dachshund

Promoter

Drosophila eye development is controlled by a conserved network of retinal determination (RD) genes. The RD genes encode nuclear proteins that form complexes and function in concert with extracellular signal-regulated transcription factors. Identification of the genomic regulatory elements that govern the eye-specific expression of the RD genes will allow a better understanding of how spatial and temporal control of gene expression occurs during early eye development. Conserved non-coding sequences (CNCSs) between five Drosophilids were compared along the ~40 kb genomic locus of the RD gene dachshund (dac). This analysis uncovers two separate eye enhancers, in intron eight and the 3' non-coding regions of the dac locus, defined by clusters of highly conserved sequences. Loss- and gain-of-function analyses suggest that the 3' eye enhancer is synergistically activated by a combination of eya, so and dpp signaling, and only indirectly activated by ey, whereas the 5' eye enhancer is primarily regulated by ey, acting in concert with eya and so. Disrupting conserved So-binding sites in the 3' eye enhancer prevents reporter expression in vivo. These results suggest that the two eye enhancers act redundantly and in concert with each other to integrate distinct upstream inputs and direct the eye-specific expression of dac (Anderson, 2006).

The smallest fragment in the 3' dac eye enhancer that can respond to dpp, eya and so is 3EE^{194 bp}, which is centered around two CNCS blocks of ~40 bp and 20 bp. These two CNCS blocks are also common to all active fragments of the 3' eye enhancer. These two evolutionarily conserved stretches were scanned for known, genetically upstream transcription factor binding sites. The 40 bp conserved stretch contains two putative consensus So-binding sites, S1-5'-CGATAT and S2-5'-CGATAC, compared with the consensus 5'-(C/T)GATA(C/T) described previously. Each of these putative So-binding sites in 3EE were mutated individually and in combination to test their requirement for normal enhancer activity in vivo. Mutation of individual So-binding sites causes a severe reduction, but not complete elimination, of enhancer activity in vivo. However, simultaneous mutation of both So binding sites completely abolishes enhancer activity in vivo. These results, coupled with loss-and gain-of-function analyses with dpp, eya and so, suggest that So binds to the 3' eye enhancer directly and nucleates a protein complex that includes Eya to regulate 3EE. However, despite much effort using a wide variety of binding conditions, it was not possible to demonstrate specific, direct binding of So protein to oligos that contain these So-binding sites. The 5' eye enhancer, which has four CNCS blocks, were scanned for potential upstream transcription factor binding sites and no strong candidate binding sites were found within the CNCS blocks (Anderson, 2006).

Loss- and gain-of-function analyses with the two eye enhancers suggest that each enhancer is regulated by a distinct set of protein complexes. The 5' eye enhancer is activated by a combination of ey, eya and so, but is not activated by Dpp signaling. 5EE is activated by ectopic ey expression even in eya and so mutants, suggesting that it is regulated exclusively by ey. However, somewhat paradoxically, expression of 5EE, the intron 8 enhancer, is lost in eya and so mutants even though ectopic expression of a combination of dpp, eya and so does not activate this enhancer. Furthermore, driving high levels of ey in so¹ mutant eye discs restores 5EE-lacZ expression. Coupled together, these results suggest that 5EE is primarily regulated by ey but that the regulation of 5EE by ey also requires eya and so (Anderson, 2006).

By contrast, the 3' dac eye enhancer is regulated by a combination of eya, so and dpp signaling, but is not directly dependent on ey. 3EE-GFP expression is lost in eya² and so¹ mutant eye discs, and in posterior margin mad^1-2 mutant clones. Furthermore, ey cannot bypass the requirement for eya and so to activate 3EE. Conversely, 3EE is strongly induced by co-expression of eya and so. Moreover, dpp signaling via the tkv receptor can synergize with eya and so to induce 3EE in ectopic expression assays. Furthermore, neither Mad nor Medea, the intracellular transducers of Dpp signaling, is sufficient to bypass the requirement for activation of the Dpp receptor Tkv in these assays. Thus, it is concluded that events downstream of Dpp-Tkv signaling, such as the phosphorylation of Mad, are essential for the synergistic activation of the 3' dac eye enhancer by eya and so. Taken together, these results suggest that there are distinct requirements for the activation of the 5' and 3' dac eye enhancers. However, the exact nature of the protein complexes that regulate 5EE and 3EE remain to be determined (Anderson, 2006).

Morphogenetic furrow (MF) initiation is completely blocked in posterior margin dac³-null mutant clones. However, dac³ clones that do not include any part of the posterior margin develop and do not prevent MF progression, but do cause defects in ommatidial cell number and organization. This dichotomy in dac function is reflected in the two eye enhancers characterized in this study. Analysis of dac⁷ homozygotes demonstrates that the 3' eye enhancer is dispensable for MF initiation and progression. It is proposed that in dac⁷ mutants, the intact 5EE enhancer is sufficiently activated by ey to drive high enough levels of dac expression to initiate and complete retinal morphogenesis. However, dac⁷ mutants have readily observable defects in ommatidial organization. Thus, it is further proposed that this lack of normal patterning in dac⁷ mutants is most likely due to the loss of 3EE, which normally acts in concert with 5EE after MF initiation, to integrate patterning inputs from extracellular signaling molecules such as Dpp with tissue-specific upstream regulators such as ey, eya and so. However, it is not known if the 3' eye enhancer is sufficient to initiate dac expression in the absence of the 5' eye enhancer (Anderson, 2006).

Based on the results, a two-step model is proposed for the regulation of dac expression in the eye. First, the initiation of dac expression in the eye disc is dependent on Ey binding to 5EE. However, Ey is fully functional only when So and Eya are present. It is possible that Ey recruits So and Eya to 5EE, but a model is favored in which Ey bound to 5EE cooperates with an So/Eya complex bound to 3EE to initiate dac expression in the eye. After initiation of the MF, dac expression is maintained by an Eya and So complex bound to 3EE. In addition, 3EE can integrate patterning information received via dpp signaling, thereby allowing the precise spatial and temporal expression of dac in the eye. This two part retinal enhancer ensures that dac expression is initiated only after ey activates eya and so expression. Thus, the dac eye enhancers provide a unique model with which the sequential activation of RD proteins allows the progressive formation of specialized protein complexes that can activate retinal specific genes (Anderson, 2006).

The redundancy in dac enhancer activity also explains the inability to isolate eye-specific alleles of dac, despite multiple genetic screens. The modular nature of the two enhancers and their potential ability to act independently or in concert suggest that both enhancers must be disrupted to block high levels of transcription of dac. Thus, two independent hits in the same generation, a phenomenon that occurs infrequently in genetic screens, would be required to obtain an eye-specific allele in dac (Anderson, 2006).

Despite much investigation, very few direct targets of RD proteins, especially for Eya and So, have been identified. One study suggests that So can bind to and regulate an eye-specific enhancer of the lz gene. However, lz is not expressed early during eye development and is required only for differentiation of individual cell types. The results suggest that regulation of dac expression occurs via the interaction of two independent eye enhancers that are likely to be bound by Ey, Eya and So, and respond to dpp signaling. This analysis of the 3' eye enhancer suggests that two putative conserved So-binding sites are essential for 3EE activity in vivo. Mutation of individual So-binding sites dramatically reduces, but does not completely eliminate, reporter expression in the eye. Mutating both predicted So-binding sites completely blocks enhancer activity in vivo. Thus, it is concluded that So binds to 3EE via these conserved binding sites. However, it has not been possible to demonstrate a direct specific interaction of either So alone or a combination of Eya and So with oligos that contain these putative So-binding sites in vitro. It is possible that other unidentified proteins are required for stabilizing the Eya and So complex. Furthermore, the 194 bp fragment that responds to ectopic expression of dpp, eya, and so contains no conserved or predicted Mad-binding sites. This raises the intriguing possibility that dpp signaling activates other genes, which then directly act with eya and so to regulate the 3' eye enhancer. Alternatively, a large complex that includes Eya, So and the intracellular transducers of dpp signaling, such as Mad and Medea, may be responsible for activation of 3EE. Similarly, the results suggest that the 5' eye enhancer is regulated primarily by ey. However, it is unclear whether Ey directly binds 5EE. Furthermore, Ey is fully functional only in the presence of Eya and So. Thus, Ey either independently recruits Eya and So into a 5' complex or is activated by virtue of its proximity to the So/Eya complex bound to the 3' enhancer or both (Anderson, 2006).

