mirror expression is first detected at the cellular blastoderm stage. It appears as an anterior ventral patch at the site of the presumptive anterior midgut invagination. Shortly thereafter, mrr is expressed at the dorsal folds prior to their formation. mrr is expressed in a segmental pattern by the beginning of stage 10, and then later in delaminating neuroblasts. mrr is expressed adjacent and posterior to Engrailed in every segment. This places mrr expression at the anterior border of each segment. mrr is also expressed at this time in more dorsal groups of cells. As the embryo undergoes germ-band retraction, mrr expression is retained in the nerve cord and appears transiently in the proventriculus as it undergoes folding (McNeill, 1997).

See Chris Doe's Hyper-Neuroblast map site for information on the expression of mirror in specific neuroblasts. The following text is adapted from that site: mirror-lacZ (mir-lacZ) is a P-element insertion in the mirror gene (H. McNeill and M. Simon, pers. comm. to C. Doe). At stage 9, the anterior row of S1 NBs (1-1, 3-2, 2-5) are mir-lacZ+. At stage 10, the S2 NBs 1-2 and 2-2 begin to express. During stages 10-11, the NBs 6-1, 3-4, the glial precursor, the median NB, and all row 2 NBs express. All mir-lacZ+ NBs delaminate from mir-lacZ+ ectoderm. The NBs and the overlying ectoderm show reproducible differences in the levels of beta-gal protein, with the most medial NBs (1-1, 2- 2) and all row 2 NBs showing the highest level of expression. mir-lacZ is found in a subset of neurons and glia in the stage 16 nerve cord including aCC and pCC and the longitudinal glia. The expression in these and other cells suggests that mir-lacZ might be lineally maintained.

For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.

The insect brain is traditionally subdivided into the trito-, deuto- and protocerebrum. However, both the neuromeric status and the course of the borders between these regions are unclear. The Drosophila embryonic brain develops from the procephalic neurogenic region of the ectoderm, which gives rise to a bilaterally symmetrical array of about 100 neuronal precursor cells, called neuroblasts. Based on a detailed description of the spatiotemporal development of the entire population of embryonic brain neuroblasts, a comprehensive analysis was carried out of the expression of segment polarity genes (engrailed, wingless, hedgehog, gooseberry distal, mirror) and DV patterning genes (muscle segment homeobox, intermediate neuroblast defective, ventral nervous system defective) in the procephalic neuroectoderm and the neuroblast layer (until stage 11, when all neuroblasts are formed). The data provide new insight into the segmental organization of the procephalic neuroectodem and evolving brain. The expression patterns allow the drawing of clear demarcations between trito-, deuto- and protocerebrum at the level of identified neuroblasts. Furthermore, evidence is provided indicating that the protocerebrum (most anterior part of the brain) is composed of two neuromeres that belong to the ocular and labral segment, respectively. These protocerebral neuromeres are much more derived compared with the trito- and deuto-cerebrum. The labral neuromere is confined to the posterior segmental compartment. Finally, similarities in the expression of DV patterning genes between the Drosophila and vertebrate brains are discussed (Urbach, 2003).

In the trunk, mirror (mirr)-lacZ is expressed in segmental ectodermal stripes giving rise to mirr-lacZ-positive NBs of row 2 and several NBs that flank row 2 at stage 11. The pattern of mirr-lacZ expression in the procephalic neuroectoderm and brain NBs differs significantly from the trunk. No evidence is found of a segmental arrangement of mirr-lacZ expression in the procephalon. Interestingly, regarding the DV axis, mirr-lacZ is mainly limited to the ventral part of the procephalic neuroectodermal region (pNR) and corresponding NBs (as confirmed by mirr-lacZ/Vnd double staining, although there is a faint dorsal mirr-lacZ expression, in the region of the later invaginating optic lobe anlage, and is, at stage 9/10, roughly complementary to en, wg and gsb-d expression, the domains of which are mainly confined to intermediate and dorsal regions of the pNR. At stage 11, expression extends towards the dorsal part of the antennal neuroectoderm and is observed in all NBs of the ventral deutocerebrum, as well as in two tritocerebral (Tv5, Td8) and four ventral, protocerebral NBs (Pad1, Pcv1, Pcv2, Pcv3). Although expression is also found in the clypeolabrum, no mirr-lacZ-positive labral NBs were identified (Urbach, 2003).

The Iroquois Complex Is Required in the Dorsal Mesoderm to Ensure Normal Heart Development in Drosophila

Drosophila heart development is an invaluable system to study the orchestrated action of numerous factors that govern cardiogenesis. Cardiac progenitors arise within specific dorsal mesodermal regions that are under the influence of temporally coordinated actions of multiple signaling pathways. The Drosophila Iroquois complex (Iro-C) consists of the three homeobox transcription factors araucan (ara), caupolican (caup) and mirror (mirr). The Iro-C has been shown to be involved in tissue patterning leading to the differentiation of specific structures, such as the lateral notum and dorsal head structures and in establishing the dorsal-ventral border of the eye. A function for Iro-C in cardiogenesis has not been investigated yet. Loss of the whole Iroquois complex, as well as loss of either ara/caup or mirr only, affect heart development in Drosophila. The data indicate that the GATA factor Pannier requires the presence of Iro-C to function in cardiogenesis. A detailed expression pattern analysis of the members of the Iro-C revealed the presence of a possibly novel subpopulation of Even-skipped expressing pericardial cells and seven pairs of heart-associated cells that have not been described before. Taken together, this work introduces Iro-C as a new set of transcription factors that are required for normal development of the heart. As the members of the Iro-C may function, at least partly, as competence factors in the dorsal mesoderm, these results are fundamental for future studies aiming to decipher the regulatory interactions between factors that determine different cell fates in the dorsal mesoderm (Mirzoyan, 2013).

Tissue patterning requires the spatial and temporal coordinated action of signals providing instructive or permissive cues that result in the specification of different cell types and their subsequent differentiation into different lineages. This analyses of Iro-C deficient embryos demonstrate that ara/caup and mirr are required in the dorsal mesoderm for normal heart development. The heart phenotypes could be caused by alterations of the fine balance of the interactions between factors of the cardiac signaling network. In early stage Drosophila embryos the mesoderm is patterned along the anterior-posterior (AP) axis with cardiac and somatic mesodermal domains alternating with visceral mesodermal domains. The tin-positive mesoderm is specified as cardiac and somatic mesoderm under the influence of combined Dpp and Wg signaling. Subsequently, the cardiac and somatic mesodermal domains are further subdivided by the action of the Notch pathway and MAPK signaling activated by EGFR and FGFR. The Eve-expressing cell clusters that give rise to pericardial and DA1 somatic muscle cells, as well as the Doc expression pattern, distinguish the cardiac and somatic mesodermal domain from the visceral mesodermal domain. The early expression pattern of Ara/Caup and Mirr at stages 10/11 suggests a role for Iro-C in patterning the dorsal mesoderm along the AP axis. Consistent with their previously described functions in other developmental contexts, members of the Iro-C may integrate signaling inputs and interact with other transcription factors to specify different dorsal mesodermal derivatives. Activation of the Iro-C by the EGFR pathway is required for the specification of the notum. Mirr was shown to interpret EGFR signaling by eliciting a specific cellular response required for patterning the follicular epithelium. During Drosophila eye development, mirr expression can be regulated by Unpaired, a ligand that activates JAK/Stat signaling. In fact, the JAK/Stat signaling pathway has only recently been added to the signaling pathways that function in Drosophila cardiogenesis. In chromatin immunoprecipitation experiments caup was identified as a target of Stat92E, which is the sole transcriptional effector of the JAK/Stat signaling pathway in Drosophila. Interestingly, the increase of Odd-pericardial cells and the additional Tin-expressing cells that were the characteristic phenotypes in ara/caup (iroDFM1) and in mirr (mirre48) mutants are highly similar to the phenotypes in stat92E mutants described by Johnson (Johnson, 2011). Also, as described for stat92E mutants, cell adhesion defects were noticed in a number of embryos as determined by the distant location of some Tin-expressing cells from the forming heart tube. As for establishing a possible link between JAK/Stat and Iro-C in the dorsal mesoderm and specifically in cardiogenesis, it would be necessary to determine for example whether caup and mirr can rescue the heart phenotype of stat92E mutants. Also, it would be interesting to compare the expression of the other crucial heart marker genes, Tup, Doc and Pnr, in stat92E mutants at early stages to determine to what extent the phenotypes of embryos mutant for Iro-C and for JAK/Stat signaling are similar (Mirzoyan, 2013).

