araucan and caupolican


Promoter Structure

The equal patterns of ara and caup in wild type and their modifications in iro1 and iroDFM2 (breaking caup between the first and second exons and deleting the first two exons of ara respectively) suggest that expression of both genes in the vein L3 and notum regions is driven by common enhancers. The vein L3 enhancer would be located to the left of the iroDFM2 breakpoint, while the notum enhancer would be found to the right of the iro1 breakpoint (Gómez-Skarmeta, 1996a).

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

The Iro-C genes ara and caup show similar patterns of expression in the wing disc. In early second instar larvae, they are expressed in the whole prospective mesothorax region. Later, in the third instar, their expression is restricted to the lateral notum. In addition, at this developmental stage, novel domains of expression appear in the prospective regions of the L1, L3 and L5 veins, tegula, dorsal radius, dorsal and ventral pleura and alula. The expression of mirr is slightly different, being absent from the L3, L5 and tegula domains but present at the other domains. The Iro-C harbours two additional transcription units, lincoyan (linc), whose pattern of expression at the notum is identical to that of ara and/or caup and quilapan (quil), which is ubiquitously expressed. Previous genetic analysis suggested the existence of enhancer-like REs that would drive the coincident expression of ara and caup in the wing disc. Thus, In(3L)iroDFM2, associated with a breakpoint within the ara transcription unit, removes ara expression in the wing disc except in the L3 vein domain, in contrast to caup expression which is only lost from that domain. This suggests the existence of vein L3-specific RE(s) distal to the In(3L)iroDFM2 breakpoint and other RE(s), specific for the remaining domains of Iro-C expression, located proximal to such breakpoint. To identify notum-specific REs, the regulatory potential of 31 different genomic fragments, spanning approximately 110 kb of genomic Iro-C DNA was analyzed (Letizia, 2007).

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

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

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

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

The transcriptional regulation of the Iro-C genes is modular. Thus, the non-coding Iro-C genomic DNA contains a series of five separate enhancers that control the expression of a reporter gene in sub-domains within the realm of Iro-C expression in the prospective notum region of the wing disc. None of the identified fragments reproduces on its own the entire pattern of expression of Iro-C in the prospective notum. However, IroRE1 and IroRE2 promote expression in complementary domains that entirely cover the territory of the presumptive lateral notum. Furthermore, IroRE2-mediated transcription recapitulates expression of Iro-C at the whole prospective notum at the second larval instar. It is hypothesized that the combined activity of both REs would be responsible for a great part of the regulation of Iro-C expression in the notum territory. Moreover, although IroRE3, IroRE4 and IroRE5 mediate lacZ expression in patterns only partly related to that of the Iro-C genes, these REs probably contribute to the complex regulation of the Iro-C. In addition, the possibility cannot be excluded of other RE(s) located outside the tested region that would help to establish the final pattern of Iro-C expression. Indeed, IroDFM3, a deficiency obtained by imprecise excision of the irorF209 P-lacZ element that extends up to the mirr promoter, maintains some lacZ expression in part of the central notum (Letizia, 2007).

The identified REs might act simultaneously on ara and caup expression to give rise to their almost coincident patterns of expression. Such coincidence cannot be attributed to cross-regulation between ara and caup since in irorF209 mutant discs (irorF209 is an ara null allele expression of caup is unmodified. Regulation of ara/caup would be, accordingly, similar to that of the achaete-scute genes of the AS-C, which show identical patterns of expression due to the use of shared enhancers. Expression of the vertebrate Iroquois (Irx) genes appears to be similarly regulated. Thus, the analysis of the regulatory potential of highly and ultra conserved non-coding regions present in the intergenic regions of the Irx clusters suggests these genes to be regulated by partially redundant enhancers shared by the components of each cluster (Letizia, 2007).

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

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

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

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

Despite the Dpp-mediated repression through IroRE2, a high level of Iro-C proteins accumulates in the lateral region of the notum, near the strong source of Dpp at the AP border. Furthermore, in this region of the wing disc Iro-C expression is refractory to Dpp-dependent repression. It is noteworthy that, IroRE1 mediates lacZ expression exclusively in that region of the wing disc and it appears to provide a regulatory mechanism for the co-existence of Iro-C expression and Dpp pathway activity, since the Dpp pathway does not repress but, on the contrary, activates IroRE1-mediated lacZ expression. Activation is restricted to the lateral notum, most likely because of the presence, in the hinge and medial notum territories, of repressors [Muscle segment homeobox, Msh; also known as Drop and Pnr/Ush, respectively] that would counteract activation. Putative binding sites for both Msh (consensus sequence G/C TTAATTG) and GATA proteins are indeed present in IroRE1. Thus, IroRE1 and IroRE2 represent two different REs in the same gene that respond in opposite ways to the same positional information, i.e. Dpp signalling. In addition a Dpp-independent mechanism based in the mutual repression between Iro-C and the homeoprotein Msh helps to maintain the distal border of Iro-C expression. This repression could be mediated by direct binding of Msh to one putative Msh binding site present in the Iro-RE2-B sequence (Letizia, 2007).

Transcriptional Regulation

Cell fate decisions in the early Drosophila wing disc assign cells to compartments (anterior or posterior and dorsal or ventral) and distinguish the future wing from the body wall (notum). Egf receptor signaling stimulated by its ligand, Vein, has a fundamental role in regulating two of these cell fate choices: (1) Vn/EGFR signaling directs cells to become notum by antagonizing wing development and by activating notum-specifying genes; (2) Vn/EGFR signaling directs cells to become part of the dorsal compartment by induction of apterous, the dorsal selector gene, and consequently also controls wing development, which depends on an interaction between dorsal and ventral cells (Wang, 2000).

To determine when Vn/EGFR signaling is required for notum development, the temperature-sensitive alleles, Egfrtsla and vntsWB240 were used. Inactivating Vn/Egfr activity during the second instar (a 24 hr period) causes loss of the notum. The wing develops but shows pattern abnormalities characteristic of vn hypomorphs. Later shifts during the third instar does not cause loss of the notum. This demonstrates that Vn/Egfr activity is required for notum development in the second instar when wg is required to specify the wing. Thus, Vn and Wg appear to have complementary roles and this relationship has been examined by following their expression in mutants (Wang, 2000).

To test the role of Vn/Egfr signaling in specifying notum an examination was carried out to see whether the Iroquois complex (Iro-C) genes, ara and cap are targets of the pathway. The Iro-C genes have been implicated in specifying notum cell fate because loss of function causes a transformation of notum to hinge. Furthermore, misexpression of ara causes loss of the wing and a duplication of notum. Ectopic expression of an activated form of the receptor, Egfrlambdatop4.2 greatly reduces the size of the wing and a small ectopic notum forms. vn is expressed in the presumptive notum in early second instar discs and Caup/Ara are expressed in the presumptive notum at the end of the second instar. In early third instar wing discs, Caup/Ara are expressed in a domain that overlaps with vn. In vn mutants, this expression of Caup/Ara is lost and loss of Egfr signaling, in Egfrts clones, in the medial notum results in a loss of Caup/Ara expression. However, clones in the lateral notum continued to express Caup/Ara, suggesting other factors regulate Iro-C gene expression in these cells at this stage (Wang, 2000).

Activation of Iro-C genes could account for the requirement for Egfr activity to specify the notum at the end of the second instar as this correlates with when these genes are first expressed. However, loss of Egfr signaling at a slightly earlier time (mid-first instar to mid-second instar, see below), prior to activation of the Iro-C genes, also results in loss of the notum. A possible explanation for this comes from the finding that vn expression is lost in vn mutants. This suggests Egfr activity must be sustained, via a positive feedback loop involving transcriptional activation of vn, during the second instar, to activate the Iro-C genes and hence specify notum at the end of this period. Interestingly, the vn gene is also a target of Egfr signaling in the embryo (Wang, 2000 and references therein).

ara-caup expression is restricted to two symmetrical patches located one at each side of the dorsoventral compartment border of the wing imaginal disc. ara-caup expression in these patches is necessary for the specification of the prospective vein L3 and associated sensory organs. Here, ara-caup expression is mediated by the Hedgehog signal through its induction of high levels of Cubitus interruptus in anterior cells near the the AP compartment border. The high levels of CI activate decapentaplegic expression, and together, CI and DPP positively control ara-caup. patched overexpression is equivalent to a reduced hh function in that accumulation of CI and DPP at the AP border are strongly depressed. The wing pouch of patched mutants have much reduced or absent ara-caup L3 patches. dpp by itself is insufficient to account for ara-caup expression. wingless is expressed in a narrow strip of cells straddling the DV compartment boundary of the wing disc, corresponding to the prospective wing margin. The dorsal and ventral ara-caup L3 patches are separated by a gap that corresponds to the cells that accumulate detectable amounts of WG. Clones of mutant wg expressing cells spanning the gap between the L3 patches extend these patches toward the DV border and a narrow gap of only one or two cell diameters remains. Thus WG represses ara-caup expression at the prospective wing margin domain. Likewise repression by Engrailed is most likely to be responsible for the posterior border of ara-caup expression in the L3 patches (Gómez-Skarmeta, 1996b)

The white gene was used as an enhancer trap and reporter of chromatin structure. white+ transgene insertions presenting a peculiar pigmentation pattern in the eye were collected: white expression is restricted to the dorsal half of the eye, with a clear-cut dorsal/ventral (D/V) border. This D/V pattern is stable and heritable, indicating that phenotypic expression of the white reporter reflects positional information in the developing eye. Localization of these transgenes led to the identification of a unique genomic region encompassing 140 kb in 69D1-3 subject to this D/V effect. This region contains at least three closely related homeobox-containing genes that are constituents of the iroquois complex (IRO-C). IRO-C genes are coordinately regulated and implicated in similar developmental processes. Expression of these genes in the eye is regulated by the products of the Polycomb-group (Pc-G) and trithorax-group (trx)-G genes but is not modified by classical modifiers of position-effect variegation. These results, together with the report of a Pc-G binding site in 69D, suggest a novel cluster of target genes for the Pc-G and trx-G products has been identified. It is proposed that ventral silencing of the whole IRO-C in the eye occurs at the level of chromatin structure in a manner similar to that of the homeotic gene complexes, perhaps by local compaction of the region into a heterochromatin-like structure involving the Pc-G products (Netter, 1998).