The exact order and dynamics of protein complex assembly at 5EE and 3EE requires further investigation. However, the two dac eye enhancers are extremely useful tools with which to investigate fundamental issues about the mechanism of RD protein action. One significant issue concerns the mechanism of Eya function during eye development. Eya consists of two major conserved domains, an N-terminal domain that has phosphatase activity in vitro and a C-terminal domain that can function as a transactivator in cell culture assays. So contains a conserved Six domain and a DNA binding homeodomain. However, it is unclear if Eya provides phosphatase activity, transactivator function, or both, in this complex. Characterization of the components of the protein complexes that regulates dac expression may uncover the targets of Eya phosphatase activity during eye development. Thus, the isolation of two eye enhancers with distinct regulation provides very useful tools with which to study protein complex formation and function during Drosophila retinal specification and determination (Anderson, 2006).

Transcriptional Regulation

Dachshund and the eye/antennal disc

Three independent results suggest that dachshund functions downstream of eyeless: (1) Misexpression of ey in the antennal, leg and wing imaginal discs is sufficient to induce ectopic dac expression in all discs. These results suggest that EY positively regulates dac expression. (2) Targeted expression of ey is unable to induce ectopic eye formation in a dac mutant background. (3) ey is expressed in a mutant dac background, indicating that dac is not required for ey expression (Shen, 1997).

The eyeless, dachshund, and eyes absent genes encode conserved, nuclear proteins that are essential for eye development in Drosophila. Misexpression of eyeless or dachshund is also sufficient to induce the formation of ectopic compound eyes. Like ey and dac, targeted expression of eya alone is sufficient to induce ectopic eye formation. However, in contrast to ey, the penetrance of the ectopic eye phenotype induced by either dac or eya alone is incomplete and, when induced, such eyes are small. When dac expression was strongly induced in all imaginal discs, ectopic eye development was observed only on the anterior surface of the fly head ventral to the antenna, in just 56% (61/109) of animals examined. In contrast to the low penetrance of ectopic eye formation induced by dac or eya expressed alone, coexpression of dac and eya induces substantial ectopic eyes on the head, legs, wings, and dorsal thorax of 100% of animals examined. On the head, the cuticle between the normal eye field and antennae is transformed into retinal cells such that the normal retinal field is expanded. Large patches of pigment are induced on the dorsal surface of the femur and tibia of all legs, which are severely truncated. Ectopic eya alone can induce small patches of glass expression in the pouch area of the wing disc with 25% penetrance. In no case has ectopic Glass staining been observed in leg discs with either dac or eya alone. However, when dac and eya are coexpressed, ectopic Glass staining is induced with 100% penetrance along the ventral margin of the eye-antennal disc, the dorsal half of the leg disc along the anterior-posterior compartment (A/P) boundary, and along the A/P boundary of the dorsal wing disc. In each case, the sites of ectopic glass expression in discs correspond to the positions of ectopic retinal development observed in adults. Taken together, these data demonstrate that dac and eya show strong genetic synergy to induce ectopic retinal development in Drosophila (Chen, 1997).

Ectopic Elav-positive cells are induced in the antennal, leg, and wing discs, in response to dac and eya coexpression, suggesting ectopic neural differentiation. These ectopic neurons must be photoreceptor cells, since the visual system-specific Glass protein is also induced in the same pattern. Ectopic eyes observed in adults corresponding to these positions contain all of the normal cell types associated with the wild-type eye, including pigment cells, lens-secreting cone cells, and interommatidial bristles. The ectopic neurons induced by dac and eya misexpression send out axonal projections. The axons of ectopic photoreceptors in the eye-antennal disc form a bundle that extends posteriorly into the eye imaginal disc. These axons appear to fuse with the axon tracts sent out by photoreceptors of the normal retinal field that exit through the optic stalk to synapse in the brain. It is likely, therefore, that the fly can perceive light through ectopic photoreceptors formed in the eye-antennal disc as a result of dac and eya coexpression. Coexpression induces ectopic dpp expression in the eye-antennal disc adjacent to the field of ectopic photoreceptors. In the leg disc, dpp expression is split and forms a ring around the ectopic photoreceptors, again suggesting that an ectopic MF is initiated and propagates (Chen, 1997).

While eya expression in the eye disc does not depend on dac function, dac expression is greatly reduced in an eya2 mutant background, demonstrating that dac expression requires eya activity. Similarly, eyeless (ey) induction of ectopic dac expression is greatly reduced in an eya2 mutant background. These results suggest that dac may function downstream of eya. Consistent with this interpretation, eya is unable to induce ectopic eye formation in a dac mutant background. eyeless misexpression is sufficient to induce eya, suggesting that eya may be required for ey function. Indeed, ectopic retinal development driven by targeted ey expression fails to occur in an eya2 mutant background. Induction of eya expression by ey does not depend on dac activity, consistent with the idea that eya functions downstream of ey but upstream of dac. However, these genes do not act in a simple, linear pathway; targeted expression of dac and eya strongly induce the expression of one another, and eya is required for ectopic eye induction by dac. Misexpression of dac or eya is also sufficient to induce ectopic ey expression in the antennal disc. These results suggest that multiple positive-feedback loops exist among these genes during normal eye development and raises the possibility that ey may be required for ectopic retinal induction by eya and dac. Indeed, ectopic eye formation driven by coexpression of dac and eya is completely blocked in an eyeless2 mutant background, indicating that induction of ey is essential. It is proposed that a conserved regulatory network, rather than a linear hierarchy, controls retinal specification and involves multiple protein complexes that function during distinct steps of eye development (Chen, 1997).

Retinal cell fate determination in Drosophila is controlled by an interactive network of retinal determination (RD) genes, including eyeless, eyes absent, sine oculis and dachshund. The role of decapentaplegic in this pathway was investigated. During eye development, while eyeless transcription does not depend on dpp activity, the expression of eyes absent, sine oculis and dachshund are greatly reduced in a dpp mutant background. dpp signaling acts synergistically with, and at multiple levels within, the retinal determination network to induce eyes absent, sine oculis and dachshund expression and ectopic eye formation. These results suggest a mechanism by which a general patterning signal such as Decapentaplegic cooperates reiteratively with tissue-specific factors to determine distinct cell fates during development (Chen, 1999).

During ectopic photoreceptor determination there is a tight correlation between the location of ectopic eyes and the endogenous pattern of dpp expression. In particular, the dpp-GAL4 driver is the most efficient means of retinal induction by any of the RD genes: ubiquitous eyeless (ey) expression induces downstream genes only in the vicinity of the anteroposterior (AP) compartment boundary of discs where dpp is normally expressed. These results suggested that dpp signaling may be essential for the RD genes to specify retinal cell fates. dpp is normally expressed along the AP boundary of the larval wing disc. The GAL4 line 30A drives gene expression in a ring that surrounds the wing pouch, which will become the wing blade in the adult. The 30A ring pattern corresponds to tissue that will form the hinge of the adult wing and overlaps endogenous dpp at only two spots. When ey is misexpressed using 30A-GAL4, ectopic eye formation is induced only at two positions: dorsal and ventral to the pouch at the AP boundary. One explanation for this phenomenon is that dpp activity is essential for ey to induce ectopic eye development. Coexpression of dpp and ey is sufficient to expand the domain of ectopic retinal development induced by ey alone. To test whether dpp and ey act synergistically to induce RD genes, mRNA levels of ey, eya, so and dac were measured in a dpp loss-of-function background. ey is normally expressed throughout the entire eye disc prior to MF initiation and anterior to the furrow during MF progression. In dpp mutants, the eye-antennal disc is much smaller than in wild-type due to a proliferation defect, and MF initiation and photoreceptor development does not occur. Nevertheless, EY mRNA is still detectable in dpp mutant eye discs throughout second and third instar larval development. In contrast, although eya is still expressed in the ocellar region, almost no EYA, SO or DAC mRNA is detected in dpp mutant eye discs prepared from second or third instar larvae. These data indicate that dpp is not essential for ey expression but is required upstream of eya, so and dac in the eye disc (Chen, 1999).