Members of the Iro-C were shown to be positively or negatively regulated by signaling pathways that play crucial roles in heart development. Conversely, the Iro-C factors can also regulate the activity of at least one of these pathways. Specifically, Ara/Caup, as well as Mirr were shown to regulate the expression of the glycosyltransferase fringe and as a consequence modulate Notch signaling activity in the eye. In the dorsal mesoderm, the lateral inhibitory function of Notch signaling establishes the proper number of heart and muscle progenitors. Given the fact that Iro-C can regulate Notch activity it may be that the loss of Iro-C leads to an imbalance of progenitor cell specification resulting in an abnormal number of heart cells. Further studies are required to decipher the molecular mechanism by which Iro-C could integrate diverse signaling inputs and thereby function in the specification and differentiation of the different dorsal mesodermal derivatives (Mirzoyan, 2013).

To determine whether Iro-C can be positioned into the early transcriptional network that determines a cardiac lineage, this study investigated the interdependency between crucial cardiac factors and Iro-C during cardiogenesis. Analyses of the expression of Ara/Caup and Mirr in tin346, Df(3L)DocA, pnrVX6 and tupisl-1 embryos demonstrated the dependency of Ara/Caup and Mirr on all four factors. The strongest loss of Ara/Caup and Mirr expression was observed in tin346 and Df(3L)DocA mutants, which clearly places tin and Doc upstream of Ara/Caup and Mirr. In tupisl-1 and in pnrVX6 mutant embryos, Ara/Caup and Mirr were strongly downregulated, however regarding Ara/Caup, some expression remained in segmental patches suggesting a different level of regulation. The currently available data indicates a positive and a negative regulatory effect of pnr on Iro-C. Whereas pnr restricts Iro-C expression in the dorsal ectoderm and in the wing disc, there is also evidence that pnr can positively regulate Iro-C in the wing disc. Whether Pnr activates or represses Iro-C appears to depend on the presence of U-shaped (Ush), a protein that modulates the transcriptional activity of Pnr. In the wing disc it was shown that an Iro-C-lacZ (IroRE2-lacZ) construct was activated in cells that contained Pnr but were devoid of Ush. The data demonstrate that in the dorsal mesoderm, the expression of Ara/Caup and Mirr depends on pnr. Additionally this analyses show that pnr expression is independent of Iro-C. This finding is intriguing with respect to the downregulation of Tup and Doc in Iro-C mutants. Pnr is required for the maintenance of Doc and for the initiation and/or maintenance of Tup. Since Iro-C mutants exhibit a reduction in Doc-positive cells despite the presence of pnr, members of the Iro-C appear to be required independently or in addition to pnr to maintain expression of Doc. This could be investigated by expressing ara, caup and/or mirr in the mesoderm of pnr mutants to determine whether these factors are able to restore Doc expression. Alternatively, it may be that Iro-C is required indirectly meaning that its main function is to provide a molecular context in which Pnr can be active. For example, it is known that Ush can bind to Pnr thereby inactivating Pnr function. It is conceivable that the absence of Iro-C affects the spatial expression of Ush. If, in the absence of Iro-C, the expression domain of Ush shifts into the Pnr expression domain, Ush could bind to Pnr and inactivate it in the region where Pnr is required to maintain the expression of Tup and Doc. Adding to the complexity of the interpretation of the observed phenotypes is the finding that the majority of embryos that are mutant for ara/caup or for mirr were characterized by supernumerary Tin-positive cells in the cardiac region by stage 11/12. This phenotype could still be observed at later stages when the heart tube forms. The additional Tin-positive cells are pericardial cells as determined by the expression of Prc around the Tin-expressing cells. Also, no increase was observed of Dmef2-positive myocardial cells. Hence, the data suggests a different level of regulation of Tin by the Iro-C. Similar to the findings of Johnson (2011), it may be that Iro-C is normally required to restrict Tin expression at an early stage. The regulation of Tin expression can be divided into four phases. The phenotype this study observed occurs when Tin expression becomes restricted to the myo- and pericardial cells in the cardiac region. In summary, the data adds Iro-C to tin, pnr, Doc and tup whose concerted actions establish the cardiac domains in the dorsal mesoderm. Further studies are required to re-evaluate the current understanding of the interactions between factors of the cardiac transcriptional network (Mirzoyan, 2013).

According to the expression pattern of Ara/Caup and Mirr it was possible to distinguish between an early and late role for these factors, the latter being a role in the differentiation of heart cells (Mirzoyan, 2013).

This analyses of the expression of Ara/Caup and Mirr during embryogenesis led to the identification of hitherto unknown heart-associated cells. Seven pairs of Ara/Caup and Mirr expressing cells and seven pairs of Mirr only expressing cells were detected that were located along the dorsal vessel. No co-expression was detected with any of the known pericardial cell markers. Because there are seven pairs of these cells segmentally arranged, it was tempting to speculate that these cells may function, for example, as additional attachment sites for the seven pairs of alary muscles. The alary muscles attach the heart to the dorsal epidermis and their extensions can be visualized by Prc. Due to the lack of markers little is known about the development of the alary muscles. Previous work demonstrated that the alary muscles attach to the dorsal vessel in the vicinity of the Svp pericardial cells and, in addition, more laterally to one of two distinct locations on the body wall. Maybe it is the Mirr-positive cells that identify the more lateral locations. Clearly, a detailed analysis is needed to identify the function of the Ara/Caup- and Mirr- as well as Mirr-expressing cells that are positioned along the heart tube and whose existence has now been revealed. Additionally, on each side at the anterior end of the dorsal vessel four pericardial cells were identified that co-express Ara/Caup and Eve. Their location at the anterior tip of the heart is intriguing. Further analysis is required to unambiguously determine whether these cells are, for example, the wing heart progenitor cells or the newly identified heart anchoring cells. It is also possible that they represent a yet undefined, novel subpopulation of pericardial cells. In any case, this finding suggests that Ara/Caup plays a role in the diversification of pericardial heart cell types. Future experiments aim to determine the developmental fate of these cells (Mirzoyan, 2013).

Taken together, this investigation of a role for Iro-C in heart development introduces ara/caup and mirr as additional components of the transcriptional network that acts in the dorsal mesoderm and as novel factors that function in the diversification of heart cell types (Mirzoyan, 2013).

The results show that the role of the Iro-C and its individual members, respectively, appears to be rather complex and awaits in-depth analyses. Nevertheless, this work raises important questions regarding the current understanding of interactions between the well-characterized transcription factors that will be addressed in future studies (Mirzoyan, 2013).

Larval and Adult

mirror is expressed in a complex pattern in larvae and adults. This pattern includes proteins of the wing, haltere, and genital imaginal discs, as well as the dorsal anterior follicle cells in the ovary. A role for mirror function in the development of these tissues is reflected by defects in these structures in mutant flies. These defects include held-out wings, missing thoracic bristles, crumpled halters, and the production of eggs lacking dorsal appendages and anterior chorion (McNeill, 1997). It is interesting to note that araucan and caupolican are, like mirror, expressed in dorsal regions of the eye (Gomez-Skarmeta and Modellel, personal communication to McNeill, 1997).