Spalt and Spalt-related encode conserved Zn-finger proteins that mediate the function of the TGF-beta molecule Decapentaplegic during the positioning of veins in the Drosophila wing. Spalt and Spalt-related regulate the vein-specific expression of the transcription factors of the knirps and iroquois gene complexes, delimiting their domains of expression in the wing pouch. The effects of spalt/spalt-related mutations on knirps and iroquois expression are cell-autonomous, suggesting that they could be direct. The regulation of iroquois involves transcriptional repression by Spalt and Spalt-related, whereas the regulation of knirps involves a combination of transcriptional activation and repression mediated by the same genes. It is suggested that the regulation of the iroquois and knirps gene complexes by Spalt and Spalt-related translates the Decapentaplegic morphogenetic gradient into precisely spaced pattern elements (de Celis, 2000).

Although the development of the four longitudinal veins of Drosophila (L2-L5) involves the same signaling systems, and vein cells show the identical type of differentiation, there are several characteristics that distinguish one vein from another. Thus, the veins L2 and proximal L4 differentiate predominantly in the ventral wing surface, whereas the veins L3, L5 and distal L4 do so in the dorsal surface. Furthermore, several genes are required for the formation of individual veins, suggesting that each vein is individually specified. Vein-specific genes include the transcription factors of the iroquois gene complex (iro-C), which are only expressed and required in L3 and L5, and the transcription factors of the knirps gene complex (kni-C), which are only expressed and required in L2. Vein-specific genes could be part of a combinatorial code of signals that activate a common vein-differentiation program in different parts of the wing. In addition, vein specific genes could also confer individual qualities to each longitudinal vein (de Celis, 2000 and references therein).

The heterogeneity in the levels of Sal/Salr could have a functional significance during vein patterning, and suggests that other factors, in addition to Dpp, participate in the regulation of sal/salr in the wing pouch. The expressions of Kni (L2) and Iro (L3 and L5) are also related to developing vein regions. Iro proteins are localized in L3 and L5, and are present in the vein and in the associated stripes of E(spl)m beta expression, both during imaginal and pupal development. In the larval disc, Kni is expressed in a domain broader than the vein L2 that corresponds to the region where E(spl)m beta is expressed at low levels. These observations indicate that Iro and Kni are expressed in vein competent regions, and that the loss of L2 and L3/L5 veins in kni and iro mutants, respectively, is due to failures in the specification of the corresponding vein competent region (de Celis, 2000).

The spatial relationships between the distribution of Kni, Iro and Sal were examined directly using appropriate antibodies. Kni is expressed within the anterior edge of the Sal/Salr expression domain, in the region where Sal/Salr are detected at lower levels. This differs from a previous report that placed the limit of Sal expression adjacent to but not overlapping with kni expression. Iro expression in the L5 vein competent region is, in contrast, immediately adjacent to the posterior limit of Sal expression. Thus, each individual vein expresses a unique combination of transcription factors (vein L2: Kni-C + Sal/Salr; veinL3: Iro + Sal/Salr; vein L4: Sal/Salr; L5: Iro) that are required for its formation and could confer individual characteristics to each longitudinal vein (de Celis, 2000).

The contrasting effects of sal/salr on the formation of specific veins (promoting L2 and suppressing L3, L4 and L5) indicate that these genes could both stimulate and antagonize the expression or activity of other vein-promoting genes. Thus, there is no evidence to indicate that sal and salr regulate vein differentiation directly; rather, they appear to influence vein development indirectly through regulating the expression of other genes that define individual veins. This regulation would require low levels of Sal/Salr to promote L2 and higher levels of Sal/Salr to inhibit L3 and L5 development. Two good candidates to be regulated by Sal/Salr are the kni-C and iro-C, because they are expressed in L2 and L3/L5, respectively. Furthermore, the function of kni-C and iro-C is required for the formation of these veins (de Celis, 2000).

To characterize the relationships of sal/salr with kni and iro-C, the effects of sal/salr mutant clones on kni and iro expression were examined. The expression of kni is eliminated in sal/salr clones that overlap the domain of Kni expression. These effects are cell-autonomous and can be observed in very small sal/salr clones. This suggests that, in contrast to the non-autonomous effect of Sal on kni, Sal/Salr regulate kni expression in a cell-autonomous fashion. All sal/salr clones localized between veins L2/L3 and L4/L5 are associated with ectopic expression of Iro proteins. Again, this effect is strictly cell-autonomous, suggesting that the repression of iro-C genes by Sal/Salr could be direct. Ectopic expression of Iro in sal/salr mutant clones is not observed in regions close to the dorso-ventral boundary, presumably because in this region iro-C expression is repressed by wingless. The effects of sal/salr on kni-C and iro-C expression have also been analyzed in experiments in which sal and salr are expressed ectopically using the GAL4 system. Widespread expression of sal or salr in the wing blade eliminates L2 and L5, and prevents expression, respectively, of Kni and Iro in the L2 and L5 territories of the corresponding imaginal discs. The expression of Iro in L3, which depends on Hh activity, is not affected by removal sal/salr functions and only slightly decreased by their ectopic expression. Taken together, these observations indicate that Sal/Salr negatively regulates iro-C expression in cells not exposed to Hh protein, and suggests that precise levels of Sal/Salr proteins are needed to activate kni expression in L2 (de Celis, 2000).

The Hedgehog (Hh) signal has an inductive role during Drosophila development. Patched is part of the Hedgehog-receptor complex and shows a repressive function on the signaling cascade, which is alleviated in the presence of Hh. The first dominant gain-of-function allele of patched has been identified: Confused (patchedCon). Analysis of the patchedCon allele has uncovered novel features of the reception and function of the Hh signal. At least three different regions of gene expression were identified and a gradient of cell affinities was established in response to Hh. A new state of Cubitus interruptus activity, responsible for the activation of araucan and caupolican genes of the iroquois complex, is described. This state has been shown to be independent of Fused kinase function. In the disc, patchedCon behaves like fused mutants and can be rescued by Suppressor of fused mutations. However, fused mutants are embryonic lethal while patchedCon is not, suggesting that Patched could interpret Hedgehog signaling differently in the embryo and in the adult (Muller, 2000).

In ptcCon clones, a unique experimental situation is presented in that reception of Hh signaling is severely impaired, allowing the accumulation of Ci in the cytoplasm without the activation of dpp. ptcCon clones in the wing differentiate vein 3 when close to the P compartment and substitute vein 4 for vein 3. This is in accordance with the activation of Caup in ptcCon clones, which is involved in determining vein 3 in the wing imaginal disc. When lowering the concentration of Hh by removing a copy of hh, vein 3 is not induced in ptcCon clones and the levels of cytoplasmic Ci are low, similar to smo clones that do not differentiate vein 3. In the same line, ptcCon clones close to but not touching the A-P border do not develop vein 3 nor express Caup. Since ptcCon cells interpret high Hh levels as low, these results ascribe the role of determining the position and differentiation of vein 3 to low levels of Hh. Furthermore, Ci accumulation in the cytoplasm indicates the activation of Ci to induce expression of Caup and differentiation of vein 3 (Muller, 2000).

The unmasking of a third level of apparent Ci activity is reported that is independent of the other two levels. This new state of Ci activity is responsible for the activation of iro and the differentiation of vein 3 in the wing. The other two levels of Ci activity arise from high levels of Hh and depend on Fu activity. The new state of Ci is activated by low levels of Hh and is Fu independent. Thus, Hh signaling activates two different pathways through inhibition of Ptc function. Fu would be involved in mediating transduction of the signal in one of these pathways. The second pathway would modify Ci to activate it in a Fu-independent manner. It has been suggested that low levels of Hh activate a new form of Ci, named 'Ci default', which does not depend on Fu activity (Muller, 2000).

Growth and patterning of the Drosophila wing imaginal disc depends on its subdivision into dorsoventral (DV) compartments and limb (wing) and body wall (notum) primordia. Evidence is presented that both the DV and wing-notum subdivisions are specified by activation of the Drosophila Epidermal growth factor receptor (Egfr). Egfr signaling is necessary and sufficient to activate apterous (ap) expression, thereby segregating the wing disc into D (ap-ON) and V (ap-OFF) compartments. Similarly, Egfr signaling directs the expression of Iroquois Complex (Iro-C) genes in prospective notum cells, rendering them distinct from, and immiscible with, neighboring wing cells. However, Egfr signaling acts only early in development to heritably activate ap, whereas it is required persistently during subsequent development to maintain Iro-C gene expression. Hence, as the disc grows, the DV compartment boundary can shift ventrally, beyond the range of the instructive Egfr signal(s), in contrast to the notum-wing boundary, which continues to be defined by Egfr input (Zecca, 2002a).

Prospective notum cells are distinguished from wing cells by the activity of the Iroquois Complex (Iro-C) genes. These results demonstrate (1) that activation of Egfr/Ras pathway is both necessary and sufficient to drive Iro-C gene expression in wing disc cells, and (2) that wing disc cells persistently monitor their level of Egfr/Ras input and are allocated to the wing or notum primordium on an ongoing basis, depending on the level of Egfr/Ras input they receive. This means that the wing-notum subdivision is not a stable compartmental partition between differently committed cell types, but rather a labile demarcation that reflects the current distribution of an instructive Egfr ligand (Zecca, 2002a).

