, a determinant of intermediate fates along the P/D axis. At intermediate levels of Wg and Dpp signaling, dac, but not Dll, is activated. The distal edge of hth expression coincides with the proximal edge of dac expression, suggesting that the threshold of Dpp and Wg signaling required to activate dac is similar to that required to repress hth. To test this idea, either Wg or Dpp signaling was elevated in the hth expression domain by generating clones of cells that express either a membrane-tethered form of Wg or an activated Dpp receptor, Thickveins QD (TKV QD). When Wg-expressing clones were generated dorsally, where endogenous Wg levels are low but where Dpp is present at high concentrations, there was a loss of Hth protein and a shift of Exd protein to the cytoplasm. This suggests that sufficient levels of both Wg and Dpp signaling are required to repress Hth (Abu-Shaar, 1998).

High levels of Wg and Dpp signaling are shown to affect Hth and Exd, at least in part, by repressing hth transcription. The ability of Wg and Dpp to repress hth appears to be indirectly mediated by Dll and dac. Like Dll, Dac appears to have the capacity to repress hth. The expression patterns of Hth and Dll were examined in leg discs that were entirely devoid of dac function. In these discs, the hth domain appears expanded distally and the Dll domain appears to be expanded proximally, consistent with the idea that dac normally represses both hth and Dll (Abu-Shaar, 1998).

The domains of gene expression for Hth, Dac and Dll, as well the regulatory interactions between them, suggest that the leg is functionally divided into two major domains. The first is a proximal domain, which expresses hth, has nuclear Exd and does not express at least some of the potential target genes of the Wg- or Dpp-signaling pathways. The second is a distal domain, which does not express hth, has Exd localized to the cytoplasm, and expresses the targets of Wg, Dpp and Wg+Dpp signaling. These data suggest that the proximal domain is what has been referred to as the coxopodite, or an extension of the body wall, and is distinct from the distal domain, the telopodite. hth expression and nuclear Exd in the coxopodite would restrict the ability of the Wg and Dpp signals to activate their target genes. This idea is consistent with the observation that these two domains differ with respect to their requirement for Hh signaling: unlike the telopodite, which exhibits severe truncations upon the reduction of hh function, the coxopodite is less severely affected. These two domains also appear to have different cell surface properties; cells from one domain prefer not to mix with cells from the other domain. For example, Dll mutant clones almost always relocalize to the hth-expressing domain and hth mutant clones frequently sort into distal regions of the leg disc. This phenomenon is not observed in the wing disc, where hth and Dll are restricted to outside and within the wing pouch, respectively: hth or Dll mutant clones are positioned randomly in this tissue. The mutant phenotypes displayed by the loss of coxopodite gene function are qualitatively different from those displayed by the loss of telopodite gene function. Removal of coxopodite genes such as exd results in either ‘nonsense’ or proximal to distal cell fate transformations, whereas removal of telopodite gene functions such as Dll and dac results in deletions of the appendage. In summary, the data support the idea that the proximal and distal regions of the leg have independent origins and differ from each other primarily due to the expression of hth, which limits or alters the ability of proximal cells to respond to Wg and Dpp signaling (Abu-Shaar, 1998 and references).

dac and Dll are shown to mediate Wg and Dpp mediated repression of hth. The demonstration that Wg and Dpp signaling repressed hth transcription and Exd’s nuclear localization was surprising, because these two signaling molecules induce Exd’s nuclear localization in the endoderm of the embryonic midgut. An investigation was carried out into the possibility that the repression of hth by Wg and Dpp is indirect and perhaps mediated by dac and Dll, which are not expressed in the midgut. TKV QD-expressing clones were generated and Hth, Dll and Dac were examined. Loss of function clones of Dll and dac were generated. When Dll- clones were generated before ~72 hours of development, hth was found to be derepressed and Exd was nuclear. However, clones generated after ~72 hours have no effect on hth or Exd, suggesting that there is an alternative mechanism for maintaining hth repression. Like Dll, Dac appears to have the capacity to repress hth. The ability of Dac to repress hth expression was confirmed by generating dac- clones. These dac- clones suggest that there might be other regulators of hth in addition to dac and Dll. Completely removing dac function results in viable animals that have deletions along the P/D axes of their legs. The expression patterns of Hth and Dll were examined in leg discs that were entirely devoid of dac function. In these discs, the hth domain appears expanded distally and the Dll domain appears to be expanded proximally, consistent with the idea that dac normally represses both hth and Dll. It is an apparent paradox that Wg and Dpp repress hth in the leg disc while these same signals activate hth expression and nuclear Exd in the midgut endoderm. This may be explained because in the leg, Wg and Dpp repress hth indirectly, by activating the hth repressors Dll and dac. In the absence of Dll or dac, hth is derepressed in the leg disc, even in cells that receive high levels of the Wg and Dpp signals. In contrast, in the embryonic endoderm, dac and Dll are not activated by Wg and Dpp, nor are any other known hth repressors, allowing hth to be activated in these cells (Abu-Shaar, 1998).

Proximal to distal cell communication in the Drosophila leg provides a basis for an intercalary mechanism of limb patterning

Patterning in insect legs is organized along anteroposterior (AP), dorsoventral (DV) and proximodistal (PD) axes. In the case of Drosophila, AP and DV axes of the leg imaginal discs are established along the embryonic AP and DV axes, which are set up based on maternal positional information. The PD axis, however, is zygotically specified by cellular interactions involving the secreted signaling molecules Wingless and Decapentaplegic (Goto, 1999 and references).

PD axis formation in the leg disc first becomes evident when cells expressing either Escargot (Esg) or Distal-less (Dll) are arranged in a circular pattern. Dll expression defines the central, distal domain. Esg-expressing cells become the proximal domain, which surrounds the distal domain. The Meis family homeodomain protein Homothorax (Hth) is expressed in the proximal domain as well as in the surrounding body wall. Hth regulates nuclear localization of another homeodomain protein, Extradenticle (Exd). Exd is active in the nucleus but inactive in the cytoplasm. The genetic requirements for Dll, Exd and Hth suggest that the distal domain gives rise to the majority of the adult leg including tarsus, tibia, femur and trochanter and that the proximal domain gives rise to the coxa and the ventral thoracic body wall. Initial PD subdivision in the embryonic leg disc becomes elaborated during larval stages by activation of additional genes, such as dachshund (dac), in a circular intermediate domain between the distal and proximal domains. dac is required for specification of the intermediate fate (Goto, 1999 and references).

The leg imaginal disc is also divided into a posterior compartment, which expresses the secreted molecule Hedgehog (Hh) and an anterior compartment, which responds to Hh by expressing Wg and Dpp along the AP compartment boundary. Mutual repression between Wg and Dpp limits Wg expression to the ventral side and Dpp expression to the dorsal side. This spatial restriction of Wg and Dpp expression is essential for DV patterning of the leg. In addition, graded activities of Wg and Dpp are required for the expression of Dll and dac and repression of hth in the distal domain. In the proximal domain, target gene activation by Dpp and Wg is inhibited by Hth and Exd, suggesting that the distal and proximal domains have distinct characters to respond to Dpp and Wg (Goto, 1999 and references).

Based on the above observations, it was proposed that the circular patterns of gene expression along the PD axis in the distal domain are organized by the gradient of the combined activity of Dpp and Wg. In the central, distal region, where combined activity of Dpp and Wg would be high, Dll is activated and dac is repressed. An intermediate level of Wg and Dpp activities would allow dac expression in the intermediate domain. Ectopic expression of Dll in the dorsal-proximal region induces wg, which is thought to interact with dpp to specify a new PD axis. These results suggest that the combination of Wg and Dpp constitute a 'distalizing' signal for the PD axis (Goto, 1999 and references).

Although these results suggest that the combination of Wg and Dpp activities centered at the distal tip is essential for PD patterning, it is not known whether Wg and Dpp are sufficient to account for all aspects of PD positional information. In fact, the grafting and regeneration experiments using larval cockroach legs suggest that the reciprocal communication between distal and proximal parts of a leg segment promotes regeneration of the intermediate part. Thus it can be speculated that a proximal to distal cell communication may also be used in PD patterning of the leg during development. Esg is expressed in the proximal domain throughout leg development. Ectopic expression of Esg and its activator Hth in the distal domain induces the intermediate fate in surrounding cells by inducing dac expression. Esg and Hth-expressing cells in the distal domain undergo a change in their adhesive property to sort out from surrounding cells. The proximal to distal inductive communication is unexpected from the model based on the graded activity of Dpp and Wg. Thus an intercalary mechanism that elaborates the PD axis pattern of the leg has been proposed. During the transition from the second to third instar, dac expression in the intermediate domain is induced by (1) a combination of a signal from proximal cells, and (2) Wg and Dpp signaling from the AP compartment boundary. The range of each signaling limits dac expression to the intermediate domain. The proximal to distal signaling dependent on Esg and Hth may provide a molecular basis for the intercalary expression of dac (Goto, 1999 and references).

Thus, it has not been clear whether Wingless and Decapentaplegic are sufficient for the circular pattern of gene expression in the Drosophila leg. A proximal gene escargot and its activator homothorax have been shown to regulate proximodistal patterning in the distal domain. Clones of cells expressing either escargot or homothorax placed in the distal domain induce intercalary expression of dachshund in surrounding cells and reorient the planar cell polarity of those cells. escargot and homothorax-expressing cells also sort out from other cells in the distal domain. Thus, inductive cell communication between the proximodistal domains is the cellular basis for an intercalary mechanism, involving expression of dachshund, during proximodistal axis patterning of the limb (Goto, 1999).

Proximal cell identity is, at least in part, controlled by the homeodomain protein Hth, which regulates nuclear localization of Exd. When expressed ectopically in the tarsal region, Hth causes non-cell-autonomous induction of dac expression and reversal of bristle and cell polarity. These phenotypes are very similar to those caused by Esg. Unlike esg-expressing clones, which secrete a smooth cuticle, hth-expressing clones in the distal part of the leg sometimes form thick socketed bristles without bracts, a characteristic of the bristles in the proximal part of the leg. Hth strongly activates a reporter gene under the control of the esg enhancer in the distal domain, but it does so weakly, if at all, in the proximal domain. This effect is cell-autonomous, suggesting that Hth may directly regulate transcription of esg. In contrast, neither a loss nor a gain of esg expression affects the activity of Hth/Exd as assessed by the expression of Hth and nuclear localization of Exd, nor is esg expression affected by the expression of another proximal gene, teashirt. These results suggest that Esg acts downstream of Hth/Exd to regulate proximodistal patterning (Goto, 1999).

The esg- or hth-expressing clones in the distal region are round in shape with smooth borders and often invaginated basally to form vesicles in the adult legs and in the larval discs. In contrast, control clones expressing non-functional esg, which lacks the zinc-finger domain, and esg-expressing clones located in the coxa and trochanter, have ragged borders. The epithelial-type homophilic cell adhesion molecule DE-cadherin is expressed throughout the leg discs and its apical localization is maintained normally in esg-expressing clones, suggesting that these cells keep their epithelial character. These results of ectopic expression studies, together with the loss of function studies on hth, indicate that Hth and Esg regulate a cell surface property that distinguishes the proximal and distal domains. It is suggested that inductive cell communication between the proximodistal domains, which is maintained in part by a cell-sorting mechanism, is the cellular basis for an intercalary mechanism of the proximodistal axis patterning of the limb (Goto, 1999).

The establishment of segmentation in the Drosophila leg

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).

A general theme in patterning during development is the subdivision of tissues initially by genes expressed in broad, partially overlapping domains, which through combinatorial control, subsequently regulate the expression of downstream genes to generate a repeating pattern. The studies presented here demonstrate that leg segmentation follows this same theme. The 'leg gap genes' Hth, Dac, and Distal-less are expressed in broad domains in the leg disc that encompass more than a single segment. Initially expression of these genes is largely nonoverlapping, but as the leg disc grows, the expression patterns of the leg gap genes change such that five different domains of gene expression are established. The analysis of the regulation of Notch ligand and fringe expression during leg development reveals two fundamental aspects of leg development. (1) These leg gap genes are key components in regulating the expression of the molecules controlling segmentation. Indeed, the effect of these leg gap genes on leg segmentation and growth can be accounted for by their regulation of Serrate, Delta and fringe expression. (2) The expression of each ring of Serrate, Delta and fringe is controlled by its own unique combination of regulators, apparently acting through independent enhancers (Rauskolb, 2001).

How do these three transcription factors regulate the formation of nine segments? Since the requirements for and the expression of the leg gap genes encompasses all leg segments, it is unlikely that there are additional leg gap genes yet to be identified. Rather, a collection of distinct combinatorial approaches is used to establish a segmental pattern of Serrate, Delta and fringe expression (Rauskolb, 2001).

In early third instar leg discs, there are two domains of gene expression: proximal cells express Hth and distal cells express Distal-less. Hth autonomously promotes the expression of Serrate, while Distal-less may prevent expression more distally, giving rise to a ring of expression in the coxa. Additionally, Distal-less-expressing cells may signal to the Hth-expressing cells to restrict Serrate expression to the distal edge of the Hth domain. As the leg disc grows, cells in an intermediate position, lying between the Hth and Distal-less domains, begin to express Dac. Dac, as shown in this study, is both necessary and sufficient to induce the expression of Serrate, Delta and fringe within the femur. Since they are not expressed in all Dac-expressing cells, other factors appear to be required to promote their expression in the proximal femur. The nonautonomous induction of Serrate expression by Hth suggests that this may be accomplished by a signal (X) emanating from the neighboring Hth-expressing cells. By mid third instar stages, expression of Serrate, Delta and fringe is also observed in tarsal segments 2 and 5, within cells expressing Distal-less but not Dac. Given that Distal-less is necessary and sufficient to repress their expression, Serrate, Delta and fringe expression within the tarsus appears to be induced by a mechanism that overrides the repressive effects of Distal-less. Subsequently, expression of Serrate, Delta and fringe is observed within the tibia, in cells expressing both Dac and Distal-less. Dac is necessary for expression of Serrate within the tibia, and its role here may be to overcome the repressive effects of Distal-less. It is also worth noting that the tibia ring of expression is not established at the time when cells first express both Dac and Distal-less. This may be because Dac levels may not be sufficiently high enough to overcome the repression by Distal-less. Clearly levels of Dac expression are critical because simply increasing Dac levels is sufficient to promote Serrate expression in cells already expressing endogenous levels of Dac. This observation can be explained if high levels of Dac expression in cells already expressing Dac override the function of inhibitory regulators of Serrate expression, such as Distal-less, where the expression of these genes overlap. Although late stages of leg segmentation were not investigated in this study, it has been noted that Hth, Dac and Distal-less are co-expressed in the presumptive trochanter late in leg development. It is thus hypothesized that Serrate, Delta and fringe expression is established by the combined activities of the three leg gap genes in the trochanter (Rauskolb, 2001).

Although these here have focused on the regulation of Serrate expression, it is thought that not only Serrate, but also Delta and fringe, receive primary regulatory input from the leg gap genes. Delta and fringe expression, like Serrate, is positively regulated by Dac. Moreover, Dl and fringe mutants have stronger leg segmentation phenotypes than Ser mutants, and thus Delta and fringe expression cannot simply be regulated downstream of Ser. The identification of two separate Ser enhancers, directing expression in the proximal versus distal leg, argues against Serrate being regulated downstream of Dl and fringe. Thus, the simplest model is that expression of all three genes is regulated directly by the leg gap genes. The regulation of Serrate, Delta and fringe expression in each segment appears to occur through independent and separable enhancer elements, supported by the analysis of the Ser reporter genes. This is reminiscent of what occurs during Drosophila embryonic segmentation, where separable enhancer elements direct different stripes of pair-rule gene expression (Rauskolb, 2001).

Most of the tarsus of the Drosophila leg derives from cells expressing Distal-less, but not Dac or Hth. Surprisingly, the studies presented here have shown that Distal-less actually represses Notch ligand expression. This negative regulatory role for Distal-less contrasts with the positive promoting role of Dac and Hth, and further indicates that a distinct molecular mechanism must promote segmentation within the tarsus. One key gene is spineless-aristapedia (ss), since simple, unsegmented tarsi develop in ss mutant flies. Moreover, ss regulates the expression of bric-à-brac (bab), which is also required for the subdivision of the tarsus into individual segments. Together, ss and bab must, in some way, ultimately overcome the repression of Notch ligand and fringe expression by Distal-less. If the sole function of ss and bab is to overcome the inhibitory effects of Distal-less, then in the absence of ss and/or bab, Serrate expression is expected to remain repressed (Rauskolb, 2001).

Intriguingly, the only notable variation between insect species is in the number of tarsal segments, with an unsegmented tarsus believed to be the ancestral state. Thus, the combinatorial regulation of segmentation by the leg gap genes may represent an ancient mechanism common to all insect species, a hypothesis supported by the conserved expression of Hth, Dac and Distal-less in the developing legs of many insect species (Rauskolb, 2001 and references therein).

Coexpression of the homeobox genes Distal-less and homothorax determines Drosophila antennal identity

The Drosophila antenna is a highly derived appendage required for a variety of sensory functions including olfaction and audition. To investigate how this complex structure is patterned, the specific functions of genes required for antenna development were examined. The nuclear factors, Homothorax, Distal-less and Spineless, are each required for particular aspects of antennal fate. Coexpression of Homothorax, necessary for nuclear localization of its ubiquitously expressed partner Extradenticle with Distal-less is required to establish antenna fate. This study tests which antenna patterning genes are targets of Homothorax, Distal-less and/or Spineless. Antennal expression of dachshund, atonal, spalt, and cut requires Homothorax and/or Distal-less, but not Spineless. It is concluded that Distal-less and Homothorax specify antenna fates via regulation of multiple genes. Phenotypic consequences of losing either dachshund or spalt and spalt-related from the antenna are reported. dachshund and spalt/spalt-related are essential for proper joint formation between particular antennal segments. Furthermore, the spalt/spalt-related null antennae are defective in hearing. Hearing defects are also associated with the human diseases Split Hand/Split Foot Malformation and Townes-Brocks Syndrome, which are linked to human homologs of Distal-less and spalt, respectively. It is therefore proposed that there are significant genetic similarities between the auditory organs of humans and flies (Dong, 2002).

As with Dll and hth loss-of-function mutants, loss of spineless (ss) also results in antenna to leg transformations. The genetic relationship among these genes was investigated. The expression of both Dll and hth appears relatively normal in the ss null antennal disc. It is therefore concluded that ss is not required for either the activation or the maintenance of Dll or hth expression in the antenna. It has been reported that Dll is required for the antennal expression of ss. To test whether Hth is also required to activate antennal ss expression, the effect of ectopic hth was examined. Ectopic Hth where Dll is expressed, for example in the wing pouch and leg disc, can activate ss expression. Conversely, loss of hth in the antenna results in loss of ss expression. Taken together, these results indicate that ss functions downstream of both Dll and hth in the antenna (Dong, 2002).

There are only a few genes expressed in either the antenna or the leg but not in both. Among these are sal and salr, which are identically expressed in a ring pattern in presumptive a2, but are detected at low levels only in leg imaginal disc cells that contribute to the body wall and not to the leg itself (Dong, 2002).

In contrast, there are other genes expressed in both antenna and leg precursors that have distinct patterns in the two appendages. Among these are dac, ato, ct and ss. The domain of dac expression in the antenna (a3) is much smaller than in the leg where it is expressed in multiple segments. The function of dac in antennal development has not been described previously (Dong, 2002).

The bHLH transcription factor encoding gene, ato, is expressed in a ring in presumptive a2, but restricted to small spots in the dorsal leg disc. ato is required for the formation of most chordotonal organs in the fly. In the antenna, ato is required for formation of Johnston's organ (JO), a complex sense organ composed of a large number of chordotonal organs that is used for sensing acoustic vibrations transmitted from the arista through a3 (Dong, 2002).

cut, which is required for differentiation of external sensory (ES) class neurons, is expressed throughout the presumptive proximal antenna (a1 and a2) and head capsule but is expressed in small clusters of cells throughout the leg disc (Dong, 2002).

ss is expressed in a circular pattern in the antenna covering the presumptive a2 through the arista. In the leg disc, ss is transiently expressed in a ring pattern in the presumptive tarsal region and subsequently becomes restricted to leg bristle precursors. Consistent with the ss expression domain, cuticular defects in ss null mutants can be found from a2 through the arista. These include the elongation of a2, loss of olfactory sensilla from a3, and transformation of a4, a5, and arista to tarsal segments (Dong, 2002).

The large differences in the expression patterns of these genes between the antenna and the leg begs the question of whether these differences are due to differential regulation by antenna-determining genes such as Dll and hth. To test whether Dll or hth are responsible for the antenna-specific expression patterns of these genes, the effects on their patterns were examined in Dll and hth loss-of-function mutants. Whether Dll and hth are regulating their antenna-specific targets via ss was tested by examining their expression in ss null antennal discs (Dong, 2002).

In contrast to the leg, in the antenna dac expression is restricted primarily to a single segment (a3). Trace levels of Dac can be detected in areas of the antennal disc immediately distal and proximal to a3. Because no antennal phenotypes have been reported for loss-of-function dac mutants, it is unclear whether dac plays a role in patterning this appendage. In transheterozygous dac null mutants, a fusion of the a5 segment with the arista occurs, accompanied by a reduction in the width of the a5 segment. This fusion phenotype is similar to what is observed in dac hypomorphic and null legs. However, unlike the leg phenotype, no obvious reductions in length or loss of segments is found in the dac mutant antenna. In addition, this antennal phenotype is observed in dac null animals but not in strong hypomorphic combinations such as daclacZ/dac4. Therefore, high levels of Dac are probably not necessary for dac function in the antenna (Dong, 2002).

If Dac levels are elevated in the antenna, expression of Dll and hth is repressed and medial leg structures are induced. Therefore if Dac levels are too high, antenna development is compromised. Because bab mutants exhibit phenotypes similar to those of dac, and dac regulates bab expression in the antenna, it is likely that antennal dac function is mediated via its regulation of bab (Dong, 2002).