Soma-dependent modulations contribute to divergence of rhomboid expression during evolution of Drosophila eggshell morphology

Patterning of the respiratory dorsal appendages (DAs) on the Drosophila melanogaster eggshell is tightly regulated by epidermal growth factor receptor (EGFR) signaling. Variation in the DA number is observed among Drosophila species; D. melanogaster has two DAs and D. virilis has four. Diversification in the expression pattern of rhomboid (rho), which activates EGFR signaling in somatic follicle cells, could cause the evolutionary divergence of DA numbers. Here we identified a cis-regulatory element of D. virilis rho. A comparison with D. melanogaster rho enhancer and activity studies in homologous and heterologous species suggested that these rho enhancers did not functionally diverge significantly during the evolution of these species. Experiments using chimeric eggs composed of a D. virilis oocyte and D. melanogaster follicle cells showed the evolution of DA number was not attributable to germline Gurken (Grk) signaling, but to divergence in events downstream of Grk signaling affecting the rho enhancer activity in somatic follicle cells. A transcription factor, Mirror, which activates rho, could be one of these downstream factors. Thus, evolution of the trans-regulatory environment that controls rho expression in somatic follicle cells could be a major contributor to the evolutionary changes in DA number (Nakamura, 2007).

Mirr was identified as a candidate for the difference in the landscape of trans-regulatory factors between D. melanogaster and D. virilis. The distribution of the mirr transcript was significantly different between these species. mirr induces rho expression, and regulates N signaling by repressing fringe, probably thereby regulating rho. Although whether or not Mirr function is also involved in the regulation of rho transcription in D. virilis remains to be tested, it is conceivable that changes in the expression patterns of mirr may account, at least in part, for the divergence in the activation patterns of Dvir rho4.2 and Dmel rho2.2 in D. melanogaster and D. virilis (Nakamura, 2007).

Determination of EGFR signaling output by opposing gradients of BMP and JAK/STAT activity

A relatively small number of signaling pathways drive a wide range of developmental decisions, but how this versatility in signaling outcome is generated is not clear. In the Drosophila follicular epithelium, localized epidermal growth factor receptor (EGFR) activation induces distinct cell fates depending on its location. Posterior follicle cells respond to EGFR activity by expressing the T-box transcription factors Midline and H15, while anterior cells respond by expressing the homeodomain transcription factor Mirror. This study shows that the choice between these alternative outputs of EGFR signaling is regulated by antiparallel gradients of JAK/STAT and BMP pathway activity and that mutual repression between Midline/H15 and Mirror generates a bistable switch that toggles between alternative EGFR signaling outcomes. JAK/STAT and BMP pathway input is integrated through their joint and opposing regulation of both sides of this switch. By converting this positional information into a binary decision between EGFR signaling outcomes, this regulatory network ultimately allows the same ligand-receptor pair to establish both the anterior-posterior (AP) and dorsal-ventral (DV) axes of the issue (Fregoso Lomas, 2016).

This study shows that the choice between two alternative Grk/EGFR signaling outcomes in the follicular epithelium depends on positional input provided by Upd and Dpp. At the posterior, the presence of Upd allows Grk to induce Mid/H15 while, at the anterior, Grk together with Dpp positively regulates Mirr. In this context, Upd and Dpp serve to define the response to Grk/EGFR signaling, since they are not sufficient to induce Mid/H15 and Mirr, respectively, in the absence of Grk (Fregoso Lomas, 2016).

Mutual repression is demonstrated between Mid/H15 and Mirr that is proposed to generate a double-negative feedback circuit that toggles the system between anterior and posterior outcomes. Moreover, in addition to their mutual regulation, analysis of double-mutant clones reveals that Upd and Dpp each regulate both Mid/H15 and Mirr and, thus, each provides input to both sides of this circuit. Upd is required for the expression of Mid and H15 even in the absence of a functional mirr gene, demonstrating that Upd is required for Mid/H15 expression independent of its ability to repress Mirr. Similarly, Dpp signaling can repress Mid independently of its positive effect on Mirr. The choice of Grk/EGFR signaling outcome in this context thus depends not only on mutual repression between these alternate targets but also on their opposing regulation by Upd and Dpp (Fregoso Lomas, 2016).

It is proposed that these elements define a bistable network that controls the choice between two alternative outcomes of Grk/EGFR signaling. These outcomes are irreversible -- e.g., posterior EGFR signaling in later stages cannot induce Mirr unless Mid and H15 are absent - and mutually exclusive, and the factors described in this study include two key elements found in bistable networks: feedback and non-linearity. The feedback in this case is provided by the reciprocal repression between Mirr and Mid/H15, generating a double-negative feedback loop that reinforces the choice of signaling outcome. In addition, bistability requires non-linearity in the response of the circuit to its upstream regulators, which makes the switch more sensitive to graded inputs. It is proposed that, in the follicular epithelium, this is achieved by the joint opposing regulation of the feedback circuit by both Dpp and Upd; each activates one side of the switch while repressing the other, biasing the outcome in the same direction (Fregoso Lomas, 2016).

These alternative responses to Grk are separated in time, as the source of Grk moves from posterior to anterior during the course of development. An important element that determines the choice between them is the early pattern of Mirr expression. In early stages of oogenesis, Mirr is Grk independent and is restricted to the main body follicle cells, due to its repression in the terminal regions by Upd. These main-body follicle cells correspond to the future anterior region of the columnar epithelium, and it is proposed that this early expression of Mirr predisposes them to express Mirr instead of Mid/H15 when Grk adopts its final dorsal anterior localization. Such a role for the early phase of Mirr expression is also consistent with the DV asymmetry of the Mid expression domain; as Grk moves anteriorly, leaving the range of posterior Upd and entering this domain of early Mirr expression and Dpp pathway activity, only the peak dorsal levels of Grk are capable of inducing Mid (Fregoso Lomas, 2016).

These observations also provide an example of how antiparallel signaling gradients can be integrated during epithelial patterning. Tissue patterning by opposing morphogen gradients is observed in developmental contexts as diverse as the Drosophila blastoderm and vertebrate neural tube, where they engage downstream transcriptional networks whose dynamic properties generate reproducible gene expression boundaries. Mutual repression between downstream transcription factors helps define the position and boundaries of cell fate domains, but how the opposing gradients are integrated is not well understood. This study shows that, in the follicular epithelium, the opposing Upd and Dpp gradients are integrated at the level of the Mirr-Mid/H15 feedback circuit. This integration occurs not only at the level of the mutual repression between Mid/H15 and Mirr but also by the ability of each gradient to regulate both sides of this circuit (Fregoso Lomas, 2016).

Together, these elements define the framework of a regulatory network that integrates localized positional information to regulate a binary choice of EGFR signaling outcome. The results allow construction of a model that both accounts for how an individual cell responds to Grk/EGFR signaling and explains how these spatial inputs are integrated across the epithelium to generate a defined pattern of Mid/H15 and Mirr expression, ultimately defining the pattern of the eggshell. Mirr is required for the generation of the high- and low-Broad domains, and Mid/H15 expression is required to define the posterior limit of these domains. The ability of Dpp and Upd to influence the outcome of EGFR signaling allows a single signaling input, namely localized secretion of Grk by the oocyte, to generate multiple distinct outputs that are localized in space and time, thus establishing both the AP and DV polarity of the epithelium and generating a complex and reproducible pattern of cell fates (Fregoso Lomas, 2016).

Effects of Mutation or Deletion

mirror mutant cuticles are smaller than wild type and have sparse denticles. Often, anterior denticles are missing from each segment, and denticle rows 2 and 3 often are fused together. A small percentage of embryos also have fusions of adjacent segments, suggesting that mirror may also play a role in defining the border between segments (McNeill, 1997).