Despite the provisional nature of the wing-notum segregation, the boundary between the two primordia is relatively straight and sharp. By manipulating Egfr/Ras signaling, it was shown that presumptive notum cells that lose the capacity to maintain Iro-C gene expression sort out of the notum primordium. Conversely, presumptive wing cells that ectopically activate the Iro-C genes sort out of the wing primordium. Similar results have been obtained by altering Iro-C gene function directly, rather than through the manipulation of Egfr/Ras signaling. Taken together, these results suggest that Iro-C gene activity, under Egfr control, programs prospective notum cells to have a different affinity from prospective wing cells, thereby straightening and sharpening the boundary between the two primordia. Further support for such a mechanism comes from experiments in which clones of cells were generated that ectopically express an activated form of Spi, an Egfr ligand, in the prospective wing hinge. All of the cells within these clones express the Iro-C genes and interdigitate freely with neighboring wild-type cells that are also induced to express the Iro-C genes. However, cells located further away do not receive sufficient Spi to activate Iro-C gene expression and these form a smooth boundary encircling the ectopic Iro-C-expressing cells (Zecca, 2002a).

The notum primordium, once established by the activation of Iro-C gene expression, is itself subdivided into distinct lateral and medial primordia by the localized activity of the pnr gene. pnr encodes a transcription factor that represses Iro-C gene expression and specifies medial as opposed to lateral notum differentiation. pnr activity also causes medial cells to adopt a distinct affinity that prevents them from mixing with lateral cells. It is tempting to speculate that pnr expression, like that of the Iro-C genes, is governed by Egfr signaling, e.g., being activated at a higher threshold concentration than the Iro-C genes, and hence in a smaller, more dorsally restricted domain. However, it was found that cells do not require peak levels of Egfr/Ras activity to remain and develop normally within the medial primordium. Conversely, enhanced activation of the Egfr/Ras pathway does not appear to cause lateral cells to sort into the medial primordium or adopt medial characteristics (e.g., the loss of Iro-C gene expression). Instead, it seems that pnr expression and subdivision of the notum into medial and lateral domains may depend on other signals, such as Dpp (Zecca, 2002a).

The relationship between pnr and the Iro-C gene expression in the notum is conserved in corresponding dorsolateral and dorsomedial regions of most of the adult segments, as well as in the embryonic and larval ectoderm. Hence, it has been proposed that the deployment of these genes reflects a fundamental partitioning process reiterated in most or all body segments. However, there are significant differences in the way that the Iro-C genes are deployed in the wing disc compared with the eye-antenna disc, the only other context in which an equivalent analysis has been performed. (1) During eye development, Iro-C gene expression is not governed by persistent signaling, in contrast to the wing disc. Instead, these genes are heritably activated early in eye development and behave as classical selector genes, performing a role that corresponds in most respects to that of ap in the wing. (2) Iro-C gene expression is activated in the eye disc by Hedgehog and Wingless signaling, rather than by Egfr signaling. Thus, it appears that the Iro-C genes are activated by different signals and govern different types of partitioning events in these two contexts, raising the possibility that their deployment in other segments, and at other stages, may reflect similarly diverse inputs and developmental roles (Zecca, 2002a).

As in the case of the Iro-C genes, Egfr/Ras signaling is both necessary and sufficient to activate ap expression in early wing disc cells. Furthermore, evidence is provided that each wing disc cell chooses to express, or not to express, ap at this time, depending on its level of Egfr/Ras activation. However, in contrast to the Iro-C genes, the descendents of each cell then inherit this initial choice without further reference to Egfr/Ras signaling. The results of eliminating Egfr/Ras activity before the establishment of the DV compartments are particularly striking. Early loss of Egfr activity causes dorsally positioned cells within the disc to choose, incorrectly, to become V compartment founders. These cells and their descendents generally sort into the existing V compartment or out of the disc epithelium. In rare cases, they can form an ectopic V compartment within the D compartment. By contrast, later loss of Egfr activity has no effect on the DV compartmental segregation. These findings establish that Egfr signaling is responsible for establishing the D and V compartments through the heritable activation of ap (Zecca, 2002a).

Although the Iro-C and ap genes are activated in overlapping dorsoproximal sectors of the early wing disc, the domain of ap expression expands relative to that of Iro-C gene expression during subsequent development, causing the DV boundary to be positioned up to 30 cell diameters ventral to the notum-wing boundary. It is suggested that this shift occurs because ap-expressing cells no longer depend on Egfr/Ras input to continue to express ap. Hence, as ap-expressing cells within the notum primordium proliferate, some will move out of range of the instructive Egfr ligand, cease to express Iro-C genes and enter the wing primordium. In the accompanying paper (Zecca and Struhl, 2002), evidence is provided that this shift must occur in order for D and V compartment cells to interact to induce Wg and stimulate wing growth and differentiation (Zecca, 2002a).

The subdivision of the Drosophila wing imaginal disc into dorsoventral (DV) compartments and limb-body wall (wing-notum) primordia depends on Epidermal growth factor receptor (Egfr) signaling, which heritably activates apterous (ap) in D compartment cells and maintains Iroquois Complex (Iro-C) gene expression in prospective notum cells. The source, identity and mode of action of the Egfr ligand(s) that specify these subdivisions has been examined. Of the three known ligands for the Drosophila Egfr, only Vein (Vn), but not Spitz or Gurken, is required for wing disc development. Vn activity is required specifically in the dorsoproximal region of the wing disc for ap and Iro-C gene expression. However, ectopic expression of Vn in other locations does not reorganize ap or Iro-C gene expression. Hence, Vn appears to play a permissive rather than an instructive role in organizing the DV and wing-notum segregations, implying the existance of other localized factors that control where Vn-Egfr signaling is effective. After ap is heritably activated, the level of Egfr activity declines in D compartment cells as they proliferate and move ventrally, away from the source of the instructive ligand. Evidence is presented that this reduction is necessary for D and V compartment cells to interact along the compartment boundary to induce signals, like Wingless (Wg), which organize the subsequent growth and differentiation of the wing primordium (Zecca, 2002b).

During development, the imaginal wing disc of Drosophila is subdivided along the proximal-distal axis into different territories that will give rise to body wall (notum and mesothoracic pleura) and appendage (wing hinge and wing blade). Expression of the Iroquois complex (Iro-C) homeobox genes in the most proximal part of the disc defines the notum, since Iro-C- cells within this territory acquire the identity of the adjacent distal region, the wing hinge. How is the expression of Iro-C confined to the notum territory? Neither Wingless signaling, which is essential for wing development, nor Vein-dependent EGFR signaling, which is needed to activate Iro-C, appears to delimit Iro-C expression. A main effector of this confinement is the TGFß homolog Dpp, a molecule known to pattern the disc along its anterior-posterior axis. At early second larval instar, the Dpp signaling pathway functions only in the wing and hinge territories, represses Iro-C and confines its expression to the notum territory. Later, Dpp becomes expressed in the most proximal part of the notum and turns off Iro-C in this region. This downregulation is associated with the subdivision of the notum into medial and lateral regions (Cavodeassi, 2002).

In third instar wing discs, the expression of dpp in both proximal and distal territories does not suggest a function in regulating the domain of Iro-C. However, in the second instar disc dpp is expressed in distal regions but it is absent from the Iro-C domain. Dpp is a diffusible molecule and, therefore, its range of activity was determined by monitoring the phosphorylated form of the Mad protein (pMad), an intermediate of the Dpp transduction pathway. pMad accumulates in the cells near the source of Dpp, but it is reduced or absent within the Iro-C domain. Another useful indicator of Dpp activity is the type I TGFß receptor Thick veins (Tkv), since its expression is negatively regulated by Dpp signaling. In addition, high levels of Tkv can limit Dpp diffusion and help to confine the region in which the pathway will be activated. The Iro-C domain is located within a region of high accumulation of Tkv, a result compatible with Dpp activity being strongly reduced or absent from that domain (Cavodeassi, 2002).

The complementary territories of Iro-C and dpp signaling activity (pMad) have suggested that the Dpp pathway might repress Iro-C at the early stages of wing disc development. The levels of Dpp signaling were therefore manipulated and the expression of Iro-C was monitored. In the strong hypomorphic dppd12/dppd14 combination, the Iro-C domain comprises most cells of the early wing disc and its distal border is very close to a small area that corresponds to the wing pouch, as identified by the Nubbin (Nub) marker. Since the Iro-C and the Nub domains are well separated in wild-type discs of similar age, this suggested that the Iro-C domain is distally expanded in the dppd12/dppd14 discs and covers at least part of the hinge/proximal-wing territory. However, it might be argued that the expansion of the Iro-C territory is an illusion caused by the apposition of an essentially normal notum to a hinge/wing territory dwarfed by reduced Dpp signaling. This was not the case. By following the development of these discs, it was observed that Iro-C proteins were gradually removed from part of the putative ectopic domain. The region in which Iro-C was gradually switched off was identified as hinge territory by two criteria: (1) it accumulates the Tsh protein very strongly, and (2) it develops a group of several sensory organ precursor cells; such characteristic groups develop in the hinge, but never in the notum. However, ectopic Iro-C expression is maintained in other distal regions. Consistent with the distal expansion of Iro-C in second instar dppd12/dppd14 discs, the Iro-C domain is coextensive with that of Tsh, which includes the territory fated to become hinge. This coexpression is never observed in wild-type discs. Note that the gradual removal of Iro-C protein from the prospective hinge in dppd12/dppd14 discs indicates that, even under conditions of strong Dpp insufficiency, the distal border of the Iro-C domain can be generated, at least in part. This could be due to residual Dpp signaling and/or to additional uncharacterized factors, which would normally contribute to maintain and refine this border. To help distinguish between these alternatives, the effect of the complete loss of reception of the Dpp signal was examined by generating, during the first instar, clones mutant for the null tkva12 allele. Owing to the difficulty of detecting cell clones in second instar discs, they were examined in third instar discs. In these tkva12 clones, the domain of Iro-C expression appears distally expanded, as detected by comparison with the domain of Tsh expression. This, however, is not the case for clones located in the more anterior part of the disc. Note again that this region coincides with that in which Iro-C is first expressed and later removed in dppd12/dppd14 discs. This suggests that after the initial restriction of Iro-C by Dpp signaling, additional factors contribute to maintain the anterior part of the Iro-C border (Cavodeassi, 2002).