If eya and dac are the primary downstream targets of dpp during eye development, then it should be possible to bypass the requirement for dpp and induce ectopic eye formation by overexpressing ey with eya or dac. While targeted expression of either eya or dac alone driven by 30A-GAL4 is unable to induce photoreceptor development, strong synergistic induction of ectopic eye formation is observed when ey is coexpressed with either dac or eya. Although there is clear synergy between ey and dac or eya, ectopic photoreceptor induction in both imaginal discs and adults is still limited to the vicinity of the AP boundary and the source of dpp signaling. Moreover, photoreceptor differentiation is still restricted to the vicinity of the AP boundary when ey, dac, eya and so are simultaneously induced by 30A-GAL4, indicating that dpp and ey must regulate other essential targets in this process (Chen, 1999).

It is possible that dpp signaling might cooperate directly and exclusively with ey. Alternatively, dpp could interact at multiple levels within this pathway. To distinguish these two models, a test was performed to see whether dpp functions synergistically with eya and so to regulate the expression of dac. No ectopic dac expression is induced by so alone: targeted expression of eya induces ectopic dac expression only at a single ventral spot on the AP boundary of the wing disc when driven by 30A-GAL4. Consistent with the idea that the Eya and So proteins function cooperatively as a complex, strong synergistic induction of dac is observed when eya and so are coexpressed. However, dac expression is still restricted mainly to places where endogenous dpp is present. In contrast, when dpp is coexpressed with eya, strong dac expression is induced all along the ventral-posterior pouch margin Moreover, ectopic Dac is detected around the entire circumference of the wing pouch as a result of dpp, eya and so coexpression. Since coexpression of dpp, eya and so is sufficient to induce dac expression in places where dpp and ey cannot, it is concluded that dpp interacts with the network at multiple levels to control the expression of retinal determination genes. Consistent with this interpretation, no induction of ey transcription could be detected in response to misexpression of dpp, eya and so with 30A-GAL4 (Chen, 1999).

Thus dpp signaling is reiteratively used to regulate gene expression within the retinal cell fate determination pathway in Drosophila. Specifically, dpp signaling enables ey to induce strong eya, so and dac expression in the posterior, but not anterior, wing disc compartment. In contrast, dpp functions synergistically with eya and so to activate the expression of dac in both compartments. This activation of dac expression by dpp, eya and so is unlikely to result from feedback induction of ey for two reasons: (1) targeted expression of ey and dpp is unable to induce dac in the anterior wing disc compartment, and (2) ectopic ey transcription is not detected in response to misexpression of dpp, eya and so driven by 30A-GAL4 in the wing disc. Thus, these data suggest that dpp signaling interacts with the retinal determination pathway at (at least) two levels to regulate RD gene expression. Interestingly, while targeted expression of dpp, eya and so with 30A-GAL4 is unable to induce ey expression or ectopic photoreceptor development in the wing disc, coexpression of eya and so using dpp-GAL4 is sufficient to induce ey expression and photoreceptor development in the antennal disc. These differences most likely reflect the unique transcriptional environments present in the specific portions of each imaginal disc tested in these assays (Chen, 1999).

teashirt was initially identified as a gene required for the specification of the trunk segments in Drosophila embryogenesis and encodes a transcription factor with zinc finger motifs. Targeted expression of teashirt in imaginal discs is sufficient to induce ectopic eye formation in non-eye tissues, a phenotype similar to that produced from targeted expression of eyeless, dachshund, and eyes absent. The expression of so and dac are induced in the antennal disc by the ectopic expression of tsh, suggesting that tsh may act upstream of these genes in eye development. Furthermore, teashirt and eyeless induce the expression of one another, suggesting that teashirt is part of the gene network that functions to specify eye identity (Pan, 1998).

However, these results do not prove that tsh does play a role in specifying the eye identity during normal development. To address this issue, an examination was carried out to see if tsh is expressed at the right time and the right place to have a role in specifying the eye identity. Indeed, TSH mRNA is expressed in the eye disc, with the strongest expression anterior to the morphogenetic furrow. This pattern of expression is similar to that of ey, a gene that is known to play an essential role in specifying eye identity. An examination was carried out to see if loss-of-function mutations of tsh affect eye development. Several weak loss-of-function tsh alleles were examined and no eye defects were found. X-ray-induced mitotic recombination was used to generate mutant clones of a null tsh allele. tsh mutant clones were recovered at a frequency similar to the wild-type control, and sections through the mutant clones revealed a normal ommatidial organization. These data suggest that tsh may play a redundant role during normal eye development, and the requirement for tsh may be masked by other factor(s) that play a role similar to tsh (Pan, 1998).

decapentaplegic mediates the effects of hedgehog in tissue patterning by regulating the expression of tissue-specific genes. In the eye disc, the transcription factors eyeless, eyes absent, sine oculis and dachshund participate with these signaling molecules in a complex regulatory network that results in the initiation of eye development. Analysis of functional relationships in the early eye disc indicates that hh and dpp play no role in regulating ey, but are required for eya, so and dac expression. Ey is expressed throughout the eye portion of the wild-type eye disc during early larval stages, prior to MF initiation. Eya and Dac are expressed throughout the posterior half of the eye imaginal disc, with stronger expression at the posterior margin. Ey is expressed normally in homozygous Mad^1-2 clones that touch the posterior margin and in clones that are positioned internally in the disc, indicating that Dpp signaling is not required for Ey expression prior to MF initiation. In contrast, neither Eya nor Dac is expressed in homozygous Mad^1-2 clones that touch the margin of the eye disc. In addition, Eya and Dac are not expressed, or are expressed weakly, in internal clones that lie well anterior of the posterior margin. However, strong Eya and Dac expression is observed in internal clones that lie within a few cell diameters of the posterior margin. Like Eya and Dac protein, SO mRNA is expressed in the posterior region of the eye disc prior to MF initiation. Mad^1-2 posterior margin clones fail to express so. These results suggest that dpp function is required to induce or maintain Eya, SO and Dac expression, but not Ey expression, at the posterior margin prior to MF initiation. This function is consistent with the pattern of DPP mRNA expression along the posterior and lateral margins at this stage of eye disc development. Whereas dpp is not necessary for Eya and Dac expression in internal, posterior regions of the early eye disc, it does play a role in regulating Eya and Dac expression in internal, anterior regions of the disc. Although DPP mRNA expression does not extend to the very center of the eye disc, it is expressed in a significant proportion of the interior of the disc. The possibility that dpp may regulate gene expression in more central regions may be attributed to the fact that it encodes a diffusible molecule (Curtiss, 2000).

Restoring expression of eya in loss-of-function dpp mutant backgrounds is sufficient to induce so and dac expression and to rescue eye development. Thus, once expressed, eya can carry out its functions in the absence of dpp. These experiments indicate that dpp functions downstream of or in parallel with ey, but upstream of eya, so and dac. Additional control is provided by a feedback loop that maintains expression of eya and so and includes dpp. The fact that exogenous overexpression of ey, eya, so and dac interferes with wild-type eye development demonstrates the importance of such a complicated mechanism for maintaining proper levels of these factors during early eye development. Whereas initiation of eye development fails in either Hh or Dpp signaling mutants, the subsequent progression of the morphogenetic furrow is only slowed down. However, clones that are simultaneously mutant for Hh and Dpp signaling components completely block furrow progression and eye differentiation, suggesting that Hh and Dpp serve partially redundant functions in this process. Interestingly, furrow-associated expression of eya, so and dac is not affected by double mutant tissue, suggesting that some other factor(s) regulates their expression during furrow progression (Curtiss, 2000).

The lack of eya, so and dac expression in Mad^1-2 clones that lie at the margins of the eye disc prior to MF initiation reflects a role for dpp in controlling early eye gene expression at these stages of eye development. Evidence from several studies suggests that ey acts together with dpp at or near the top of the hierarchy: (1) ey expression is not regulated by dpp; (2) ey and dpp are both required for eya, so and dac expression prior to MF initiation; (3) ey is not capable of rescuing dpp^blk eye development or of inducing ectopic eyes in regions of imaginal discs in which dpp is not already expressed. These observations suggest that ey functions upstream of or in parallel with dpp. The possibility that ey is responsible for dpp expression, leading indirectly to eya, so and dac expression, is unlikely. Since ey cannot induce ectopic eyes without a source of dpp, it probably cannot induce dpp expression, at least not in the absence of factors that are specific to the eye disc. Moreover, Ey protein binds to the regulatory region of so, suggesting it is directly involved in so regulation. Thus, it is likely that ey and dpp cooperate to induce expression of the other early eye genes (Curtiss, 2000).