The mirrorP1 insertion, used initially to identify mirror gene, gives rise to smooth eyes, with no disruptions in the packing of the ommatidial facets. However, closer analysis reveals subtle patterning defects. In 20% of the mirrorP1 eyes, at least one ommatidium close to the equator is misspecified, having ventral chirality and polarity but residing in the dorsal field, or vice versa. In addition, mirrorP1 eyes have defects in the path of the equator across the eye. In wild-type eyes, the equator moves up and down one ommatidial width as it traverses the eye and rarely if ever moves more than two ommatidial widths at one time. In contrast, about 10% of mirrorP1 eyes display an irregular equator. These irregularities are either three consecutive steps in a single direction or a single step of three ommatidial widths. These defects in mirrorP1 flies suggest that mirror has a role in defining the equator (McNeill, 1997).

The Drosophila eye is patterned by a dorsal-ventral organizing center mechanistically similar to those in the fly wing and the vertebrate limb bud. Here it is shown how this organizing center in the eye is initiated -- the first event in retinal patterning. Early in development, the eye primordium is divided into dorsal and ventral compartments. The dorsally expressed homeodomain Iroquois genes are true selector genes for the dorsal compartment; their expression is regulated by Hedgehog and Wingless. The organizing center is then induced at the interface between the Iroquois-expressing and non-expressing cells at the eye midline. It was previously thought that the eye develops by a mechanism distinct from that operating in other imaginal discs, but this work establishes the importance of lineage compartments in the eye and thus supports their global role as fundamental units of patterning (Cavodeassi, 1999).

In the Drosophila compound eye, the hexagonal array of dorsal and ventral ommatidia is extremely accurate; only rarely the path of the equator deviates from the midline and never by more than one ommatidium. The path of the equator takes alternating dorsal and ventral steps, while the path of the DV midline is straight. The accuracy of the location of the equator may depend on the shape and position of the DV midline, which itself is defined at an earlier stage. The midline can be visualized by the expression of the transcription factor Mirr in the dorsal cells. The two other Mirr-related proteins, Ara and Caup, are also expressed in a dorsal-specific domain.The functions of the three IRO-C proteins in establishing the DV midline have been examined by generating clones of cells deficient for the whole complex (IRO-C clones); these clones were then compared with similarly induced mirr minus clones (Cavodeassi, 1999).

Dorsal lRO-C clones are frequently associated with extensive outgrowths of the eye (73 out of 90 clones examined), including both mutant and adjacent wild-type cells. A subset of these IRO-C clones develops a clearly independent eye, consisting of both IRO-C- and IRO-C+ cells. Sections through such mosaic eyes show that the border of IRO-C expression always defines an ectopic equator: wild-type ommatidia, located as far as 7 ommatidial rows away from the clonal border, are repolarized toward the new border of IRO-C expression. The ectopic equators and the independent eye fields are also revealed in eye discs stained with ommatidial cell markers. All the above described phenotypes of IRO-C clones are only observed when the clone abuts the disc margin; in addition, IRO-C clones in the dorsal head capsule cause autonomous transformations to ventral cuticle structures. Clones mutant in mirr alone affect ommatidial polarity, suggesting that Ara and/or Caup may partially compensate for the absence of Mirr in such clones. Ectopic borders of IRO-C expression can also be generated by targeted expression of mirr, ara or caup. In the ventral region of the eye disc, ara + or caup + ectopic borders, like mirr borders, reorganize DV polarity and promote formation of ectopic eye fields, albeit at a low frequency. Dorsally situated clones overexpressing ara or caup also induce ectopic eyes at the same low frequency, suggesting that confrontation of cells with different amounts of IRO-C proteins may be sufficient to generate an organizing border. In summary, the boundaries of IRO-C expression exert long range organizing activity and can promote the formation of an independent eye, consisting of both IRO-C + and IRO-C - cells. These boundaries are relatively straight, suggesting that IRO-C mutant cells do not intermix with neighboring IRO-C-expressing cells (Cavodeassi, 1999).

IRO-C mutant cells differentiate ommatidia normally but they form compact patches with smooth borders, as if mutant cells minimize their contact with surrounding cells. In contrast, their twin wild-type clones have wiggly borders. The smooth boundaries are probably caused by the confrontation of IRO-C-expressing and non-expressing cells. The ventral part of the eye disc lacks IRO-C expression and hence ventral IRO-C clones have wiggly contours. Moreover, IRO-C clones of dorsal origin (according to the position of their twin clones) locate in the ventral part of the disc. Such clones form straight boundaries with dorsal cells but wiggly boundaries with ventral cells. The failure of two populations to mix has been ascribed to an autonomous function of a selector gene in specifying a characteristic 'affinity' to the compartment cells where the gene is expressed; this causes maximization of contact among cells of the same compartment, while minimizing cellular contact between compartments. The properties of the IRO-C clones suggest that the homeodomain IRO-C proteins confer a dorsal-specific cell 'affinity' (Cavodeassi, 1999)

The formation of the DV midline has been postulated to appear de novo in an initially homogeneous eye field via a mechanism that involves gradients of secreted signaling molecules, like Wg, expressed at the disc margin. Accordingly, the position and shape of the eye midline are defined at the point of lowest concentration of a dorsal (Wg) and a ventral (unidentified) signal and prior to the subdivision of the disc into dorsal and ventral expression domains. One way these signals might enable the DV midline to become the organizer is by inducing the expression of IRO-C genes in the dorsal cells. According to the results presented in this study, affinity differences between dorsal (IRO-C +) cells and ventral (IRO-C -) cells may be the main mechanism responsible for maintaining the straight DV midline. To investigate how the two models are reconcilable, the expression of IRO-C and wg was examined at first/early second instar stages and their putative regulatory relationships were studied by clonal analysis. At late first/early second instar, IRO-C expression is already restricted to the dorsal half of the disc. A groove marking the limit of IRO-C expression, resembles that found along the presumptive DV boundary in the early third instar eye discs, as previously described. Differences in affinity between dorsal and ventral cells probably induce this groove because dorsal IRO-C clones are also transiently surrounded by a fold. At early second instar wg is expressed in the presumptive dorsal region of the eye disc. Later this expression evolves to dorsal and ventral anterior marginal domains. Expression of IRO-C was assayed in cells lacking dishevelled activity and therefore unable to transduce Wg signaling. Early and late induced dsh clones autonomously lack ara/caup expression, indicating that Wg is required continuously for IRO-C expression. This expression is normally downregulated in cells posterior to the morphogenetic furrow but it is maintained in dorsal-posterior clones of shaggy mutant cells, where the Wg pathway is constitutively active. However, activation of IRO-C in ventral sgg M1-1 clones is seen only occasionally in a subset of the mutant cells. Hence, it is concluded that Wg signaling is necessary but not sufficient to activate IRO-C expression (Cavodeassi, 1999).

Another factor required for IRO-C expression is Hh. Similar to wg, hh is expressed in a dorsally restricted domain at late first/early second larval instar. Regulation of IRO-C by the Hh pathway was assayed in clones of cells deficient for the Hh receptor complex formed by Smoothened (Smo) and Patched (Ptc). In ptc mutant cells, a situation equivalent to constitutive activation of the Hh pathway in the receiving cells, mirr-lacZ and ara/caup expression are ectopically activated within the mutant cells and in some wild-type adjacent cells. Late induced ptc clones (at 72-96 hours AEL) do not derepress mirr-lacZ. In smo clones, where Hh reception is blocked, ara/caup expression is absent in the center of the clone and strongly decreased in its periphery. This result, and the non-autonomous effect of ptc clones, suggest that a secreted signal, induced by Hh, rescues the loss of hh in the smo mutant cells. This factor could be Wg, as wg is derepressed in ptc clones in the anterior region of the eye disc (Cavodeassi, 1999).