The antennal dac expression domain expands in Dll hypomorphs and in hth null clones. This expansion of dac expression in Dll and hth mutant antennae resembles the leg pattern of dac expression. In contrast, in the ss null antenna, there appears to be neither expansion nor reduction of dac expression. The only detectable difference in the ss null antennal disc is overgrowth in the central (distal) area such that the ring of dac expression has a larger radius. This correlates with the transformation phenotype of the ss null arista into a tarsus, which is a larger structure. Since the expression of dac relative to other genes appears normal in ss null antennae, ss is not thought to regulate dac (Dong, 2002).

The expression of ato is required for the formation of the JO. The JO is a structure unique to the antenna and is required to sense sound vibrations transmitted from the arista. ato function is generally associated with neuronal differentiation, so it is interesting that cuticular defects are associated with ato null antennae. It may be that formation of the JO is required for the normal morphology of the a2/a3 joint. The circular outline of the a2/a3 joint is lost in hth and Dll loss-of-function mutants, but is present in ss null mutants. Consistent with this, the antennal expression of ato is lost in hth null clones and in Dll hypomorphs, but persists in the ss null antenna discs. Thus although ss null mutants exhibit cuticular defects in a2 and a3, the a2/a3 joint to which the JO is attached is present. It is noted that the Dll hypomorphic combination used, DllGAL4/Dll3, does not lead to loss of a2. Thus the absence of ato expression in these antennae is not due to death of the cells that would normally express it (Dong, 2002).

sal and salr have similar sequences and are identically expressed in the antennal imaginal disc in presumptive a2. However, functions for sal and salr in the antenna have not yet been described. To investigate whether sal and/or salr are required for normal antenna development, clones null either for sal alone or for both sal and salr in the adult head were examined. Clones null for only sal in the antenna have no obvious cuticular phenotypes. However Df(2L)32FP-5 clones, which are null for both sal and salr, exhibit cuticular defects in the antenna. This supports the view that sal and salr have some redundant functions. The areas affected in the mutants are correlated with their expression domains in the antennal disc (Dong, 2002).

a2 normally forms a cup, in which a3 sits and must rotate along the PD axis, to transmit sound vibrations from the arista. An overall reduction in a2 is observed in salFCK–25/Df(2L)32FP-5 transheterozygous null antennae. In addition, a2 appears to be fused to a3 and a portion of the stalk that connects a3 to a2 is exposed. The circular outline of the a2/a3 joint, to which the chordotonal organs of the JO attach, is defective in Df(2L)32FP-5 clones and lost in salFCK–25/Df(2L)32FP-5 mutant antennae. Furthermore, a3 is unable to rotate in a2. The same antenna phenotypes are observed in salFCK–25 homozygous flies. However, these phenotypes are not observed in sal null clones generated using a sal16 FRT40A chromosome or in salFCK–25/sal16 transheterozygous antennae, that do not express sal but do express salr in the antenna. Together, the loss of the a2/a3 joint and the loss of the freedom of rotation of a3 in a2 indicate that sal/salr null antennae are defective in hearing and implicate both sal and salr in normal development of the auditory organ (Dong, 2002).

Since ato is expressed within a subset of the sal/salr domain and is activated later than sal and salr in the antenna, tests were performed to see whether Dll and hth activate ato via sal/salr. No detectable reduction of ato expression is found in a2 either in Df(2L)32FP-5 clones or in salFCK–25/Df(2L)32FP-5 transheterozygous animals. This allelic combination lacks detectable sal and salr expression in the antenna, but retains sal and salr expression in the eye. The normal expression of ato in the antennae of these mutants suggests that the activation of ato expression by Dll and hth is independent of sal/salr. Antennal sal/salr expression is also unaffected in ato null imaginal discs. Therefore, sal/salr and ato are required in parallel for development of antennae that are functional in audition (Dong, 2002).

Dll and hth are required for the expression of sal in the antenna. sal expression does not appear to be affected in ss null antenna. The fact that Ss is not required for the expression of either ato or sal in a2 is consistent with the observation that the a2/a3 joint is still present in the ss null antenna (Dong, 2002).

Expression of the homeodomain transcription factor encoded by ct almost completely fills the hth expression domain of the third instar antennal disc. In contrast, the ct and hth expression patterns in the leg disc are distinct from one another. This makes ct a strong antenna-specific candidate target for Hth. The antennal expression of ct is lost in hth null clones indicating that ct is indeed downstream of hth. To test whether the a2 expression of ct also requires Dll, ct expression was examined in Dll mutants. ct expression is not reduced in Dll null clones or in Dll hypomorphs. Therefore, although Dll and hth are both required for antennal fate, cut is an antenna-specific target of Hth activation that is independent of Dll. As with other antenna-specific targets of Dll and Hth, ct expression is also not lost in ss null antenna (Dong, 2002).

This study serves to initiate an understanding of the different roles that these homeotic genes are playing in antenna specification. During imaginal disc development, the expression of Dll and ss is found from a2, a3, a4, a5 and arista. Expression of hth is dynamic and retracts from the distal-most segments by late third instar, but hth is expressed and cell-autonomously required throughout the antenna from a1 through to the arista (Dong, 2002).

The Dll mutant phenotypes indicate that Dll is required both for the distal limb development and for antenna fate. Dll hypomorphs exhibit distal limb deletions as well as antenna to leg transformation. The transformation phenotypes of hypomorphic Dll antennae can be observed from a2 through to the arista. In these mutants, hth expression is not lost or detectably reduced. Thus medial leg structures can develop in the presence of Hth. This suggests that although loss of hth expression from the distal and medial leg, via Antennapedia-mediated repression, occurs during normal leg development, loss of Hth is not essential for leg differentiation. It also suggests that the requirement for Antennapedia in normal leg development is not only to regulate hth (Dong, 2002).

Since ss is not required to activate antenna-specific expression of genes such as sal/salr and ato that are involved in antenna differentiation, the question arises as to what ss does do in the antenna. ss represses tarsus and tarsal claw organ formation in the antenna. Since loss of ss also leads to loss of olfactory sensillae on a3, ss probably potentiates the formation of these sensillae, either cooperating with or mediating Dll and hth activities in a3. Similarly, since ectopic expression of ss elsewhere in the body can lead to the formation of ectopic aristae, ss may also cooperate with or mediate Dll and hth activities in arista differentiation (Dong, 2002).

sal and salr, like ato, are required for normal auditory functions. Since both Dll and hth are required for the antennal expression of ato and sal, Dll and hth mutant antennae are also hearing defective. In contrast, ss null antennae exhibit normal expression of both ato and sal and normal morphology of the a2/a3 joint, leading to the idea that ss mutants are likely to be functional in audition (Dong, 2002).

Homeotic genes, Dll and hth, regulate multiple targets during antennal development. These targets function in specifying antenna structures and/or in repressing leg development. For example, the ss mutant phenotype suggests that it represses leg tarsal differentiation. But ss is also required for the formation of olfactory sensory sensilla normally found in a3. Although Dll and hth repress distal leg development via activation of ss, their repression of medial leg development appears to be, at least in part, independent of ss. Instead, this is achieved via their regulation of the medial leg gene, dac, to a narrower domain of expression with lower levels in the antenna as compared to the leg. sal/salr and ato are required for proper differentiation of a2. However, no transformation phenotypes are associated with the sal/salr and ato null antenna. This indicates that while sal/salr and ato are required to make particular antenna-specific structures, they do not appear to repress leg fates. Therefore homeotic genes such as Dll and hth repress the elaboration of other tissue fates in addition to activating genes required for the differentiation of particular tissues (Dong, 2002).

In third instar imaginal discs, coexpression of Dll and Hth activates sal/salr and ato in a2 where they, in turn, are needed for JO development. The expression of ato is required for the formation of the JO and the a2/a3 joint to which it is attached. Although sal and salr are not required for the expression of ato, the a2/a3 joint is lost in the sal/salr null antenna. It is expected that this leads to improper formation of the JO, although it is also possible that defects in a2/a3 joint formation preclude JO differentiation. In addition, because sal is not lost in ato null antennae, it is concluded that sal/salr and ato are required in Drosophila parallel for proper formation of the JO. Furthermore, in the sal/salr null antenna, a3 cannot freely rotate within a2. This rotation is necessary for transmission of sound vibrations from the arista to the JO. Taken together, these findings implicate sal/salr in Drosophila audition. Interestingly, mutations associated with the human homolog of sal, SALL1 cause the human autosomal dominant developmental disorder, Townes-Brocks Syndrome (TBS). Auditory defects are also associated with the human genetic disorder, Split Hand/Split Foot Malformation (SHFM), and the SHFM1 locus is linked to the Dll homologs, DLX5 and DLX6. The sensorineural hearing defects associated with the Distal-less and spalt genes in both Drosophila and Homo sapiens, in conjunction with a recent finding that atonal functions in mouse as well as fly audition, leads to the proposal that insect and vertebrate hearing share a common evolutionary origin. Further developmental genetic dissection of the Drosophila auditory system should therefore provide additional insights into human ear development and suggest that Drosophila could provide a useful model system for studying both TBS and SHFM (Dong, 2002).

In the wing wingless is expressed in a complex and dynamic pattern that is controlled by several different mechanisms. These involve the Hedgehog and Notch pathways and the nuclear proteins Pannier and U-shaped. The mechanisms that drive wingless expression in the wing hinge have been analyzed. Evidence is presented that wingless is initially activated by a secreted signal that requires the genes vestigial, rotund and nubbin. Later in development, wingless expression in the wing hinge is maintained by a different mechanism, which involves an autoregulatory loop and requires the genes homothorax and rotund. The role of wingless in patterning the wing hinge is discussed (Rodriguez, 2002).

The adult wing is formed by a continuous monolayer of epidermal cells that folds to form the dorsal and ventral surfaces of the wing pouch. The two surfaces contact at the margin of the wing and extend proximally through the wing hinge to the dorsal notum and the ventral pleura. In the presumptive wing region of the wing disc, wg is expressed in a narrow stripe of cells that runs all along the wing margin and in two rings that surround the wing pouch. The phenotypes and wg expression have been examined in several mutants in which the wing hinge is deleted (Rodriguez, 2002).

Several results presented here indicate that Wg signaling activates hth expression, which is in turn required to maintain wg expression. wg and hth are co-expressed in the IR (inner ring) and OR (outer ring), and wg expression precedes hth expression. spd mutations are a type of wg allele that specifically removes wg expression from the IR, with no effects on other expression domains. hth expression is missing in spdfg discs, and wg expression is lost in hth mutant clones. Nevertheless, spdfg discs show activation of the IR enhancer, as revealed by the spd-lacZ construct and wg expression is not affected in hth mutant clones when observed in early third instar larvae. This indicates that Hth, while required to maintain IR activation, is not required to initiate wg expression (Rodriguez, 2002).

distal antenna and distal antenna related encode nuclear proteins containing pipsqueak motifs involved in antenna development in Drosophila

Legs and antennae are considered to be homologous appendages. The fundamental patterning mechanisms that organize spatial pattern are conserved, yet appendages with very different morphology develop. The distal antenna (dan) and distal antenna-related (danr) genes encode novel 'pipsqueak' motif nuclear proteins that probably function as DNA binding proteins serving as sequence-specific transcription factors but may serve instead as more general chromatin modification factors. dan and danr are expressed in the presumptive distal antenna, but not in the leg imaginal disc. Ectopic expression of dan or danr causes partial transformation of distal leg structure toward antennal identity. Mutants that remove dan and danr activity cause partial transformation of antenna toward leg identity. Therefore it is suggested that dan and danr contribute to differentiation of antenna-specific characteristics. Antenna-specific expression of dan and danr depends on a regulatory hierarchy involving homothorax and Distal-less, as well as cut and spineless. It is proposed that dan and danr are effector genes that act downstream of these genes to control differentiation of distal antennal structures (Emerald, 2003).

The overlap between Hth and Dll has been proposed to define antennal identity, because co-expression of the two proteins in ectopic locations can induce formation of ectopic arista structures in other discs. To ask whether Hth and Dll have a role in defining the non-overlapping expression domains of Cut and Dan/Danr, clones of cells were examined lacking hth or Dll activity in the antenna. Dan expression is lost in cells mutant for hthc1 in the region where the two expression domains overlap. This suggests that Hth activity is required for Dan expression. Likewise, clones of cells lacking Dll activity have lost Dan expression in the distal region of the disc. Many Dll mutant clones were found adjacent to the edge of the Dan domain, suggesting that loss of Dan may cause these clones to sort out proximally. Thus both Dll and Hth are required for Dan expression (Emerald, 2003).

Ectopic expression of Hth in the leg disc under dppGal4 control, induces Dan expression in distal, Dll-expressing cells. It is known that Hth-expressing cells sort out from the distal region of the disc. This is also visible in GFP labeled cells in this study. Nonetheless, dppGal4 directed expression of Hth induces Dan expression in distal cells. This raises the possibility of a non-autonomous effect of Hth expression leading to sustained Dan expression. Ectopic expression of Dll in the leg disc under dppGal4 control, induces Dan expression in proximal, Hth-expressing cells. In this case, ectopic Dan was limited to cells expressing the Gal4 driver (Emerald, 2003).

These observations indicate that the regulatory relationship between Hth, Dll and Dan (Danr) is complex. Dll and Hth are each required for Dan expression. However, it is clear that Dan is not expressed in every cell in which Hth and Dll are co-expressed in the antenna. All Dan-expressing distal antenna cells express Dll but not all express Hth. These observations point to a non-autonomous effect of Hth on Dan expression, which may explain how Hth can be required for sustained expression of Dan in distal cells where Hth is not expressed (Emerald, 2003).

The hernandez and fernandez genes of Drosophila specify eye and antenna

The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of 'eye-specifying' genes, among them eyeless, which can direct eye development ectopically. hernandez (distal antenna related or danr) and fernandez (distal antenna or dan) are expressed in the antennal and eye primordia of the eye-antenna imaginal disc (see Dan and Danr). Hernandez and Fernandez are the names of twin brothers in Tintin comic-books. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: such expression can form distal antenna, activating genes like homothorax, spineless, and spalt, and can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since both the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. These results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically (Suzanne, 2003).

To test whether hern and fer are sufficient to induce eye or antennal development, they were expressed ectopically using the GAL4/UAS system. When either the hern or the fer genes are misexpressed in the leg discs with dpp-GAL4 or Dll-GAL4 (EM212) drivers, distal legs are transformed to aristae. These transformations are accompanied by the ectopic expression of hth, sal, and ss, three genes expressed in the antennal primordium but not in the distal region of mature wild-type leg disc. Clones expressing either the hern or the fer genes in the leg or wing disc have smooth borders and frequently activate the sal and hth genes cell-autonomously. In dpp-GAL4/UAS-fer or ptc-GAL4/UAS-hern leg (or wing) discs, the expression of ss is also activated. Curiously, although ss is downstream of hth in the antenna and leg, ectopic ss in the leg disc can also activate hth in a few cells (Suzanne, 2003).

The hth or ss genes, together with Dll, are sufficient to develop ectopic distal antennae when expressed in different regions of the adult. The hern or fer genes are also able to elicit this transformation in the leg and they activate hth and ss. Conversely, when high levels of the Hth or Ss products are induced in the leg discs, ectopic expression of the hern and fer genes is found. To study the interactions between these genes in normal development, the relationship between Dll, hth, ss, and hern/fer in the antennal primordium was examined. A reduction of Hth activity using a dominant negative form of hth (UAS-EN-HTH1-430) results in a decreased activity of the MD634 and AC116 GAL4 lines, which reveal hern and fer expression, respectively. Similarly, in antennal discs of a Dll strong hypomorph or a ss null mutation, the expression of hern and fer disappears. These results suggests that hth, Dll, and ss are required to maintain hern and fer expression in the antenna. By contrast, high levels of hern or fer may reduce hth expression. In dpp-GAL4/UAS-fer or dpp-GAL4/UAS-hern larvae, the expression of hth (and sal) in the third antennal segment is eliminated or strongly reduced dorsally (where levels of hern and fer are high) and does not change or is ectopically activated ventrally (where levels of hern and fer are low). Similarly, fer-expressing clones are able to downregulate hth expression in the antennal primordium. These results suggest that levels of hern and fer expression may be important for a normal antennal development (Suzanne, 2003).

The differentiation of legs or antennae depends on the activity of the hth and Antp genes. The ss gene, however, is also able to transform distal leg (and also maxillary palp and rostral membrane) into distal antenna, and the absence of ss, like that of hth, transforms antenna into leg. Although ss seems to be downstream of Dll and hth in antenna specification, ectopic ss can activate hth in some cells of the leg disc. Similarly, misexpression of ss in the rostral membrane induces Dll expression. It seems, therefore, that ss can trigger an antennal genetic program when misexpressed in certain places (Suzanne, 2003).

The fer and hern genes are both required and sufficient to make part of the distal antenna. Four different genes, hth, ss, hern, and fer, are able to form distal antenna, together with Dll, when ectopically expressed. Their mutual regulation seems to differ when misexpressed in the leg disc or when normally expressed in the antennal primordium. In the leg disc, hern or fer activates hth and ss and, reciprocally, hth and ss induce hern and fer expression. Moreover, even ss can promote hth transcription, although just in a few cells. Taken together, these results suggest that the four genes can form distal antenna by activating each other's transcription when ectopically expressed (Suzanne, 2003).

In the third antennal segment, Dll, hth, and ss are required to activate hern/fer expression. Since ss is downstream of Dll and hth in the antenna, the activation of hern/fer by Dll and hth could be mediated by ss. It is noted, however, that the levels of hern and fer may modulate hth expression. Moderately increased levels of fer can activate hth in dpp-GAL4/UAS-fer discs but, when the levels of hern or fer in the antenna are highly increased, the transcription of hth is prevented. These results suggest that the total amount of hern and fer expression may be regulated in the antennal primordium. Accordingly, in clones mutant for danr (hern), the expression of dan (fer) is upregulated. Also supporting the conclusion that levels of hern and fer have to be regulated, it was found that, in ey-GAL4/UAS-hern or ey-GAL4/UAS-fer flies, where levels of either hern or fer are highly increased in the eye–antennal disc, both the eye and the antenna disappear (Suzanne, 2003).

The hern and fer genes can form ectopic aristae and eye tissue, but only in a limited number of regions of the adult cuticle. This is similar to what happens with other genes making ectopic antennae (hth, ss) or eye (eye-specification genes). This is due to the particular developmental context of the region where the genes are ectopically activated (Suzanne, 2003).

Specificity of Distalless repression and limb primordia development by Abdominal Hox proteins

In Drosophila, differences between segments, such as the presence or absence of appendages, are controlled by Hox transcription factors. The Hox protein Ultrabithorax (Ubx) suppresses limb formation in the abdomen by repressing the leg selector gene Distalless, whereas Antennapedia (Antp), a thoracic Hox protein, does not repress Distalless. The Hox cofactors Extradenticle and Homothorax selectively enhance Ubx, but not Antp, binding to a Distalless regulatory sequence. A C-terminal peptide in Ubx stimulates binding to this site. However, DNA binding is not sufficient for Distalless repression. Instead, an additional alternatively spliced domain in Ubx is required for Distalless repression but not DNA binding. Thus, the functional specificities of Hox proteins depend on both DNA binding-dependent and -independent mechanisms (Gebelein, 2002).

This work begins with a characterization of a Ubx binding site in the Dll gene that is critical for Dll repression; both Exd and Hth play a role in Ubx binding and repression. The Dll304 enhancer is sufficient to recapitulate the early expression pattern of Dll in the embryonic leg primordia. In addition to activation functions, Dll304 contains two Hox binding sites, Bx1 and Bx2, that repress enhancer activity in the abdomen and thereby restrict Dll expression to the thorax. Most of the repression activity is conferred by Bx1, a sequence bound by Ubx and Abd-A. In agreement with this result, a Distalless minimal element (DME) that lacks the Bx2 site accurately recapitulates the expression of Dll304 in the embryonic thorax. The DME enhancer also shows no derepression within the abdomen, suggesting that Bx1 is sufficient to fully repress Dll (Gebelein, 2002).

To better understand how Bx1 represses Dll, the presence of Exd and Hth binding sites were sought near the previously characterized Hox binding site. A consensus Exd site and a near consensus Hth site are in close proximity to the Hox site of Bx1. The Hox/Exd site (5'-AAATTAAATCA-3'), however, is unlike other previously characterized Hox/Exd binding sites because it contains an additional base pair in between the Hox and Exd half-sites. The Bx1 region containing this Hox/Exd/Hth site is referred to as the Distalless repression element (DllR). To determine whether DllR is required to repress DME expression in the abdomen, it was deleted from the DME enhancer (DMEact) and its ability to activate a reporter gene was tested in vivo. DMEact drives gene expression in all abdominal segments as well as in the thoracic region. Because the thoracic expression driven by DMEact is similar to that of DME, the DllR region is not required for DME activation but solely functions in the repression of Dll in the abdomen (Gebelein, 2002).

To determine whether Exd and Hth stimulate Hox binding to DllR, electrophoretic mobility shift assays (EMSAs) were performed with purified Ubx, Exd, and Hth proteins. Unless stated otherwise, all of these experiments were performed with UbxIa, the most widely expressed of several Ubx isoforms. By themselves, Ubx or an Exd/Hth heterodimer are capable of weakly interacting with DllR. The combination of all three proteins results in a slower migrating band indicating the formation of a Ubx/Exd/Hth/DNA complex. The formation of this protein/DNA complex is highly cooperative when compared to the amount of binding observed with Ubx or Exd/Hth alone. To test the contribution of each binding site, point mutations were introduced within the individual Hox, Exd, and Hth sites. Mutation of any one of these sites results in a decrease in the formation of the trimeric protein/DNA complex, suggesting that all three are required for optimal binding to DllR (Gebelein, 2002).

To test whether the Hox, Exd, and Hth binding sites are also required for Dll repression in vivo, reporter constructs were created containing the lacZ gene under the control of mutant versions of the DME enhancer. Mutation of the Hox site (DMEHox) results in a similar level of derepression of reporter gene expression throughout the abdomen, as does the the complete deletion of DllR. Mutation of the Exd (DMEExd) and Hth (DMEHth) sites individually also results in derepression, albeit slightly weaker than mutation of the Hox site. However, if both the Exd and Hth sites are mutated together, full derepression is observed. Taken together, these results demonstrate that the efficient formation of a Hox/Exd/Hth trimeric complex on DllR is required for Dll repression within the abdomen (Gebelein, 2002).