Dpp signaling was next increased by misexpressing UAS-dpp in the proximal region of the disc (MS248-Gal4 driver); it downregulates Iro-C in a large part of the notum territory. Misexpression in cell clones of a constitutively activated form of Tkv (UAS-tkvQD) also suppresses Iro-C expression autonomously, although not completely in some regions. It is concluded that Dpp signaling must be absent (or strongly reduced) from the notum territory for Iro-C expression. Consistently, misexpression of the Dpp pathway antagonists UAS-brinker or UAS-daughters against dpp within this territory (MS248-Gal4) does not detectably affect the expression of Iro-C in second instar discs (Cavodeassi, 2002).

During the third instar, after Iro-C has specified the prospective notum, dpp is turned on in this territory and helps effect its patterning. The activation of dpp in the proximal-most region of the prospective notum is accompanied by a gradual removal of Iro-C, a repression essential to specify the medial versus the lateral notum. Dpp is responsible for this downregulation, since it is prevented by decreasing (dppd12/dppd14 mutant) or abolishing (clones mutant for a null tkv allele) Dpp signaling. In contrast, constitutive activity of the Dpp pathway in cell clones autonomously inhibits Iro-C in the lateral notum, except in a region overlapping or very close to an endogenous source of Dpp. Thus, while in the medial notum there is a correspondence between Dpp expression and Iro-C repression, this correlation does not hold everywhere in the lateral notum, where the appearance of Dpp expression may not result in turning off Iro-C. Interestingly, vn is also maximally expressed in the region of overlap of dpp and Iro-C expressions, and might antagonize, through the activation of EGFR signaling, the repression of the Iro-C genes by the Dpp pathway. It is concluded that, in the third instar disc, the levels of Dpp signaling are critical to establish the medial-lateral subdivision of the notum by its negative regulation of Iro-C in the medial region. This negative regulation should be mediated by pannier, which is activated by dpp in the medial notum (Cavodeassi, 2002).

Mutual repression between msh and Iro-C is an essential component of the boundary between body wall and wing in Drosophila

During development, the imaginal wing disc of Drosophila is subdivided into territories separated by developmental boundaries. The best characterized boundaries delimit compartments defined by cell-lineage restrictions. This study analyzes the formation of a boundary that does not rely on such restrictions, namely, that which separates the notum (body wall) and the wing hinge (appendage). It is known that the homeobox genes of the Iroquois complex (Iro-C) define the notum territory and that the distal limit of the Iro-C expression domain demarks the boundary between the notum and the wing hinge. However, it is unclear how this boundary is established and maintained. msh, a homeobox gene of the Msx family, is strongly expressed in the territory of the hinge contiguous to the Iro-C domain. Loss- and gain-of-function analyses show that msh maintains Iro-C repressed in the hinge, while Iro-C prevents high level expression of msh in the notum. Thus, a mutual repression between msh and Iro-C is essential to set the limit between the contiguous domains of expression of these genes and therefore to establish and/or maintain the boundary between body wall and wing. In addition, msh is found to be necessary for proper growth of the hinge territory and the differentiation of hinge structures. msh also participates in the patterning of the notum, where it is expressed at low levels (Villa-Cuesta, 2005).

msh is known to be involved in different processes. Thus, it participates in regional specification of muscle progenitors/founders; together with vnd and ind, it helps subdivide the embryonic neuroectoderm along the dorsoventral axis, and it confers dorsal identity to the dorsal bristles of the anterior margin of the wing. This study reports additional functions of msh, namely, the formation/maintenance of the subdivision between the territories of the wing disc that will give rise to the notum (dorsal mesothoracic trunk) and the dorsal hinge (appendix), the proper growth of the dorsal hinge, and the patterning of this region and of the notum (Villa-Cuesta, 2005).

In the developing wing disc, msh is expressed most strongly in the territory of the dorsal hinge, the region between the notum and the dorsal wing blade territories. Removal of msh in clones results in malformations that range from small defects, such as an outheld wing, to partial or even complete loss of most hinge structures. In the latter cases, the hinge may be posteriorly misplaced and ectopically attached to the scutellum. In addition, in a fraction of flies ectopic notum tissue appears contiguous to the extant hinge. Because at least a large part of the hinge tissue is still present, it is surmised that the absence of recognizable hinge structures is due to the failure of their proper differentiation. This phenotype correlates well with that observed in third instar wing discs displaying msh- clones. Indeed, even large clones that remove msh from most of the dorsal hinge territory allow the specification of this territory, as demonstrated by the relatively unmodified characteristic patterns of expression of genes such as wg, zfh-2, hth and tsh, and the presence of recognizable proneural clusters of sc expression. Moreover, the presence in mutant hinges of relatively well resolved clusters of sc expression indicate that the prepatterning of the hinge can proceed to a large extent in the absence of msh. It is concluded that msh is largely dispensable for specification of the dorsal hinge territory, but it is required for the final stages of its patterning and differentiation (Villa-Cuesta, 2005).

Mosaic analyses aimed at studying the patterns of cell proliferation in the wing disc have disclosed the presence of the anterior, posterior, dorsal and ventral compartments of the wing with borders that imposed absolute restrictions to cell proliferation. A border of this type has been suggested to exist between the notum and dorsal hinge, as well as between the pleura and the ventral hinge, but the complex morphology of these regions and the unavailability of appropriate cuticular markers made the proposal uncertain. In fact, analyses performed in the wing disc, has shown that clones can straddle the notum/dorsal hinge boundary, this being defined by the distal border of the Iro-C domain. Hence, at this boundary, the descendants of a cell adopt their developmental fate not according to lineage, but depending on the side of the boundary they were located. The issue thus arises of how the boundary between the notum and the dorsal hinge territories is established and maintained. Considering that the extent of the notum territory is defined by the expression of the Iro-C, this issue can be largely resolved by explaining how the distal border of the Iro-C domain of expression is defined (Villa-Cuesta, 2005).

So far, several genetic interactions have been identified that together permit to suggest a mechanism that partially answers this question. In the second instar disc, the EGFR pathway activates ap and Iro-C. The distinct but overlapping domains of expression of these genes, the dorsal compartment (ap) and the notum territory (Iro-C), may be defined by differential sensitivity to EGFR signaling or, alternatively, in the case of Iro-C, by Dpp signaling. In these early stages, Dpp signaling is active only in the distal part of the disc, where it represses the Iro-C and sets its distal limit of expression. Hence the antagonistic actions of the EGFR and the Dpp pathways define the position of the distal limit of the Iro-C domain, and therefore the position of the notum/hinge subdivision (Villa-Cuesta, 2005).

At approximately the time Iro-C starts to be expressed in the more proximal part of the disc, i.e., that which will become the notum, expression of msh (by means of ap) is turned on in the adjacent dorsal hinge territory. These essentially complementary patterns of expression are maintained, with some qualifications, in the third instar disc. Loss- and gain-of-function experiments show that msh prevents ara/caup from being expressed in the hinge, and ara/caup restrain msh from being expressed in the notum at the high levels typical of the hinge (although it is expressed at a low level in part of the notum). This mutual repression also occurs late during development (Villa-Cuesta, 2005).

How relevant is this mutual repression for the establishment of the notum/dorsal hinge territorial subdivision? As indicated above, in ~19% of flies with msh clones, the removal of Msh from the hinge induces extra notum tissue. In the remaining cases, this removal does not substantially affect the identity of the hinge territory. Thus, the mutual repression between msh and Iro-C is crucial for the notum/hinge territorial subdivision in only a small but substantial fraction of the discs. This indicates that additional agents, probably expressed in the hinge, participate in effecting the subdivision. By contrast, notum cells that lose Iro-C always change their fate to hinge cells and, consequently, depending on position, they modify the notum/hinge subdivision or create an ectopic notum/hinge boundary. Hence, the relevance of the msh/Iro-C mutual repression to define/maintain that subdivision relies mainly on its defining/maintaining the border of the Iro-C domain, and thereby preventing the expression of hinge genes within the notum territory. Thus, a 'pronotum' gene (Iro-C) and a 'hinge differentiation' gene (msh), despite their different positions within the genetic hierarchies that govern the development of their respective domains, cross-regulate each other and participate in the early definition of their respective territories. The current data do not permit distinguishing between the possibilities that the mutual repression between msh and Iro-C is instrumental in establishing this territorial border, or, alternatively, that it stabilizes a previous border defined by the antagonistic actions of EGFR and Dpp on the Iro-C (Villa-Cuesta, 2005).

The relevance of the mutual repression between Iro-C and msh is also manifested by their respective overexpression. Ectopic Iro-C products in the hinge impair the proper differentiation of hinge structures (R. Diez del Corral, PhD thesis, Universidad Autónoma de Madrid, 1998). High levels of Msh in the notum turn on a hinge-specific marker like zfh-2 and are detrimental for notum development (Villa-Cuesta, 2005).

In the third instar disc, the distal border of the Iro-C domain is no longer straight and displays a pronounced 'bay' where ara/caup are downregulated. This roughly coincides with the area of highest expression of msh in the lateral notum. msh is probably responsible for this downregulation of ara/caup, as the 'bay' disappears in msh clones. Moreover, the abutting domains of msh and Iro-C in the ventral hinge and pleura, respectively, suggest that a similar mutual repression may occur there to establish the subdivision between these neighboring regions. Finally, the removal of msh does not activate Iro-C in the anterior part of the hinge territory, suggesting again that agents other than msh and Dpp help maintain Iro-C expression confined to the notum territory (Villa-Cuesta, 2005).

