araucan and caupolican

DEVELOPMENTAL BIOLOGY

Embryonic

Throughout development, ara and caup evince very similar patterns of expression. Embryonic expression of these genes begins at stage 11 in the lateral epidermis; later expression occurs in the proventriculus and in several parts of the procephalon (Gómez-Skarmeta, 1996a).

Larval

In larvae, ara and caup are expressed in strongly in imaginal discs and weakly in the ventral ganglion of the brain. In the late second larval instar, expression starts at the presumptive notum region of the wing imaginal disc; during the third instar it is strongly increased in two large areas of the presumptive lateral heminotum. From the mid-third instar, expression occurs at the presumptive distal tegulat, the dorsal redius, proximal vein L1, veins L3 and L5, the allula, and the pleura (Gómez-Skarmeta, 1996a).

The Bar homeobox genes function as latitudinal prepattern genes in the developing Drosophila notum. In Drosophila notum, the expression of achaete-scute proneural genes and bristle formation have been shown to be regulated by putative prepattern genes expressed longitudinally. The two Bar locus genes may belong to a different class of prepattern genes expressed latitudinally: it is suggested that the developing notum consists of checker-square- like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate the formation of microchaetae within the region of BarH1/BarH2 expression through activating achaete-scute. Presutural macrochaetae formation also requires Bar gene activity. Bar gene expression is restricted in dorsal and posterior regions by Decapentaplegic signaling, while the ventral limit of the expression domain of Bar genes is determined by wingless, whose expression is under the control of Decapentaplegic signaling (Sato, 1999).

The Drosophila notum is considered genetically divided into several longitudinal, side by side, domains whose boundaries are determined by pannier, wingless and iroquois expression (listed respectively from medial to lateral). To further clarify relative locations of pnr, wg and iro expression areas, third-instar larval and pupal future notum were stained with various combinations of molecular markers. In larval and pupal future notum, pnr-Gal4 is expressed medially and iro-lacZ laterally. pnr-Gal4 and iro-lacZ domains partially overlap one another, and wg-lacZ (or Wg) expression is noted in the pnr-iro overlapping region and its immediate neighbors. Bar homeobox genes may belong to an additional class of notal subdivision genes. Staining for BarH1 indicates that BarH1 is expressed latitudinally (anterior vs. posterior) in the anterior-most region of future notum and postnotum. BarH1 expression begins at early to mid third instar. Anti-Ac antibody staining and neur-lacZ expression indicates PS macrochaetae are situated in the vicinity of posterior-ventral corners of the anterior BarH1 expression domain. BarH1 and BarH2 are referred to as Bar collectively and the anterior portion of the prescutum or its precursor expressing Bar is referred to as Bar prescutum. The Bar expression domain overlaps that of pnr, wg and iro. Bar expression similar to that in wing discs is observed in haltere discs (Sato, 1999).

It is concluded that a checker-board-like subdivision of future notum is regulated by putative prepattern gene expression. Future notum may be divided into square subdomains in a checker-board-like manner, each with its own unique combinations of prepattern gene expression. Putative prepattern genes, iro and pnr, form longitudinal domains. Bar homeobox genes form the anterior-most domain. This is the first demonstration of the presence of latitudinal, front to back, prepattern genes in the notum. Bristle formation in each subdomain may be positively regulated by a region-specific combination of prepattern genes. Consistent with this, microchaetae formation in the anterolateral prescutum (the lateral Bar prescutum), where Bar and iro are coexpressed, requires the concerted action of Bar and iro (Sato, 1999).

tailup, a LIM-HD gene, and Iro-C cooperate in Drosophila dorsal mesothorax specification