These above data support a model in which a Ubx/Exd/Hth complex bound to DllR is necessary for Dll repression. Whether a single copy of DllR is sufficient to repress a heterologous enhancer element was tested. An artificial enhancer, called fkh(250con), is activated by Scr, Antp, and Ubx (with Exd and Hth), and thus provides a useful heterologous activator to test for DllR function. A reporter construct under the control of both fkh(250con) and DllR was created. Unlike fkh(250con), which is expressed in parasegments (PS) 2-6, the composite enhancer (fkh250con-DllR) is not expressed in PS 6, where Ubx is expressed. Ubx-mediated repression of fkh(250con)-DllR is more obvious in embryos mutant for abd-A, which derepress Ubx and, consequently, fkh(250con) throughout the abdomen. In this genetic background, fkh(250con)-DllR is still active only in PS 2-5. Furthermore, misexpression of Ubx throughout the embryo activates fkh(250con) but represses fkh(250con)-DllR. Taken together, these results indicate that DllR is sufficient to confer Ubx-mediated repression of a heterologous enhancer. In addition, these results also illustrate that Ubx/Exd/Hth complexes can mediate repression through DllR in the same cells as it mediates activation through fkh(250con) (Gebelein, 2002).

A general question for all transcription factors is how they achieve specificity in vivo. For the Hox proteins, a large number of studies have implicated sequences both within and outside the homeodomain as being important for their in vivo specificities. But how do these sequences function? Because DNA binding domains, including homeodomains, can also be protein interaction domains, studies that map the domains necessary for target gene regulation cannot answer this question by themselves. Instead, direct transcriptional targets must be identified and, once binding sites are characterized, DNA binding, in addition to target gene regulation, must be measured. The results allow two steps to be discriminated in the repression of Dll by Ubx. First, Exd and Hth stimulate Ubx, but not Antp, binding to DllR. In contrast, Ubx/Exd/Hth and Antp/Exd/Hth have similar affinities for a different "consensus" binding site (5'-CCATAAATCA-3'), suggesting that subtle differences in the DNA sequence, in addition to differences between Ubx and Antp, contribute to specificity. A C-terminal peptide in Ubx stimulates this cofactor-dependent binding to DllR. DNA binding, however, is not sufficient for Dll repression. Instead, an additional linker domain included in only a subset of Ubx isoforms is required for repression. Thus, a second step, the recruitment of additional factors to the Ubx/Exd/Hth complex bound to DllR, is implied by these data. In addition to the UbxIa linker, this step also requires the specific sequences and conformation imposed on the Ubx/Exd/Hth trimer by DllR (Gebelein, 2002).

Although the Ubx C terminus plays an important role in cofactor-dependent binding to DllR, additional domains contribute to optimal binding. In the presence of Exd and Hth, the AAUU chimera, but not heterologous AAUA or AAAU, binds DllR, suggesting that both the Ubx homeodomain and C terminus are important for optimal DNA binding to this site. The C terminus is not absolutely required for binding because a Ubx protein that lacks this domain (UUU*) is still able to bind well to DllR. Last, the finding that UUU*, but not AAU*, binds DllR suggests that a domain N terminal to the homeodomain also enhances DllR binding. Based on the crystal structures of Hox/Exd/DNA complexes, this difference could be due to the YPWM motif. Taken together, the data suggest that multiple regions of Ubx contribute to binding DllR and that no one domain is sufficient for full binding activity. This finding may be understood in light of the fact that the entire Ubx coding sequence has been constrained over millions of years of insect evolution to maintain leg (and Dll) repression in the abdomen (Gebelein, 2002).

Although these experiments focused on understanding why Antp is different from Ubx, the results provide some insights into the mechanism of transcriptional repression. The data strongly argue that a DNA-bound Ubx/Exd/Hth complex is necessary, but not sufficient, for repression. First, in addition to repressing Dll, Ubx/Exd/Hth activates fkh(250con). When both fkh(250con) and DllR simultaneously regulate the same reporter gene, DllR is able to repress gene expression in the same cells in which fkh(250con) normally activates gene expression. This result suggests that the repressor proteins required for DllR activity are not cell type specific and are widely expressed in the embryo. Further, these results suggest that differences between the fkh(250con) and DllR sequences determine whether transcription is activated or repressed. These sequences may recruit additional DNA binding factors that interact with the trimeric complex. These factors, which have not yet been identified, might provide or reveal a latent activation or repression domain within the Hox/Exd/Hth complex. Alternatively, another DNA binding factor may not be needed. Instead, the unique arrangement or spacing of the Hox, Exd, and Hth sites in these two elements may result in distinct conformations of the trimeric complex that recruit different coactivators or corepressors. Such a mechanism has been suggested for the nuclear receptor family of transcription factors and for the POU domain protein Pit-1, where a difference in spacing in a Pit-1 dimer binding site regulates the recruitment of a corepressor. Consistent with such a mechanism, it was found that the DllRcon binding site, which has one less base pair between the Hox and Exd half-sites than the DllR binding site, fails to repress transcription despite having a higher affinity for Ubx/Exd/Hth complexes. In addition, although repression activity for the UbxIa linker and C terminus in S2 cells can be measured, the experiments suggest that their activities are context dependent. The abdominal expression of DMEcon-lacZ suggests that the mere presence of these domains is not sufficient for repression. Thus, the data suggest that transcription factor domains have distinct properties when assayed by themselves versus when they are part of a multiprotein complex. Further, it is concluded that the unique architecture of the complex assembled on DllR is necessary for efficient repression (Gebelein, 2002).

Direct integration of Hox and segmentation gene inputs during Drosophila development

During Drosophila embryogenesis, segments, each with an anterior and posterior compartment, are generated by the segmentation genes while the Hox genes provide each segment with a unique identity. These two processes have been thought to occur independently. This study shows tha abdominal Hox proteins work directly with two different segmentation proteins, Sloppy paired and Engrailed, to repress the Hox target gene Distalless in anterior and posterior compartments, respectively. These results suggest that segmentation proteins can function as Hox cofactors and reveal a previously unanticipated use of compartments for gene regulation by Hox proteins. The results suggest that these two classes of proteins may collaborate to directly control gene expression at many downstream target genes (Gebelein, 2004).

The segregation of groups of cells into compartments is fundamental to animal development. Originally defined in Drosophila, compartments are critical for providing cells with their unique positional address. The first compartments to form during Drosophila development are the anterior and posterior compartments and the key step to defining them is the activation of the gene engrailed (en). Expression of en, which encodes a homeodomain transcription factor, results in a posterior compartment fate, and the absence of en expression results in an anterior compartment fate. Once activated by gap and pair-rule genes, en expression and, consequently, the anterior-posterior compartment boundary later become dependent upon the protein Wingless (Wg), which is secreted from adjacent anterior compartment cells. Concurrently with anterior-posterior compartmentalization and segmentation, the expression of the eight Drosophila Hox genes is also initially established by the gap and pair-rule genes. The Hox genes, however, which also encode homeodomain transcription factors, do not contribute to the formation or number of segments but instead specify their unique identities along the anterior-posterior axis (Gebelein, 2004).

This flow of genetic information during Drosophila embryogenesis has led to the idea that anterior-posterior compartmentalization and segment identity specification are independent processes. In contrast to this view, this study shows that these two pathways are interconnected in previously unrecognized ways. Evidence is provided that Hox factors directly interact with segmentation proteins such as En to control gene expression. Moreover, Hox proteins collaborate with two different segmentation proteins in anterior and posterior cell types to regulate the same Hox target gene, revealing a previously unknown use of compartments to control gene expression by Hox proteins (Gebelein, 2004).

Distalless (Dll) is a Hox target gene that is required for leg development in Drosophila. In each thoracic hemisegment, wg, expressed by anterior cells adjacent to the anterior-posterior compartment boundary, activates Dll in a group of cells that straddle this boundary. A cis-regulatory element derived from Dll, called DMX, drives accurate Dll-like expression in the thorax. The abdominal Hox genes Ultrabithorax (Ubx) and abdominalA (abdA) directly repress Dll and DMX-lacZ in both compartments, thereby blocking leg development in the abdomen. DMX is composed of a large activator element (DMXact) and a 57-base-pair (bp) repressor element referred to here as DMX-R. Previous work demonstrated that Ubx and AbdA cooperatively bind to DMX-R with two homeodomain cofactors, Extradenticle (Exd) and Homothorax (Hth). In contrast, the thoracic Hox protein Antennapedia (Antp) does not repress Dll and does not bind DMX-R with high affinity in the presence or absence of Exd and Hth. Thus, repression of Dll in the abdomen depends in part on the ability of these cofactors to selectively enhance the binding of the abdominal Hox proteins to DMX-R (Gebelein, 2004).

Exd and Hth, as well as their vertebrate counterparts, are used as Hox cofactors at many target genes. Moreover, Hox/Exd/Hth complexes are used for both gene activation and repression, raising the question of how the decision to activate or repress is determined. One view posits that these complexes do not directly recruit co-activators or co-repressors, but instead are required for target gene selection. Accordingly, other DNA sequences present at Hox/Exd/Hth-targeted elements would determine whether a target gene is activated or repressed. Consistent with this notion, DMX-R sequences isolated from six Drosophila species show extensive conservation outside the previously identified Hox (referred to here as Hox1) Exd and Hth binding sites, suggesting that they also play a role in Dll regulation (Gebelein, 2004).

To test a role for these conserved sequences, a thorough mutagenesis of DMX-R was performed. Each mutant DMX-R was cloned into an otherwise wild-type, full-length DMX and tested for activity in a standard reporter gene assay in transgenic embryos. Thoracic expression was normal in all cases. However, surprisingly, many of the DMX-R mutations, such as X5, resulted in abdominal de-repression only in En-positive posterior compartment cells, whereas other mutations, such as X2, resulted in abdominal de-repression only in En-negative anterior compartment cells. Single mutations in the Hox1, Exd, or Hth sites also resulted in de-repression predominantly in posterior cells. In contrast, deletion of the entire DMX-R (DMXact-lacZ), or mutations in both the X2 and X5 sites (DMX[X2 + X5]-lacZ), resulted in de-repression in both compartments. These results suggest that distinct repression complexes bind to the DMX-R in the anterior and posterior compartments and that segmentation genes play a role in Dll repression (Gebelein, 2004).

One clue to the identity of the proteins in these repression complexes is that the sequence around the Hth site is nearly identical to a Hth/Hox binding site that had been identified previously by a systematic evolution of ligands by exponential enrichment (SELEX) approach using vertebrate Hox and Meis proteins. This similarity suggested the presence of a second, potentially redundant Hox binding site, Hox2. In agreement with this idea, mutations in both the Hox1 and Hox2 binding sites resulted in de-repression in both the anterior and posterior compartments of the abdominal segments. Similarly, although individual mutations in the Exd and Hth binding sites lead predominantly to de-repression in the posterior compartment, mutation of both sites resulted in de-repression in both compartments. These results suggest that a Hox/Exd/Hth/Hox complex may be used for repression in both compartments. Furthermore, they suggest that although single mutations in these binding sites are sufficient to disrupt the activity of this complex in the posterior compartment, double mutations are required to disrupt its activity in the anterior compartment (Gebelein, 2004).

To provide biochemical evidence for a Hox/Exd/Hth/Hox tetramer, DNA binding experiments were performed using DMX-R probes and proteins expressed and purified from E. coli. Previous experiments demonstrated that a Hox/Exd/Hth trimer cooperatively binds to the Hox1, Exd and Hth sites. The function of the Hox2 site was tested in two ways. First, binding was measured to a probe, DMX-R2, that includes the Exd, Hth and Hox2 sites, but not the Hox1 site. It was found that Exd/Hth/AbdA and Exd/Hth/Ubx trimers cooperatively bind to this probe and that mutations in the Hth, Exd or Hox2 binding sites reduced or eliminated complex formation (Gebelein, 2004).

Second, if both the Hox1 and Hox2 sites are functional, the full-length DMX-R may promote the assembly of Hox/Exd/Hth/Hox tetramers. Using a probe containing all four binding sites (DMX-R1 + 2), the formation of such complexes was observed. Mutation of any of the four binding sites reduced the amount of tetramer binding whereas mutation of both Hox sites or both the Exd and Hth sites eliminated tetramer binding. Furthermore, Antp, which does not repress Dll, formed tetramers with Exd and Hth that were approximately tenfold weaker than with Ubx or AbdA, but bound well to a consensus Hox/Exd/Hth trimer binding site. Because mutation of both Hox sites or both the Exd and Hth sites resulted in de-repression in both compartments, these experiments correlate the binding of a Hox/Exd/Hth/Hox complex on the DMX-R with the ability of this element to mediate repression in both compartments (Gebelein, 2004).

Although binding of a Hox/Exd/Hth/Hox tetramer is sufficient to account for the necessary abdominal Hox-input into Dll repression, it does not explain the compartment-specific de-repression exhibited by some DMX-R mutations. The X2 and X5 mutations, for example, result in abdominal de-repression but do not prevent the formation of the Hox/Exd/Hth/Hox tetramer. Sequence inspection of the DMX-R revealed that the X2 mutation, which resulted in de-repression specifically in the anterior compartment, disrupts two partially overlapping matches to a consensus binding site for Forkhead (Fkh) domain proteins. With this in mind, the expression pattern of Sloppy paired 1 (Slp1), a Fkh domain factor encoded by one of two partially redundant segmentation genes, slp1 and slp2, was examined. The two slp genes are expressed in anterior compartment cells adjacent and anterior to En-expressing posterior compartment cells. In the thorax, cells expressing Dll and DMX-lacZ co-express either Slp or En at the time Dll is initially expressed. In the abdomen, the homologous group of cells, which express DMXact-lacZ (a reporter lacking the DMX-R), co-express either Slp in the anterior compartment or En in the posterior compartment. The expression patterns of Slp and En were compared with Ubx and AbdA. Ubx levels are highest in anterior, Slp-expressing cells whereas AbdA levels are elevated in posterior, En-expressing cells. In contrast, both Exd and Hth are present at similar levels in both compartments throughout the abdomen (Gebelein, 2004).

On the basis of these data, a model is presented for Hox-mediated repression of Dll in both the anterior and posterior compartments of the abdominal segments. In the anterior compartment it is proposed that Slp binds to DMX-R directly with a Ubx/Exd/Hth/Ubx tetramer. In the posterior compartment it is suggested that En binds to DMX-R directly with an AbdA/Exd/Hth/AbdA tetramer. One important feature of this model is that Antp/Exd/Hth/Antp complexes fail to form on this DNA, thereby accounting for the lack of repression in the thorax. Furthermore, the model proposes that Slp and En should, on their own, have only weak affinity for DMX-R sequences because repression does not occur in the thorax, despite the presence of these factors. The Hox/Exd/Hth/Hox complex, perhaps in conjunction with additional factors, is required to recruit or stabilize Slp and En binding to the DMX-R. Both Slp and En are known repressor proteins that directly bind the co-repressor Groucho. Thus, the proposed complexes in both compartments provide a direct link to this co-repressor and, therefore, a mechanism for repression. DNA binding and genetic experiments are presented that test and support this model (Gebelein, 2004).

To test the idea that En is playing a direct role in Dll repression, the ability of En and Hox proteins to bind to DMX-R probes was examined. On its own, En binds to DMX-R very poorly. Surprisingly, it was found that En binds DMX-R with the abdominal Hox proteins Ubx or AbdA in a highly cooperative manner. The thoracic Hox protein Antp does not bind cooperatively with En to this probe. Mutations in the Hox1 or X5 binding sites block AbdA/En binding in vitro, consistent with these mutations showing posterior compartment de-repression in vivo. In contrast, the X6, X7 and Hth mutations do not affect AbdA/En complex formation (Gebelein, 2004).

On the basis of DMX-R's ability to assemble a Hox/Exd/Hth/Hox tetramer, whether En could bind together with an AbdA/Exd/Hth/AbdA complex was tested. Addition of En to reactions containing AbdA, Exd and Hth resulted in the formation of a putative En/AbdA/Exd/Hth/AbdA complex. This complex contains En because its formation is inhibited by an anti-En antibody. A weak antibody-induced supershift is also observed. Moreover, this complex fails to form on the X5 mutant, which causes posterior compartment-specific de-repression. It is noted that En/Exd/Hth complexes also bind to the DMX-R and that it cannot be excluded that an En/Exd/Hth/AbdA complex may be important for Dll repression. The model emphasizes a role for an En/AbdA/Exd/Hth/AbdA complex because it better accommodates the cooperative binding observed between En and AbdA on the DMX-R (Gebelein, 2004).

Repression in the anterior compartments of the abdominal segments requires the sequence defined by the X2 mutation, which is similar to a Fkh domain consensus binding site. The model predicts that this sequence is bound by Slp. Consistent with this view, Slp1 binds weakly to wild type, but not to X2 mutant DMX-R probes. However, in contrast to En, no cooperative binding was observed between Slp and Hox or Hox/Exd/Hth/Hox complexes, suggesting that additional factors may be required to mediate interactions between Slp and the abdominal Hox factors (Gebelein, 2004).

Together, these results suggest that En and Slp play a direct role in DMX-lacZ and Dll repression. However, these experiments do not unambiguously determine the stoichiometry of binding by these factors. Furthermore, in vivo, additional factors may enhance the interaction between these segmentation proteins and Hox complexes, thereby increasing the stability and/or activity of the repression complexes (Gebelein, 2004).

The model for Dll repression is supported by previous genetic experiments that examined the effect of Ubx and abdA mutants on Dll expression in the abdomen. Ubx abdA double mutants de-repress Dll in both compartments of all abdominal segments. In contrast, Ubx mutants de-repress Dll in the anterior compartment of only the first abdominal segment, which lacks AbdA. abdA mutant embryos de-repress Dll in the posterior compartments of all abdominal segments, where Ubx levels are low (Gebelein, 2004).

Several genetic experiments were performed to provide in vivo support for the idea that Slp and En work directly with Ubx and AbdA to repress Dll. The design of these experiments had to take into consideration that the activation of Dll in the thorax depends on wg, and that wg expression depends on both slp and en. Consequently, Dll expression is mostly absent in en or slp mutants, making it impossible to characterize the role that these genes play in Dll repression from examining en or slp loss-of-function mutants. However, some of the mutant DMX-Rs described here provide the opportunity to test the model in alternative ways (Gebelein, 2004).

According to the model, DMX[X5]-lacZ is de-repressed in the posterior compartments of the abdominal segments because it fails to assemble the posterior, En-containing complex. Repression of DMX[X5]-lacZ in the anterior compartments still occurs because it is able to assemble the anterior, Slp-containing complex. According to this model, DMX[X5]-lacZ should be fully repressed if Slp is provided in posterior cells. A negative control for this experiment is that ectopic Slp should be unable to repress DMX[X2]-lacZ because this reporter gene does not have a functional Slp binding site. To mis-express Slp, paired-Gal4 (prd-Gal4), which overlaps both the Slp and En stripes in the odd-numbered abdominal segments, was used. As predicted, ectopic Slp repressed DMX[X5]-lacZ but not DMX[X2]-lacZ, providing strong in vivo support for Slp's direct role in Dll repression in the anterior compartments (Gebelein, 2004).

Conversely, the model posits that DMX[X2]-lacZ is de-repressed in the anterior compartment because it cannot bind Slp, but remains repressed in the posterior compartment because it is able to assemble the En-containing posterior complex. Thus, providing En in the anterior compartment should repress DMX[X2]-lacZ. A complication with this experiment is that En is a repressor of Ubx, which is the predominant abdominal Hox protein in the anterior compartment. It was confirmed that prd-Gal4-driven expression of En represses Ubx and that AbdA levels remain low at the time Dll is activated in the thorax. Consequently, ectopic En expression is not sufficient to repress DMX[X2]-lacZ, consistent with the observation that low levels of abdominal Hox proteins are present. Therefore, to promote the assembly of the posterior complex in anterior cells, En was co-expressed with AbdA using prd-Gal4. As predicted, this combination of factors repressed DMX[X2]-lacZ but not DMX[X5]-lacZ, providing strong in vivo evidence for En playing an essential role in Dll repression in the posterior compartments (Gebelein, 2004).

Several observations provide additional support for the model. First, ectopic expression of AbdA or Ubx in the second thoracic segment (T2) represses DMX[X5]-lacZ in the anterior compartment, but not in the posterior compartment. Conversely, expression of AbdA or Ubx in T2 represses DMX[X2]-lacZ only in posterior compartment cells. Second, co-expression of Slp with Ubx completely represses DMX[X5]-lacZ in T2 but does not repress DMX[X2]-lacZ in T2. Third, in those cases where repression is incomplete (for example, En + AbdA repression of DMX[X2]-lacZ in the abdomen), cells that escape repression have low levels of either an abdominal Hox protein or Slp/En. Together, these data provide additional evidence that the abdominal Hox proteins work together with Slp and En to repress Dll (Gebelein, 2004).

The segregation of cells into anterior and posterior compartments during Drosophila embryogenesis is essential for many aspects of fly development. The results presented in this study reveal an unanticipated intersection between anterior-posterior compartmentalization by segmentation genes and segment identity specification by Hox genes. Specifically, it is suggested that the abdominal Hox proteins collaborate with two different segmentation proteins, Slp and En, to mediate repression of a Hox target gene (Dll) in the anterior and posterior compartments of the abdomen, respectively. This mechanism of transcriptional repression suggests a previously unknown use of compartments in Drosophila development. The mechanism proposed here contrasts with the alternative and simpler hypothesis in which the abdominal Hox proteins would have used the same set of cofactors to repress Dll in all abdominal cells, regardless of their compartmental origin (Gebelein, 2004).

These results provide further support for the view that Hox/Exd/Hth complexes do not directly bind co-activators or co-repressors but instead indirectly recruit them to regulatory elements. Consistent with previous analyses, it is suggested that Hox/Exd/Hth complexes are important for the Hox specificity of target gene selection. Additional factors, such as Slp or En in the case of Dll repression, are required to determine whether the target gene will be repressed or activated. In the future, it will be important to dissect in similar detail other Hox-regulated elements, to assess the generality of this mechanism (Gebelein, 2004).