Early generalized ectopic expression of hh dorsalizes the eye, severely reducing its size. Similar effects have been reported for early misexpression of wg. Together, these observations and the previous data support a model in which both Wg and Hh signaling organize DV patterning by directing IRO-C expression. However, Wg and Hh do not meet the complete requirement for the postulated gradient model: (1) their expression is already asymmetric in the early disc; (2) ubiquitous and high expression of Wg or Hh should prevent the formation of the straight DV boundary, but this is not the case (Cavodeassi, 1999).

Retinal differentiation is associated with the passage of the morphogenetic furrow, which normally begins at the intersection of the DV midline with the posterior margin. The site of furrow initiation is widely assumed to be specified at the lowest point of concentration of Wg activity. IRO-C expression borders can non-autonomously recruit mutant and wild-type cells to form an eye provided they are located close to the disc margin. Thus, IRO-C may induce retinal differentiation through the local repression of wg at the disc margin, causing a sink of the Wg gradient. Therefore the expression of wg was examined in relation to IRO-C borders. At late second/early third instar, wg is expressed around the anterior dorsal and ventral disc margins. wg expression is not impeded within marginal IRO-C mutant clones. Thus, it is concluded that an IRO-C expression border is sufficient to promote furrow initiation, even in the presence of wg. In the wild-type eye, this process requires the positive action of Decapentaplegic (Dpp) and Hh. dpp is expressed around the posterior and posterior-lateral disc margin, symmetrically across the IRO-C expression border. Similarly, dpp-lacZ is activated straddling the border of an IRO-C clone abutting the disc margin. hh is expressed along the dorsolateral and posterior margin of the early third instar eye disc. Just before morphogenetic furrow initiation, hh expression increases at the posteriormost region, which is the site where the IRO-C border intersects with the disc margin. This modulation of hh expression was investigated in eye discs where the IRO-C border has been eliminated (by generalized expression of ara using the ey-GAL4 driver). hh-lacZ expression initiates normally, but its levels fail to increase at the posteriormost domain. At mid/late third instar, hh-lacZ expression is completely eliminated from the posterior disc margin, a loss not due to generalized cell death, since wg expression around the posterior margin is not impeded in the mutant late third instar discs. Nor is the failure to maintain hh expression a consequence of the absence of ommatidial differentiation, because hh-lacZ posterior expression is not eliminated in atonal mutant eye discs, where eye neurogenesis fails to initiate. Thus, an IRO-C expression border is needed to maintain and upregulate hh expression at the posteriormost margin, which is necessary for furrow initiation (Cavodeassi, 1999).

These analyses demonstrate that an IRO-C border is essential and instructive for growth, DV polarity, and initiation of eye morphogenesis at both sides of the border. Nevertheless, the IRO-C is only expressed at the dorsal half of the eye disc and encodes transcription factors. Consequently, their non-autonomous effects should be mediated through a signaling pathway with long-range activity. It has been proposed that fringe acts downstream of the IRO-C in the formation of the DV organizer. Consistently, dorsal IRO-C mutant cells exhibit autonomous derepression of fng expression. Thus, eye patterning requires a dorsal expression of IRO-C that establishes a fng expression border. This leads to the localized activation of Notch along the DV midline. Accordingly, the artificial elimination of the fng expression border or the block of Notch activation produces a loss-of-eye phenotype equivalent to that caused by misexpression of caup. This effect on eye development is likely caused by the failure to maintain hh. Here, the Fng/Notch pathway has been shown to act downstream of the IRO-C border (Cavodeassi, 1999).

The teashirt (tsh) gene has dorso-ventral (DV) asymmetric functions in Drosophila eye development: promoting eye development in dorsal and suppressing eye development in ventral regions by Wingless mediated Homothorax (HTH) induction. A search was carried out for DV spatial cues required by tsh for its asymmetric functions. The dorsal Iroquois-Complex (Iro-C) genes and Delta (Dl) are required and sufficient for the tsh dorsal functions. The ventral Serrate (Ser), but not fringe (fng) or Lobe (L), is required and sufficient for the tsh ventral function. It is proposed that DV asymmetric function of tsh represents a novel tier of DV pattern regulation, which takes place after the spatial expression patterns of early DV patterning genes are established in the eye (Singh, 2004).

The three genes of the Iro-C (ara, caup and mirr) are expressed in the dorsal domain of the eye disc and are functionally redundant. Misexpression of ara (driven by bi-GAL4 and abbreviated as bi>ara) results in eye suppression on both dorsal and ventral margins. However, coexpression of tsh and ara in bi>tsh+ara results in overall enlargement of the eye. Clonal induction of ara (abbreviated as Act>ara) and coexpression of tsh and ara (Act>tsh+ara) gives the same results. Thus, ara provides the dorsal cue for tsh to induce eye enlargement on both margins (Singh, 2004).

The requirement for ara was further confirmed by misexpressing tsh (bi>tsh) in Df(3L) iroDFM3/+ background. This deficiency uncovers ara, caup and the promoter region of mirr. In this background, bi>tsh suppresses eye development in both ventral and dorsal. Thus, when the Iro-C dosage is reduced, the dorsal function of tsh can be reversed to its ventral function. These results suggest that the dorsal function of tsh is dependent on the Iro-C genes (Singh, 2004).

Dl is expressed preferentially in the dorsal eye. Misexpression of Dl anterior to morphogenetic furrow in the hairy domain (hairy>Dl) accelerates photoreceptor differentiation but does not result in eye enlargement. bi>Dl (bifid-Gal4, UAS Delta) does not affect eye size. However, coexpression of tsh with Dl (bi>tsh+Dl) results in eye enlargements on both dorsal and ventral margins. Act>tsh+Dl clones in both dorsal- and ventral-eye also causes enlargements. These results suggest that Dl can provide the dorsal cue for tsh function (Singh, 2004).

Dl function was blocked by a dominant-negative form of DL, DLDN. bi>DlDN causes reduction of eye on both dorsal and ventral margins whereas coexpression of tsh and DlDN (bi>tsh+DlDN) further enhances this phenotype. A dorsal Act>tsh+DlDN clone suppresses eye development. Act>tsh+DlDN clones also non-autonomously suppress eye development, a phenotype also seen in Act>DlDN clones. These phenotypes suggest that Dl is also required for the dorsal function of tsh in eye. In the absence of Dl, tsh exerts its ventral function in dorsal eye (Singh, 2004).

Ser is preferentially enriched in the ventral eye until late second instar of larval development. Misexpression of Ser (bi>Ser) does not suppress eye development whereas coexpression of tsh+Ser(bi>tsh+Ser) suppresses eye development on both dorsal and ventral margins. Despite the suppression of photoreceptor differentiation, bi>tsh+Ser eye disc shows overall enlargement. The adult eyes were also enlarged and folded despite the suppression of photoreceptors differentiation on the dorsal and ventral margins. These results suggest that Ser can provide the ventral cue for the eye suppression function of tsh but does not affect its early function in promoting growth (Singh, 2004).

The dominant-negative form of Ser, SerDN was used to block Ser function. In bi>SerDN, the eyes are suppressed on both dorsal and ventral margins. This phenotype is partially blocked in bi>tsh+SerDN eye. Similar results were observed in Act>tsh+SerDN clones. Thus, tsh requires Ser for its ventral function (Singh, 2004).