The LIM-HD gene tailup has been categorised as a prepattern gene that antagonises the formation of sensory bristles on the notum of Drosophila by downregulating the expression of the proneural achaete-scute genes. tup has an earlier function in the development of the imaginal wing disc; namely, the specification of the notum territory. Absence of tup function causes cells of this anlage to upregulate different wing-hinge genes and to lose expression of some notum genes. Consistently, these cells differentiate hinge structures or modified notum cuticle. The LIM-HD co-factors Chip and Sequence-specific single-stranded DNA-binding protein (Ssdp) are also necessary for notum specification. This suggests that Tup acts in this process in a complex with Chip and Ssdp. Overexpression of tup, together with araucan, a `pronotum' gene of the iroquois complex (Iro-C), synergistically reinforces the weak capacity of either gene, when overexpressed singly, to induce ectopic notum-like development. Whereas the Iro-C genes are activated in the notum anlage by EGFR signalling, tup is positively regulated by Dpp signalling. These data support a model in which the EGFR and Dpp signalling pathways, with their respective downstream Iro-C and tup genes, converge and cooperate to commit cells to the notum developmental fate (de Navascues, 2007).

Tup has been categorised as a prepattern factor that controls the expression of the proneural achaete-scute genes in the third instar wing disc. This study shows that tup functions earlier in the development of the dorsal mesothorax. Loss of tup causes a range of phenotypes, which taken together indicate interference with the assignment of cells to form notum. Thus, depending on the time of induction of the clones and their location multiple effects are observed; the formation of notum-like cuticle with altered cell-cell adhesion properties, the generation of ectopic wing-hinge structures including tegulae, sclerites or sensilla typical of the proximal wing, or even the loss of the entire heminotum. Consistent with these adult phenotypes, in third instar wing discs tup mutant cells can upregulate genes typically expressed at high levels in the wing-hinge territory of the disc, such as zfh2, msh, sal and the lacZ insertion line l(2)09261. Concomitantly, notum-expressed genes such as eyg, ush and pnr are generally repressed, although in some cases tup cells may abnormally express notum and hinge genes together. These data indicate that notum tup cells undergo transformation towards either an altered notum fate or a hinge fate. Moreover, the activation of hinge markers in wild-type cells surrounding some tup clones might reflect the presence of ectopic notum/hinge borders, which are known to promote non-autonomous effects (de Navascues, 2007).

Unequivocal notum-to-hinge transformations are consistently observed in clones induced during the first larval instar. In later-induced clones, this phenotype becomes less manifest and the modified notum cuticle phenotype becomes prevalent. Accordingly, the upregulation of hinge marker genes and the converse downregulation of notum genes in the notum territory are most consistently observed in first instar-induced clones. This suggests that the requirement for the 'pronotum' function of tup progressively decreases as development advances. Lesions associated with tup clones can appear anywhere within the notum, although each particular phenotype shows a degree of topographic specificity. Interestingly, the activation of hinge genes and the repression of notum genes are best shown in early-induced clones located in the presumptive medial notum. Probably, these clones, which are normally large, do not yield adult structures, since the expected large regions of mutant cuticle have not been recovered. The clones might give rise to flies lacking part or most of a heminotum. The dynamic expression pattern of tup fits well with the spatial distribution of these phenotypes and the early requirement for tup function for the development of the notum. Indeed, tup is expressed very early in the wing disc, when it has less than 100 cells, and the expression occurs within the region that will form the notum. It is concluded that, similar to other LIM-HD factors such as Ap and the vertebrate Tup homologue Isl1, Tup is required for the proper specification of not only cell types, but also developing territories (de Navascues, 2007).

Tup is known to bind the co-factor Chip. Since, in dorsal compartment specification, Chip functions in a 2Ap-2Chip-2Sspd hexamer, it was asked whether a similar 2Tup-2Chip-2Sspd complex might mediate Tup function in notum specification. The results support this interpretation. The loss of either Chip or Ssdp upregulates hinge genes (zfh2, msh), represses a notum marker (eyg), and induces cuticular defects similar to those associated with tup clones. Moreover, an excess of Chip would be expected to titrate Tup and/or Ssdp in incomplete complexes and mimic the loss-of-function phenotype of notum-to-hinge transformation, as was experimentally observed (de Navascues, 2007).