These results also broaden the spectrum of cofactors used by Hox proteins to regulate gene expression. Although the analysis of Exd/Hth in Drosophila and Pbx/Meis in vertebrates has provided some insights into how Hox specificity is achieved, there are examples of tissues in which these proteins are not available to be Hox cofactors and of Hox targets in which Exd and Hth seem not to play a direct role. This study shows that En, a homeodomain segmentation protein, is used as a Hox cofactor to repress Dll in the abdomen. Although the complex defined at the DMX-R includes Exd and Hth, the DNA binding studies demonstrate that Hox and En proteins can bind cooperatively to DNA in the absence of Exd and Hth. These findings suggest that En may function with Ubx and/or AbdA to regulate target genes other than Dll, and perhaps independently of Exd and Hth. Consistent with this idea are genetic experiments showing that, in the absence of Exd, En can repress slp and this repression requires abdominal Hox activity. Although these experiments were unable to distinguish whether the Hox input was direct or indirect, the results suggest that En may bind directly with Ubx and AbdA to repress slp, and perhaps other target genes (Gebelein, 2004).

Finally, these results raise the question of why a compartment-specific mechanism is used by Hox factors to repress Dll. The activation of Dll at the compartment boundary by wg may be important for accurately positioning the leg primordia within each thoracic hemisegment, but this mode of activation requires that Dll is repressed in both compartments in each abdominal segment. The utilization of segmentation proteins such as En and Slp may be the simplest solution to this problem. Compartment-specific mechanisms may also provide additional flexibility in the regulation of target genes by Hox proteins by allowing them to turn genes on or off specifically in anterior or posterior cell types. For these reasons, compartment-dependent mechanisms of gene regulation may turn out to be the general rule instead of the exception (Gebelein, 2004).

Transcriptional activation by Extradenticle and Homothorax in the Drosophila visceral mesoderm

decapentaplegic is a direct target of Ultrabithorax (Ubx) in parasegment 7 (PS7) of the embryonic visceral mesoderm. This study demonstrates that extradenticle (exd) and homothorax (hth) are also required for dpp expression in this location, as well as in PS3, at the site of the developing gastric caecae. A 420 bp element from dpp contains Exd binding sites necessary for expressing a reporter gene in both these locations. Using a specificity swap, Exd was demonstrated to directly activate this element in vivo. Activation does not require Ubx, demonstrating that Exd can activate transcription independently of homeotic proteins. Restoration is restricted to the domains of endogenous dpp expression, despite ubiquitous expression of altered specificity Exd. Nuclear Exd is more extensively phosphorylated than the cytoplasmic form, suggesting that Exd is a target of signal transduction by protein kinases (Stultz, 2006).

Previous studies (Sun, 1995) demonstrated that Ubx directly regulates dpp in PS7 of the VM using a specificity swap strategy. Subsets of six Ubx binding sites were mutated in a 420 bp reporter construct (PX) from binding sites for Q50 homeodomains to binding sites for K50 homeodomains. For example, the wild-type UBX site 5/EXD site e2 was AGGCCTATCAATTAGCACC (with the EXD site underlined) and the mutant UBX site 5/EXD site e2 was AGGCCTAGGGATTAGCACC. It was then possible to restore the expression of these constructs by changing Q50 to K50 in the Ubx protein (called Ubx K50). However, it was not possible to restore expression of a reporter in which all six Ubx sites were altered. This suggested that an additional factor was required, and it was noted that the alterations in the fully substituted PX reporter also disrupted closely apposed Exd binding sites, suggesting that Ubx and EXD may co-regulate dpp (Stultz, 2006).

In previous work (Sun, 1995), it was not possible to restore expression of the fully substituted PX4–9 reporter using Ubx K50. This study shows that ubiquitous and simultaneous induction of Ubx K50 and Exd K50 restores expression of PX4–9 in a manner that is similar to wild-type PX. Induction of Exd K50 alone also restores PX4–9 in these domains but changes the balance of staining intensity between them, with PS7 expression appearing less prominent. This reflects a sufficiency of Exd K50 for activation of gene expression at both sites, but with an additional requirement for Ubx K50 to achieve wild-type levels in PS7. These experiments identify Exd as a direct activator of dpp's VM expression in both PS3 and PS7 (Stultz, 2006).

No HOX proteins are expressed in PS3. Thus, Exd K50 activates gene expression independently of HOX family proteins in this location. In cases where Ubx K50 restored partially substituted PX constructs, restoration was never seen in PS3 (Sun, 1995), further indicating that dpp expression here does not require HOX proteins. In addition, in PS7, where it is clearly established that Ubx contributes to activation of dpp expression, the results demonstrate that Ubx is not absolutely required for Exd K50 to activate transcription. Ubx increases the level of dpp expression, as demonstrated by the reduced PS7 expression in Exd K50-alone restorations, but is not required for Exd function. This point is further reinforced by the ability of Exd K50 to activate PX4–9 gene expression, even in Ubx homozygous mutants. On simple Exd binding sites, PBX proteins have not demonstrated transcriptional activation, but the data suggest that Exd can participate in gene activation without a HOX gene. Other unidentified factors in PS3 or PS7 could also be involved, and one candidate would be HTH, which is genetically required for dpp's VM expression and capable of binding to the PX element in concert with Exd. Genetic evidence for the ability of Exd/HTH to act in the absence of HOX proteins has been steadily accumulating, based on mutant phenotypes that cannot be attributed to HOX genes, and both genetic and in vitro data suggest that HTH/MEIS may have transcriptional activation capabilities (Stultz, 2006).

Two models for the role of Exd in regulating HOX targets have been proposed. The data indicate that the PX element is directly regulated by both Exd and Ubx, allowing evaluation of these two models based on the results. The 'co-selective binding' model proposes that Exd enhances the specificity and affinity of its HOX partner for a DNA binding site. This model requires that Exd and HOX proteins bind cooperatively as heterodimers to closely spaced Exd and HOX binding sites. This model predicts that the relative spacing and orientation of PBX/Exd and HOX binding sites must be tightly constrained, as has been shown by in vitro studies. Although the dpp cis-regulatory PX element contains multiple Ubx and Exd sites identified by DNA footprinting (Sun, 1995), only site e2 resembles the optimal site for binding by a PBX1/HOXB7 heterodimer. Even this site is not a perfect match, and data indicate that this site may be more likely to bind Exd/HTH in vivo. The electrophoretic mobility shift data demonstrate that Exd K50 can bind TAATCCC sites (the optimal site for HOX K50) that replace Ubx sites, as well as unaltered Exd sites. Thus, Exd K50 restores PX4–9 by binding to some or all of these sites. This demonstrates that an Exd protein altered only in its binding specificity can act in vivo through sites of altered spacing and orientation and is not necessarily constrained to act in close proximity to a HOX protein (Stultz, 2006).

The second model, 'widespread binding', proposes that Exd determines the outcome of HOX protein action. According to this model, either Exd or HOX proteins in isolation can bind DNA and act as transcriptional repressors. When both proteins are present, a complex that activates transcription is formed. For Deformed, Exd activates an otherwise silent transcriptional activation domain within the Deformed protein. The physical association between the proteins stabilizes their binding to DNA, but they do not have to bind as heterodimers. This model is more consistent with both the spacing of Exd and HOX sites in the dpp PX element and the apparent flexibility in the location of Exd-responsive sites observed in the experiments. However, this model predicts that independent Exd action is repressive, based on a Deformed-responsive target. In contrast, the data indicate that Exd can also activate reporter gene expression without a HOX partner, suggesting that repression is not the default action of Exd in the absence of HOX proteins (Stultz, 2006).

This study has shown that Exd is a direct activator of dpp expression in the VM. In PS3, the normal action of Exd does not require input from any homeotic protein. In PS7, input from Ubx is critical to achieving the correct level of gene expression, but the data do not support a model where Ubx is absolutely necessary for transcriptional activation. The data suggest that Exd can activate transcription in the absence of HOX proteins but that, in many cases, it also collaborates with HOX proteins, allowing the complex to achieve a more robust level of transcriptional activation. The current notion is that Exd is an essential cofactor for homeotic proteins. An equally tenable model for gene activation is that HOX proteins are the cofactors of Exd, imparting additional spatial regulation, site specificity, and activity to this transcriptional regulator (Stultz, 2006).

The striking restriction of reporter restoration to domains influenced by kinase-mediated signaling pathways led to an examination Exd protein for evidence of phosphorylation. The primary sequence of Exd contains more than 15 potential sites for various protein kinases, including Protein Kinase A (PKA) and Casein Kinase II upstream of its NLS. Protein kinase action is required for gene activation by PBX proteins in tissue culture cells, PKA converts HOX/PBX complexes from repressors to activators on the Hoxb1 autoregulatory element, and phosphorylation by PKA induces nuclear import of PBX1 independently of the PBX/MEIS nuclear localization mechanism. While it was not possible to establish a connection between DPP signaling and Exd phosphorylation, nonetheless, Exd clearly exists in multiple phosphoprotein forms, and the increased phosphorylation is clearly correlated to subcellular localization in Drosophila as well. Thus, Exd must be a target of kinase action, although whether this activity is solely required for nuclear translocation or for activity once in the nucleus is unresolved (Stultz, 2006).

dpp requires both its own expression and that of wg to achieve normal gene expression in the VM. These data led to a hypothesis that the spatial restriction observed in the restoration must be connected to DPP or WG signaling. However, the data do not support this hypothesis, and it is more likely that the major inputs generating dpp's localization in the VM are repressive in nature. In previous work, it was postulated that dpp's spatial regulation in the VM was the result of dual modes of regulation involving both general activation and spatially specific repression and spatially restricted activation (Sun, 1995). The general activator has been identified as biniou (bin), a member of the FoxF/forkhead family of transcription factors. This factor is capable of inducing dpp expression throughout the posterior half the VM, including PS7, when its action is not specifically repressed. This repression comes from multiple inputs. dpp is a direct target of posterior repression via Abd-A. dpp is also repressed outside of PS3 and PS7 via the action of Drosophila T Cell Factor (dTCF) in the absence of WG signaling. The ectopic PS4–6 expression of longer dpp constructs in exd or hth null embryos identifies exd and hth or a downstream target of these genes as another repressor of dpp in PS4–6. Such a downstream target could be teashirt, a known repressor whose VM expression is lost in exd null embryos and is expressed in PS4–6 (Stultz, 2006).

To this model of multiple general activators and spatially specific repressors is added the spatially localized strong activator Ubx. Ubx directly regulates dpp and may also have indirect inputs to dpp's PS7 gene expression, as the reduced restoration in Ubx9.22 null embryos indicates. Ubx is itself repressed via chromatin factors such as Polycomb and osa in the anterior midgut and Abd-A posterior to PS7. dpp autoregulation provides additional weak activation via inputs from SMAD proteins and through DPP-mediated schnurri repression of the repressor brinker. Thus, dpp expression is the cumulative result of general activation constrained by spatially specific repression and augmented by spatially specific activation. Clearly, evolution has deemed the formation of the embryonic midgut of sufficient importance to create a highly buffered, reinforced system of gene expression (Stultz, 2006).

Regulation of the Drosophila distal antennal determinant spineless by Homothorax

The transformation of antenna to leg is a classical model for understanding segmental fate decisions in Drosophila. The spineless (ss) gene encodes a bHLH-PAS transcription factor that plays a key role in specifying the identity of distal antennal segments. This report identifies the antennal disc enhancer of ss and then uses enhancer-lacZ reporters to work out how ss antennal expression is regulated. The antennal determinants Distal-less (Dll) and homothorax (hth) are key activators of the antennal enhancer. Dll is required continuously and, when present at elevated levels, can activate the enhancer in regions devoid of hth expression. In contrast, homothorax (hth) is required only transiently both for activation of the enhancer and for specification of the aristal portion of the antenna. The antennal enhancer is repressed by cut, which determines its proximal limit of expression, and by ectopic Antennapedia (Antp). Repression by Antp is not mediated by hth, suggesting that ss may be a direct target of Antp. ss+ is not a purely passive target of its regulators: ss+ partially represses hth in the third antennal segment and lies upstream of Dll in the development of the maxillary palp primordia (Emmons, 2007; full text of article).

This study used lacZ reporters to identify the enhancers responsible for most aspects of ss expression during embryonic and imaginal development. Antennal expression is driven by two large fragments from the ss 5' region, B6.9 and EX8.2. Both of these fragments drive expression in the antennal segment of the embryo and in the distal portion of the pupal antenna. B6.9 is also expressed in the antennal disc through most or all larval development. Dissection of B6.9 allowed localization of the larval antennal enhancer to a fragment of 522 bp. The B6.9 and 522 reporters were used as a proxy for ss expression in experiments to determine the effects of potential upstream regulators of ss. This strategy has its strengths and weaknesses, but has been made necessary by an inability to generate antisera against Ss. A major strength of the approach is that it was possible to assess the effects of regulators on individual enhancers. It is likely that monitoring endogenous ss expression would give results that are less clear cut since both the antennal and tarsal enhancers of ss are active within the antenna. A potential weakness is that the reporters may not faithfully reproduce the normal expression of ss. However, as far as is possible to tell, the antennal reporters reproduce ss expression very well. The expression of B6.9 and EX8.2 in the embryonic antennal segment and the pupal antenna corresponds very closely to that of endogenous ss. Expression of B6.9 and 522 in the larval antennal disc appears very similar or identical to that of ss+, and the transient requirement for hth+ in the activation of these reporters corresponds well to the transient requirement for hth+ in aristal specification. The tarsal enhancer P732 likely also reproduces the spatial pattern of ss+ expression as its tarsal expression domain corresponds well to the region deleted in ss mutants (Emmons, 2007).

The results of this dissection of the B6.9 fragment were surprising. Removal of the left-hand 2 kb of B6.9 to produce S4.9 resulted in the loss of antennal specificity; S4.9 reporters are expressed in both antennal and leg discs. The E2.0 subfragment of S4.9 shows a similar expression pattern, and expression of this fragment in both leg and antennal discs is independent of Hth, but requires Dll continuously. On further subdivision of the E2.0 fragment, it was found that antennal and leg expression are separable; the 522 fragment is largely specific for the antenna, whereas the 531 fragment drives expression primarily in leg discs. To summarize, antennal specificity is present in B6.9, lost in S4.9 and E2.0 and regained in 522. How can sense be made of this? The region deleted from B6.9 to produce S4.9 clearly plays an important role in enforcing antennal specificity. Since this region contains a PRE, one might suspect that it functions in larval stages to maintain repression of the enhancer outside of the antennal segment. However, that the E2.0 fragment has lost the requirement for Hth in both the antenna and leg (S4.9 has not been tested) suggests that the PRE-containing region might function in both locations. One possibility is that this region represses the enhancer in both antennal and leg discs. In the antenna, this repression can be overcome by the combined action of Hth and Dll, while in the leg Dll alone is not sufficient for activation. When the PRE-containing region is deleted, repression is absent or reduced, so that Dll can activate the enhancer without assistance from Hth, and expression is seen in both antennal and leg discs. Why then is antennal specificity restored in the 522 subfragment? Perhaps this fragment is lacking a subset of Dll interaction sites so that it can no longer be activated by Dll alone, but requires combined activation by Hth and Dll. Although this model is consistent with many of the results, it does not provide a ready explanation for the leg specificity of the 531 fragment (Emmons, 2007).

In addition to activation by combined Hth and Dll, the ss antennal disc enhancer is repressed by Cut and by ectopic Antp. Each of these regulators will be discussed separately. It was found that hth+ is required only transiently for activation of the B6.9 reporter. hth clones induced in the embryo or first instar lose expression of B6.9 autonomously in both A3 and the aristal primordia. However, some time in the second of early third instar. Regulatory instar expression of B6.9 becomes independent of hth. Consistent with this transient requirement, it is shown that hth+ is required only early in larval development for specification of the arista. hth clones induced in the first and second instars show a transformation of the entire antenna to a leg-like appendage. However, clones induced after this time show normal aristal development. These temporal requirements are reflected in the expression pattern of hth: hth is expressed throughout the antennal primordium early in development, but in the second or early third instar is repressed in the central domain, which will produce the arista (Emmons, 2007).

The stable activation of B6.9 by Hth suggests that this fragment contains a 'cellular memory module'. The presence of a PRE within B6.9 is consistent with this idea. The ss locus binds Polycomb protein in salivary gland chromosomes and was recently shown to contain PREs by chromatin immunoprecipitation. In the latter work, ss PREs were localized to within the E1.6 subfragment of B6.9 as well as the EX8.2 fragment, both of which showed pairing dependent suppression in this work. PREs are generally thought of as functioning to stably repress genes. However, PREs can also be associated with activating elements to form memory modules that mediate stable activation. It seems likely that B6.9 contains such a module that responds to Hth. Like a memory module from the hedgehog gene, activity of the ss module is set sometime around the second instar. Surprisingly, it was found that activation of the 522 reporter by Hth can also be persistent, although not as stable as for B6.9. The 522 fragment does not appear to contain a PRE, suggesting that Hth may directly recruit factors to the 522 element that cause semi-stable transcriptional activation (Emmons, 2007).

ss is not a completely passive target of hth; ss partially represses hth in antennal discs, which causes hth to be expressed at a lower level in A3 than in A2. This repression appears to be important for normal development as ectopic expression of Hth can delete A3. Moreover, clones ectopically expressing Hth are largely blocked from entering A3 from the proximal (A2) side, suggesting that the different levels of Hth present in A2 and A3 cause a difference in cell affinities between these segments. Hth-expressing clones are similarly restricted to the two most proximal segments in leg discs, although here there is no endogenous expression of hth more distally (Emmons, 2007).

In contrast to hth, Dll is required continuously for expression of both B6.9 and 522 as Dll clones induced even very late in development lose expression of these reporters. This continuous requirement for Dll indicates that stable activation of the B6.9 memory module by Hth does not by itself commit the reporter to expression; rather, activation by Hth appears to render B6.9 open to interaction with Dll and perhaps other positive factors (Emmons, 2007).

Three lines of evidence suggest that Dll is the primary activator of the ss antennal enhancer. (1) It was found that expression of B6.9 and 522 is sensitive to the dosage of Dll+. Expression of both reporters is reduced in animals carrying only one dose of Dll+, and for 522, expression is enhanced in clones having extra doses of Dll+. This dose sensitivity suggests that ss is a direct target of Dll. (2) It was found that expression of both reporters is often induced within clones expressing ectopic Dll, even in the apparent absence of Hth expression. Such activation is seen in clones in the distal leg, wing and elsewhere. (3) It was found that the embryonic antennal enhancer carried by B6.9 is absolutely dependent upon Dll+, but independent of hth. Taken together, these observations suggest that Dll is a primary activator of the ss antennal enhancers. Hth may provide antennal specificity by boosting the level of activation by Dll in the antennal disc (Emmons, 2007).

Surprisingly, it was found that the regulatory relationship between ss and Dll is reversed in the maxillary palp. Here, ss is expressed prior to Dll and is required for the normal initiation of Dll expression. Although some Dll expression ultimately takes place in the palp primordium in ss animals, this expression is weak and occurs in only a few cells. It has not been worked out how ss is activated in the palp. However, it seems likely that dpp plays a role as the 531 subfragment of B6.9 drives expression in a stripe in the region of the palp that roughly coincides with a stripe of dpp expression. The positioning of ss upstream of Dll in the palp may explain why the region ventral to the antenna is so sensitive to ectopic expression of Ss. Strong activation of Dll here by ectopic Ss combined with endogenous expression of hth might be expected to cause frequent induction of ectopic antennae, as is observed. Since ss is normally expressed in the palp, why should earlier ectopic Ss cause the palp primordium to develop as antenna? It seems likely that timing is key, but level of Ss expression could also be important (Emmons, 2007).

The reciprocal regulatory roles of ss and Dll in the antenna and palp suggest a particularly close relationship between these genes. This relationship is reinforced by the finding that ss is required for the development of bracts in the femur, as is Dll (Emmons, 2007).

The finding that Dll and Hth are both activators of the ss antennal reporters is consistent with the proposal that antennal identity is defined by the combined activity of these regulators. However, the results indicate that this model is an oversimplification. Examination of clones expressing Dll, Hth, or both proteins together revealed little correlation between activation of the B6.9 and 522 antennal reporters and combined expression of Dll and Hth. Strikingly, Dll-expressing clones often activate the reporters ectopically without any apparent concomitant expression of Hth, and clones expressing both proteins usually do not activate the reporters. These experiments also reveal strong context dependence. Examples include the leg, where Dll-expressing clones can activate the reporters distally, but not proximally (where endogenous hth expression occurs) and the wing disc, where clones expressing Dll or both Dll and Hth activate the reporters in the wing pouch, but not at all in the notum. The level of expression of both proteins also appears to be key as high levels of Dll can activate the reporters in the leg in the absence of Hth and elevated levels of Hth can repress expression in the normal antennal domain. Previous results have shown that antennal structures can be induced by ectopic expression of Dll in the wing hinge region or proximal leg (which express hth endogenously) or by combined expression of Dll and Hth elsewhere. While this is true, the results indicate highly variable effects in such ectopic expression experiments and fail to detect the strongly synergistic activation of antennal identity by combined Hth and Dll implied by the model. The results indicate that Dll is the primary activator of the ss antennal reporters, that Hth serves to promote this activity and that activation by Dll and Hth is highly context-dependent (Emmons, 2007).

Consistent with direct control of the antennal reporters by Dll and Hth, two highly conserved regions within the 522 fragment contain apparent binding sites for Dll, Hth, and the Hth dimerization partner Extradenticle. The functional importance of these binding sites is currently being tested (Emmons, 2007).

This study has show that the proximal boundary of B6.9 and 522 expression is defined by repression by cut. This repression likely explains why ectopic Cut causes a transformation of arista to tarsus. cut has been shown to define the proximal expression limit of distal antenna (dan) and distal antenna related (danr); since ss lies upstream of these genes, it seems very likely that their regulation by cut is indirect. The mechanism of action of Cut is not well understood, since only one direct target has been characterized in Drosophila (Emmons, 2007).