Such cooperation could achieve two ends. (1) ey is expressed throughout the eye disc and from embryonic stages of development through MF initiation. However, induction of eya, so and dac expression and MF initiation occurs approximately 48 hours later, around the time of the transition between second and third instars. Moreover, eya, so and dac are not expressed throughout the eye disc as ey is, but have stronger levels of expression around the margins than in other regions. The initiation of dpp expression at the posterior margin at approximately the same time suggests that it could be the spatiotemporal signal that sets the MF in motion. (2) dpp induces expression of tissue-specific genes as part of its role in patterning many diverse structures in Drosophila. An interaction with ey could be essential to ensuring that in the eye imaginal disc dpp initiates factors that are appropriate to eye development, such as eya, so and dac (Curtiss, 2000).

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).

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).

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 dac^lacZ/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).

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

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

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

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

The complementary patterns of Hth versus So, Eya, and Dac at the transition between domain II and the PPN domain suggested that these factors may also be playing a role in hth repression. To test this idea, clones of cells mutant for eya were examined. eya^- clones de-repress hth. Part of this de-repression is probably due to the fact that dpp expression requires eya. However, the de-repression of hth is observed in all eya^- cells, even in cells that are next to wild-type, dpp-expressing cells. Thus, Dpp expressed in wild-type neighboring cells is not able to repress hth in adjacent eya^- cells. These data suggest that eya is required for Dpp to repress hth in the PPN domain. hth was also de-repressed in dac^- clones, suggesting that dac also plays a role in hth repression (Bessa, 2002).

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

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

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

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

The Wingless protein plays an important part in regional specification of imaginal structures in Drosophila, including defining the region of the eye-antennal disc that will become retina. Wingless signaling establishes the border between the retina and adjacent head structures by inhibiting the expression of the eye specification genes eyes absent, sine oculis and dachshund. Ectopic Wingless signaling leads to the repression of these genes and the loss of eyes, whereas loss of Wingless signaling has the opposite effects. Wingless expression in the anterior of wild-type discs is complementary to that of these eye specification genes. Contrary to previous reports, it has been found that under conditions of excess Wingless signaling, eye tissue is transformed not only into head cuticle but also into a variety of inappropriate structures (Baonza, 2002).

In order to analyse the effect of ectopic activation of the Wingless pathway during the development of the eye-antennal imaginal disc, clones either mutant for the negative regulator of Wingless signaling, Axin, or expressing an activated form of Armadillo (Arm*) were induced. The loss of eye identity caused by the ectopic activation of Wingless, suggests a possible function for Wingless in the regulation of the eye selector genes. The top of the genetic hierarchy involved in eye specification appears to be the Pax6 homolog, Eyeless. In the third instar eye disc the expression of Eyeless is restricted to the region anterior to the furrow and, despite the Wingless-induced inhibition of eye development, the expression of Eyeless in this region is not affected by axin^- clones. This lack of an effect anterior to the furrow, despite the overgrowth and abnormal Distal-less expression in the same region, implies that misregulation of Eyeless is not the primary cause of the transformations caused by ectopic Wingless activity (Baonza, 2002).

Downstream of Eyeless (although feedback relationships makes the epistatic relationship complex) are other transcription factors required for eye specification, including Eyes absent, Sine oculis and Dachshund. A phenotype similar to axin^- clones of excess proliferation and consequent overgrowth is caused by loss of Eyes absent and Sine oculis. Moreover, as in axin^- clones, clones mutant for sine oculis ectopically express Eyeless in the region posterior to the furrow. The similar mutant phenotypes shown by the loss of function of these genes and the ectopic activation of Wingless signaling make them good candidates to be regulated by the Wingless pathway (Baonza, 2002).

The expression patterns of Eyes absent, Sine oculis and Dachshund, in axin^- and/or arm^* mutant clones, were examined in third instar eye discs. At this stage, Dachshund is expressed at high levels on either side of the morphogenetic furrow, whereas Eyes absent and Sine oculis are expressed in all the cells of the eye primordium. In order to produce large patches of mutant tissue, the Minute technique was used. In axin^- M⁺ clones the expression of Eyes absent in front of the furrow is always autonomously eliminated. This effect is not only seen in large clones that touch the eye margin but also in small internal clones. Identical results were obtained with Sine oculis and Dachshund: their expression was autonomously lost from anterior axin^- M⁺ clones. Consistent with these results, in arm^*-expressing clones Eyes absent, Dachshund and sine oculis (detected with a lacZ reporter construct) are similarly autonomously eliminated. It is therefore concluded that Wingless signaling represses the expression of the eye selector genes eyes absent, dachshund and sine oculis anterior to the morphogenetic furrow. Posterior to the furrow, however, some clones express high levels of Eyes absent, and Dachshund. This effect is always associated with overgrowth, and this expression is restricted to only some cells in these clones (Baonza, 2002).

The conclusion that Wingless signaling negatively regulates the expression of Eyes absent, Dachshund and Sine oculis anterior to the furrow leads to the prediction that in normal development, domains of high Wingless activity in the anterior region of the eye disc will be associated with low expression of these genes. Previous work indicates that their expression is broadly non-overlapping, but to analyse this precisely, discs were double-labelled to detect the expression of Wingless and Eyes absent or Sine oculis throughout the third instar larval stage. The expression of these eye specification genes is precisely complementary to that of Wingless in the anterior lateral margins of the eye throughout the third instar. This is consistent with a role for Wingless signaling in initiating the borders between eye and other head structures. Note that in posterior lateral regions slight overlap is observed between the expression of Wingless and these genes; this is presumably analagous to the expression of eye specification genes seen in some posterior axin^- clones, and confirms that in posterior regions of the eye disc, Wingless signaling is not incompatible with the expression of these genes (Baonza, 2002).

These results indicate that Wingless regulates the final size of the eye field of cells by controlling the expression of eyes absent, sine oculis and dachshund. The expression pattern of these genes in the anterior eye margin is complementary to the expression of Wingless throughout the third instar, indicating that in anterior regions, high activity of Wingless signaling corresponds to absence of these gene products. Moreover, ectopic activation of Wingless signaling represses their expression anterior to the furrow (where they act to specify the eye field) throughout eye development. Finally, the loss of Wingless signaling causes ectopic expression of Eyes absent and Dachshund (Baonza, 2002).

It is proposed that the initial expression of Eyes absent, Sine oculis and Dachshund is negatively regulated by Wingless signaling in the eye disc, and that this regulation initiates the border between the eye field and adjacent head cuticle. Attempts were made to define whether Wingless represses the eye specification genes independently or whether eyes absent is the primary target but the data confirms earlier reports of the complexity of the regulatory relationships between eyes absent, sine oculis and dachshund. The observation that Eyes absent is able partially to restore the expression of the other two genes but cannot rescue the overgrowth and differentiation phenotype of axin^- clones has two possible explanations. Either Wingless represses eye development through at least one additional gene, or high level Wingless signaling blocks eye development later in the developmental program -- e.g., it is known to inhibit morphogenetic furrow initiation, even after its earlier effects are rescued by eyes absent expression (Baonza, 2002).

Dachsund expression in the wing imaginal disc

The absence of Drosophia Frizzled-3 produces no apparent phenotype. Binding studies reveal that Wg can interact with Dfz3 in cultured cells. In order to reveal a role for Dfz3 in development, the possiblity of a genetic interaction of Dfz3 with wingless has been investigated. Dfz3 may be involved in Wg signaling required for adult appendage formation. For example, Dfz3 may serve as an attenuator of Wg signaling, at least in a wg hypomorphic mutant background; the absence of Dfz3 may increase Wg signaling and stimulate wing formation. For analysis of this possiblility, a study was made to find possible interaction between Dfz3 and Wg signaling in various wg mutant backgrounds. Wing blades are frequently absent from flies mutant for wg ¹. Thus, the first question to be examined was is the wg ¹ phenotype affected by the absence of Dfz3? The absence of wing blades is partially rescued through the elimination of Dfz3 activity. On a wg ¹/wg ^CX4 background, fractions of flies with two wings increased from 46% to 87%, while those flies with one wing and wing-less flies, respectively, reduced from 44% and 10% to 13% and 0.5%. The wing-less phenotype of wg ¹ is enhanced in a heterozygous apterous (ap) mutant background: no wing blade is generated at approx. 90% of the presumptive wing-blade-forming sites in wg ¹ homozygous flies heterozygous for ap. Wing blade formation increases 3-fold in the absence of Dfz3 activity. Since wg ^CX4 and wg ¹ are null and regulatory mutant alleles, respectively, these effects are not due to possible change in Wg protein conformation. Thus, wild-type Dfz3 may serve as an attenuator of Wg signaling at least in a wg hypomorphic mutant background; the absence of Dfz3 may increase Wg signaling and stimulate wing formation (Sato, 1999).