Iro-C clones located within the medial notum not only undergo an autonomous transformation to dorsal hinge. They also become surrounded by a fold similar to that which separates the notum and hinge territories, and they modify the expression of several markers in the surrounding wild-type tissue in a way consistent with a transformation of this tissue towards lateral notum. These nonautonomous effects suggest that signals emerge from the Iro-C clones, and that these signals alter the fate of the aposed notum tissue. Hence, it was inferred that, in the wild-type disc, signaling would take place across the hinge/notum boundary and this would help pattern at least the lateral notum. This is reminiscent of the DV and AP compartment boundaries, where signaling mediated by the diffusible molecules Wg, and Hh and Dpp, respectively, are key to stimulating the growth and pattern of the wing disc. However, in the hinge/notum boundary, the signaling agents have not been identified. They could be either diffusible molecules or cell-bound molecules that mediate this cell to cell communication (Villa-Cuesta, 2005).

The imaginal disc territories flanking the notum/hinge border are reduced in size when they are mutant for msh. It is not known whether this effect is due to decreased cell proliferation, increased cell death or both, and whether it mostly affects the hinge or the lateral notum. However, it is clear that by removing msh and allowing Iro-C to be expressed in the hinge, the msh clones suppress the confrontation of proper hinge cells with notum cells. It is tempting to speculate that this could affect the net growth of the territory by removing positional values and/or by suppressing or making ineffective the postulated signaling associated with the hinge/notum border. Consistently, a reduced size of the notum plus hinge region (and a simplification of the patterning) is also observed in discs overexpressing UAS-ara in the dorsal compartment, a condition that removes most msh expression from the hinge. The failure of Iro-C clones within the notum territory to grow and survive when they are also depleted of Msh might result from the absence of proper signaling across a boundary where wild-type notum cells confront Iro-C msh cells. Considering that the activity of the EGFR signaling pathway is necessary for notum cell proliferation, it would be of interest to examine whether this pathway is involved in, or is modulated by, the presence of the notum/hinge boundary (Villa-Cuesta, 2005).

In Drosophila, the Iro-C genes and msh respectively participate in the DV subdivision of the eye and of the neuroectoderm. In vertebrates, although no instance of mutual repression between homologs of Iro-C and msh has been described, members of each family participate in establishing borders by repression with other genes in the spinal cord, the brain and between rhombomeres. Clearly, both genes are used frequently to subdivide territories and establish alternative differentiation pathways at each side of the border that separates them (Villa-Cuesta, 2005).

Throughout the third instar, msh is expressed at relatively low levels in the posterior notum territory. Here, removal of msh most often results in impaired growth of the scutellum, absence of the scutellum/scutum suture and alterations of the bristle pattern. Interestingly, the lateral/anterior notum macrochaetae are often missing, even though they arise in a region apparently devoid of msh expression. This suggests that either msh is expressed there at very low but functional levels, or that the suppression of macrochaetae results from non-autonomous effects of the absence of Msh from neighboring territories. It should be noted that non-autonomous macrochaetae suppression is also associated with Iro-C clones that cause notum to hinge transformations. This has suggested that modification of the putative signaling across the notum/hinge boundary interferes with macrochaetae patterning at the notum. It is possible that the msh clones might also interfere, as indicated above, with signaling from this border. If so, the presence of clusters of sc expression at the anterior lateral notum within large msh clones suggest that this interference might occur at a stage later than the emergence of the proneural clusters (Villa-Cuesta, 2005).

The absence of msh function does not modify the expression of Iro-C in the lateral notum or the characteristic patterns of expression of eyg and hth, genes that are high in the hierarchy that control notum development. But it removes the scutum/scutellar suture and promotes development of extra bristles in the dorsocentral and scutellar regions. Again, these are phenotypes suggestive of an interference with the late patterning and differentiation of these structures (Villa-Cuesta, 2005).

Defective proventriculus specifies the ocellar region in the Drosophila head: Dve represses ara

A pair of the Drosophila eye-antennal disc gives rise to four distinct organs (eyes, antennae, maxillary palps, and ocelli) and surrounding head cuticle. Developmental processes of this imaginal disc provide an excellent model system to study the mechanism of regional specification and subsequent organogenesis. The dorsal head capsule (vertex) of adult Drosophila is divided into three morphologically distinct subdomains: ocellar, frons, and orbital. The homeobox gene orthodenticle (otd) is required for head vertex development, and mutations that reduce or abolish otd expression in the vertex primordium lead to ocelliless flies. The homeodomain-containing transcriptional repressor Engrailed (En) is also involved in ocellar specification, and the En expression is completely lost in otd mutants. However, the molecular mechanism of ocellar specification remains elusive. This study provides evidence that the homeobox gene defective proventriculus (dve) is a downstream effector of Otd, and also that the repressor activity of Dve is required for en activation through a relief-of-repression mechanism. Furthermore, the Dve activity is involved in repression of the frons identity in an incoherent feedforward loop of Otd and Dve (Yorimitsu, 2011).

This study presents evidence that Dve is a new member involved in ocellar specification and acts as a downstream effector of Otd. The results also revealed a complicated pathway of transcriptional regulators, Otd-Dve-Ara-Ci-En, for ocellar specification (Yorimitsu, 2011).

Transcription networks contain a small set of recurring regulation patterns called network motifs. A feedforward loop (FFL) consists of three genes, two input transcription factors and a target gene, and their regulatory interactions generate eight possible structures of feedforward loop (FFL). When a target gene is suppressed by a repressor 1 (Rep1), relief of this repression by another repressor 2 (Rep2) can induce the target gene expression. When Rep2 also acts as an activator of the target gene, this relief of repression mechanism is classified as a coherent type-4 feedforward loop (c-FFL). During vertex development, Ara is involved in hh repression, and the Dve-mediated ara repression is crucial for hh expression and subsequent ocellar specification. However, the cascade of dve-ara-hh seems to be a relief of repression rather than a cFFL, because Dve is not a direct activator of the hh gene. Furthermore, dve RNAi phenotypes were rescued in the ara mutant background, suggesting that a linear relief of repression mechanism is crucial for hh maintenance (Yorimitsu, 2011).

In photoreceptor R7, Dve acts as a key molecule in a cFFL. Dve (as a Rep1) represses rh3, and the transcription factor Spalt (Sal) (as a Rep2) represses dve and also activates rh3 in parallel to induce rh3 expression. Interestingly, Notch signaling is closely associated with the relief of Dve-mediated transcriptional repression in wing and leg disks. These regulatory networks may also be cFFLs in which Dve acts as a Rep1, although repressors involved in dve repression are not yet identified. In wing disks, expression of wg and ct are repressed by Dve, and Notch signaling represses dve to induce these genes at the dorso-ventral boundary. The Dve activity adjacent to the dorso-ventral boundary still represses wg to refine the source of morphogen. In leg disks, Dve represses expression of dAP-2, and Notch signaling represses dve to induce dAP-2 at the presumptive joint region. The Dve activity distal to the segment boundary still represses dAP-2 to prevent ectopic joint formation. Taken together, these results suggest that Dve plays a critical role as a Rep1 in cFFLs in different tissues. In the head vertex region, it is likely that the repressor activity of Dve is repressed in a cFFL to induce frons identity (Yorimitsu, 2011).

The homeodomain protein Otd is the most upstream transcription factor required for establishment of the head vertex. During second larval instar, Otd is ubiquitously expressed in the eye-antennal disk and it is gradually restricted in the vertex primordium until early third larval instar. Expression of an Otd-target gene, dve, is also detected in the same vertex region at early third larval instar. Otd is required for Dve expression, and the Otd-induced Dve is required for repression of frons identity through the Hh signaling pathway in the medial region. However, Otd is also required for the frons identity in both the medial and mediolateral regions (Yorimitsu, 2011).

This regulatory network is quite similar to the incoherent type-1 feedforward loop (iFFL) in photoreceptor R7. Otd-induced Dve is involved in rh3 repression, whereas Otd is also required for rh3 activation. iFFLs have been known to generate pulse-like dynamics and response acceleration if Rep1 does not completely represses its target gene expression. However, the repressor activity of Dve supersedes the Otd-dependent rh3 activation, resulting in complete rh3 repression in yR7. In pR7, Dve is repressed by Sal, resulting in rh3 expression through the Otd- and Sal-dependent rh3 activation. Thus, Dve serves as a common node that integrates the two loops, the Otd-Dve-Rh3 iFFL and the Sal-Dve-Rh3 cFFL (Yorimitsu, 2011).

In the head vertex region, Otd and Dve are expressed in a graded fashion along the mediolateral axis with highest concentration in the medial region. It is assumed that Otd determines the default state for frons development through restricting the source of morphogens Hh and Wg, and also that high level of Dve expression in the medial ocellar region represses the frons identity through an iFFL. It is likely that repression of dve by an unknown repressor X occurs in a cFFL and induces the frons identity in the mediolateral region (Yorimitsu, 2011).

Interlocked FFLs including Otd and Dve appear to be a common feature in the eye and the head vertex. However, other factors are not shared between two tissues. In R7, a default state is the Otd-dependent Rh3 activation, an acquired state is (1) Rh3 repression through the Otd-Dve iFFL and (2) Spineless-dependent Rh4 expression. In the vertex, a default state is Otd-dependent frons formation, an acquired state is (1) frons repression through the Otd-Dve iFFL and (2) Hh-dependent ocellar specification associated with En and Eya activation (Yorimitsu, 2011).