Ectopic expression of Antp in the antenna represses the B6.9 and 522 reporters. This finding was expected, since it is well known that expression of Antp or other Hox genes in the antenna causes a transformation to leg. The conventional view is that this transformation results from the repression of hth by ectopic Hox proteins. Repression of hth early in development would be expected to lead secondarily to loss of ss expression and loss of distal antennal identity. However, it was found that clones expressing Antp repress the B6.9 and 522 reporters even when these clones are induced very late in development, long after the requirement for activation by hth has passed. Late repression of the antennal reporters by Antp must therefore occur independently of hth and could be direct. One possibility, currently being tested, is that Antp might compete with Dll for binding to the 522 enhancer. Late repression of the ss antennal enhancer by Antp is consistent with the effects of Antp-expressing clones on antennal identity: such clones induced in the mid to late third instar cause transformations of distal antenna to leg (Emmons, 2007).

Clones induced late that ectopically express Antp in a sustained fashion were examined. In contrast, previous work studied the effects of pulses of Antp expression induced by one-hour heat shocks in a heat shock/Antp line. It had been found that transformations of arista to tarsus were induced by such pulses only when they were administered at the end of the second instar. Why do pulses of Antp at this time cause a stable, heritable transformation of the distal antenna? The current results suggest an explanation. The period sensitive to Antp pulses coincides roughly with when the ss antennal enhancer becomes independent of hth. This correlation suggests that pulses of Antp in the second instar cause heritable transformations by interfering with the stable activation of ss by Hth. Recently, it has been reported that ectopic Antp does not repress hth in the antenna early in larval development. This observation suggests that Antp might act directly on the ss antennal enhancer to prevent its stable activation by Hth (Emmons, 2007).

The regulation of ss by ectopic Antp suggests that Antp may normally play a significant role in repressing ss antennal enhancer activity in the legs. Although this idea has not been tested directly, it seems unlikely that Antp is primarily responsible for keeping the ss antennal enhancers inactive in the leg. Antp null clones do cause activation of the ss target gene dan in leg discs, implying ectopic activation of ss. However, this activation occurs only proximally, with the distal leg appearing to develop independently of Antp. Expression of Antp in the proximal leg may account for why Dll-expressing clones fail to activate B6.9 or 522 in this location. Ectopic activation of the ss antennal enhancers in the leg primordia of the embryo is not seen in an Antp null mutant (Emmons, 2007).

These studies suggest that antennal structures are specified in a combinatorial fashion by Hth, Dll, Ss and probably other factors. In A3, all three proteins are required for normal antennal identity. In ss antennae, hth continues to be expressed in A3 (although at elevated levels), as does Dll. Despite this continued expression of hth and Dll, A3 develops without antennal characteristics and produces only naked cuticle. Thus, Hth and Dll are unable to specify A3 characters in the absence of Ss. Conversely, assuming that ss is stably activated in the antenna by Hth, as is B6.9, then hth clones induced late would show persistent expression of both ss and Dll in A3. Such clones are transformed to leg, implying that Ss and Dll have no ability to direct A3 identity in the absence of Hth. Taken together, these observations suggest that Hth, Dll and Ss must act together to specify A3 identity. This requirement for combined action accounts for why ectopic expression of Ss does not induce A3 tissue in the medial leg, since hth is not normally expressed here. The view of combinatorial control suggests that many A3-specific target enhancers might be identifiable in genome searches as regions that contain clustered binding sites for Hth, Dll and Ss; tests of this prediction will be presented elsewhere (Emmons, 2007).

In contrast to A3, the aristal primordium appears to be specified by ss and Dll acting together in the absence of hth expression. hth is expressed in the aristal region early in development, where it functions to establish ss expression, but it is soon repressed here. Therefore, for most of development, the arista is specified by Ss and Dll acting without input from Hth. Consistent with this picture, the arista adopts leg identity in ss null mutants, and ectopic expression of ss causes the distal tip of the leg to develop as arista (Emmons, 2007).

In ss mutants, the distal antenna is terminated by a single tarsal segment (the fifth). In contrast, in ss mutants that lack only antennal enhancer activity (e.g. the breakpoint mutations ssD114.3 and ssD114.7, the distal antenna develops with a near complete set of tarsal segments. This difference likely reflects the activity of the tarsal enhancer in the antenna. In support of this view, the ss tarsal enhancer drives expression in the segmented base of the arista, a region known as the basal cylinder. This region transforms to tarsal segments 2-4 in Antp-induced transformations of antenna to leg. However, the question arises as to why normal antennal expression of ss causes the proximal arista to develop as basal cylinder, whereas ss expression driven by the tarsal enhancer alone causes this same region to develop as tarsal segments. Likely, the key difference is that expression driven by the tarsal enhancer is transient, whereas expression driven by the antennal enhancer is sustained. Perhaps transient expression of ss allows growth and subsegmentation to produce a full set of tarsal segments, whereas sustained expression inhibits growth, producing the basal cylinder. Consistent with this idea, sustained expression of ss driven by the GAL4 method can cause deletion of tarsi in the legs. The levels of expression driven by the tarsal and antennal enhancers may also be important as flies having only one dose of ss show a partial transformation of the basal cylinder to tarsus. The ss tarsal enhancer drives weak expression in A3 as well as in the basal cylinder, likely accounting for the presence of some specialization of A3 in ss mutants lacking the antennal enhancers (Emmons, 2007).

The view that antennal identity is specified by the combined action of Hth, Dll and Ss contradicts the now prevalent view that antennal identity is determined solely by hth. The major evidence supporting the latter view is that early hth clones transform the entire antenna to leg, and ectopic expression of Hth can induce ectopic antennal structures in the anal plates. Moreover, Dll shows little antennal specificity, being expressed in the distal portions of all of the ventral appendages, and ss expression in the antenna is dependent upon hth+. Should hth be viewed as the antennal 'selector' gene? hth does not seem to be a selector in the same sense as the Hox genes; it is expressed very broadly in the embryo and in other imaginal discs and plays no role in activating ss in the antennal segment of the embryo. Moreover, the ability of ectopic Hth to induce antennal structures is very limited: transformations of anal plate to distal antenna have been reported following ectopic expression of Hth or Meis1, a mammalian homolog. However, others have been unable to reproduce this effect by ectopic expression of Hth, matching the results of this study. That anal plates are susceptible to transformation at all is likely due to the fact that Dll and ss are coexpressed here in normal development. A further dissimilarity is that hth acts only as an establishment regulator of ss in the antennal disc, unlike the continuous requirements usually seen for the Hox genes. Ultimately, assessment of the importance of hth will depend on whether its function in the antenna is conserved. The expression pattern of hth in the antenna does appear to be conserved in the milkweed bug Oncopeltus. However, localization of nuclear Exd (a proxy for Hth expression) indicates that Hth is not differentially expressed in the antenna and leg of the cricket. Expression of hth in the crustacean Porcellio also appears to be identical in the second antenna and the legs. Characterization of hth, Dll and ss expression and function in additional arthropods will be required to assess properly the importance of these genes in antennal specification (Emmons, 2007).

Hox and senseless antagonism functions as a molecular switch to regulate EGF secretion in the Drosophila PNS

Hox factors are key regulators of distinct cells, tissues, and organs along the body plan. However, little is known about how Hox factors regulate cell-specific gene expression to pattern diverse tissues. This study shows an unexpected Hox transcriptional mechanism: the permissive regulation of EGF secretion, and thereby cell specification, by antagonizing the Senseless transcription factor in the peripheral nervous system. rhomboid expression in a subset of sensory cells stimulates EGF secretion to induce hepatocyte-like cell development. A rhomboid enhancer was identified that is active in these cells; an abdominal Hox complex directly competes with Senseless for enhancer binding, with the transcriptional outcome dependent upon their relative binding activities. Thus, Hox-Senseless antagonism forms a molecular switch that integrates neural and anterior-posterior positional information. As the vertebrate Senseless homolog is essential for neural development as well as hematopoiesis, it is proposed Hox-Senseless antagonism will broadly control cell fate decisions (Li-Kroeger, 2008).

Hox genes have long been known to specify distinct cell types along the body axes of both vertebrates and invertebrates. However, it has remained elusive how Hox factors regulate transcription in a tissue- or cell-specific manner. In this study, a Hox-regulated enhancer (Rho654) active within a subset of PNS cells was identified. Rho654 drives gene expression in abdominal C1-SOP cells to induce oenocytes, and an Exd/Hth/Abd-A complex stimulates gene expression by directly competing with Sens for this enhancer. These findings have three main implications: (1) They demonstrate how a Hox selector gene integrates A-P positional information with a PNS factor to differentially regulate gene expression along the body plan. (2) They uncover a permissive rather than instructive role for Hox factors in regulating transcription. (3) As Hox and Sens binding sites share a common core sequence, they suggest that additional target genes will be regulated through this mechanism. Moreover, genetic studies in mice have linked Gfi1 and Hox factors to both neural and blood cell development, and this study found that vertebrate Hox and Gfi1 factors compete for binding sites in blood cells (Li-Kroeger, 2008).

Sensory organs within the fly head, thorax, and abdomen require sens for their development. However, the type, location, and number of sensory organs that form in different body regions are regulated, at least in part, by Hox factors. The results provide new insight into how Hox factors provide positional information to modify gene expression in sensory cells. A series of point mutations was used to demonstrate that Hox-Sens competition forms a molecular switch whose outcome correlates with the binding activity of each factor. Intrinsic to this model is the following prediction: If Hox factors differ in their ability to interact with composite sites, then A-P differences in Hox-Sens target expression will be observed. Previous biochemical studies revealed that posterior Hox factors have higher affinity for DNA when bound with Pbx (Exd) than anterior Hox proteins (LaRonde-LeBlanc, 2003). Consistent with these results, this study found that a posterior Hox complex (Abd-A/Hth/Exd) that stimulates Rho654 binds 5-fold more RhoA than an anterior Hox complex (Antp/Hth/Exd) that fails to stimulate Rho654. Thus, differences in binding activities between Hox factors for Hox-Sens composite sites result in the differential regulation of gene expression along the A-P axis of the sensory system (Li-Kroeger, 2008).

Hox proteins instructively regulate gene expression by either activating and/or repressing transcription. In fact, the same Hox factor can perform both functions. Abd-A directly binds regulatory elements to activate wingless (wg) and repress decapentaplegic (dpp) in the same cells of the visceral mesoderm. So what determines if a Hox factor activates or represses transcription? Two recent studies revealed that the transcriptional outcome depends upon the binding of additional transcription factors (Gebelein, 2004; Walsh, 2007). The repression of Distal-less (Dll) by the Abd-A and Ultrabithorax (Ubx) Hox factors requires the binding of two transcription factors in addition to Exd and Hth. In posterior compartment cells, the Engrailed (En) protein collaborates with Abd-A/Exd/Hth to bind DNA and repress Dll. In anterior compartment cells, the Sloppy-paired (Slp) protein binds DNA near the Hox complex to repress Dll (Gebelein, 2004). As both En and Slp interact with the Groucho (Gro) corepressor, their recruitment by Hox factors suggests a mechanism to repress transcription. Similarly, Walsh and Carroll found that Ubx and Smad binding are required to repress spalt-major (salm) in the wing. In this case, the Smad proteins recruit the Schnurri corepressor to inhibit transcription. Thus, Hox factors collaborate with additional factors to determine the transcriptional outcome (Li-Kroeger, 2008).

Studies on Abd-A stimulation of a rho enhancer reveal an unexpected mechanism by which Hox factors control gene expression: through competition with the Sens repressor for DNA binding sites. Sens binds RhoA to repress thoracic gene expression, whereas in the abdomen Exd/Hth/Abd-A is permissive for activation by out-competing Sens. Importantly, mutations that disrupt both Sens and Hox binding to RhoA (SensM/HoxM) are expressed in the thorax and abdomen, revealing that Exd/Hth/Abd-A binding is not required to activate gene expression. In addition, coexpression of Exd, Hth, and Abd-A in cultured cells failed to stimulate Rho654- or RhoAAA-luciferase unless Abd-A is fused to a potent activation domain. Thus, unlike other Hox target genes, Hox complexes on RhoA are permissive rather than instructive and stimulate Rho654 by interfering with the binding of a transcriptional repressor (Li-Kroeger, 2008).

A comparison of consensus Sens, Hox/Exd, and Exd/Hth sites reveal a shared core sequence, suggesting that additional target genes will be regulated through Hox-Sens antagonism. In fact, bioinformatics reveals many Hox-Sens composite sites throughout the Drosophila and mammalian genomes. However, both the Sens and Hox sites extend beyond this core sequence, indicating that only a subset of target genes will comprise composite sites. Thus, three types of target genes for those factors are proposed: (1) those regulated by only Hox factors, (2) those regulated by only Sens/Gfi1, and (3) those regulated by both Hox and Sens/Gfi1. For example, many of the previously characterized Hox target genes in the Drosophila embryo are controlled in tissues that do not express Sens, suggesting they are only regulated by Hox genes. However, the Hox and Sens/Gfi1 factors are coexpressed in many neural cells of the developing PNS in both flies and vertebrates, indicating that similarly to rho regulation in abdominal SOP cells, additional targets will be coregulated by Hox and Sens (Li-Kroeger, 2008).

Like Hox genes, the Sens gene family is conserved in C. elegans (Pag-3), Drosophila, and vertebrates (Gfi1 and Gfi1b). These zinc finger transcription factors are essential for nervous system development in all three organisms. In addition, Gfi1 plays a critical role in hematopoiesis, where it participates in regulating stem cell renewal as well as specific blood cell lineages. Interestingly, Hox factors also regulate blood cell differentiation, proliferation, and stem cell renewal. HoxA9, for example, is required for normal hematopoiesis in mice, and alterations in HoxA9 expression have been implicated in acute myeloid leukemia (AML). In fact, a study analyzing the expression profile of 6817 genes in AML patients who either responded or did not respond to treatment found the highest correlated gene associated with poor prognosis is HoxA9. To determine if the Hox-Sens mechanism uncovered in Drosophila is conserved in mammals, in vitro DNA binding assays were used to show that HoxA9 forms a complex with Pbx and Meis that competes with Gfi1 for common binding sites. Moreover, mouse genetic studies support the hypothesis that Hox-Gfi1 factors antagonize each other to regulate gene expression and blood cell development. Thus, Hox-Sens/Gfi1 competition for composite binding sites is likely a conserved mechanism for the regulation of gene expression in organisms from flies to humans (Li-Kroeger, 2008).

Transcription factor choice in the Hippo signaling pathway: homothorax and yorkie regulation of the microRNA bantam in the progenitor domain of the Drosophila eye imaginal disc

The accurate control of cell proliferation and survival is critical for animal development. The Hippo tumor suppressor pathway regulates both of these parameters by controlling the nuclear availability of the transcriptional coactivator Yorkie (Yki), which regulates downstream target genes together with Scalloped (Sd), a DNA-binding protein. This study provides evidence that Yki can also regulate target genes in conjunction with Homothorax (Hth) and Teashirt (Tsh), two DNA-binding transcription factors expressed in the uncommitted progenitor cells of the Drosophila eye imaginal disc. Clonal analyses demonstrate that Hth and Tsh promote cell proliferation and protect eye progenitor cells from apoptosis. Genetic epistasis experiments suggest that Hth and Tsh execute these functions with Yki, in part by up-regulating the microRNA bantam. A physical interaction between Hth and Yki can be detected in cell culture, and this study shows that Hth and Yki are bound to a DNA sequence approximately 14 kb upstream of the bantam hairpin in eye imaginal disc cells, arguing that this regulation is direct. These data suggest that the Hippo pathway uses different DNA-binding transcription factors depending on the cellular context. In the eye disc, Hth and Tsh provide spatial information to this pathway, promoting cell proliferation and survival in the progenitor domain (Peng, 2009).

The evidence suggests that Hth and Tsh function as partners to carry out two main functions in anterior eye disc cells: They repress the expression of the later-acting retinal determination factors, and they promote cell proliferation. That these functions require hth is supported by both loss-of-function studies as well as gain-of-function studies. For example, hthP2 clones fail to survive anterior to the MF, and Tsh's ability to induce overgrowths when ectopically expressed is abolished in the absence of hth. The involvement of Tsh is supported by gain-of-function experiments and the finding that Hth and Tsh directly interact with each other in vivo. Carrying out loss-of-function genetics for tsh is difficult because this gene is located proximal to the standard Flp recombination targets (FRTs) used to generate mitotic recombination. In addition, the highly related gene tio, which is closely linked to tsh, functions redundantly with tsh in several instances, including some aspects of eye development. Nevertheless, knocking down tsh using RNAi in a tio-null background results in poor survival in the progenitor domain. Taken together, these data provide a compelling argument for Hth + Tsh functioning together to promote cell survival in the anterior eye disc (Peng, 2009).

A functional relationship between Hth and Tsh also exists in other tissues in Drosophila, most notably in both wing and leg imaginal discs, where they are coexpressed in cells that will give rise to the proximal domains of these appendages. In both wings and legs, Tsh has the capacity to regulate hth when expressed in clones, and both tsh and hth have the ability to suppress distal appendage development when misexpressed. However, in these tissues, and unlike the eye disc, Hth + Tsh expression is not correlated with proliferation, which occurs uniformly throughout these discs. Consistently, the expression pattern exhibited by the bantam sensor does not correlate with Hth or Tsh in the leg or wing. The special relationship between proliferation and Hth + Tsh in the eye may be due in part to the Drosophila Pax6 homolog Eyeless (Ey), which is critical for eye identity. Moreover, Ey is found in a complex with Hth in vivo and participates with Hth and Tsh in the repression of retinal determination genes. Thus, it may also be the case that Ey directly participates in the regulation of bantam together with Hth and Yki (Peng, 2009).

Although hthP2 clones fail to survive in the eye progenitor domain, the data demonstrate that hth is not absolutely required for cells in this domain to proliferate. The effects observed on the bantam sensor are consistent with the idea that hth promotes, but is not essential for, cells to proliferate in the eye progenitor domain. In hthP2 clones, bantam sensor levels increased above those normally observed in progenitor cells, but not as high as the levels observed in differentiated photoreceptors. Thus, if the level in photoreceptors represents the complete absence of bantam, these data imply that hth only up-regulates bantam over a basal hth-independent level. Moreover, the levels of the bantam sensor in other tissues, such as the wing disc, rarely approach those observed in photoreceptors, suggesting that most cells have some bantam expression, and that bantam regulators, such as hth, only serve to modulate bantam levels (Peng, 2009).

If eye progenitor cells have the capacity to proliferate in the absence of hth, how important is the proliferation-promoting function of hth? Although normal eyes can develop in animals in which hthP2 clones are generated, this is likely due to the ability of neighboring wild-type cells to compensate in this mosaic situation. In contrast, when wild-type and heterozygous cells are killed (using the EGUF method [ey-Gal4/UAS-flp/GMF-hidwe find that the remaining hthP2 tissue is unable to produce normal-sized eyes. This experiment suggests that the proliferation-promoting functions of hth in the eye progenitor domain are critical for normal eye development, likely by providing a sufficient pool of progenitor cells prior to differentiation (Peng, 2009).

The Hippo pathway has emerged recently as an important regulator of cell proliferation and survival in both vertebrates and invertebrates. In Drosophila, this pathway appears to regulate proliferation in nearly all tissues. For example, wts- clones or Yki+ clones have the capacity to induce overgrowths throughout the body. As Yki and its mammalian ortholog Yap are transcriptional coactivators that do not have their own DNA-binding domain, they are thought to partner with DNA-binding transcription factors to regulate gene expression. Prior to this work, the only transcription factor proposed to work directly with Yki was Sd, a member of the TEAD/TEF family of DNA-binding proteins. However, unlike other components of the Hippo pathway, the available data suggest that Sd plays a more limited role in cell proliferation and survival in Drosophila. In contrast to its essential role in the wing pouch, sd- clones survive well in other tissues, including the region of the wing disc that will give rise to the notum of the fly. Similarly, sd-null clones grow well in the eye progenitor domain. Thus, unlike in the wing pouch, sd is not required for cell survival and proliferation in the eye progenitor domain (Peng, 2009).

In contrast to the survival of sd clones in this domain, hthP2 clones fail to survive in the eye progenitor domain. Thus, analogous to sd in the wing pouch, hth is required for cells to survive and proliferate in the anterior eye imaginal disc. This observation suggests that hth could play an analogous role to sd in this progenitor domain, a view that is supported by the results. This evidence includes (1) Hth can interact with Yki when coexpressed in S2 cells, (2) Hth + Tsh regulate the Yki target bantam, and (3) Hth and Yki are both bound to the same region of the bantam locus in eye discs. Genetically, it was shown that the Hippo pathway is unable to induce overgrowths in the eye progenitor domain without hth, and that Hth + Tsh cannot induce overgrowths in the absence of Yki. These results suggest that Hth + Tsh comprise the DNA-binding transcription factors that function with Yki to regulate proliferation and survival genes, such as bantam. Thus, analogous to Sd in the wing pouch, Hth + Tsh are transcription factors used by the Hippo signaling pathway in eye progenitor cells (Peng, 2009).

The finding that Hth + Tsh play an analogous role in the eye progenitor domain as Sd does in the wing pouch has several implications for how the Hippo pathway is regulated in vivo. For one, the use of different DNA-binding transcription factors to regulate Hippo target genes suggests a previously unknown degree of specificity available to this pathway. Hth, a TALE family homeodomain protein, and Tsh, a Zn finger protein, are likely to bind very different target DNA sequences than Sd, a TEAD/TEF domain DNA-binding factor. Accordingly, it was found that ectopic Hth + Tsh clones in the eye disc do not consistently up-regulate diap1 or expanded, known Sd-Yki targets in the wing disc (Peng, 2009).

These results also imply that the transcriptional regulation of hth, tsh, and sd has the potential to change the output of the Hippo pathway. Because hth and tsh are transcriptionally repressed by signals coming from the MF, these factors are not available to work with the Hippo pathway posterior to the MF. However, loss of Hippo kinase activity can lead to proliferation of differentiated cells posterior to the MF. In these cells, sd is expressed, suggesting that Yki may use this transcription factor in this context. Analogously, loss of Hippo kinase activity can cause overgrowths in the notum as well as in the wing pouch. As sd- clones grow well in the notum, but not in the wing pouch, these data suggest that the notum overgrowths may be mediated by a transcription factor other than Sd. hth clones also survive well in the notum, implying that yet another transcription factor or factors may work with Yki in this tissue. In sum, it is suggested that Yki, and thus the Hippo pathway, may be able to work with multiple transcription factors to regulate target genes. In principle, the use of several transcription factors that are themselves developmentally regulated allows the Hippo pathway to be interpreted in different ways in different contexts (Peng, 2009).