To confirm that Dfz3 attenuates Wg signaling, an examination was made of the effects of Dfz3 absence in a different developmental context. Nearly all wg^11en/wg^CX4 flies lack antennal structures. This antenna-less phenotype is significantly rescued by removing Dfz3 activity; complete antennal structures, as well as incomplete ones, areregenerated at more than 70% of putative antennal sites. Distal antennal segment formation requires the circular expression of Bar homeobox genes. Dachshund (Dac) is required for the formation of proximal leg structures and expressed circularly in leg and antenna discs. Thus, wg ^11en/wg^CX4 fly discs with or without Dfz3 activity were stained for Wg, BarH1 and Dac. When there is Dfz3 activity, antennal discs are small and no or little expression of BarH1 and Dac is detected. In the absence of Dfz3 activity, about 10% of the discs, probably corresponding to the completely rescued type, exhibit circular BarH1 and Dac expression similar to that of wild-type discs. In about 50% of discs, presumably corresponding to the partially rescued type, Dac expression is partially restored without recovery of BarH1 expression. In contrast to BarH1 and Dac, no Wg expression is detected in the rescued mutant discs, indicating that wg expression is not enhanced by the absence of Dfz3. That wg^CX4 and wg ^11en are regulatory mutant alleles of wg suggests again that the genetic interactions found here would not be due to possible change in Wg protein structure, but simply to reduction in transcription products of wg. Thus it follows that in wg hypomorphic mutants, Dfz3 reduces Wg signaling activity required for antennal formation without changing wg expression; accordingly, Dfz3 would appear to function as a negative factor or attenuator of Wg signaling at least on a wg hypomorphic mutant background (Sato, 1999).

Dachsund expression in the leg imaginal disc

Drosophila proboscipedia (HoxA2/B2 homolog) mutants develop distal legs in place of their adult labial mouthparts. How pb homeotic function distinguishes the developmental programs of labium and leg has been examined. The labial-to-leg transformation in pb mutants occurs progressively over a 2-day period in mid-development, as viewed with identity markers such as dachshund (dac). This transformation requires hedgehog activity, and involves a morphogenetic reorganization of the labial imaginal disc. These results implicate pb function in modulating global axial organization. Pb protein acts in at least two ways. (1) Pb cell autonomously regulates the expression of target genes such as dac; (2) Pb acts in opposition to the organizing action of hedgehog. This latter action is cell-autonomous, but has a nonautonomous effect on labial structure, via the negative regulation of wingless and decapentaplegic. This opposition of Pb to hedgehog target expression appears to occur at the level of the conserved transcription factor cubitus interruptus/Gli that mediates hedgehog signaling activity. These results extend selector function to primary steps of tissue patterning, and leads to the notion of a homeotic organizer (Joulia, 2005).

The labial palps, the drinking and taste apparatus of the adult fly head, are highly refined ventral appendages homologous to legs and antennae. As for most adult structures, these mouthparts are derived from larval imaginal discs, the labial discs. Wild-type pb selector function acts together with a second Hox locus, Scr, to direct the development of the labial discs giving rise to the adult proboscis. In the absence of pb activity, the adult labium is transformed to distal prothoracic (T1) legs, reflecting the ongoing expression and function of Scr in the same disc. Though the pb locus shows prominent segmental embryonic expression, as for the other Drosophila homeotic genes of the Bithorax and Antennapedia complexes, it is unique in that it has no detected embryonic function and null pb mutants eclose as adults that are unable to feed. Thus, normal pb selector function is required relatively late, in the labial imaginal discs that proliferate and differentiate during larval/pupal development to yield the adult labial palps. Though the genetic pathway guiding development of the ventral labial imaginal discs to adult mouthparts remains relatively unexplored both in flies and elsewhere, study of P-D patterning has identified several genes subject to pb regulation in the labial discs (notably Dll, dac, and hth) and a distinct organization of normal labial discs has been indicated compared to other imaginal discs (Joulia, 2005).

This study pursued an investigation of how pb homeotic function distinguishes between labial and leg developmental programs. The results implicate pb function at the level of global axial organization. Employing identity markers such as dachshund (dac), a 2-day period late in larval development has been identified when normal pb function is required for labial development. The labial-to-leg transformation occurs during the third larval instar stage, involves a progressive morphogenetic reorganization of the labial imaginal disc, and is hedgehog-dependent. This analysis of the transformation indicates that normal pb action is required at least at two distinct levels. One is in the cell-autonomous regulation of target genes such as dac likely to be implicated in cell identity. A second level involves an autonomous action with a nonautonomous effect on labial structure, through the negative regulation of wingless and decapentaplegic downstream of hh signaling. This opposition to hh targets is likely to occur at the level of the transcription factor cubitus interruptus/Gli, a crucial and conserved mediator of hh signaling activity. These results led to a proposal that homeotic function may exist in intimate functional contact with the hedgehog organizer signaling system: the 'homeotic organizer' (Joulia, 2005).

Segmental organization in the imaginal discs involves the reiterated deployment of segment polarity genes that organize the fundamental segmental form. This involves a cascade proceeding from posteriorly expressed Engrailed protein through a short-range Hh morphogen gradient in anterior cells favoring the activator form of Ci transcription factor, which in turn activates wg and dpp to establish two concurrent, instructive concentration gradients that structure gene expression along the proximo-distal axis. In contrast with this elaborate choreography of the segment polarity genes, the homeodomain proteins encoded by Hox genes are expressed in a segmental register, which obscures how they can direct the differentiation of distinct cell types within the segment. The present investigation of homeotic proboscipedia function during labial palp formation indicates a multipronged action for pb in the labial disc. Pb acts cell-autonomously in the negative regulation of target genes including dac, which is normally extinguished in Pb-expressing cells of labial or leg imaginal discs but is activated in labial discs in the absence of pb activity. This activation of dac in mutant labial cells is hh-dependent and is likely a response to wg and dpp morphogen signals as for leg discs. The data further indicate that pb acts cell autonomously to regulate the level of both wg and dpp expression in response to hh. Thus, pb appears to negatively regulate dac expression directly, but also by withholding positive instructions from Wg and Dpp morphogens. The interweaving of homeotic selector proteins with strategic target genes including morphogens (wg, dpp) and targets of signaling activity (dac, Dll) may influence segment patterning from global size and shape to specific local pattern and cell identity. This positioning offers a powerful yet economical mode of selector function that helps to better understand how a single selector gene can integrate global patterning with cellular identity (Joulia, 2005).

Dachsund expression in the genital disc

In both sexes, the Drosophila genital disc contains the female and male genital primordia. The sex determination gene doublesex controls which of these primordia will develop and which will be repressed. In females, the presence of Doublesex^F product results in the development of the female genital primordium and repression of the male primordium. In males, the presence of Doublesex^M product results in the development and repression of the male and female genital primordia, respectively. This report shows that Doublesex^F prevents the induction of decapentaplegic by Hedgehog in the repressed male primordium of female genital discs, whereas Doublesex^M blocks the Wingless pathway in the repressed female primordium of male genital discs. It is also shown that Doublesex^F is continuously required during female larval development to prevent activation of decapentaplegic in the repressed male primordium, and during pupation for female genital cytodifferentiation. In males, however, it seems that Doublesex^M is not continuously required during larval development for blocking the Wingless signaling pathway in the female genital primordium. Furthermore, Doublesex^M does not appear to be needed during pupation for male genital cytodifferentiation. Using dachshund as a gene target for Decapentaplegic and Wingless signals, it was also found that Doublesex^M and Doublesex^F both positively and negatively control the response to these signals in male and female genitalia, respectively. A model is presented for the dimorphic sexual development of the genital primordium in which both Doublesex^M and Doublesex^F products play positive and negative roles (Sanchez, 2001).