These results show that tsh requires several early DV eye patterning genes for its dorsal and ventral specific functions in the eye. The requirement for these DV patterning genes is specific, because not all the DV patterning genes have similar effects. Eye suppression by tsh is prevented in the dorsal eye region. This function requires the normal dosage of both Iro-C and Dl genes, because the reduction of either Iro-C or Dl allows tsh to suppress eye development even in the dorsal eye. However, when ectopically expressed in the ventral eye, either Iro-C genes or Dl can block the ventral function of tsh, suggesting that the two genes may play similar roles (Singh, 2004).

The genes involved in early DV eye patterning can be categorized in two classes: (1) genes that are preferentially expressed in dorsal (e.g., Iro-C, Dl) or ventral (e.g. Ser, fng) and (2) genes that are uniformly expressed but function only in one domain (e.g., L). It is proposed that tsh comprises a new class of genes, which is expressed symmetrically but perform asymmetric functions in dorsal and ventral eye (Singh, 2004).

Although tsh is expressed ubiquitously in the early eye disc, the DV asymmetric functions of tsh can be uncovered only after the expression of early DV patterning genes is established. These results suggest that early expression of tsh may be responsible for its growth function only, whereas for the DV asymmetric functions the expression of early DV patterning genes is a prerequisite. Therefore, TSH function in eye represents a new tier of DV pattern regulation, which functions in interpreting the DV spatial cues in eyes. It would thus be interesting to identify other members of this class. Interestingly, two orthologs of tsh have been identified in mouse but their function in eye is not yet known. Since there is evolutionary conservation in patterns of gene expression and functions, it would be interesting to look for the role of tsh during eye development in higher organisms (Singh, 2004).

Wnt4 regulates the dorsoventral specificity of retinal projections in the Drosophila melanogaster visual system: iroquois may attenuate the competence of frizzled2 to respond to Wnt4

In Drosophila, the axons of retinal photoreceptor cells extend to the first optic ganglion, the lamina, forming a topographic representation. DWnt4, a secreted protein of the Wnt family, is the ventral cue for the lamina. In DWnt4 mutants, ventral retinal axons misproject to the dorsal lamina. DWnt4 is normally expressed in the ventral half of the developing lamina and DWnt4 protein is detected along ventral retinal axons. Dfrizzled2 and dishevelled, respectively, encode a receptor and a signaling molecule required for Wnt signaling. Mutations in both genes caused DWnt4-like defects, and both genes are autonomously required in the retina, suggesting a direct role of DWnt4 in retinal axon guidance. In contrast, iroquois homeobox genes are the dorsal cues for the retina. Dorsal axons accumulate DWnt4 and misproject to the ventral lamina in iroquois mutants; the phenotype is suppressed in iroquois:Dfrizzled2 double mutants, suggesting that iroquois may attenuate the competence of Dfrizzled2 to respond to DWnt4 (Sato, 2005).

The spatial order of projecting neurons is preserved in the spatial order of their targets to establish the topographic maps in the nervous system. In the visual system, precise topographic mapping of photoreceptor neurons to their targets in the brain, termed retinotopic mapping, is necessary for the correct interpretation of visual information received in the retina. The Drosophila visual system includes the retina, the compound eye and the optic lobe, which is the visual center of the brain and is connected to the eye via the optic stalk. Each of the approximately 750 ommatidial units in the retina consists of eight unique types of photoreceptor neurons called R cells (R1-R8). During larval development, R cells sequentially differentiate behind the morphogenetic furrow, progressing in a posterior-to-anterior order in the third larval instar retina, and send their axons through the optic stalk to the most distal part of the optic lobe, the lamina. R1-R6 axons terminate in the lamina layer, whereas R7 and R8 axons project through the lamina to terminate in the medulla layer. Although all the retinal axons pass through the narrow optic stalk, they distribute evenly and project to their correct targets along the anteroposterior and the dorsoventral axes. Thus, R cell axon connections between the retina and the lamina (or the medulla) are precisely retinotopic in the adult. Similarly, R axon connections established during the third instar are anatomically retinotopic (Sato, 2005 and references therein).

Like the retina, the lamina must also be patterned along the dorsoventral axis so that retinal axons can project precisely to their targets. It is assumed that selector genes and genes encoding guidance molecules are asymmetrically expressed along the dorsoventral axis in the developing lamina and it was found that DWnt4, one of seven D. melanogaster Wnt family genes, is specifically expressed in the ventral half of the developing lamina during the third larval instar (Sato, 2005).

The present study investigated the involvement of DWnt4 in D. melanogaster retinotopic mapping along the dorsoventral axis. DWnt4 is normally expressed in the ventral half of the developing lamina and DWnt4 protein has been detected on the surface of ventral retinal axons. In DWnt4 mutant backgrounds, ventral axons misproject to the dorsal lamina. Conversely, ventral axons are redirected by an ectopic source of DWnt4, suggesting that DWnt4 is a ventral cue for retinal axon projections in the lamina. Furthermore, ventral axon projections are regulated by noncanonical Wnt signaling in R cells, which is most likely under the control of DWnt4. These genetic data also suggest the involvement of JNK signaling in this process. Finally, iro may attenuate the competence of Dfz2 in dorsal axons to respond to DWnt4, since dorsal-to-ventral misroutings in iro clones are significantly suppressed in iro:Dfz2 double mutant clones (Sato, 2005).

As a first step to investigating retinotopic mapping in D. melanogaster, focus was placed on the iroquois (iro) complex genes, three related homeobox genes that act as selector genes for the dorsal retina. To test if iro regulates retinotopic mapping, cells homozygous for an iro deficiency were generated and labeled with green fluorescent protein (GFP) to visualize axons using the MARCM system. Dorsal mutant R axons were occasionally observed projecting to the ventral lamina. A similar phenotype was observed by generating large iro clones using ey-flp. Both outer- and inner-photoreceptor axons visualized with ro-lacZ and ato-myc were affected by iro. Thus, iro genes seem to function as dorsal cues for the retina (Sato, 2005).

Although the planar cell polarity (PCP) pathway is categorized as a noncanonical Wnt pathway transduced by the Fz receptor, no PCP defects were observed in DWnt4 and Dfz2 mutant retinae. In addition, the retinotopic phenotype was not observed in fz null mutant backgrounds. These results suggest that DWnt4 regulates R axon projections via a noncanonical Wnt signaling distinct from the PCP pathway. wingless (wg) is involved in the specification of the dorsal retina through the activation of iro expression. The distinct chiral forms of ommatidia in the dorsal and ventral retina reflect the dorsoventral specification of the retina and the PCP signaling. The normal iro expression and the normal ommatidial chirality suggest that axonal misroutings occur independently of the retinal dorsoventral specification in DWnt4 and Dfz2 backgrounds. Since dsh is required for PCP signaling and the specification of the dorsal retina, ommatidial chirality was disorganized and dorsal iro expression was eliminated in dsh retinae. However, the expression of Serrate (Ser), which is specific to the ventral retina in wild-type backgrounds, was not affected, suggesting that the ventral cell fate is correctly specified in dsh homozygotes. Additionally, UAS-dsh and UAS-dshDEP expression under the control of GMR-Gal4 did not affect the dorsoventral specification of the retina as visualized by iro and Ser expression. Note that GMR-Gal4 is expressed behind the morphogenetic furrow well after the dorsoventral specification at earlier stages. The data shown above suggest that dsh also regulates R axon projections independently of the dorsoventral patterning of the retina (Sato, 2005).