Although the data suggest that the Hippo pathway uses Hth + Tsh to up-regulate bantam, they also suggest that both Hth + Tsh and Yki have additional, independent targets. For example, the loss of Hippo kinase activity leads to the up-regulation of diap1 throughout the eye disc. Because diap1 is not affected when Hth + Tsh are coexpressed, the Hippo pathway has the capacity to regulate some genes independently of Hth + Tsh, even in the eye progenitor domain. Moreover, at least when Yki is ectopically expressed, sd appears to be required in all regions of the eye disc for diap1 activation. Thus, although it has not been shown that sd is required for endogenous diap1 expression in this tissue, these data suggest that Yki may use both Sd and Hth + Tsh to regulate gene expression in the eye disc. In fact, it has been suggested that sd is also a modifier of bantam expression in the eye disc and that sd is required for normal-sized eyes. However, these clones, which used RNAi to knock down Sd, grew well in the eye progenitor domain. In addition, the smaller eyes observed when sd was knocked down could be due to the earlier embryonic expression of the Gal4 driver used in these experiments. In contrast, when generated during larval stages, hth- clones, but not sd- clones, fail to survive in the eye progenitor domain, arguing that, at least post-embryonically, gene regulation by Hth + Tsh, not Sd, is critical for cell survival in this tissue. This conclusion is also supported by the finding that Hth + Tsh can induce proliferation in the absence of sd (Peng, 2009).

As shown previously, Hth + Tsh play a key role in blocking eye differentiation by repressing the retinal determination genes eya and so. The available data do not yet resolve whether this repression works independently of the Hippo pathway. In contrast, the loss of Hippo kinase activity leads to overgrowths without blocking differentiation, arguing that nuclear Yki promotes proliferation without changing cell fate. Consistently, it was found that wts- or Yki+ clones do not alter Elav expression in differentiated photoreceptors. Curiously, however, ectopic expression of Hth + Tsh did not block differentiation in the absence of Yki. Although these data could be interpreted to suggest that Yki is directly required for repressing differentiation, they could alternatively suggest that repression requires cell proliferation. Consistently, Hth + Tsh were also unable to block differentiation in the absence of bantam. These observations raise the possibility that the absence of bantam or yki indirectly inhibits Hth + Tsh's ability to repress differentiation by compromising the proliferation of these cells, although other indirect affects are also possible (Peng, 2009).

Hth + Tsh are also likely to regulate genes in addition to bantam to promote proliferation and survival in the eye progenitor domain. This is most strongly supported by the observation that ectopic expression of bantam only partially rescues the survival of hthP2 clones. In addition, it was found that the overgrowths generated by ectopic expression of Hth + Tsh are only partially suppressed by the coexpression of Hpo, whose overexpression removes Yki from the nucleus. These data suggest that some of the Hth + Tsh targets that mediate growth and survival in the eye progenitor domain are regulated independently of Yki (Peng, 2009). In summary, these results suggest that the transcriptional regulation of hth and tsh along the anterior-posterior axis of the eye disc changes the output of the Hippo pathway. In the eye progenitor domain, where Hth and Tsh are both present, the pathway uses these transcription factors to promote proliferation and cell survival, at least in part by up-regulating bantam. Once hth and tsh are repressed by signals coming from the MF, the Hippo pathway may use other transcription factors, such as Sd, to regulate a different set of target genes. Thus, together with other functions carried out by these transcription factors, their regulation across the anterior-posterior axis coordinates the complex switch from proliferation to differentiation during eye development (Peng, 2009).

Abdominal-A mediated repression of Cyclin E expression during cell-fate specification in the Drosophila central nervous system

Homeotic/Hox genes are known to specify a given developmental pathway by regulating the expression of downstream effector genes. During embryonic CNS development of Drosophila, the Hox protein Abdominal-A (AbdA) is required for the specification of the abdominal NB6-4 lineage. It does so by down regulating the expression of the cell cycle regulator gene cyclin E (CycE). CycE is normally expressed in the thoracic NB6-4 lineage to give rise to mixed lineage of neurons and glia, while only glial cells are produced from the abdominal NB6-4 lineage due to the repression of CycE by AbdA. This study investigated how AbdA represses the expression of CycE to define the abdominal fate of a single NB6-4 precursor cell. Both in vitro and in vivo, the regulation was examined of a 1.9 kb CNS-specific CycE enhancer element in the abdominal NB6-4 lineage. CycE was shown to be a direct target of AbdA and it binds to the CNS specific enhancer of CycE to specifically repress the enhancer activity in vivo. These results suggest preferential involvement of a series of multiple AbdA binding sites to selectively enhance the repression of CycE transcription. Furthermore, the data suggest a complex network to regulate CycE expression where AbdA functions as a key regulator (Kannan, 2010).

All progenies of both thoracic and abdominal NB6-4 can be traced using Eagle (Eg) as a lineage marker, and Reversed polarity (Repo) for differentiating glial cells. Thus, Eg-only expression marks neuronal fate. The thoracic variant of NB6-4 (NB6-4t) gives rise to both neuronal and glial cells, whereas the abdominal variant (NB6-4a) gives rise to only glial cells. The Hox gene Antennapedia (Antp) is expressed in the NB6-4t lineage of thoracic segments (T1-T3) whereas abdominal A (abdA) and Abdominal B (AbdB) are expressed in the NB6-4a lineage of abdominal segments A1-A6 and A7-A8, respectively. However, loss of Antp function does not affect the lineage development in contrast to loss of abdA or AbdB, which results in NB6-4a to NB6-4t homeotic transformations. Thus, thoracic identity of NB6-4 lineage acts as a default state without the requirement of any Hox gene input, while abdominal identity of the lineage is imposed by the function of abdA and AbdB. AbdA and AbdB function by suppressing the expression of CycE, a cell cycle molecule necessary for G1-S phase transition. This study has focused on the mechanism by which AbdA regulates CycE expression (Kannan, 2010).

Exd and AbdA cooperatively bind as a heterodimer to a consensus DNA sequence. During development, nuclear localization of Exd is regulated by interaction with another homeodomain protein Homothorax. The expression pattern of the cofactors Exd and Hth was examined in NB6-4 lineages of wild-type embryos. Exd expression was detected in glial precursors of the NB6-4a lineage, Interestingly, it was not found in the NB6-4t lineage, although in the ectoderm expression levels of nuclear Exd are higher in thoracic segments than in the abdominal segments. In the case of Hth, the protein was detected in NB6-4a glial cells only after late stage 11, and also weakly in NB6-4t derived glia. Thus, consistent with their requirement to modulate the function of Hox proteins, Exd was found expressed in the NB6-4a lineage and not in NB6-4t lineage, although weak expression of Hth was detected in glial cells of NB6-4t lineage. Assuming that both are required together to modulate Hox function, modulation of Hox function is predicted only in NB6-4a lineage (where abdA and AbdB are expressed) and not in NB6-4t (where Antp is expressed). Indeed, loss of abdA and AbdB show NB6-4a to NB6-4t transformations, while loss of Antp has no phenotypic consequence (Kannan, 2010).

To gain additional evidence on the relevance of exd and hth expression in the NB6-4a lineage, their loss of function mutations were analyzed. exd mutant embryos showed an increase in the number of NB6-4a progeny. Some of these cells migrated medially in a pattern similar to glial cells of NB6-4t, while others migrated to the dorso-lateral cortex, suggesting neuronal identity. Abdominal hemisegments of hth mutant embryos did not show an increase in glial progeny, but generated ectopic neurons in the dorsal lateral cortex suggesting homeotic transformation of NB6-4a to NB6-4t. The fact that mutations in both the cofactors of AbdA independently induced homeotic transformations, although at much lesser degree (11% in exd mutants and 7% in hth mutants, compared to 100% in abdA mutants), suggests that this observation is a phenocopy of the abdA loss of function phenotype. The mildness of the phenotypes could be an indication of their role as cofactors to enhance the effect of AbdA rather than essential factors to regulate cell-fate specification (Kannan, 2010).

Next the expression pattern of CycE transcripts was investigated in the transformed abdominal NB6-4 lineage in exd mutant embryos. Consistent with the phenotype at the cellular level, CycE mRNA was observed exclusively in neuronal cells of transformed NB6-4a (Eg expressing cells) in exd mutant background as is the case for the thoracic NB6-4. In hemisegments that show no transformation and thus represent the wild-type NB6-4a, absence of CycE mRNA was found (Kannan, 2010).

The complex cis-regulatory region of zygotic CycE comprises of tissue and stage specific activator and repressor elements within an at least 10 kb genomic region including upstream and downstream elements. Based on the expression pattern of a 1.9 kb lacZ reporter gene (CycE-lacZ) in transgenic assays, it is evident that this region includes cis-acting sequences that drive zygotic CycE transcription both in epidermis (mitotic cycles 14-16) and CNS and regulatory elements responsible for CycE down regulation at the end of st11. Similar to CycE transcripts and CycE protein, CycE-lacZ is not expressed in NB6-4a and is activated in abdA, AbdB double mutant embryos. Thus, this 1.9 kb lacZ reporter reliably reflects the CycE expression in the abdominal NB6-4 lineage (Kannan, 2010).

To elucidate the mechanisms that AbdA specifies in collaboration with Exd/Hth, the 1.9 kb CNS-specific CycE regulatory element for known AbdA, Exd and Hth-binding sites. The element harbours at least 3 binding sites for AbdA, and one each for Exd and Hth in close association. Since strong repression of β-Gal expression from the 1.9 kb CycE-lacZ regulatory element was observed in the NB6-4a lineage, it was wondered whether this regulation could be due to the presence of AbdA and Exd/Hth-binding sites. Therefore, the 1.9 kb CycE-lacZ fragment in more detail both in vitro and in vivo (Kannan, 2010).

Binding of AbdA, Exd and Hth to the regulatory sequences of CycE was tested by electro-mobility shift assays (EMSAs) on three spatially separated AbdA binding sites named AbdA-1, -2 and -3. AbdA-2 is in close association with binding sites of cofactors Exd and Hth, named as 68 bp fragment. EMSA suggested physical association of AbdA protein with all the three putative binding sites (AbdA-1, -2 and -3) when tested independently with corresponding oligosequences. In addition, the association of AbdA, Exd and Hth complex was observed in the 68 bp fragment. To check whether the putative AbdA, Exd and Hth sites identified within the CycE enhancer are responsible for assembling the complex, the core sequences that make critical contact to each of these factors was mutated. Mutations in Hox binding sites resulted in the loss of association of AbdA, Exd and Hth to the core sequence. These results suggest cooperative binding of AbdA and its cofactors Exd and Hth to the CycE enhancer element (Kannan, 2010).

To test the functional relevance of binding of AbdA, Exd and Hth in vivo, reporter gene constructs were constructed, with presence or absence of either of three AbdA binding sites, upstream of a minimal promoter driving β-Gal expression. Transgenic flies were generated by P-element mediated transformation (Kannan, 2010).

Embryos homozygous for lacZ transgenes (two independent insertion lines for each transgene to rule out position variation effects) were stained for Eg, Repo and β-Gal to visualize the regulatory behaviour of the CNS-specific CycE element in the abdominal NB6-4 lineage. The enhancer elements deleted independently for putative binding sites AbdA-1 and AbdA-2 drive β-Gal expression in abdominal NB6-4 cells, suggesting the preferential requirement of both sites for transcriptional repression of CycE. In contrast, the transgene deleted for AbdA-3 showed the wild-type 1.9 kb CycE-lacZ expression pattern i.e. no expression in NB6-4a, suggesting that AbdA-3 may not be a preferred binding site for repressive activity. As expected, regulatory elements deleted for both AbdA-1 and AbdA-3 or AbdA-2 and AbdA-3 drive lacZ expression in abdominal NB6-4 progenies. However, deletion of both the repressor elements abdA1 and abdA2 (CycE-lacZAbdA-1&2) did not result in de-repression of lacZ in NB6-4a. Interestingly, deletion of all the three elements resulted in the activation of lacZ in NB6-4a. This suggests that AbdA-3 may act as a cryptic repressor, functional only in the absence of both AbdA-1 and AbdA-2. In addition, while deletion of AbdA-2 alone resulted in the activation of β-Gal expression in NB6-4a, CycE-lacZ68bp element deleted for Exd/Hth and AbdA-2 mimicked wild-type β-Gal expression pattern suggesting that in its absence, AbdA-1 and AbdA-3 may maintain repression of CycE in NB6-4a (Kannan, 2010).

These results do not rule out the possibility of other sequences in the regulatory region of CycE that contribute to the AbdA-mediated repression. The fact that lacZ is strongly repressed in CycE-lacZAbdA-1&2 and CycE-lacZ68bp embryos, but de-repressed in CycE-lacZAbdA-1,2&3, CycE-lacZAbdA-1&3 and CycE-lacZAbdA-2&3 embryos, suggest that other regulators may function together with this enhancer in vivo. There is a possibility that this regulatory region is between AbdA-1 and AbdA-2, which is required to assemble a repressor complex. Computational screening of the 1.9 kb enhancer fragments revealed the existence of at least 3 Engrailed (En)-binding sites. Two of the En sites are between AbdA-1 and AbdA-2. It is likely that a minimum of two AbdA binding sites along with this regulatory region is required to assemble a repressor complex that also involves Exd/Hth and probably En. When either AbdA-1 or AbdA-2 is deleted, this repressor complex fails to assemble and hence leads to activation of CycE-lacZ in NB6-4a. In the absence of both AbdA-1 and AbdA-2, the putative En-binding region may come closer to AbdA-3 and still be able to assemble a repressor complex. Interestingly, repression of lacZ was observed when the whole 68 bp region is deleted. This could also be due to the fact that AbdA-3 is now much closer to the putative En-binding sites. Unfortunately, this could not be tested in the background of loss of function of en since NB6-4 itself is not born in those embryos. Nevertheless, the above mentioned model appears to be identical to the way Dll expression is repressed in the epithelial cells, which is mediated by Ubx and En. Further investigation in this direction involves Chromatin immunoprecipitation for AbdA or En followed by Western blot analyses for the other protein under different conditions (Kannan, 2010).

To conclude, these results suggest the preferential involvement of a series of multiple AbdA binding sites for enhanced repression of CycE transcription. These data suggests a complex network to regulate CycE expression where AbdA functions as a key regulator. This may have evolved to ensure tight repression of CycE as it is a potent regulator of cell fate in NB6-4 and possibly other CNS lineages (Kannan, 2010).

Control of the spineless antennal enhancer: direct repression of antennal target genes by Antennapedia

It is currently thought that antennal target genes are activated in Drosophila by the combined action of Distal-less, homothorax, and extradenticle, and that the Hox gene Antennapedia prevents activation of antennal genes in the leg by repressing homothorax. To test these ideas, a 62bp enhancer was isolated from the antennal gene spineless that is specific for the third antennal segment. This enhancer is activated by a tripartite complex of Distal-less, Homothorax, and Extradenticle. Surprisingly, Antennapedia represses the enhancer directly, at least in part by competing with Distal-less for binding. Antennapedia is required in the leg only within a proximal ring that coexpresses Distal-less, Homothorax and Extradenticle. It is concluded that the function of Antennapedia in the leg is not to repress homothorax, as has been suggested, but to directly repress spineless and other antennal genes that would otherwise be activated within this ring (Duncan, 2010).

This report examines the regulation of an enhancer from the antennal gene ss that drives expression specifically in the third antennal segment (A3). The work provides the first look at how the homeodomain proteins Dll, Hth, and Exd function in the antenna to activate antennal target genes. These proteins form a trimeric Dll/Hth/Exd complex on the enhancer, suggesting that Dll acts much like a Hox protein in antennal specification. This work also reveals how the Hox protein Antp functions in the leg to repress antennal development. The conventional view has been that the primary function of Antp is to repress hth in the distal leg, which then prevents the activation of all downstream antennal genes. However, this study found that Antp represses the ss A3 enhancer directly. This repression is essential within a proximal ring in the leg that coexpresses the antennal gene activators Dll, Hth, and Exd. Antp competes with Dll for binding to the enhancer, and this competition is part of a molecular switch that allows the ss A3 element to be activated in the antenna, but represses its activation in the leg. The results suggest that repression of antenna-specific genes in the proximal ring is the sole function of Antp in the leg imaginal disc (Duncan, 2010).

At 62 bp, the ss A3 enhancer (called D4) is one of the smallest enhancers to be identified in Drosophila, and yet it is quite strong; only a single copy is required to drive robust expression of lacZ reporters. The enhancer is also very specific, driving expression in A3 and nowhere else in imaginal discs. It has been proposed that antennal identity in Drosophila is determined by the combined action of Dll, Hth, and Exd. Consistent with this proposal, all three of these factors were found to be required for D4 expression. Although these activators are coexpressed in both A2 and A3, D4/lacZ expression is restricted to A3 by Cut, which represses the enhancer in A2. Like ss itself, D4/lacZ is also repressed by ectopically expressed Antp (Duncan, 2010).

A previous report (Emmons, 2007) showed that the antennal expression pattern of ss is reproduced by lacZ reporters containing a 522 bp fragment from the ss 5' region. This fragment contains five conserved (41%-90% identity) domains, each of which was deleted and tested for effect on expression in vivo. Expression in the arista and the third antennal segment (A3) prove to be under separate control; expression in the arista requires domains 1, 3 and 5, whereas expression in A3 is lost only when domain 4 is deleted. Moreover, reporters containing domain 4 alone show expression in A3 and nowhere else in imaginal discs. Thus, domain 4 is both necessary and sufficient for A3-specific expression. Domain 4 (D4) is 62 bp in length and is highly conserved, being invariant at 50/62 base pairs in the 12 Drosophila species sequenced (Duncan, 2010).

Surprisingly, Dll, Hth, Exd, Cut, and Antp all act directly upon D4. The activators Hth and Exd bind with strong cooperativity to directly adjacent sites. Their joint binding site matches the optimum site for in vitro binding of the mammalian homologs of Hth and Exd (Meis and Prep), consistent with the robust activity of the enhancer in vivo. Mutation of either of these sites abolishes activity of the enhancer. The coactivator Dll binds three sites in D4; one of these sites (Dlla) is required for almost all activity of the enhancer. Dll shows strong cooperativity with Hth and Exd for binding to D4, indicating that Dll interacts physically with these proteins. This interaction requires DNA binding, as Dll protein containing a missense change that blocks DNA binding (a change of asn51 to ala in the homeodomain) shows no ability to associate with D4-bound Hth and Exd. A curious feature of the cooperativity seen in the binding studies is that although Hth and Exd increase the affinity of Dll for D4, Dll appears to have little effect on the affinity of Hth and Exd for the enhancer. Since Hth and Exd already bind cooperatively with one another, it may be that additional cooperative interactions with Dll have little effect. Alternatively, it may be that Hth and Exd interact with Dll only after binding DNA. If so, Hth and Exd would be expected to increase Dll binding to D4, but Dll would have little effect on the binding of Hth and Exd, as observed. Interactions between Dll and Hth in the absence of DNA have been reported in immunoprecipitation experiments. However, this study was unable to repeat these observations. Moreover, the finding that the asn51 mutant of Dll fails to associate with D4-bound Hth and Exd argues strongly against such interactions (Duncan, 2010).

The repressor Cut also acts directly upon D4. Binding of Cut requires two sites, one overlapping Dlla and the other overlapping the joint Hth/Exd site. These binding sites suggest that D4 is controlled by Cut in much the same way that a structurally similar Abdominal-A (Abd-A) regulated enhancer from the rhomboid gene is controlled by the repressor Senseless (Sens). In the rhomboid enhancer, adjacent Hth and Exd sites are also present, and these create a binding site for Sens. Activity of the rhomboid enhancer is controlled by a competition between binding of the Sens repressor and binding of the activators Abd-A, Hth, and Exd. It seems likely that D4 is controlled similarly, with the repressor Cut competing for binding with the activators Dll, Hth, and Exd. It will be of interest to determine whether enhancers similar to D4 are used more widely to control Cut targets involved in its role as an external sense organ determinant (Duncan, 2010).

A key finding in this work is that Antp represses D4 by direct interaction. Antp binds a single site in D4, which overlaps or is identical to the Dlla binding site. Like Dll, Antp binds cooperatively with Hth and Exd. Using purified proteins, it was showm that binding of Dll and Antp to the Dlla site is mutually exclusive. This indicates that Antp represses the enhancer at least in part by competing with Dll for binding. Similar competition may occur at other enhancers; when Antp expression is driven artificially in the distal leg, variable deletions of the tarsal segments occur. These defects might arise because Antp competes with Dll for binding to its target genes in the distal leg. In most other contexts examined, Antp is an activator of transcription; why it fails to activate D4 is not clear. The similar behavior of Dll and Antp in binding to D4 supports the idea that Dll behaves like a Hox protein in activating D4 (Duncan, 2010).

Although the initial focus of this study was on the antenna, the finding that Antp interacts directly with D4 led to an examination of D4 regulation in the leg, where Antp is normally expressed. In second leg imaginal discs, Antp is required only in a proximal ring of cells that coexpresses Dll and Hth. This ring appears in the early third instar, and is of uncertain function. Large Antp clones in T2 leg discs that do not enter this ring appear to develop completely normally, regardless of whether they are located distal or proximal to the ring. However, clones that overlap the ring show activation of D4/lacZ within the ring cells. Importantly, such clones have no effect on the expression of Dll or Hth within the ring. By examining Antp clones of increasing age the following sequence of events is inferred. First, D4/lacZ is activated in cells of the ring that are included within Antp clones. Second, many such clones begin expressing the antennal markers Ss and Cut, indicating a transformation to antenna, and round up as if they have lost affinity for neighboring cells. Third, such clones appear to extend and move distally in the disc (Duncan, 2010).