The gene dachsund (dac) is also a target of the Hh pathway in the leg and antenna. In the present study, it was found that dac is differentially expressed in female and male genital discs. In the female genital discs, which have Dsx^F product, dac expression mostly coincides with that of wg in both the growing female primordium and the RMP. In contrast, in male genital discs, which have Dsx^M product, dac is not similarly expressed to wg but its expression partially overlaps that of dpp and no expression is observed in the RFP. In pkA minus clones, which autonomously activate Wg and Dpp signals in a complementary pattern, dac was ectopically expressed only in mutant pkA minus cells at or close to the normal dac expression domains in male and female genital discs. In pkA minus;dpp minus double clones, which express wg, dac is not ectopically induced in the male primordium of the male genital disc, but is still ectopically induced in both the growing female genital primordium and the RMP of female genital disc. Conversely, in pkA minus wg minus double clones, which express dpp, dac is not ectopically induced in the growing female or in the RMP of female genital discs, but is ectopically induced in the growing male primordium of the male genital disc. These results indicate that dac responds differently to Wg and Dpp signals in both sexes (Sanchez, 2001).

In dsx^Mas/+ intersexual genital discs, which have both Dsx^M and Dsx^F products, and in dsx¹ intersexual genital discs, which have neither Dsx^M nor Dsx^F products, dac is expressed in Wg and Dpp domains although at lower levels than in normal male and female genital discs. These results suggest that Dsx^M plays opposing, positive and negative roles in dac expression in male and female genital discs, respectively; and that Dsx^F plays opposing, positive and negative roles in dac expression in female and male genital discs, respectively. To test this hypothesis, tra2 clones (which express only Dsx^M ) were induced in female genital discs. The expression of dac is repressed in tra2 clones located in Wg territory. Therefore, Dsx^F positively regulates dac expression in the Wg domain, and Dsx^M negatively regulates dac expression in this domain, otherwise dac would be expressed in tra2 clones at the low levels found in dsx intersexual genital discs. However, when the tra2 clones are induced in the RMP, in the territory competent to activate dpp, they show ectopic expression of dac (Sanchez, 2001).

Therefore, Dsx^M positively regulates dac expression in the Dpp domain, whereas Dsx^F negatively regulates dac expression in this domain, since in normal female genital discs with Dsx^F dac is not expressed in Dpp territory. This is further supported by the induction of dac in the Wg domain and repression of dac in the Dpp domain by ectopic expression of Dsx^F in the male genital primordium of male genital discs. It is concluded that in male genital discs, Dsx^M positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, Dsx^F positively and negatively regulates dac expression in Wg and Dpp domains, respectively (Sanchez, 2001).

Homozygous tra2^ts larvae with two X-chromosomes develop into female or male adults if reared at 18°C or 29°C, respectively, because at 18°C they produce Dsx^F and at 29°C they produce Dsx^M. A shift in the temperature of the culture is accompanied by a change in the sexual pathway of tra2_ts larvae. Analysis of the growth of genital primordia and their capacity to differentiate adult structures of tra2_ts flies was performed using pulses between the male- and the female-determining temperatures in both directions during development (Sanchez, 2001).

Regardless of the stage in development at which the female-determining temperature pulse was given (transitory presence of functional Tra2_ts product; i.e. transitory presence of Dsx^F product and absence of Dsx^M product), the male genital disc develops normal male adult genital structures and not female ones. This occurs even if the pulse is applied during pupation. Pulses of 24 hours at the male-determining temperature (temporal absence of functional Tra2 ts product; i.e. transitory absence of Dsx^F product and presence of Dsx^M product) before the end of first larval stage produces female and not male genital structures. However, later pulses always give rise to male genital structures, except when close to pupation. Further, the capacity of the female genital disc to differentiate adult genital structures is also reduced when the temperature pulse is applied during metamorphosis (Sanchez, 2001).

When the effect of the male-determining temperature pulses was analyzed in the genital disc, it was found that overgrowth of the RMP is always associated with the activation of dpp in this primordium. However, this activation and the associated overgrowth only occurs when the temperature pulse is given after the end of first larval instar. This suggests that there is a time requirement for induction of dpp (Sanchez, 2001).

The activation of this gene in the RMP and the cell proliferation resumed by this primordium, as well as its capacity to differentiate adult structures is irreversible, because they are maintained when the larvae are returned to the female-determining temperature, which is when functional Tra2^ts product is again available (i.e. the presence of Dsx^F product and absence of Dsx^M product). This time requirement for induction of dpp is also supported by the fact that dsx¹¹ clones (which lack Dsx^M) induce differentiated normal male adult genital structures in the developing male genital primordium of XY; dsx¹¹/+ male genital discs (which express only Dsx^M ) after 24 hours of development. However, when the dsx¹¹ clones are induced in the time period between 0 and 24 hours of development, they do not differentiate normally and give rise to incomplete adult male genital structures. This different developmental capacity shown by the dsx¹¹ clones depending on their induction time is explained as follows. When the clones are induced after 24 hours of development, dpp is already activated. Indeed, these clones show no change in the expression pattern of dpp or their targets. Accordingly, these clones display normal proliferation and capacity to differentiate male adult genital structures. However, when the clones are induced early in development, dpp is not yet activated, since this gene is not expressed in the male genital primordium of male genital discs early in development. Therefore, when the male genital disc reaches the state in development when dpp is induced, the cells that form the clones activate this gene as in dsx mutant intersexual flies because the clones have neither Dsx^M nor Dsx^F products. Consequently, these clones do not achieve a normal proliferation rate, and then do not differentiate normal adult male genital structures (Sanchez, 2001).

As described above, it has been shown that dsx regulates the expression of gene dac. Recall that in male genital discs, Dsx^M positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, Dsx^F positively and negatively regulates dac expression in Wg and Dpp domains, respectively. The expression of the gene dac was analyzed in genital discs of tra2^ts flies using pulses between the male- and the female-determining temperatures in both directions. It was found that the dac expression pattern switches from a 'female type' to a 'male type' when male-determining temperature pulses were applied to tra2^ts larvae after first larval instar. Note that dac expression is reduced in the Wg domain of the RMP and is progressively activated in the Dpp domain. It should be remembered that these pulses lead to the transient presence of Dsx^M instead of Dsx^F product. Thus, these results are consistent with the previously proposed suggestion that Dsx^M activates dac in the Dpp domain and represses it in the Wg domain (again the converse is true for Dsx^F). When the pulse is given during first larval instar, dac is not activated in the Dpp domain of RMP, in spite of the fact that there is also a transient presence of Dsx^M instead of Dsx^F. This is explained by the lack of competence of cells to express Dpp, which is acquired after first larval instar. When the tra2^ts larvae reach such a developmental stage, these cells now produce Dsx^F because they have returned to the female-determining temperature (Sanchez, 2001).

Dsx^F prevents activation of dpp in the RMP, and consequently no induction of dac expression occurs. In the female genital primordium, dac expression is strongly reduced in the Wg domain and absent in the Dpp domain. Taken together, these results suggest that the development of male and female genital primordia have different time requirements for Dsx^M and Dsx^F products (Sanchez, 2001).

The integration of multiple developmental cues is crucial to the combinatorial strategies for cell specification that underlie metazoan development. In the Drosophila genital imaginal disc, which gives rise to the sexually dimorphic genitalia and analia, sexual identity must be integrated with positional cues, in order to direct the appropriate sexually dimorphic developmental program. Sex determination in Drosophila is controlled by a hierarchy of regulatory genes. The last known gene in the somatic branch of this hierarchy is the transcription factor doublesex (dsx); however, targets of the hierarchy that play a role in sexually dimorphic development have remained elusive. The gene dachshund (dac) is differentially expressed in the male and female genital discs, and plays sex-specific roles in the development of the genitalia. Furthermore, the sex determination hierarchy mediates this sex-specific deployment of dac by modulating the regulation of dac by the pattern formation genes wingless (wg) and decapentaplegic (dpp). The sex determination pathway acts cell-autonomously to determine whether dac is activated by wg signaling, as in females, or by dpp signaling, as in males (Keisman, 2001).