Both Otd and Dve are K50-type homeodomain transcription factors, and they bind to the rh3 promoter via canonical K50 binding sites (TAATCC). The Otd-Dve iFFL in the eye depends on direct binding activities to these K50 binding sites, but the iFFL in the vertex seems to be more complex. Although target genes for frons determination are not identified, the iFFL in the vertex includes some additional network motifs. For instance, in the downstream of Dve, Hh signaling is critically required for repression of the frons identity (Yorimitsu, 2011).

Since iFFLs also act as fold-change detection to normalize noise in inputs, interlocked FFLs of Dve-mediated transcriptional repression may contribute to robustness of gene expression by preventing aberrant activation. It is an intriguing possibility that, in wing and leg disks, Dve also serves as a common node that integrates the two loops as observed in the eye and the vertex. Further characterization of regulatory networks including Dve will clarify molecular mechanisms of cell specification (Yorimitsu, 2011).

Targets of Activity

Ara and Caup regulate the pattern elements (sensory organs and veins) in wing imaginal discs by spatially restricting the domains of expression for the proneural genes achaete and scute and the provein gene rhomboid. It is suggested that Ara alone is sufficient for ac-sc expression at the vein L3; conversely, Caup alone is competent to promote ac-sc expression in the notum and vein L3. (Gómez-Skarmeta, 1996a).

Expression of ac-sc at the presumptive vein L3 depends on enhancer sequences located 0.2-0.6 kb upstream of the scute transcriptional start. This enhancer also drives expression at the twin sensilla of the wing margin (TSM) proneural cluster, located on the proximal vein L1, a site of ara/caup expression. These sequences contain binding sites for IROC proteins. Two contiguous short stretches of DNA are revealed in a DNase1 protection assay. One of these contains the TAAT motif found in the consensus binding sites of many homeoproteins. A mutagenized enhancer fails to be expressed in both vein L3 and TSM territories (Gómez-Skarmeta, 1996a).

Expression of araucan and caupolican in the wing imaginal disc starts during the second larval instar at the presumptive notum region and is increased in two large areas of the presumptive lateral heminotum. Like ara and caup, mirror is expressed in the lateral notum region of the wing disc, but with a pattern different from that of ara/caup. Thus mirror may also participate in sensory organ formation in the lateral notum. In contrast, mirror is not expressed in the prospective veins L3 and L5 or in the allula. Thus, it should not contribute to the activation of ac-sc and rhomboid/veinless at these sites or to the development of these structures. Nevertheless, in some instances, mirror can substitute for ara/caup function (Gómez-Skarmeta, 1996a).

The functions of pannier during Drosophila embryogenesis

The pannier gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. Novel aspects of the regulation and function of pnr during embryogenesis are reported in this study. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but it has been found that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila (Herranz, 2001).

In early development, pnr is activated in response to dpp activity in a broad dorsal domain, which extends from parasegments 2/3 to the border between 13/14, although the borders are not strictly parasegmental. The control by dpp is consistent with the effect of brk mutations on early pnr expression. The original expression domain is substantially modified during embryogenesis. By germ band extension (stage 10) pnr activity is limited dorsally by the border between the epidermis and the amnioserosa, and laterally by the dorsal border of iro. It is not known which factor(s) is responsible for the loss of expression in the amnioserosa, although likely candidates are several genes specifically active in this region, such as Race, zen, hindsight or serpent. In addition, it is not known how the late expression is regulated at the lateral border. It is not achieved by iro, since the loss of the entire Iroquois complex does not affect pnr expression (Herranz, 2001).

The transformation of ventral and dorsolateral epidermis towards dorsomedial observed after ectopic pnr expression is also reflected in the activity of marker genes of the distinct regions. Characteristic genes of the ventral neuroectoderm such as BP102 for the CNS or buttonhead are suppressed. In addition, pnr is able to suppress iro activity, a property that, as in the adult cells, is important to keep the dorsomedial and dorsolateral domains separate during embryogenesis (Herranz, 2001).

The Pax-homeobox gene eyegone is involved in the subdivision of the thorax of Drosophila

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

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

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

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

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

Different classes of photoreceptors (PRs) allow animals to perceive various types of visual information. In the Drosophila eye, the outer PRs of each ommatidium are involved in motion detection while the inner PRs mediate color vision. In addition, flies use a specialized class of inner PRs in the 'dorsal rim area' of the eye (DRA) to detect the e-vector of polarized light, allowing them to exploit skylight polarization for orientation. Homothorax plays a critical role for DRA development: hth is expressed specifically in maturating inner PRs of the DRA and maintained through adulthood. homothorax is both necessary and sufficient for inner PRs to adopt the polarization-sensitive DRA fate instead of the color-sensitive default state. Loss of hth results in the transformation of the DRA into color-sensitive ommatidia, and misexpression of hth forces color-sensitive inner PRs to acquire the typical features of polarization-sensitive DRA cells. Homothorax increases rhabdomere size and uncouples R7-R8 communication to allow both cells to express the same opsin rather than different ones as required for color vision. Homothorax expression is induced by the Iroquois complex and the Wingless (Wg) pathway. However, crucial Wg pathway components are not required, suggesting that additional signals are involved (Wernet, 2003).

Although the IRO-C genes have been suggested to act only before the MF, the current experiments reveal that IRO-C genes are able to induce dorsal-specific morphological changes at later time points. Evidence has been found that members of the IRO-C complex indeed act as selector genes to specify the dorsal compartment of the developing eye. They fulfill at least two additional typical features proposed for such selector genes: persistence of expression and induction of transformations when misexpressed in the ventral compartment (Wernet, 2003).

caup persists at very low levels during pupal stages before returning to high levels in adults. One possible explanation for such transient downregulation could be that high levels of IRO-C genes are toxic for the developing PRs. Indeed, massive cell death is observed when ara, caup, or mirr are overexpressed under the control of a strong GMR-GAL4 driver. Weaker drivers expressed posterior to the MF, however, give rise to healthy PRs and a ventral rim area. Therefore, during early pupal stages, low levels of dorsally expressed IRO-C genes might restrict induction of Hth expression to the dorsal half of the rim. The results suggest that the IRO-C complex acts together with a factor induced by high levels of Wg signaling. Indeed, overexpression of both ArmS10 and ara posterior to the morphogenetic furrow induces Hth expression in inner PRs throughout the eye. Since loss of all three IRO-C genes does not result in a loss of the DRA, a fourth unknown factor might be partially redundant with the IRO-C genes, or alternatively the deficiency used to eliminate the three genes might bear residual activity (Wernet, 2003).

Araucan regulates decapentaplegic expression during Drosophila wing veins pupal development

The differentiation of veins in the Drosophila wing relies on localised expression of decapentaplegic in pro-vein territories during pupal development. The expression of dpp in the pupal veins requires the integrity of the shortvein region (shv), localised 5' to the coding region. It is likely that this DNA integrates positive and negative regulatory signals directing dpp transcription during pupal development. A minimal 0.9 kb fragment has been identified giving localised expression in the vein L5 and a 0.5 kb fragment giving expression in all longitudinal veins. Using a combination of in vivo expression of reporter genes regulated by shv sequences, in vitro binding assays, and sequence comparisons between the shv region of different Drosophila species, binding sites were found for the vein-specific transciption factors Araucan, Knirps and Ventral veinless, as well as binding sites for the Dpp pathway effectors Mad and Med. It is concluded that conserved vein-specific enhancers regulated by transcription factors expressed in individual veins collaborate with general vein and intervein regulators to establish and maintain the expression of dpp confined to the veins during pupal development (Sotillos, 2006).

The expression of dpp in the wing disc is restricted to a narrow stripe of anterior cells localised at the anterior/posterior compartment boundary. This expression is regulated by sequences localised 3′ to the dpp coding region, and the function of the gene at this stage is required for the growth and patterning of the wing. The expression of dpp is still detected at the A/P boundary during the 8 h of pupal development. Later, at 14 h APF novel domains of dpp expression appear corresponding to the developing wing veins. The function of dpp during pupal development requires the integrity of the shv region, which is localised 5′ to the dpp coding region. There are two different transcripts expressed during pupal development, transcripts dpp-RA and dpp-RC, whose promoters (P5 and P3, respectively) are separated by approximately 20 kb of DNA. This DNA includes the first exon of transcript dpp-RC and corresponds to the place where all dpps alleles map. Because the strength of dpps alleles correlates with their distance to the P3 promoter, it is likely that dpp function in pupal development is mediated mainly by transcript dpp-RC. This suggests that dpps mutations affect regulatory sequences necessary to drive dpp expression in presumptive vein territories during pupal development. This possibility was confirmed by analysing the expression of a 8.5 kb construct containing most of the shv region fused to the reporter gene lacZ (shv8.5lacZ). The expression of βGal in shv8.5lacZ is detected exclusively in the pupal veins, indicating that this region includes all dpp wing veins regulatory regions (Sotillos, 2006).

Several constructs were made using different sub-fragments from the original 8.5 kb dpps DNA to identify with more precision the sequences that regulate dpp expression during pupal development. These fragments were cloned in front of a dicistronic lacZGal4 reporter gene and the activity of these constructs was analysed by looking at the expression of βGal in pupal wings from transgenic flies. In addition, to amplify the signal of the dicistronic lacZGal4 gene, the expression was monitored of a reporter gene regulated by UAS sequences. This expression should reveal all places where the Gal4 protein is present. Several regulatory regions were detected that control dpp expression in the veins during pupal development. One regulatory sequence is localised in a 1.1 kb fragment localised 6.5 kb from P3, and drives high levels of expression in most pupal veins and low levels of expression in some interveins. Additional regulatory sequences that efficiently drive expression in most veins are localised in an adjacent 0.5 kb fragment, and further vein-specific regulatory sequences for L5 are localised in the 0.9 kb SalI/KpnI fragment (Sotillos, 2006).