The events described for Antp clones in the leg make sense of several previously enigmatic observations. It has been noted that many Antp clones in the leg do not transform to antenna and appear to develop normally. The finding that only clones that overlap the proximal ring undergo transformation accounts for this observation. Antp clones that do contain transformations usually show apparent nonautonomy in that not all cells in the clone are transformed to antenna. The current results account for this observation as well, since within an Antp leg clone only those cells located in the proximal ring undergo transformation to antenna; cells located elsewhere in the clone retain normal leg identity. Most importantly, these observations provide an explanation for why ss is controlled directly by Antp. Antp clones have no effect on hth or Dll expression in the proximal ring. Therefore, Antp must function in the ring at the target gene level to repress antennal genes that would otherwise be activated by combined Hth and Dll (and Exd). Since several such targets are known, it seems likely that several, perhaps many, antennal genes in addition to ss are repressed directly by Antp (Duncan, 2010).

Transformed Antp clones in the leg often show ectopic hth expression in distal locations. If hth is not directly controlled by Antp in the leg, as this study suggests, then why is hth ectopically expressed within such clones? A likely explanation is that downstream antennal genes that have become activated in such clones feed back to activate hth. This interpretation is strongly supported by the finding that ectopic expression of the antennal genes ss, dan, or danr in the distal leg causes ectopic activation of hth. Thus, the distal expression of hth seen in Antp leg clones is likely a consequence rather than a cause of the transformation to antenna. Whether repression of hth in the antenna by ectopic Antp is also indirect is not clear. Dll is also expressed ectopically in transformed Antp leg clones, suggesting that it is also subject to feedback activation by downstream antennal genes (Duncan, 2010).

The function of the proximal Dll- and Hth-expressing ring in the proximal leg is not well understood. The ring is highly conserved among the insects, and may serve as a boundary between the proximal and distal portions of the legs. In the context of this work, a striking feature of the ring is that it contains a microcosm of gene expression domains corresponding to the three major antennal segments. Thus, proceeding from proximal to distal through the ring, cells express hth alone, hth + Dll, and hth + Dll + strong dachshund. These expression combinations are characteristic of the A1, A2, and A3 antennal segments, respectively. Looked at in this way, the ring would appear to resemble a repressed antennal primordium within the leg (Duncan, 2010).

It has been known for almost thirty years that Antp is required in the leg to repress antennal identity. However, an understanding of how this repression occurs has been lacking. The current results indicate that Antp functions within the proximal ring to directly repress antennal genes that would otherwise be activated by combined expression of Dll, Hth, and Exd. This appears to be the only function of Antp in the leg, at least during the third instar larval stage. The results are entirely consistent with the idea that second leg is the 'ground state' ventral appendage (the limb type that develops in the absence of identity specification) and that the role of Antp in the leg is to preserve this ground state by repressing the activation of 'head-determining' genes (Duncan, 2010).

Segment-specific regulation of the Drosophila AP-2 gene during leg and antennal development

Segmentation involves subdivision of a developing body part into multiple repetitive units during embryogenesis. In Drosophila and other insects, embryonic segmentation is regulated by genes expressed in the same domain of every segment. Less is known about the molecular basis for segmentation of individual body parts occurring at later developmental stages. The Drosophila transcription factor AP-2 gene, dAP-2, is required for outgrowth of leg and antennal segments and is expressed in every segment boundary within the larval imaginal discs. To investigate the molecular mechanisms generating the segmentally repetitive pattern of dAP-2 expression, transgenic reporter analyses was performed and multiple cis-regulatory elements were isolated that can individually or cooperatively recapitulate endogenous dAP-2 expression in different segments of the appendages. An enhancer specific for the proximal femur region, which corresponds to the distal-most expression domain of homothorax (hth), was analyzed in the leg imaginal discs. Hth is known to be responsible for the nuclear localization and, hence, function of the Hox cofactor, Extradenticle (Exd). Both Hth and Exd were shown to be required for dAP-2 expression in the femur, and a conserved Exd/Hox binding site was found to be essential for enhancer activity. These loss- and gain-of-function studies further support direct regulation of dAP-2 by Hox proteins and suggest that Hox proteins function redundantly in dAP-2 regulation. This study reveals that discrete segment-specific enhancers underlie the seemingly simple repetitive expression of dAP-2 and provides evidence for direct regulation of leg segmentation by regional combinations of the proximodistal patterning genes (Ahn, 2011).

The segmentally repeated expression of dAP-2 in the developing leg and antennal discs may suggest that its expression in each segment is regulated in a similar manner by upstream segmentation genes. Alternatively, each domain (ring) of dAP-2 expression could result from the combinatorial activities of multiple transcription factors, which themselves are not expressed in a repeated pattern, but instead occupy distinct and broader domains along the PD axis of the appendages. Current data provide strong evidence that the latter strategy is utilized to establish dAP-2 expression in all but the tarsal segments. It seems that the tarsus has adopted a strategy different from that of other leg segments to regulate dAP-2 expression (Ahn, 2011).

In an effort to understand molecular mechanisms controlling dAP-2 expression during leg development, the regulatory potential of dAP-2 genomic fragments was tested using transgenic reporter analyses. Multiple enhancers were successfully isolated which can independently direct reporter expression in specific leg segments and together recapitulate, almost completely, the endogenous expression pattern. It is intriguing that the relative positions of these enhancers on the chromosome are well correlated with the position of their activity along the PD axis of the leg. Importantly, the presence of segment-specific enhancers suggests that dAP-2 expression is differentially regulated in each leg segment. It is likely that each domain of dAP-2 expression in the true joints is regulated by a combination of upstream regulators involved in PD patterning using segment-specific enhancers similar to the distinct enhancers used to regulate expression of the pair-rule gene, even-skipped, in every other parasegment during embryonic segmentation. Interestingly, dAP-2 expression in the coxa is differentially regulated along the DV axis and depends on two region-specific enhancers. In addition, the EB fragment displayed relatively weaker activity in the ventral region compared to the larger E6 fragment. These data raise the possibility that DV patterning genes are also involved in dAP-2 regulation in the proximal segments. It is possible that the use of multiple region-specific enhancers is a general mechanism establishing expression of segmentation genes during leg development (Ahn, 2011).

Current data indicate that dAP-2 expression in antennal discs also requires multiple region-specific enhancers. Some of the leg enhancers showed an antennal expression pattern similar to their leg patterns with respect to the PD axis. However, there are also enhancers specific for either antennal or leg discs implying that the genes required for normal identity of the two homologous appendages might be involved in regulation of dAP-2 expression in some segments. One of the features that distinguish antennae from legs is that in antennal discs, hth expression is expanded to the intermediate region where dac expression is missing. In contrast, the expression patterns of Dll, dac and hth are very similar in the proximal and distal regions of the two appendages. It is interesting to note that the dAP-2 enhancers for the most proximal and distal regions are shared between the two appendages while the intermediate region utilizes distinct enhancers. This implies that dAP-2 expression in the intermediate region is more likely to be regulated by antennal- or leg-specific regulatory pathways. For example, although the femur and the AIII are homologous structures, the Hox-dependent proximal femur enhancer is active in the leg, but not in the antenna. Likewise, the BE enhancer is active in the proximal AIII of the Hox-free antenna, but not in the leg (Ahn, 2011).

The Hox gene Antp has been considered to be a key factor in determining leg identity since Antp mutant clones in the T2 leg cause a leg-to-antenna transformation, mainly outside of the Hth domain. Previous studies suggested that Antp performs its selector function by acting as a repressor of hth and other antennal genes in the intermediate leg. In contrast, both Antp and hth are expressed in the proximal leg, and are required for growth and segmentation of this region. Therefore, it has been proposed that the role of Antp as a repressor of hth is limited to the intermediate leg, and that both Antp and Hth contribute to proper development of the proximal leg (Ahn, 2011).

The similar loss-of-function phenotypes of hth and exd suggest that Hth and Exd act on common target genes during development of the proximal leg. In certain developmental contexts, Hth can directly bind to DNA through its homeodomain in a ternary complex including Exd and Hox proteins to regulate expression of target genes. However, it has been shown that a Hth isoform lacking the homeodomain can execute the function of Hth in PD patterning of Drosophila leg discs indicating that direct DNA binding is not necessary for its function in proximal leg discs. Since no conserved, consensus Hth binding site were found in the proximal femur enhancer of dAP-2, Hth is likely functioning in dAP-2 expression through a mechanism independent of its direct binding to DNA through its homeodomain. Instead, Hth may regulate dAP-2 expression in the proximal femur by facilitating the nuclear localization of Exd or by interacting with other transcription factors which bind DNA (Ahn, 2011).

As a cofactor of Hox proteins, Exd, and its mammalian homolog Pbx, cooperatively bind DNA with Hox proteins and regulate expression of their target genes which are involved in a variety of developmental processes in both vertebrates and Drosophila. Although previous genetic analyses have revealed essential functions of Exd and Hox proteins in leg development, it has been unclear whether these factors act together on common target genes during this process. This study has identified a conserved Exd/Hox binding site which is required for activity of the proximal femur enhancer of dAP-2. Through clonal analyses, it was demonstrated that hth, exd and Antp are necessary for dAP-2 expression in the presumptive proximal femur of leg imaginal discs. This is the first example of a direct target gene of an Exd/Hox complex in Drosophila limb development. This study also provides insight into the molecular mechanism integrating the combinatorial actions of PD patterning genes in the regulation of region-specific expression of leg segmentation genes (Ahn, 2011).

Although Antp is expressed in all three pairs of legs, most of the prothoracic (T1) and metathoracic (T3) legs with Antp mutant clones appeared to be normal, except for a rare fusion between the femur and tibia. However, Scr/Antp double mutant clones in T1 legs and Antp/Ubx double mutant clones in T3 legs generated leg defects indistinguishable from those generated by Antp mutant clones in T2 legs. It was proposed that the low penetrance of the Antp mutant phenotypes in T1 and T3 legs is due to redundancy with Scr and Ubx, which are expressed in T1 and T3 leg discs, respectively. This idea is consistent with the previous observations that Scr and Ubx both can induce antenna-to-leg transformations when ectopically expressed in antennal discs. It is proposed that Antp, Scr and Ubx can redundantly activate dAP-2 expression in the proximal femur as Exd/Hox heterodimers based on the following observations. First, dAP-2 expression in T2 leg discs, but not in T1 and T3 leg discs, requires Antp. Secondly, EMSA results demonstrate that all three Hox proteins bind strongly to the binding site in the proximal femur enhancer as Exd/Hox heterodimers. Thirdly, all three Hox proteins can activate PrF enhancer function when ectopically expressed in antennal discs (Ahn, 2011).

The Dorsocross T-box transcription factors promote tissue morphogenesis in the Drosophila wing imaginal disc

The Drosophila wing imaginal disc is subdivided into notum, hinge and blade territories during the third larval instar by formation of several deep apical folds. The molecular mechanisms of these subdivisions and the subsequent initiation of morphogenic processes during metamorphosis are poorly understood. This study demonstrates that the Dorsocross (Doc) T-box genes promote the progression of epithelial folds that not only separate the hinge and blade regions of the wing disc but also contribute to metamorphic development by changing cell shapes and bending the wing disc. Doc expression is restricted by two inhibitors, Vestigial and Homothorax, leading to two narrow Doc stripes where the folds separating hinge and blade are forming. Doc mutant clones prevent the lateral extension and deepening of these folds at the larval stage and delay wing disc bending in the early pupal stage. Ectopic Doc expression is sufficient to generate deep apical folds by causing a basolateral redistribution of the apical microtubule web and a shortening of cells. Cells of both the endogenous blade/hinge folds and of folds elicited by ectopic Doc expression express Matrix metalloproteinase 2 (Mmp2). In these folds, integrins and extracellular matrix proteins are depleted. Overexpression of Doc along the blade/hinge folds causes precocious wing disc bending, which can be suppressed by co-expressing MMP2RNAi (Sui, 2012).

Although the morphogenesis of the Drosophila wing disc epithelium has been studied intensively, the molecular mechanisms that tie wing disc subdivision to different fates and subsequent morphogenetic processes remain poorly understood. This study demonstrates that the T-box Doc genes take part in fold formation and promote the metamorphic development of the wing disc by controlling cell shape changes and tissue remodeling (Sui, 2012).

To study the role of Doc in wing disc development, its spatiotemporal expression pattern was analyzed by antibody staining. By simultaneous recording of Doc expression and fold formation, it was found that there was a correlation between Doc expression and blade/hinge (B/H) fold formation and progression. Doc was not activated until the initiation of the B/H folds, which appeared later than the hinge-internal fold in early L3. During early pupal development, the ventral compartment of the wing disc folds underneath the dorsal compartment, leading to a basal apposition of the ventral and dorsal B/H folds. This suggests that Doc plays an important role during these morphogenetic changes (Sui, 2012).

As in the embryo, Doc in the peripheral wing pouch is activated by Dpp. The formation of the double-crescent pattern requires, in addition, Doc repression. Previous studies established that the subdivision of the wing disc into notum, hinge and blade regions is attained by the action of Iro-C, Hth/Tsh and Vg in these territories, respectively. The subdivision of hinge and blade cell fates requires mutual repression between Hth and Vg. The current data revealed that Doc is expressed in the proximal region of low vg expression adjacent to the hth expression domain. Ectopic expression of either vg or hth was sufficient to repress Doc. As with the mutual repression between vg and hth, there was feedback repression between Doc and vg and between Doc and hth, ectopic Doc being sufficient to repress both vg and hth. Therefore, the mutual antagonism between Doc/Vg and Doc/Hth defines Doc expression at the two B/H folds (Sui, 2012).

Doc was not detectable in the wing imaginal disc until the initiation of the B/H folds at ~85 hours AEL. Doc expression coincided in time and space with B/H fold formation. Lack of Doc function inhibited B/H fold extension at the larval stage and caused a delay in wing disc bending at the early pupal stage. When Doc was ectopically expressed in the dpp-Gal4 domain, it was sufficient to generate an apical fold with the same characteristics as the endogenous B/H folds (Sui, 2012).

To explore the mechanism of Doc-controlled cell shape changes and collective cell movement, the distribution of cell adhesion, cytoskeletal and basal membrane proteins was examined. The cell adhesion molecule DE-cadherin is localized at adherens junctions, which maintain the polarized architecture of epithelial cells but limit their movement. Remodeling adhesion to neighboring cells contributes to cell shape changes and cell movement. No obvious effect of Doc overexpression was found on the distribution of the cell adhesion protein E-cadherin. Similarly, the ectopic fold formed at the anterior-posterior boundary of the wing pouch when expression of the Tbx gene omb (bifid -- FlyBase) is reduced, is not associated with an altered distribution of DE-cadherin. The actin and microtubule cytoskeletons coordinately control cell shape. The elongation of columnar epithelial cells requires the assembly of aligned microtubules that form a diffuse microtubule-organizing center at the apical surface. Failure of proper microtubule organization causes columnar cells to round up and shorten along their apicobasal axis. Both in endogenous folds and in folds triggered by ectopic Doc expression, the apical microtubule web was redistributed to a basolateral position, along with an expanded cell diameter and shortened cell height. It is proposed that one mechanism by which Doc causes cell shape changes is by reorganizing the microtubule web (Sui, 2012).

During development, morphogenesis requires the coordination of cell-cell and cell-ECM adhesions, and coordination between molecules involved in these processes is essential for tissue formation and morphogenesis. The degradation of basement membrane barriers is an essential step in cancer invasion. Basement membrane modulation also plays an important role during development. A role for extracellular proteolysis in imaginal disc eversion has long been recognized. Recently, it was shown that integrin-ECM interactions are necessary to maintain the columnar shape of wing disc epithelial cells. This study has shown that overexpression of Doc2 also causes cell shortening and widening of the cell diameter. The levels of integrin and main ECM components were downregulated in Doc-overexpressing cells and MMP expression was ectopically activated in these cells. The same changes were observed in wild-type B/H folds (Sui, 2012).

Members of the MMP family are able to degrade most ECM proteins. Therefore, the changes in the distribution of ECM proteins and integrins might be secondary to increased MMP activity. Mmp2 is downstream of Doc in cell shape control, as overexpressing Doc was sufficient to induce Mmp2 and repressing Doc induced a reduction of Mmp2 in B/H fold cells. Manipulation of the Mmp2 level mimicked the effects of Doc on microtubule web redistribution and fold progression. Repression of Mmp2 by expressing its inhibitor Timp or MMP2RNAi efficiently rescued the abnormal wing disc bending induced by 30A>Doc expression. Taken together, Doc-expressing cells loosen their contacts with the underlying ECM owing to enhanced MMP activity, and changes in integrin and the ECM promote cell shape changes and facilitate subsequent tissue remodeling (Sui, 2012).

In the wing pouch bending process, cells at the dorsal-ventral boundary detach from the basal membrane, shorten and acquire a wedge-shaped morphology. This process is likely to contribute to the force that causes the doubling-up of the flat pouch epithelium. The data show that Doc is required for proper hinge development by deepening the B/H folds of the wing disc. Doc mutant clones covering the B/H fold cells lead to the delay in wing disc bending at the early pupal stage. Overexpression of Doc along the B/H folds elicits precocious wing pouch bending. These data indicate that the B/H folds contribute to the bending process in either a passive or active way. The deepening of the B/H folds could provide more pliability during wing disc bending (Sui, 2012).

Members of the MMP family are involved in tissue remodeling and contribute to cell migration by destroying the ECM and the basement membrane barrier. Elevated MMP levels cause cell shape changes and promote cell mobility by disorganizing the normal tissue architecture. Doc-expressing B/H fold cells have increased MMP levels, causing enhanced ECM degradation. This might facilitate the concerted migration of fold cells. Ectopic Doc expression in the central wing pouch induced abnormal cell migration, both in the plane and out of the plane of the epithelium. Doc could contribute to the wing disc bending process by promoting the movement of B/H fold cells, possibly emulating its role in amnioserosa development (Sui, 2012).

Engrailed cooperates directly with Extradenticle and Homothorax on a distinct class of homeodomain binding sites to repress sloppy paired

Even skipped (Eve) and Engrailed (En) are homeodomain-containing transcriptional repressors with similar DNA binding specificities that are sequentially expressed in Drosophila embryos. The sloppy-paired (slp) locus is a target of repression by both Eve and En. At blastoderm, Eve is expressed in 7 stripes that restrict the posterior border of slp stripes, allowing engrailed (en) gene expression to be initiated in odd-numbered parasegments. En, in turn, prevents expansion of slp stripes after Eve is turned off. Prior studies showed that the two tandem slp transcription units are regulated by cis-regulatory modules (CRMs) with activities that overlap in space and time. An array of CRMs that generate 7 stripes at blastoderm, and later 14 stripes, surround slp1. Surprisingly given their similarity in DNA binding specificity and function, responsiveness to ectopic Eve and En indicates that most of their direct target sites are either in distinct CRMs, or in different parts of coregulated CRMs. Cooperative binding sites for En, with the homeodomain-containing Hox cofactors Extradenticle (Exd) and Homothorax (Hth), were located within two CRMs that drive similar expression patterns. Functional analysis revealed two distinct, redundant sites within one CRM. The other CRM contains a single cooperative site that is both necessary and sufficient for repression in the en domain. Correlating in vivo and in vitro analysis suggests that cooperativity with Exd and Hth is a key ingredient in the mechanism of En-dependent repression, and that apparent affinity in vitro is an unreliable predictor of in vivo function (Fujioka, 2012).

Consistent with the fact that Eve is expressed earlier than En, with some overlap at embryonic stages 8–9, slp CRMs tended to respond to ectopically expressed Eve at earlier stages than to En. Transgenic dissections further showed that they have distinct responsive regions within CRMs, suggesting that many of their binding sites are distinct. This is somewhat surprising because they are both homeodomain-containing repressors that set the posterior borders of slp stripes, and they have been seen to have similar in vitro binding specificities. A possible explanation is that they cooperate in DNA binding with different cofactors, making their functional sites distinct. Despite detailed analyses of Eve function in segmentation, no candidate co-factors for specifying target genes have emerged (Fujioka, 2012).

A recent study showed that CRM u8172 drives ectopic expression within odd-numbered parasegments in cells that normally do not express detectable levels of slp RNA. However, when combined with the promoter-proximal CRM u3125, which drives properly restricted expression within even-numbered parasegments, ectopic expression is repressed, suggesting that an Eve-responsive element resides within this region. Consistent with these findings, transgenes containing this region responded to ectopically expressed Eve, and rescue-type transgenes carrying u8172 without this region drove ectopic Slp, causing embryonic defects (Fujioka, 2012).

Recently, a striking number of distinct CRMs surrounding the slp1 transcription unit were found to drive expression that overlaps in both space and time. Extensive dissection of this regulatory region and rescue of slp mutants with various transgenes suggested that apparent redundancy may be necessary to provide fully functional levels of expression across the various stages of slp expression. This study shows that there are functionally redundant En/Exd/Hth binding sites within CRM u1523. In vitro binding analysis identified a strongly cooperative binding site and a weaker, but still highly cooperative site. Despite the apparent difference in in vitro binding affinity, either site is sufficient to confer repression in the En domain, and both sites must be mutated to cause significant derepression. Thus, apparent redundancy exists at multiple levels in slp regulation. Whether apparent redundancy at this level has a function in increasing the robustness of functional gene expression within the organism, as does apparent redundancy among multiple enhancers regulating the same gene, remains to be determined. Furthermore, cooperativity with cofactors in vitro seems to be a significant indicator of function in vivo, in addition to affinity. It was found that while the B1b site has the same apparent affinity as A2a, A2a confers considerably stronger repression activity, and shows greater cooperativity in binding by En with Exd/Hth. The discrepancy between relative affinity and functionality may be attributed to the challenge of reproducing functional binding conditions in vitro, where protein–protein interactions leading to cooperativity may be less sensitive to the differences in conditions than are protein–DNA interactions. Relatedly, competition with a variety of DNA binding proteins in vivo for sites on the DNA may lead to a greater reliance on cooperativity in vivo for occupancy of functional sites (Fujioka, 2012).

Previous studies indicated that En requires the Hox co-factors Exd and Hth to efficiently repressslp, especially in the anterior half of the embryo, and En was found to act cooperatively on target sites in the distalless gene with both Exd/Hth and posteriorly-expressed Hox gene products. Although it remains possible that the relatively weak, yet functional binding site (A2a) within i1523 might bind En with other cofactors in addition to Exd/Hth, dissection and construction experiments with this and other sites have not revealed any clear anterior–posterior differences in their activity that might suggest a functional interaction with cofactors such as Hox proteins that are restricted in expression along the anterior–posterior axis. Nonetheless, previous studies suggested that regulation of slp by En might utilize posterior-specific factors. Further analysis will be required to more fully explore this possibility (Fujioka, 2012).