A number of obstacles make it difficult to demonstrate that the sex determination pathway is responsible for the sex-specific regulation of a gene in the genital disc. These obstacles stem from the fact that the male and female primordia, which are the primary constituents of their respective discs, differ in their segmental origin. This raises the possibility that 'sex-specific' gene regulation is really just segment-specific gene regulation, made to look sex specific by the fact that only one primordium develops in each sex. Attempts were made to address this concern by creating clones of the opposite genetic sex in chromosomally male and female genital discs. Thus, for example, dac regulation could be examined in the male (A9) primordium, in both male and female cells. By varying the genetic sex of cells in a context where segmental identity is uniform, it was hoped that the contributions of sex and segmental identity to dac regulation could be disentangled (Keisman, 2001).

In the male primordium of both male and female discs, the regulation of dac varies according to the genetic sex of the cell. Genetically female clones in the male (A9 derived) primordium of the male genital disc are unable to express dac in the lateral male (dpp-dependent) domain, but are able to express dac when they extended medially, towards the source of Wg. Conversely, in the female genital disc, genetically male clones in the repressed male primordium (A9) lose their ability to express dac in the medial, wg-dependent domain, and begin to express dac laterally, presumably in response to Dpp. Finally, dac expression is abnormal in intersexual genital discs from dsx mutant larvae: the male primordium of dsx genital discs expresses dac in both the endogenous, lateral male domains, and in a slightly weaker medial domain that corresponds roughly to the region where tra + clones are able to activate dac. Thus, it is concluded that in the male primordium, the sex determination pathway determines how a cell will regulate dac (Keisman, 2001).

In the female primordium the results fail to show a role for the sex determination pathway in dac regulation. If such a role exists, it would be expected that genetically male clones in the female primordia of a female genital disc would activate dac laterally, like their counterparts in the male primordia. They do not, even when they take up much of the presumptive dpp-expressing domain. It would also be expected that such clones would repress dac medially. Only a few clones were observed to extend into the medial wg-expressing domain, and as expected these appear to repress dac. Interpretation of these results is complicated by the fact that changing the genetic sex of a cell in the genital disc can cause it to enter the 'repressed' state. Thus, for example, if a genetically male clone represses dac when it intersects the medial dac domain in the female primordia, it can be concluded either that the sex determination pathway regulates dac expression or that the cells, which are now male, have adopted a repressed state and are generally unresponsive. A similar caveat prevents interpreting the failure of tra2IR clones to activate dac ectopically in the female primordium. That tra + clones in the male primordium of male genital discs enter such a generally non-responsive state was not of concern, because these clones both repress and activate dac expression. The expression pattern of dac in the female primordium of a dsx mutant genital disc is also difficult to interpret. dac is not activated ectopically in the lateral domains of the dsx female primordium, which is consistent with the failure of tra2IR clones to cause such activation. However, even the medial, wg-dependent dac domain is frequently absent or severely reduced in the dsx female primordium, and thus the authors are reluctant to draw any conclusions from the absence of ectopic dac laterally (Keisman, 2001).

A model is proposed for dac regulation in the male primordium, in which the different isoforms of Dsx protein modulate dac regulation by wg and dpp. In the absence of dsx, both wg and dpp can activate dac, producing the two domains of dac expression observed in the male primordium of a dsx disc. In the female, Dsx^f modulates dpp activity so that dpp becomes a repressor of dac; Dsx^f may also potentiate the activation of dac by wg. In the male, Dsx^m modulates wg activity so that it becomes a repressor of dac, leaving dpp alone to activate dac. In support of this model, it is noted that the Dsx proteins act in a similar manner to positively or negatively modulate the effect of tissue-specific regulators on the yp genes (Keisman, 2001 and references therein).

The behavior of tra + and tra2IR clones provides insight into the mechanism of repression in the undeveloped genital primordium. It was anticipated that such clones would be difficult to recover when they occurred in the male and female primordium, respectively, because they should adopt the repressed state. Instead, large tra + (female) clones were recovered in the male primordium of a male disc, and large tra2IR (male) clones were recovered in the female primordium of a female disc. Some of these clones constitute a substantial fraction of the primordium in question. Though tra + or tra2IR clones were not scored in adults, previous studies strongly suggest that such clones would fail to differentiate adult genital structures (Keisman, 2001).

It has been shown that tra - (male) clones cause large deletions in the female genitalia, indicating that genetically male cells like those in a tra2IR clone divide but cannot differentiate female genital structures. Further, it has also been shown that male structures are deleted when the mosaic border passes through the male genitalia, suggesting that female tissue cannot differentiate male structures. To reconcile these data, it is proposed that repression of the inappropriate genital primordium involves two separable processes: repression of growth and the prevention of differentiation. Thus, clones of cells of the inappropriate genetic sex cannot differentiate, but they can grow and contribute to a morphologically normal genital primordium. This poses yet another question. Cells in a tra + clone in the male primordium of a male genital disc are analogous to the cells in the repressed male primordium of a wild-type female genital disc: both are genetically female, and both have A9 segmental identity. Why do tra + clones in the male primordium grow, while the repressed male primordium in a female disc does not? One possibility is that the decision of the male primordium to grow in a male disc is made before tra + clones were induced and cannot be over-ridden by a later switch of genetic sex. However, temperature-shift experiments with tra-2 ts alleles suggest that the decision of a genital primordium to develop can be reversed later in development. Furthermore, occasional, large tra + clones can cause severe reductions in male genital discs. This observation leads to the suggestion of a model in which growth in the genital disc is regulated from within organizing zones, such as the domains of wg and dpp expression. According to this model, the sex of the cells in the organizing regions would determine how the disc grows, while cells in other regions would respond accordingly, regardless of their sex. The tra + clones that cause reduction could result when such a clone intersects with one of the postulated organizing centers within the disc. The implication is that the sex determination pathway acts in yet undiscovered ways to modulate the function of the genes that establish pattern in the genital disc. One such interaction was found in the regulation of dac; further study is needed to determine if others exist, and what role they play in producing the sexual dimorphism of the genital disc and its derivatives (Keisman, 2001).

Limb development requires the formation of a proximal-distal axis perpendicular to the main anterior-posterior and dorsal-ventral body axes. The secreted signaling proteins Decapentaplegic and Wingless act in a concentration-dependent manner to organize the proximal-distal axis. Discrete domains of proximal-distal gene expression are defined by different thresholds of Decapentaplegic and Wingless activities. distal-less is expressed in a central domain that corresponds to the presumptive tarsal segments and the distal tibia. The dachshund gene is required for development of the femur and tibia. Dac is expressed in a ring corresponding to the presumptive femur, tibia and first tarsal segment, but is absent from the more distal tarsal segments of the leg disc. Although there is little or no overlap between Dll and Dac domains at early stages, by mid third instar the combination of Dac and Dll expression defines three regions along the P-D axis. Dll and Dac are expressed in circular domains centered on the point at which the ventral Wg domain and the dorsal Dpp domain meet. Dll expression in the center of the disc depends on the combined activities of wg and dpp. Wg and Dpp act directly to induce Dll, as analysis of constitutively active Thick-veins clones has shown (Tkv is the receptor for Dpp); analysis of shaggy/zeste white 3 clones (Sgg is required for transduction of the Wingless signal) reveals that both Wg and Dpp transduction pathways are activated cell autonomously. Continuous signalling is not required to maintain Dll or Dac expression. The spatial domains of Dac and Dll expression are defined by different threshold levels of both Wg and Dpp activities. Both Dpp and Wg act to directly repress Dac in the center of the disc. Dac repression is actively maintained by Wg and Dpp signaling long after Dac and Dll have been induced and are stably expressed in the absence of further signaling. Subsequent modulation of the relative sizes of these domains by growth of the leg is required to form the mature pattern (Lecuit, 1997).

Homothorax is shown to limit Dpp and Wg expression. 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 ënonsenseí 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 Exdís nuclear localization was surprising, because these two signaling molecules induce Exdís 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).

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).