The expression of dpp during embryogenesis is highly dynamic and several independent regulatory regions controlling embryonic dpp expression have already been identified. The shv constructs included in the 8.5 kb EcoRI fragment drive reporter expression during embryonic development from stage 12/13 mainly in three regions of the mesoderm: the oesophagus, gastric caeca and midgut. Regulatory regions controlling dpp expression in the oesophagus appear to be duplicated, because they are localised in the 2.7 kb EcoRI/SalI fragment and also in the 3 kb KpnI/KpnI fragment. Similarly, regions controlling dpp expression in the gastric caeca are also present in the two adjacent fragments 0.9 kb SalI/KpnI and 3 kb KpnI/KpnI. The regions driving reporter expression in the gut are localised in the 3′ end of the shv region (Sotillos, 2006).

To better understand the regulation of dpp expression during vein development, the interactions were analyzed between a 2.5 kb region including wing veins pupal enhancers and several proteins involved in the regulatory network controlling the formation of veins. For this purpose, the 2.5 kb region was subdivided into overlapping fragments of 250-300 bp used as probes to detect the binding of different transcription factors by Electrophoretic Mobility-Shift Assays (EMSAs). Both prepattern specific genes that control vein development, such as Ventral veinless (Vvl) and the Araucan protein (Ara), and transcription factors belonging to the Dpp pathway (Mad and Medea) were analyzed (Sotillos, 2006).

The Iroquois complex includes three genes, araucan (ara), caupolican (caup) and mirror, encoding highly related homeodomain-containing proteins. The genes ara and caup are expressed in the presumptive veins L3 and L5 and their activity is required for the formation of the distal region of these two veins. The activity of these genes is required during imaginal development to regulate the expression of rhomboid in the L3 and L5 veins, but it is not known whether they are also required during pupal development. The Ara homeodomain and the different probes included in the 2.5 kb SalI/SacII region were used, finding strong binding using S1, S5, S9 and S10 as probes. Only high amounts of Ara protein caused bandshift of the S2-4 and S6-8 probes. It was possible to displace these bindings using cold DNA, suggesting specific interactions between Ara and these DNA fragments. When the binding was competed with oligonucleotides included in the S9 and S10 probes, two binding regions were found in S9 and three in S10. The sequence a/t ACAnnTGT t/a has been recently defined as a binding site for the Iro-C component Mirror in random oligo-selection experiments. This sequence is specific for Mirror, although other members of the Iro-C, Ara or its homolog Irx4, also bind this sequence with a weaker affinity. Three sites were found with similar consensus sequence in the enhancer, located in the S1, S9 and S10 probes. However, in this case the palindrome is in opposite direction. Interestingly, the consensus identified included in S9 and S10 are in a highly conserved regions between D. melanogaster and D. pseudoscura and is present in oligonucleotides that compete efficiently in the binding assays, pointing to the relevance of these sequences to mediate Ara binding to the shv enhancer. The binding of Ara to the shv enhancer suggests that the Iro-C proteins participate in the development of the L3 and L5 veins acting as transcriptional activators to regulate positively the expression of dpp during pupal development (Sotillos, 2006).

Drosophila araucan and caupolican integrate intrinsic and signalling inputs for the acquisition by muscle progenitors of the lateral transverse fate

A central issue of myogenesis is the acquisition of identity by individual muscles. In Drosophila, at the time muscle progenitors are singled out, they already express unique combinations of muscle identity genes. This muscle code results from the integration of positional and temporal signalling inputs. This study identified, by means of loss-of-function and ectopic expression approaches, the Iroquois Complex homeobox genes araucan and caupolican as novel muscle identity genes that confer lateral transverse muscle identity. The acquisition of this fate requires that Araucan/Caupolican repress other muscle identity genes such as slouch and vestigial. In addition, Caupolican-dependent slouch expression depends on the activation state of the Ras/Mitogen Activated Protein Kinase cascade. This provides a comprehensive insight into the way Iroquois genes integrate in muscle progenitors, signalling inputs that modulate gene expression and protein activity (Carrasco-Rando, 2011).

The study of myogenesis in Drosophila has increased the understanding of how the mechanisms that underlie the acquisition of specific properties by individual muscles are integrated within the myogenic terminal differentiation pathway. Thus, the current hypothesis proposes that distinct combinations of regulatory inputs leads to the activation of specific sets of muscle identity genes in progenitors that regulate the expression of a battery of downstream target genes responsible for executing the different developmental programmes. However, the analysis of the specific role of individual muscle identity genes and of their hierarchical relationships is far from complete since the characterisation of direct targets for these transcriptional regulators is very scarce (Carrasco-Rando, 2011).

ara and caup, two members of the Iroquois complex, have been identified as novel type III muscle identity genes. The homeodomain-containing Ara and Caup proteins are necessary for the specification of the lateral transverse (LT) fate. ara/caup appear to be bona fide muscle identity genes. Indeed, similarly to the identity genes Kr and slou, absence of ara/caup does not interfere with the segregation of muscle progenitors or their terminal differentiation, but modifies the specific characteristics of LT1-4 muscles, which are transformed towards VA1, VA2, LL1 and LL1 sib fates. These transformations may be due in part to the up-regulation of slou and vg in the corresponding muscles. Thus, a recent report (Deng, 2010) shows that forced expression of vg in LT muscles induces changes in muscle attachments similar to the ones observed in LT1 in ara/caup mutant embryos. However, it should be stressed that although in ara/caup mutants LT muscles are lost in more than 95% of cases, they are not completely transformed into perfect duplicates of the newly acquired fates. For instance, while the specific LT marker lms is lost in 91% of cases, ectopic slou expression is detected in only 75% of cases. These partial transformations might be due to differences in the signalling inputs acting in the mesodermal region from where these muscles segregate. Unpublished data also showed that forced pan-mesodermal expression of ara/caup alter the fates of many muscles both in dorsal and in ventral regions without converting them into LT muscles (i.e., they do not ectopically express lms). Similarly, Kr and slou ectopic expression is not sufficient to implement a certain muscle fate. The failure to recreate a given muscle identity by adding just one of the relevant muscle identity proteins reveals the importance that cell context, that is, the specific combination of signalling inputs and gene regulators present in each cell, have in determining a specific muscle identity (Carrasco-Rando, 2011).

Analysis of the myogenic requirement of ara/caup has revealed several features about how these genes act to implement LT fates. Thus, although they are expressed in six developing embryonic muscles, only four of them, LT1-4, are miss-specified in the absence of Ara/Caup. The remaining two, DT1 and SBM, seem to develop correctly, according to morphological as well as molecular criteria. It is worth noting that the requirement for ara/caup genes in these six muscles correlates with the onset of their expression. Thus, in the affected LT1-4 muscles Ara/Caup can be first detected at the earliest step of muscle lineages, that is in the promuscular clusters. In contrast, in the unaffected muscles ara/caup start to be expressed later, in the DT1/DO3 progenitor and the SBM founder. This suggests that in muscle lineages ara/caup have to be expressed very early to repress slou and vg to implement the LT fate. Several data support this interpretation. For instance, the observation that ara/caup are co-expressed with slou in DT1, whereas they repress slou in LT3-4, may be related to the fact that slou expression precedes that of ara/caup in the DT1 lineage. Should this be so, one would expect that ectopic expression of ara using the early driver mef2-GAL4, would repress slou in DT1, as it actually does, whereas this repression is not evident using the late driver Con-GAL4. Furthermore, the hypothesis of the relevance of the timing of muscle identity gene expression for muscle fate specification might also apply to the case of slou, where a similar correlation between the strength of the loss-of-function slou phenotypes in specific muscles and the onset of slou expression has also been found (Carrasco-Rando, 2011).

It should be stressed that the generation of the LT code depends not only on the early presence of Ara/Caup on the promuscular clusters but also on the absence (or strong reduction) of DER/Ras activity at that precise developmental stage and location. There is a dynamic regulation of MAPK signalling in the lateral mesoderm. Caup-expressing muscles develop from DER-independent clusters whereas the duplicated muscles observed in ara/caup mutants derive from progenitors that segregate very near the LT progenitors, but originate in DER-dependent promuscular clusters that are specified slightly later in development. Furthermore it was observed both by in vivo and in cell culture that low MAPK activity is required for Caup-dependent slou repression. Therefore, the role of Ara/Caup in the implementation of LT fate is interpreted as follows. At mid stage 11 in the myogenic mesoderm, groups of mesodermal cells acquire myogenic competence as a result of interpreting a combinatorial signalling code that reflects their position along the main body axes, as well as the state of activation of different signalling pathways. Accordingly, these clusters initiate the expression of lethal of scute and a unique code of muscle identity genes, as has been shown in great detail for eve expression in the dorsal mesoderm. In the case of the dorso-lateral mesoderm this code includes ara/caup and Kr and implements the LT fate. Since the level of activation of the Ras/MAPK cascade is low in these clusters, Ara/Caup will behave as transcriptional repressors, preventing the activation of slou or vg in LT1-2 and LT3-4 clusters, which would be otherwise activated in this location. Thus, Ara/Caup implement the LT fate by repressing the execution of the alternative fates (Kr+, Slou+, Con+, Poxm+ and Kr+, Vg+) that would give rise to duplicates of PVA1/VA2 and PLL1/LL1sib, respectively, and by allowing a different identity gene code (Kr+, Caup+, Con+, lms+) that generates the LT fate (Carrasco-Rando, 2011).

Slightly later the Ras/MAPK pathway becomes active at the dorsolateral region. This changes the combinatorial signalling code and coincides with a change in the muscle identity genes expressed by the promuscular clusters that segregate from this position, which now accumulate Kr but not Ara/Caup. Progenitors born from them will express either slou or vg and give rise to VA1-2 and LL1/LL1sib fates, all DER-dependent (Carrasco-Rando, 2011).