The relative arrangement of consensus En and Exd sites that facilitate cooperative binding appears to be quite flexible. For example, the A2a site contains no canonical consensus core for En binding (ATTA), while for the other two functional sites, the distance between the centers of the En and Exd sites is 10–12 bp for A1a and only 2 bp for B1a. The latter is reminiscent of En–Exd/Hth binding in distalless, where simultaneous Hox binding occurs, although the position of the En site is on the opposite side of the Exd core consensus ATCA. This relative arrangement of En and Exd sites (En binding 5′ of the Exd core ATCA) is seen for all of the functional sites analyzed in this study. This arrangement is similar to the relative positions of Hox and Exd binding to sites where there is no En involvement. The flexibility overall is consistent with that seen for Exd/Hth binding in conjunction with the Hox gene products, and suggests that while homeodomain family transcription factors are able to function combinatorially in vivo on a wide variety of binding sites, there are significant constraints on the positions of contact by the individual homeodomains. A full understanding of the similarities and differences between En binding in conjunction with Exd and Hth, and Hox binding with these cofactors, will require further investigation (Fujioka, 2012).

The highly cooperative, strong En/Exd/Hth binding site B1a was both necessary and sufficient for repression of u4734 in the En domain. However, it did not fully substitute for the entire repression element that contains it, located between −3.9 and −3.4 kb from the slp1 TSS. This finding suggests that there may be other functional En binding sites in this region. Consistent with this, in vitro binding suggested that other subregions (B2 and/or B3) harbor some binding activity. Thus, like i1523, there may be partial redundancy in En complex binding within u4734, despite the existence of a single essential binding site (Fujioka, 2012).

This study has established the functional significance of three cooperative En/Exd/Hth binding sites within slp. Interestingly, two of them are well conserved among the 12 species of Drosophila whose genomes have been sequenced, and the other site is conserved within the more closely related species. The duplication that generated the twin slp transcription units apparently took place before the divergence of these 12 species, as all drosophilids (but not mosquitoes) contain two tandem slp-related protein coding regions. This might suggest that the two conserved En/Exd/Hth sites were duplicated along with the locus as a whole. It has been shown that Drosophila enhancers contain clusters of conserved sequences blocks, and the two CRMs analyzed in this study contain such conserved sequence clusters. However, the patterns of conservation in the regions surrounding the conserved En/Exd/Hth sites do not suggest that they are directly related to each other. Furthermore, both CRMs are more closely linked to slp1 than to slp2. Clearly, there have been other chromosomal rearrangements in the history of the slp locus, precluding a simple description of its evolution (Fujioka, 2012).

A recent study investigating the genome-wide distribution of En binding showed a peak on i1523, but not on u4734. The data were derived from 7–24 h-old embryos, which were mostly at later stages than those at which these CRMs are active. In addition, the data show peaks where our analysis has not identified functional CRMs. Such sites may function to assist those within the core enhancer regions, or they might be functional during larval or adult stages to keep slp in the off state. Alternatively, they might not be functionally important. Further study will be required to address these issues (Fujioka, 2012).

Hox proteins coordinate peripodial decapentaplegic expression to direct adult head morphogenesis in Drosophila

The Drosophila BMP, decapentaplegic (dpp), controls morphogenesis of the ventral adult head through expression limited to the lateral peripodial epithelium (P e) of the eye-antennal disc by a 3.5 kb enhancer in the 5' end of the gene. A 15 bp deletion mutation within this enhancer was recovered that identified a homeotic (Hox) response element that is a direct target of labial and the homeotic cofactors homothorax and extradenticle. Expression of labial and homothorax are required for dpp expression in the peripodial epithelium, while the Hox gene Deformed represses labial in this location, thus limiting its expression and indirectly that of dpp to the lateral side of the disc. The expression of these homeodomain genes is in turn regulated by the dpp pathway, as dpp signalling is required for labial expression but represses homothorax. This Hox-BMP regulatory network is limited to the peripodial epithelium of the eye-antennal disc, yet is crucial to the morphogenesis of the head, which fate maps suggest arises primarily from the disc proper, not the peripodial epithelium. Thus Hox/BMP interactions in the peripodial epithelium of the eye-antennal disc contribute inductively to the shape of the external form of the adult Drosophila head (Stultz, 2012).

dpp expression in the lateral PE of the eye-antennal disc is necessary for correct morphogenesis of the adult Drosophila head. This study shows that dpp expression related to ventral head formation is part of a Hox/BMP genetic network restricted to the PE of the eye-antennal disc. The homeotic gene lab, and its cofactors hth and exd positively regulate PE dpp expression. This is supported by the observation that Lab, Exd and Hth bind in vitro to the dpphc enhancer and the consensus sites for these factors are required in vivo for expression. In addition, individually, lab and hth are both genetically necessary and together demonstrate sufficiency for expression from dpphc enhancer, as shown from both LOF and GOF clonal analyses. lab exerts positive control over Dfd expression, as indicated by loss of Dfd expression in lab LOF clones. In contrast, Lab is ectopically expressed in Dfd LOF clones, demonstrating that Dfd represses lab in domains of its expression. Dpp signalling is genetically required for the transcription of lab, as expression from a lab reporter construct is reduced in tkv LOF clones. Expression of a hth enhancer trap increases in tkv LOF clones, and is reduced when activated Tkv is ectopically expressed, indicating that hth transcription is negatively regulated by Dpp signalling. Finally, dpp directly autoregulates its own expression (Stultz, 2006), and may be spatially limited to domains of signalling by repression by brk, as demonstrated by the ability of ectopically expressed Brk to repress expression from the dpphc enhancer. Lab and Hth (acting with Exd) activate the expression of dpp. Lab also contributes to the activation of Dfd, which when expressed, represses lab, acting as a switch to limit the extent of lab expression. It is envisioned that during disc development, lab initiates both dpp and Dfd, and when Dfd reaches a certain threshold level, it turns off lab, establishing the boundary between the two Hox proteins. However, while loss of Dfd is capable of derepressing lab throughout the disc, it does not do so to dpp, so further negative regulation must exist. brk may provide this repression to further ensure the lateral boundary of PE dpp through a potential AE element in the enhancer. These inputs collaborate to define the sharp boundary of PE dpp expression. The level of dpp transcription is positively modulated by feedback between lab and dpp and autoregulation of dpp, presumably through Mad/Med binding to the AE element. Negative feedback between dpp and hth provides a brake on expression; others may exist. For example, the inhibitory Smad protein, daughters against dpp is a target of peripodial Dpp expression (Stultz, 2006). It is presumed these interactions activate dpp expression rapidly but shut it down when a certain expression level is reached (Stultz, 2012).

The Hox response region represents one of what will likely be many inputs into the expression of this 3.5 kb enhancer. Another input, opa, is homologous to the Zinc Finger Protein of the Cerebellum or Zic family of transcription factors, and was identified due to its genetic interaction with dpps-hc mutations. Other transcription factors and signalling pathways display genetic interactions, and their contribution to PE dpp expression is being actively investigated, although it is noteworthy that lab, Dfd, hth, and exd are not among them. It is expected that many transcription factors and signalling pathways impinge on the dpphc enhancer. In this regard, the dpphc enhancer may resemble the dpp visceral mesoderm enhancer, another identified Hox target, where direct Ubx, Abdominal A, Exd, and Hth homeodomain inputs collaborate with the Fox-F-related factor binou, as well as Dpp and Wingless signalling to control gene expression. Enhancers that respond to signalling pathways often demonstrate characteristic behaviours: 'activator insufficiency', 'cooperative activation', and 'default repression', and the dpphc enhancer conforms to this model. No single activator is able to induce expression over the entire disc, as shown by GOF experiments. Ectopically expressed Lab produced activation only in close proximity to the domain of endogenous dpp, while Hth activated only in the PE of the posterior eye disc. Addition of two inputs together (Lab and Hth or Lab and Dpp signalling, activated over a much broader area. Only Opa has broad ability to activate on its own over the PE but only in concert with Lab was it able to activate outside the PE. Thus each activator is insufficient individually; the enhancer requires simultaneous cooperative inputs of multiple factors to produce correct spatial expression. Brk would provide the default repression, preventing Lab and Hth individually from successfully activating in the middle of the disc, away from domains of dpp activity (Stultz, 2012).

Based on the transcriptional inputs so far identified, it is proposed that activation is controlled on the lateral side at a minimum by Lab, Hth, Exd, Mad, Med, and Opa. In the middle of the disc, the presence of only Hth and Exd is insufficient to activate the enhancer, particularly over resident default repression provided by Brk. On the medial (future dorsal) side of the disc, Dpp and phosphorylated Mad expression are observed, controlled by an unknown area of the dpp gene. Lab, Hth, Exd, and Opa are expressed there as well, so an additional repressor was hypothesised to be needed that limits expression driven by the dpphc enhancer to the lateral side. In this model, Lab is the activity required for peripodial specificity, with its cofactors Hth and Exd, while Mad/Med and Opa act as necessary collaborative activators of the enhancer (Stultz, 2012).

At the nucleotide level, the Hox response element in and adjacent to the dpps-hc1 deficiency bears sequence homology to previously identified Lab response elements: the mouse Hoxb1 autoregulatory enhancer (b1-ARE), which also generates a lab-like pattern, dependent on lab and exd activity, in Drosophila, and the lab autoregulatory enhancer. Both these enhancers have binding sites for Hox (Hoxb1, Lab), PBC (Pbx, Exd) and MEIS (Prep, Hth) proteins. The orientation of the bipartite Exd/Lab site relative to the MEIS site is the same in these elements as seen in the dpphc Hox response element, and the relative spacing between the PBC/Hox and MEIS components is very similar. However, the dpphc Hox response element has a cluster of three overlapping Hth sites, two residing on the opposite strand, and an additional functional Exd site downstream of the Hth sites, as determined by its requirement for expression in vivo). The expression of mutated reporter constructs in vivo, as well as LOF analyses of lab and hth, indicate that Hth/Exd plays a more critical role in enhancer activity than does Lab, as neither mutations in the Lab binding site nor Lab loss-of-function within somatic clones completely extinguished expression. This suggests that there may be multiple ways that homeodomain transcription factors activate the enhancer, depending on the cellular context. It is noted that the expression driven by the dpphc enhancer actually manifests as two separate lines {see also Stultz, 2006b). The level of Lab associated with each of these lines is not equivalent, therefore the control of expression may be specific to each line. This would be reminiscent of a situation seen within dpp itself, where the Ubx responsive visceral mesoderm enhancer is activated by Ubx/Exd/Hth in parasegment seven, but only requires Hth/Exd for activation in parasegment three. The in vitro EMSA data further support this, as Hth and Exd bind synergistically to more locations within the enhancer than Lab. The TALE family homeodomain proteins function independently of Hox proteins in many contexts. An additional explanation for the apparent primacy of hth may be because it plays both direct and indirect roles on enhancer expression. Hth acts with the transcription factor Yorkie (Yki) as part of the Hippo signalling pathway, and the nuclear activity of Yki and Hth are required to specify the PE of the eye-antennal disc. In the absence of hth, the PE is incorrectly fated. This may effect early gene expression upstream of the Hox/BMP interactions described in this study, magnifying the genetic affect of hth (Stultz, 2012).

The Hox/BMP network described in this study plays a prominent role in the external appearance of the adult head, yet is restricted completely to the PE of the eye-antennal disc. The terminal mutant phenotypes of dpps-hc, Dfd, and lab have similarities, but are sufficiently distinct that additional targets for each must exist, and for the cell autonomous Dfd and lab, these targets must reside in the PE. Other signalling proteins such as Wingless and Hedgehog, and the Notch pathway ligands Serrate and Delta, are expressed in the PE of the eye-antennal disc. While some adult structures derive from the PE, and PE cells likely contribute to other adult structures, it is likely that much of the effect of the PE on head morphogenesis is via inductive interactions with the DP, either through secreted signalling molecules, or targeted cell protrusions. Based on the cuticular alterations seen in dpps-hc, Dfd, and lab mutations, such interactions are capable of exerting structural modifications on the final head shape. Dipterans demonstrate great variety in the external morphology of their heads often with sexually dimorphic alterations within a species. Much of this variety involves changes in the relative proportions of eye and head capsule tissue. BMP expression has been implicated in shaping the jaws of cichlid fish and the beak shape of finches, while dpp expression itself is correlated with the growth of beetle horns, a specialized cuticular structure of the head. It is speculated that the PE specific Hox/BMP network described in this study could be a motor for such types of shape change in the Drosophila species (Stultz, 2012).

Divergent transcriptional regulatory logic at the intersection of tissue growth and developmental patterning

The Yorkie/Yap transcriptional coactivator is a well-known regulator of cellular proliferation in both invertebrates and mammals. As a coactivator, Yorkie (Yki) lacks a DNA binding domain and must partner with sequence-specific DNA binding proteins in the nucleus to regulate gene expression; in Drosophila, the developmental regulators Scalloped (Sd) and Homothorax (Hth) are two such partners. To determine the range of target genes regulated by these three transcription factors, genome-wide chromatin immunoprecipitation experiments for each factor was performed in both the wing and eye-antenna imaginal discs. Strong, tissue-specific binding patterns are observed for Sd and Hth, while Yki binding is remarkably similar across both tissues. Binding events common to the eye and wing are also present for Sd and Hth; these are associated with genes regulating cell proliferation and 'housekeeping' functions. In contrast, tissue-specific binding events for Sd and Hth significantly overlap enhancers that are active in the given tissue, are enriched in Sd and Hth DNA binding sites, respectively, and are associated with genes that are consistent with each factor's tissue-specific functions. Overall these results suggest that both Sd and Hth use distinct strategies to regulate distinct gene sets during development: one strategy is shared between tissues and associated with Yki, while the other is tissue-specific, generally Yki-independent and associated with developmental patterning (Slattery, 2013).

The control of gene expression in multicellular eukaryotes depends on a limited set of transcription factors that are reused in different contexts and combinations to execute a diverse array of cellular functions. To gain insight into this process this study used tissue-specific, genome-wide ChIP to explore the global DNA targeting properties of three transcriptional regulators – Yki, Sd, and Hth. Yki is a transcriptional coactivator that regulates tissue growth in all tissues, and it does so in part through interactions with the DNA binding TFs Sd and Hth. However, in addition to their Yki-dependent roles in promoting tissue growth, Sd and Hth also have highly tissue-specific developmental roles. Thus, this group of regulators provides an ideal starting point for addressing the logic by which TFs execute both tissue-specific and -nonspecific gene regulatory functions in vivo. The implications of the differences this study uncovered between these modes of binding for Hth and Sd are discussed, as well as the unexpectedly large number of shared binding sites for Yki (Slattery, 2013)

Drosophila Yki was initially identified as an essential transcriptional coactivator in the Hippo tumor suppressor pathway. Loss of function clones of yki grow very poorly, while gain of function Yki clones result in tissue overgrowths that are similar to those generated when the upstream kinases (Hippo and Warts) are compromised. These observations suggested that Yki, with the help of DNA binding proteins, would target genes required for cell proliferation and survival, including the known Hippo pathway targets cycE and diap1. Consistent with this expectation, this study observed Yki binding to these and other genes that are regulated by the Hippo pathway. Unexpectedly, however, in addition to known Hippo pathway genes Yki binding to several thousands of genes was observed in both the eye-antenna and wing imaginal discs, implying that Yki targets many more genes than those regulated by the Hippo pathway, or that the Hippo pathway targets many more genes than previously thought. Consistent with the latter possibility, over 1000 of the genes identified as tissue-shared Yki targets in this study are upregulated >2-fold in wts- wing discs relative to wild-type based on recently published RNA-seq data. In addition, Yki was recently shown to bind and activate several genes required for mitochondrial fusion. Moreover, the mammalian homologs of Yki, Yes-associated protein (YAP) and TAZ (transcriptional coactivator with PDZ-binding motif) are thought to regulate many genes in a wide variety of contexts, including human embryonic stem cells and several adult human tissues. Taken together, these results suggest that Yki may be a widely used transcriptional coactivator in Drosophila and vertebrates. The severe cell proliferation defects associated with yki mutant clones may have obscured its other functions in other pathways. These results are consistent with the idea that Yki and its vertebrate orthologs interact with a wide variety of transcription factor. Together, the data imply that DNA binding proteins in addition to Sd and Hth may recruit Yki to a large number of broadly active CRMs (Slattery, 2013)

The view that Yki is recruited to DNA by factors other than Sd was recently questioned by experiments suggesting that, in the eye imaginal disc, sd yki double mutant clones proliferate better than yki single mutant clones. These observations were interpreted to suggest that Sd is a default repressor of proliferation and survival-promoting genes. However, this conclusion is complicated by the observation that both Sd and Yki are also important for specifying non-retinal (peripodial epithelium) fates in the eye imaginal disc: thus, the partially rescued growth of sd yki clones could in part be due to a fate transformation. Further, this study found that the activity of the ban-eye enhancer is not affected in sd clones, but is lost in hth clones, arguing that at least for this direct Hippo pathway target Hth, not Sd, is the primary activator. It is noteworthy that although their activities can be separated, the ban wing and eye enhancers identified in this study are adjacent to each other in the native ban locus. It is plausible that Sd+Yki input provides a basal level of activity in both tissues and that Hth and Sd boost this level in the eye and wing, respectively. Regardless, the improved growth of sd yki clones does not argue against the idea that Yki is recruited to survival genes by Hth in wild type eye discs. Taken together with genome-wide binding and ban enhancer studies, it is suggested that the absence of Sd results in both a fate change and some derepression of survival genes, but that wild type proliferation and gene regulation in the eye disc requires the recruitment of Yki to the DNA by Hth (Slattery, 2013)

In contrast to the widespread and largely tissue-nonspecific binding observe for Yki, Sd and Hth exhibit both tissue-specific and tissue-shared binding events. Multiple characteristics distinguish these types of binding. First, tissue-shared binding by both Sd and Hth is frequently associated with Yki binding and often close to cell cycle and housekeeping genes, while tissue-specific binding is not. These observations are consistent with previous studies showing that Yki controls cell survival and proliferation in all imaginal discs, an activity that is regulated by the Hippo pathway. Second, compared to tissue-shared binding, DNA sequences bound by Sd and Hth in a tissue-specific manner are more conserved, more likely to contain the TF's consensus binding site, less likely to be promoter proximal, and more likely to be associated with key developmental regulatory loci. Third, tissue-specific Sd and Hth binding events are more likely to overlap with enhancers active in the corresponding tissue. To illustrate this point, the newly identified tissue-specific TF-CRM interactions at wg match the known roles for Sd and Hth. Taken together, these results suggest that regulation at the level of TF-DNA binding is a significant mechanism by which Sd and Hth regulate tissue-specific gene expression. Tissue-specific binding could be regulated through direct or indirect interactions with additional transcription factors, through tissue-specific differences in DNA accessibility, or through a combination of these factors (Slattery, 2013)

This study also found that distinct chromatin types are differentially correlated with tissue-specific and -nonspecific binding, even though these chromatin categories were defined in Kc cells. All tissue-shared binding events have a strong tendency to occur in actively transcribed chromatin states. Tissue-specific (W>EA) Sd and Hth binding is also enriched in RED chromatin (see Filion, 2010) but is uniquely enriched in BLUE chromatin. BLUE chromatin is associated with Polycomb-mediated repression. The W>EA Sd and Hth binding in Polycomb-associated chromatin indicate that these factors target tissue-specific enhancers that are also regulated by PcG proteins during development (Slattery, 2013)

Despite the importance of tissue-specific binding as a regulatory mechanism for Sd and Hth activity, both factors also displayed a significant amount of tissue-shared binding. These tissue-shared binding events can be broken down into distinct groups based on the local chromatin environment. The majority of tissue-shared binding occurs in YELLOW chromatin and is associated with ubiquitously expressed housekeeping genes. However, binding that occurs in BLUE chromatin, and to a lesser extent in RED chromatin, is more conserved and more likely to be associated with a TF's motif, both characteristics of tissue-specific binding. In the case of the bantam eye and wing enhancers, Sd and Hth binding in BLUE chromatin is direct and apparently able to drive tissue-specific, rather than ubiquitous, expression patterns. Other examples of enhancers in RED or BLUE chromatin that drive patterned expression and have tissue shared binding are shown in this study. These observations suggest that gene regulation by Sd and Hth may also be controlled at a step beyond DNA binding, perhaps via interactions with additional transcription factors at a given enhancer. Alternatively, some of the binding events called as tissue-shared may turn out to be specific binding events in distinct cell types within each imaginal disc (e.g. hinge, notum, and pouch in the wing disc and antenna, eye progenitor domain, and photoreceptors in the eye-antenna disc). Regardless, the hundreds of Sd- and Hth-CRM interactions identified in this study provide a tremendous resource for further dissecting the mechanisms by which Sd and Hth regulate patterned gene expression (Slattery, 2013)

Notably, few of the above conclusions would have been clear had genome-wide binding been measured in only one of the two tissues. Tissue-specific binding is not the most highly enriched (that is, the signal is generally weaker compared to tissue-shared events) and might have been overlooked had just one tissue been characterized, where the strongest peaks are generally the focal point. The tissue-specific binding events detected in this study may also occur in subsets of cells in the wing or eye-antennal discs, which are also heterogeneous in cell type. This would explain why tissue-specific binding signals may be weaker, because the ChIP data represent an average of all cell types in a single imaginal disc type. If correct, it would be an error to focus on only the strongest peaks when analyzing in vivo TF binding, particularly in heterogeneous tissues. It is possible that ChIP signal is more biologically meaningful in highly homogenous tissues like the blastoderm Drosophila embryo, or in cell culture. Still, distinct TF-DNA binding mechanisms (long residence time versus rapid binding turnover) with different functional outcomes can lead to indistinguishable, strong ChIP peaks, making it difficult to interpret ChIP data on strength of signal alone. Despite their lower intensity, many biologically relevant binding events, such as those identified in this study, may only stand out when looking at the influence of tissue context on binding (Slattery, 2013).

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).

homothorax: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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