JNK signaling is known to act downstream of the noncanonical Wnt pathway in many developmental contexts. The involvement of JNK signaling was examined by expressing puckered (puc), which encodes a JNK phosphatase, and a dominant-negative form of JNK encoded by basket (bsk) to block JNK signaling in the retina. Defects were observed only rarely, and it was next asked whether genetic interactions exist between hemipterous (hep) encoding a JNK kinase and DWnt4 or Dfz2. In a strong hep mutant background, or in DWnt4, DWnt4 or Dfz2 heterozygous backgrounds, little or no ventral-to-dorsal misrouting was observed. However, a reduction in the dosage of DWnt4 or Dfz2 in the hep background resulted in a marked increase in the ventral-to-dorsal phenotype. These findings provide some support for the idea that JNK signaling is involved in the DWnt4/Dfz2 pathway in retinal axon guidance. Since iro expression and ommatidial chirality were normal in retinae expressing the dominant-negative form of bsk and in hep hemizygotes in combination with DWnt4/+ and Dfz2/+, the misrouting of ventral axons observed in the brain mutant for JNK signaling appears to be caused by a failure in axon guidance and independent of the dorsoventral cell specification or PCP signaling in the retina. Note that mutations in JNK pathway components alone have no PCP phenotype (Sato, 2005).

iro is thought to be the dorsal cue in the retina. The dorsal axons project to the ventral lamina in the absence of iro, perhaps because dorsal axons are attracted by ventral cues in the lamina, such as DWnt4. When iro mutant clones were generated under the control of ey-flp, dorsal axons projected to the ventral lamina in 32.4% of them, and ectopic accumulations were observed of DWnt4 on the surface of the dorsal axons misprojecting ventrally. Since Dfz2 was expressed in the dorsal axons, iro may attenuate the competence of Dfz2 in the dorsal axons to respond to DWnt4. If this is the case, simultaneous removal of Dfz2 in iro clones should suppress the iro phenotype, which was indeed observed. In iro:Dfz2 double mutant clones, 'dorsal-to-ventral' misroutings were observed in 3.5% of the cases, and the class III phenotype was no longer observed. Instead, abnormal bundles of dorsal axons were found in iro:Dfz2 clones. This might be because iro:Dfz2 axons do not respond to either dorsal or ventral cues in the lamina. Indeed, no DWnt4 accumulation was found in those abnormal bundles found in iro:Dfz2 clones. The absence of iro expression in differentiated R cells behind the morphogenetic furrow suggests indirect modulation of Dfz2-dependent Wnt signaling by iro (Sato, 2005).

It was hypothesized that three events are required for retinotopic mapping along the dorsoventral axis in D. melanogaster: (1) dorsoventral identity is specified by selector genes expressed in the retina; (2) dorsoventral identity is specified by selector genes in the lamina; (3) guidance molecules recruit R axons to their correct targets in the lamina. The results nicely fit the hypothesis. iro expressed in the dorsal retina specifies the dorsal axon identity, and DWnt4 expressed in the ventral lamina recruits ventral axon projections. The restricted expression of DWnt4 to the ventral lamina suggests there could be unidentified dorsoventral selectors in the lamina (Sato, 2005).

Prepatterning the Drosophila notum: the three genes of the iroquois complex play intrinsically distinct roles

The Drosophila thorax exhibits 11 pairs of large sensory organs (macrochaetes) identified by their unique position. Remarkably precise, this pattern provides an excellent model system to study the genetic basis of pattern formation. In imaginal wing discs, the achaete-scute proneural genes are expressed in clusters of cells that prefigure the positions of each macrochaete. The activities of prepatterning genes provide positional cues controlling this expression pattern. The three homeobox genes clustered in the iroquois complex (araucan, caupolican and mirror) are such prepattern genes. mirror is generally characterized as performing functions predominantly different from the other iroquois genes. Conversely, araucan and caupolican are described in previous studies as performing redundant functions in most if not all processes in which they are involved. The question of the specific role of each iroquois gene in the prepattern of the notum was addressed, and it was clearly demonstrated that they are intrinsically different in their contribution to this process: caupolican and mirror, but not araucan, are required for the neural patterning of the lateral notum. However, when caupolican and/or mirror expression is reduced, araucan loss of function has an effect on thoracic bristles development. Moreover, the overexpression of araucan is able to rescue caupolican loss of function. It is concluded that, although retaining some common functionalities, the Drosophila iroquois genes are in the process of diversification. In addition, caupolican and mirror are required for stripe expression and, therefore, to specify the muscular attachment sites prepattern. Thus, caupolican and mirror may act as common prepattern genes for all structures in the lateral notum (Ikmi, 2008).

In earlier works, the iro-C genes functions were mainly assessed from studies of mutations affecting solely mirr, of deficiencies deleting the whole complex or of rearrangements susceptible to affect several genes, and on misexpression experiments. In order to unravel the respective roles of ara, caup and mirr and to analyze their possible functional redundancy in the neural prepatterning of the notum, this study has combined the analysis of LoF mutations of these genes with a functional replacement approach (Ikmi, 2008).

mirr appears to be required for the formation of four out of the seven lateral macrochaetes (PS, pSA, aPA and pPA): their loss is elicited by LoF alleles of mirr and by a dominant allele (mirrG8. The proneural clusters as well as the SOPs corresponding to the macrochaetes unaffected by mirr mutations may reside outside (aNP) or inside (pNP) the mirr domain of expression. Therefore the requirement or dispensability of mirr for the patterning of the bristles in the lateral notum appear to depend partly but not only on its domain of expression. All the phenotypes observed in this study were elicited by mild perturbations of the mirr function, compatible with viability of adults. Therefore, a role of mirr in the patterning of the other lateral macrochaetes cannot be completely excluded (Ikmi, 2008).

In the prospective lateral notum, ara and caup appear to have mostly identical domains of expression, which, at least, enclose all the SOPs for lateral macrochaetes. These authors have suggested that there is a functional redundancy of these two genes on the grounds of their similar protein-coding sequences and patterns of expression, and this is generally admitted. However no evidence of this has been reported to date. Notably no LoF mutations affecting only one of these two genes have been described previously. This study characterized mutations abolishing (or drastically reducing) caup expression without an appreciable effect on ara and mirr (e.g. iro1 or caupG3) and null (or strong hypomorph) mutations of ara that do not affect significantly caup or mirr (e.g. TSI or araG4) (Ikmi, 2008).

Six transposable element (TE) insertions were characterized in caup, and it was shown that they behave like an allelic serie of caup hypomorphic mutations: flies homozygous for these insertions are viable and display phenotypes ranging from wt or the loss of only a few macrochaetes to the loss or a strong frequency decrease of all the seven lateral macrochaetes (strong Iroquois phenotype). These phenotypes are aggravated when these TE insertions are heterozygous over an iro-C deficiency and even more over the iro1 rearrangement, which has a breakpoint in the first caup intron. Heterozygous iro1/Df-BSI L3 larvae display no caup expression (or an extremely low level) although ara and mirr levels are similar to the control conditions. Similarly, the strongest P allele of caup (caupG3) causes a drastic reduction or an absence of caup expression without affecting notably ara and mirr, neither in expression level nor in expression domain in the notum. Therefore, it can be assumed that caup is required for the patterning of all lateral macrochaetes and, presumably, for the direct or indirect activation of sc expression in the lateral notum (Ikmi, 2008).

Besides inactivating caup expression, the P[Gal4] insertions in the caup gene reproduce its expression pattern. By combining such a Gal4 driver with an UAS-caup transgene, it was shown that caup overexpression in its normal domain of expression is able to rescue the caup LoF phenotype to an almost wt situation. The frequency of occurrence of all the seven lateral macrochaetes is either normal or at least elevated as compared to the mutant situation. Taken together, loss and gain of function approaches demonstrate that caup is required for the patterning of all the lateral macrochaetes of the notum (Ikmi, 2008).

The same approach was applied to study the loss of function of ara, and the effects were analyzed of five insertions and an inversion (TSI) in the ara gene. In araG4/TSI and TSI/Df-BSI L3 larvae, ara expression is absent or strongly reduced while caup and mirr expression are not significantly affected as compared to the control situations. These larvae die as pupae, showing the requirement of ara for viability. The very rare adult escapers do not present any bristle defects. The same is true for the four other ara hypomorphic mutants studied. In summary, the sole LoF of ara is never associated with a loss of macrochaetes. Consequently, conversely to caup, ara is dispensable for the patterning of all the lateral bristles of the notum (Ikmi, 2008).