The first sign of proximodistal axis formation in the leg imaginal disc was a circular arrangement of cells expressing either Esg or Dll during embryogenesis. As the disc grows in size and evolves circular folds that separated tarsus, tibia, femur, trochanter and coxa, the pattern of esg expression is maintained. At the late stage of the third instar, more Esg protein is detected in the proximal region corresponding to the coxa and trochanter. The distal most part of the esg-expressing domain partially overlaps with the Dac-expressing domain in the trochanter. The esg expression in the overlapping domain is weaker than that in the more proximal domain, where only esg is detected. The domain of Esg expression appears to overlap with the proximal domain defined by expression of homothorax and teashirt, and nuclear localization of Extradenticle (Goto, 1999 and references).

Dll induces distal leg development when expressed ectopically in the proximal domain. To determine if any of the proximal genes have an organizing activity analogous to that of Dll, esg was induced ectopically using the flip-out technique. In the adult, Esg-positive clones marked by GFP are found as vesicles inside the leg cuticle and are often associated with malformation. In the region proximal to the clones, the bristles and epidermal hairs, which normally point distally, are often reversed. These bristles and hairs are genetically wild type, suggesting that the polarizing activity of Esg is non-cell-autonomous (Goto, 1999).

In the third instar leg disc, dac is expressed in a partially overlapping manner with the expression of Dll and esg in an intermediate ring that corresponds to the proximal tarsus, tibia, femur and trochanter. When esg expression is induced during the second instar, clones in the distal tarsal region show compact morphology; and many of them are associated with ectopic dac expression in cells within and surrounding the clone. The ectopic dac expression results in a local reversion of the proximal-distal order of the gene expression, which prefigures the change in the cell polarity in the adult leg. The esg-positive clones in the coxa spread normally and do not show induced dac. The non-cell-autonomy of the Esg function could be due to a modulation of known secreted molecules controlling anteroposterior and dorsoventral patterning. However, the expression patterns of the Hh target genes wg and dpp, and optomotor-blind (omb, 1996), a target gene of Dpp, are unaffected by misexpression of Esg (Goto, 1999).

esg^G66B null mutant clones were used to assess the requirement of Esg for dac expression. esg^G66B is a derivative of an enhancer trap and lacks the coding region of esg but retains the lacZ gene that reproduces the expression pattern of esg. esg mutant cells are marked by the loss of Myc antigen or by the high expression of beta-gal produced from the two copies of the lacZ gene. dac expression is frequently lost in clones induced at the late second instar larval stage. The partial loss of dac expression in large clones may have been due to a non-cell-autonomous rescue by esg+ cells next to the clones. The clones are sometimes associated with ectopic fold formation. Taken together with the gain-of-function analysis, these data suggest that Esg is necessary and sufficient for dac induction (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).

BarH1 and BarH2 play essential roles in the formation and specification of the distal leg segments of Drosophila. In early third instar, juxtaposition of Bar-positive and Bar-negative tissues causes central folding that may separate future tarsal segments 2 from 3, while juxtaposition of tissues differentially expressing Bar homeobox genes at later stages gives rise to segmental boundaries of distal tarsi including the tarsus/pretarsus boundary. Tarsus/pretarsus boundary formation requires at least two different Bar functions: early antagonistic interactions with a pretarsus-specific homeobox gene, aristaless, and the subsequent induction of Fas II expression in pretarsus cells abutting tarsal segment 5. Bar homeobox genes are also required for specification of distal tarsi. Bar expression requires Distal-less but not dachshund, while early circular dachshund expression is delimited interiorly by BarH1 and BarH2 (Kojima, 2000).

Circular Dac expression appears in second-instar leg discs before Bar ring appearance. This early Dac-ring is associated interiorly with Bar-positive Keilin's organ cells, which are situated along the interior circumference of or within the early Bar ring. Although they are separated from each other by a Bar-negative, Dac-negative region just before the onset of central fold formation Dac and Bar rings are immediate neighbors at earlier stages. Dac expression is derepressed in Bar minus clones observed in early third instar, while repressed by Bar misexpression, indicating that Bar is essential for distal restriction of Dac expression. Since early Bar expression normally occurs in dac minus clones, dac appears dispensable for proximal restriction of the early Bar ring. Interestingly, in dac minus mutants, Bar misexpression occurs in regions fated to become trochanter, indicating that Dac represses Bar in future trochanter (Kojima, 2000)

The proximal distal axis of the Drosophila leg is patterned by expression of a number of transcription factors in discrete domains along the axis. The homeodomain protein Homothorax and the zinc-finger protein Teashirt are broadly coexpressed in the presumptive body wall and proximal leg segments. Homothorax has been implicated in forming a boundary between proximal and distal segments of the leg. Evidence is presented that Teashirt is required for the formation of proximal leg segments, but Tsh has no role in boundary formation (Wu, 2000).

The leg disc consists of a single epithelial sheet in which the presumptive distal segments are specified in the center and the presumptive proximal segments are specified in the periphery. Cross-sections show that proximal segments, which express Hth and Tsh, fold back over the distal segments, which express Dll and Dac. Hth and Tsh expression is limited to the proximal region of the disc through repression by the combined activities of Wg and Dpp. Although the Hth and Tsh expression domains overlap through much of the proximal region, Hth expression extends more distally than Tsh. This is visible as a band of Hth expression that does not overlap Tsh in a basal optical section. This band coincides with the outer ring of Dll expression. The Tsh domain overlaps the proximal edge of the Dll ring by one or two cells. Tsh expressing cells are also found beneath the disc epithelium. Their location suggests that these may be adepithelial cells. Hth functions as a repressor to modulate Tsh expression. More distally located hth mutant clones lose Tsh expression. Loss of Tsh expression in hth correlates with ectopic expression of Dachshund. hth mutant clones cause ectopic expression of Dac close to the endogenous Dac domain, but do not do so in more proximal regions. The differential effect on Dac expression of hth clones located at different positions along the PD axis has been attributed to a role of Hth as a repressor of Wg and Dpp signaling. Thus the paradoxical loss of Tsh in more distal hth clones can be explained as an indirect effect of Hth on Dac expression. Dac can repress both Tsh and Hth when overexpressed. Thus the different distal limits of the Hth and Tsh expression domains presumably reflect a difference in their sensitivity to repression by Dac. The observation that Tsh levels increase in proximal hth clones suggests that Hth serves as a repressor of Tsh. Thus Hth modulates Tsh expression levels in the proximal leg in two ways. Hth may act directly to reduce Tsh expression levels in the proximal leg, and indirectly via repression of Dac to define the distal limit of Tsh expression (Wu, 2000).

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).

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-ß and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. This study revises understanding of limb development in flies and shows that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals (Campbell, 2002).

In addition to activating genes, EGFR signalling is required to repress genes in distal regions, and again different genes appear to be differentially sensitive, with some, such as B and rn, possibly being both activated and repressed above different thresholds. B, rn and dac are repressed in the center of wild-type discs, with dac being repressed over a wider region than B and rn. Lowering EGFR activity in Egfr^ts discs to a level sufficient only for loss of al, results in expression of B and rn in the center, but not dac. Raising the temperature still further results in extension of the dac domain to fill the center. Clonal analysis shows that Egfr acts autonomously to repress dac. Ectopic EGFR activity can also repress B, dac and rn but again predominantly in ventral regions (B is repressed mainly at later stages). Previous studies have shown that repression of dac in distal regions requires high levels of Wg and Dpp signalling, so all three pathways appear to be required to achieve this (Campbell, 2002).

The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homolog of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. A novel function of btd and Sp1 is reported: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. It is proposed that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth (Estella, 2003).

In a search for genes with restricted expression in the adult cuticle, the MD808 Gal4 line was found to direct expression in the ventral derivatives of the adult body; proboscis, antennae, legs and genitalia. In the abdomen and analia no clear expression was discerned. It was also noticed that the insertion was located in the first chromosome and associated with a lethal mutation. The mutant larvae showed a head phenotype resembling that described for mutants at the btd gene: loss of antennal organ and the ventral arms of the cephalopharyngeal skeleton, and complementation analysis indicated that the chromosome carrying the insert contained a mutation at btd. The expression pattern found in MD808/UAS-lacZ embryos was also similar to that reported for btd, suggesting that the Gal4 insertion was located at this gene. In addition, the imaginal expression of MD808 and of btd was largely coincident (Estella, 2003).

Further to the genetic analysis and the expression data, DNA fragments at the insertion site were cloned to map the position of the P-element on the genome. It is located 753 bp 5' of the btd gene. The related gene Sp