The data suggested that Ara/Caup might act as repressors of slou in the Drosophila mesoderm. Therefore whether slou might be a direct target of Ara/Caup was investigated. An 'in silico' search of a previously reported slou cis-regulatory region identified two putative Iro binding sites (BS) at positions +129 (BS1) and -1642 (BS2), relative to the transcription start site, which match the consensus ACAN2-8TGT. This regulatory region was cloned in a Luciferase reporter vector and Luciferase activity was measured in Drosophila Schneider-2 (S2) cells transiently transfected with this construct and increasing amounts of HA-tagged Caup. Contrary to expectations, it was found that addition of Caup-HA increased the basal Luciferase activity driven by the slou regulatory region in a dose dependent manner, indicating that Caup acts as a transcriptional activator of slou under these conditions. The reported regulation of the chicken Irx2 factor by MAPK (that switches it from repressor to activator) could explain this result. Since Western Blot analysis of S2 lysates using an antibody against diphospho-extracellular-signal related kinase (dpErk) showed the MAPK pathway to be active in S2 cells, and experimental evidence has been obtained showing the presence of phosphorylated Caup in S2 cells with constitutively active MAPK pathway, it was hypothesized that the activation effect of Caup in S2 cells could be due to the Ras/MAPK cascade turning Caup from transcriptional repressor into activator. Indeed, the inhibition of the Ras/MAPK pathway by the PD98059 MAP-erk kinase-1 (MEK1) inhibitor induced a Caup-dose dependent decrease in Luciferase activity driven by the slou regulatory sequences. This result could not be attributed to a direct effect of the inhibitor over the slou promoter, since its addition did not modify the basal Luciferase activity of the construct (Carrasco-Rando, 2011).

Thus S2 cell experiments suggest a molecular mechanism by which the Ras/MAPK pathway modulates the transcriptional activity of Ara/Caup on slou. Low MAPK activity and direct binding of Caup to BS1 site of the slou gene would favour strong repression of slou. BS1 could be embedded in a silencer regulatory element or its binding to Caup may block transcription of the downstream located luciferase gene. On the contrary, Caup-dependent activation of slou would be dependent on MAPK signalling. It is hypothesized that MAPK-dependent Caup phosphorylation could modulate its interaction with different transcriptional co-factors or/and its binding site affinity (Carrasco-Rando, 2011).

Furthermore, in vivo evidence indicates a repressor function of presumably non-phosphorylated Caup on slou since forced activation of the Ras pathway allows co-expression of slou and caup. On the other hand, the ectopic expression of slou induced by caup-over-expression is suggestive of a possible activator function of phosphorylated Caup (Carrasco-Rando, 2011).

The role of IRO proteins in cell fate specification is conserved in both vertebrates and invertebrates. This study has shown that the interplay between MAPK signalling and IRO activity found in vertebrate neuroepithelium is also at work in Drosophila myogenesis. This study has identifed potential direct target of Ara/Caup, slou and has proposed vg as a candidate gene to be regulated by Ara/Caup. In both cases the genes subordinated to ara/caup encode transcription factors that might in turn regulate the expression of other genes, genes that must be repressed in LT muscles in order to acquire the LT fate. These results, therefore, provide insights into the way Ara/Caup control lateral muscle identity and on the role of signalling pathway inputs to modulate the activity of these transcription factors, with consequences in their downstream targets. It also highlights the importance that the specific combination of muscle identity genes, their hierarchical relationships and their temporal activation have in determining the identity of a given muscle cell, very alike to what is at work during the acquisition of neural fates (Carrasco-Rando, 2011).

Protein Interactions

The homeodomain proteins defined by human PBX1 and Drosophila Extradenticle are notable for forming heterodimers with other homeodomain proteins and increasing the target specificity of their partner(s). As Ara and Caup belong to this family of homeodomain proteins, it is likely that they too form heterodimers with one another and/or with other Hox proteins.

The homeodomain Iroquois proteins control cell cycle progression and regulate the size of developmental fields

During development, proper differentiation and final organ size rely on the control of territorial specification and cell proliferation. Although many regulators of these processes have been identified, how both are coordinated remains largely unknown. The homeodomain Iroquois/Irx proteins play a key, evolutionarily conserved, role in territorial specification. This study shows that in the imaginal discs, reduced function of Iroquois genes promotes cell proliferation by accelerating the G1 to S transition. Conversely, their increased expression causes cell-cycle arrest, down-regulating the activity of the Cyclin E/Cdk2 complex. Physical interaction of the Iroquois protein Caupolican with Cyclin E-containing protein complexes, through its IRO box and Cyclin-binding domains, underlies its activity in cell-cycle control. Thus, Drosophila Iroquois proteins are able to regulate cell-autonomously the growth of the territories they specify. Moreover, the study provides a molecular mechanism for a role of Iroquois/Irx genes as tumour suppressors (Barrios, 2015).

The identification of genes that control cell proliferation is paramount in developmental and cancer biology. The Iroquois proteins play multiple roles in regionalization and patterning during Drosophila development. This study shows that they are also involved in the control of cell proliferation and, interestingly for homeodomain-containing proteins, they appear to do so by a non-transcriptional mechanism. This novel function of Iro genes would help developmental fields to attain their correct size and, if altered by Iro downregulation, could be a critical step for tumour progression (Barrios, 2015).

iro hypomorphic and over-expression conditions were analysed; Iro proteins were found to negatively control the G1-S transition of the cell cycle. caup over-expression impaired the activity of CycE/Cdk2 complex, while simultaneously increasing the level of CycE protein. Still, CycE appears to be a limiting factor since its exogenous administration restores cell proliferation, while its reduction enhances it. The presence of Caup in CycE-containing protein complexes led to a proposal that this physical interaction inhibits CycE/Cdk2 activity thus slowing down cell proliferation. This hypothesis is supported by the observation that Caupcyc* and CaupIRObox* mutant proteins show both impaired ability to co-immunoprecipitate with CycE and to restrict cell cycle progression. Although not experimentally demonstrated, it is speculated that Caup may interact with CycE and Cdk2 containing complexes and inhibit their activity by preventing substrate recognition and/or stabilizing p21 binding. Further work is required to determine more precisely these molecular interactions. Since Caupcyc*IRObox* still retains some ability to repress cell proliferation, it is presumed that either the functionality of these domains was not completely abolished by the mutations generated or the existence of additional unidentified interacting sites (Barrios, 2015).

Although other homeobox proteins (and also some epigenetic regulators) have been shown to modulate the activity of cell cycle regulators by protein-protein interaction, many of them do it through transcriptional regulation. A transcriptional effect of Caup on cell cycle regulation can be ruled out since transcriptionally inactive CaupHD*1 and CaupHD*2 are still able to inhibit cell cycle progression (Barrios, 2015).

Iro proteins play redundant roles in several developmental contexts. The three of them are able to repress cell cycle progression when over-expressed and this effect is abrogated by co-expression of cycE. The presence of putative Cyclin binding motives and the high conservation of the IRObox in the Iro proteins leads to the proposal that Ara and Mirr may also physically interact with CycE containing complexes. Since it was found that the penetrance of the dorsal eye enlargement phenotype increases by reducing the overall amount of Iro proteins, it is suggested that they may act in a redundant manner to modulate CycE/Cdk2 activity. Alternatively, the three Iro proteins may be functioning in a stoichiometric complex, this explaining why depletion of only one of them causes eye enlargement (Barrios, 2015).

The current results suggest a novel role of Iro proteins as cell-autonomous regulators of the growth of the domains of the imaginal discs where they are expressed. Furthermore, the results fit to a current model that suggests that growth of territorial fields modulates the response of cells to morphogens. In the eye discs, the ability of Decapentaplegic (Dpp) to induce retina differentiation is counteracted by Wg emanating from the anterior-most region of the discs until the disc attains a size such that dpp expressing cells are beyond the range of action of Wg. Accordingly, it is suggested that the enhanced cell proliferation found in iro mutant discs, would enlarge the physical separation between Wg- and Dpp-expressing cells in the dorsal domain, thus increasing the efficiency of Dpp signalling and causing dorsal eye enlargement (Barrios, 2015).

In analogy with this model for eye disc development, specification of the wing driven by Wg in the distal part of the wing disc is counteracted by the Vein morphogen, which spreads from the most proximal part of the wing disc. In this scenario, reduction of the size of the distal wing disc by inhibition of cell proliferation prevents wing development (with the concomitant generation of a notum-like tissue), by facilitating the inhibition of Wg by Vein. Interestingly, Vein activates Iro gene expression in the notum region while Wg does so in the dorsal eye disc. Thus, it is proposed that Iro genes could provide a molecular mechanism that allow the ligands Vein (in the notum) and Wg (in the dorsal eye) to regulate the size of the morphogenetic field in which they operate (Barrios, 2015).

The results further suggest that a direct regulation of cell cycle progression by Iro/Irx proteins may be relevant for tumorigenesis. Thus, tumorous-like growth was observed in the eye imaginal discs when iro function was reduced in a sensitized genetic background (such as ey>Dl or ey>Dl eyeful flies). Conversely, the ability of caup over-expression was shown to counteract the overgrowth induced by Yki in imaginal discs, and that this is partially mediated by cycE/cdk2 inactivation. These data suggest a role of Iro genes as TSGs in Drosophila and agree with the association found between loss or reduced expression of members of Irx gene family and certain types of human cancer. Note however that the role of Iro/Irx genes in tumorigenesis may be cell type-dependent since in some cases they appear to act as oncogenes. Considering the presence of the IRO box and of putative Cyclin-binding domains in Irx proteins, it is hypothesized that some Irx mutations may contribute to cancer progression in vertebrates by increasing the activity of the CycE/Cdk2 complex and thus accelerating the G1-S transition, a key step frequently affected in cancer cells (Barrios, 2015).

araucan and caupolican: Biological Overview | Evolutionary Homologs | Developmental Biology | References

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