In conclusion, the Iro proteins are different for their contribution to the neural patterning of the lateral notum. Only Caup and Mirr are required for the patterning of the lateral macrochaetes and therefore to control the activation of expression of ac and sc in the corresponding proneural clusters (Ikmi, 2008).

When it is strongly overexpressed in the caup domain of expression, Ara, although not required, is sufficient to rescue the lack of lateral macrochaetes phenotype elicited by caup LoF. Therefore, unexpectedly considering the LoF results, when overexpressed Ara can carry out the function of Caup in the neural patterning of the notum. This property may play a biological role, as can be seen in the exacerbation of the phenotypic defects elicited by ara LoF in sensitized conditions (reduction of caup and/or mirr function). Although to a much less extent, Mirr is also able to perform in part the Caup function, as seen from the limited rescue of PS, aNP and aSA macrochaetae by mirr overexpression in a caup LoF background. This functional difference between Ara and Mirr correlates well to their sequence divergence with Caup. A possible explanation for the difference observed between the LoF of ara and caup is that Ara, in physiological conditions, has a lower activity than Caup to promote proneural genes expression. The increase of the amount of the Ara protein to non-physiological levels probably increases the expression of proneural genes sufficiently to compensate for the loss of Caup activity. This is in good agreement with the findings that Drosophila Iro proteins bind in vitro the same sequence but differ in their strength of binding to this site. Consequently, owing essentially to the proteins intrinsic properties, the Iro transcription factors do not regulate in vivo the same target genes (Ikmi, 2008).

It has been suggested that a common network of prepattern genes regulates all structures of the notum. Indeed, in the prospective medial notum, pnr is required for both ac-sc and sr expression, which respectively specify the development of bristles and tendon precursors. Preliminary observations indicate that pnr also regulates pigment patterns in medial notum (Ikmi, 2008).

In the wing discs of larvae mutant for caup or for mirr, two (b and d) out of the three domains of sr expression located in the prospective lateral notum are missing. Furthermore, a mirr mutant partially affects sr expression in the two remaining domains (a and c). In addition, adult flies lacking either caup or mirr display falling wings, suggesting an abnormal attachment of indirect flight muscles. Therefore, caup and mirr activate sr expression and in consequence participate to the specification of lateral flight muscle attachment sites. It had been shown that Iro proteins can form homodimers and heterodimers that bind in vitro the same palindromic site called Iro Binding Site (IBS; Bilioni, 2005). Thus, it is possible that the activity of a Caup-Mirr heterodimer controls the regulation of sr expression in regions (b) and (d). The sr expression pattern is normal in the wing discs of larvae lacking the ara function. Hence ara is not required for the expression of sr in the prospective notum. Nonetheless, a reduction of ara function, as seen in hypomorphic mutants or escapers to the lethality, leads to heldout wings. This phenotype is often observed in flies carrying mutations that affect direct flight muscles. It is thus possible that ara regulates the specification of direct flight muscle attachment sites. In summary, here again, the iro-C genes products are not functionally equivalent in their contribution to the muscular attachment sites prepattern (Ikmi, 2008).

Although there are no strong evidences that iro-C regulates pigment patterns, preliminary reports suggest that this is the case. From all these data, the iro-C genes caup and mirr appear as common prepattern genes for the specification of all the structures in the lateral notum, similarly to pnr in the medial notum (Ikmi, 2008).

In addition, pnr and iro-C domains partially overlap each other at the virtual border between the medial and the lateral notum. The DC macrochaetes and the 'a' sr domain can be affected in caup and mirr mutants. These structures are dependant on the pnr function. Therefore, both pnr and the iro-C appear to prepattern a region of the notum, at the intersection of their expression domains, which could correspond to the medial-lateral band of wg expression overlapping these domains (Ikmi, 2008).

mirr LoF leads to embryonic lethality. Although the targets of ara are yet to be identified, ara LoF causes pupal lethality, whereas flies lacking caup function are viable and exhibit developmental defects. Thus, ara, caup and mirr play distinct biological functions during development. These different roles can be attributed in part to differences in expression pattern. For instance, mirr is the first iro-C gene that is detected during embryogenesis and it is the only iro-C gene that is expressed and plays a role in oogenesis. However, this diversity in expression domains cannot account for all the observed functional differences. The overexpression of these three proteins in the same domains (here with the same caup-Gal4 driver) have different consequences: while overexpression of caup and ara to more than 10 times the normal level is compatible with viability, the overexpression of mirr to the same level is lethal. When expressed at levels compatible with viability, mirr is unable to rescue the caup LoF while ara is. Thus, the differences in the iro-C genes roles cannot be only attributed to differences in their patterns of expression but rather should also be due to differences in their coding sequences (Ikmi, 2008).

In mammals, there are two clusters of three iroquois genes (IrxA and IrxB). Expression patterns, LoF and misexpression studies reveal a similar situation where the Irx genes may have redundant or non-redundant roles, depending on the gene and/or on the developmental process (Ikmi, 2008).

The roles of the Drosophila iro-C genes have been documented in numerous other developmental processes, for instance: eye dorsal-ventral patterning, wing veins patterning, wing-body wall boundary, dorsal-ventral axis formation in oogenesis. This study has put together the tools and conditions allowing to address the question of the specific roles and/or of the functional redundancy of the iro-C gene in these other developmental processes (Ikmi, 2008).

The long-term fates for duplicated genes include inactivation, maintenance and diversification. Maintenance of duplicated developmental genes can be the result of selective pressure exerted through dosage requirements and/or contribution to the genetic and developmental robustness necessary to reproducibly elaborate correct patterning of diverse territories. Alternatively, the subfunctionalization model proposes that, after a duplication of genes, each copy may sustain deleterious mutations in different structural and/or regulatory elements. Eventually, the ancestral functions are partitioned between the copies that are both retained (Ikmi, 2008).

An interesting example is the ac-sc complex. Similarly to ara and caup, ac and sc arose from the most recent duplication event in this complex. It has been shown that ac, but not sc, is dispensable for the development of the sensory bristles. Two hypotheses have been proposed for the reason why evolution has retained ac: first, an as yet undiscovered function; second (the favored hypothesis), a contribution to genetic robustness. However the situations of the ac-sc and iro complexes are not identical: no phenotypic consequence of the ac LoF in has been found in an otherwise wt background while this study observed ara LoF cause lethality. The reasons why the three Drosophila iro-C genes are maintained appear thus different and may proceed from the two (non-exclusive) hypotheses mentioned above. mirror has clearly evolved to perform different functions from ara and caup. This can be seen in expression pattern as well as in LoF phenotypes and in the functional properties of the protein. This study has provided evidences for the functional divergence between ara and caup: first, the LoF of ara is lethal while the LoF of caup is not; second, caup is required for the neural and the muscular prepatterning of the prospective notum, while ara is dispensable. Additionally, subtle differences exist in their expression pattern in the L3 wing disc. Other differences have yet to be characterized more precisely at other stages and in other tissues and their role investigated. The three iro-C genes appear in the process of diversification and subfunctionalization, both in their expression domains and in the functions of their encoded proteins. In addition, the partial ability of ara to perform caup functions, at least in the neural patterning of the notum, may contribute to buffer this patterning against intrinsic and extrinsic perturbations. It could thus be retained by a selective pressure at work on fly populations surviving in the wild (Ikmi, 2008).


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mirror: Biological Overview | Transcriptional Regulation | Targets of Activity | Developmental Biology | Effects of Mutation

date revised:  10 April 2010 

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