By contrast, during the later process of sensory organ formation, Tup appears to act by sequestering both Chip and Pnr, thus preventing activation of the proneural genes achaete-scute. This negative function of Tup does not seem relevant for notum specification, where both Tup and Chip work as positive effectors. Moreover, the Tup homeodomain is dispensable for titrating Chip and Pnr, but this is not the case for its 'pronotum' function. Interestingly, a missense mutation within the LIM-interacting interacting domain of Chip (Chip^E) severely reduces its ability to interact with Tup and suppresses the negative regulation by Tup of bristle formation. However, homozygous Chip^E flies have no defects in notum specification. This suggests that a residual interaction between Chip^E and Tup might persist, as additionally suggested by the suppression of the extra bristles present in Chip^E individuals by UAS-tup overexpression. A weak interaction between Tup and Chip, which might only permit the formation of low levels of hexameric complex, might still allow proper notum specification. This suggestion agrees with the fact that tup^d03613, a strong hypomorphic allele (as substantiated by its embryonic lethality over the null tup^ex4, allows proper notum formation in homozygosis (de Navascues, 2007).

Similarly to tup, Iro-C also has a 'pronotum' function. However, their roles are not entirely equivalent. Anywhere within the notum territory, loss of Iro-C during first or second instar induces a clear switch to hinge fate. By contrast, loss of tup causes an assortment of different combinations of derepressed hinge genes and repressed notum genes. Moreover, many tup clones induced during the second larval instar, and even some induced in the first, can develop recognisable notum cuticle. Thus, it is proposed that tup reinforces/stabilises the commitment of cells to develop as notum, a commitment imposed mainly by Iro-C. This reinforcement or stabilisation might be most necessary in the proximal part of the disc, where expression of ara/caup ceases after the second instar, but that of tup persists. This might account for the derepression of hinge genes being most manifest in this region. Depending on the location and time of Tup deprival, its loss may be inconsequential or lead to a partial or even a complete loss of notum commitment. Such diversity of consequences led to an exploration of whether tup might act on target genes by affecting chromatin remodelling. However, no genetic interactions have been found with Polycomb (Pc, Scr+Pcl+esc) or trithorax (trx, osa, brm, Trl, lawc) group genes (de Navascues, 2007).

In contrast to the absolute requirement for Iro-C for notum specification, overexpression of UAS-ara can impose a notum fate only on the wing anlage, and only when provided early in the development of the disc. An extra notum with mirror-image disposition versus the extant notum is generated at the expense of the wing, a phenotype identical to that resulting from early deprivation of Wg function. Since UAS-ara overexpression can interfere with wg expression, Wg deprival probably explains the formation of the extra notum. Thus, by itself, overexpression of UASara probably lacks a genuine potential for imposing the notum fate. Similar notum duplications arise upon early and strong overexpression of UAS-tup (MD638, dpp-Gal4 and ptc-Gal4 drivers) and, again, they probably result from inhibition of Wg activity. Consistent with this interpretation, weaker and later expression of either UAS-tup or UAS-ara (C765 driver) has little or no capacity to promote notum fate. However, when coexpressed, these transgenes are effective in imposing the notum fate and this should not be attributed to Wg depletion. Indeed, the transformation consists of an expansion of the notum tissue, rather than a notum duplication. Moreover, as detected by the onset of the ectopic expression of notum markers (eyg, DC-lacZ), the transformation occurs in late third instar discs (J.deN., unpublished) that have a nearly wild-type morphology and a distinguishable wing pouch. This indicates that these markers are activated in territories previously specified as wing, hinge or pleura, and subsequently forced to acquire notum identity. Moreover, overexpression of the Wg pathway antagonists UAS-Axin or UAS-dTCF^DN (dTCF or pan with the same driver failed to transform wing towards notum. Finally, the activation of eyg and the formation of notum tissue in the sternopleurite, a derivative of the leg disc, also attest to the capacity of tup plus ara to commit cells to develop as notum (de Navascues, 2007).

It is well established that signalling by the EGFR pathway is essential for notum development. Its inhibition prevents activation of Iro-C and the growth of the notum territory. By contrast, Dpp negatively regulates Iro-C and restricts its domain of expression at both its distal and proximal borders. The data indicate a novel function of Dpp in notum development; namely, the activation or maintenance of tup expression in second and third instar discs. In the notum region of the early disc, Dpp signalling occurs at low levels, but the results suggest that these are sufficient for activating tup. Expression of tup is largely independent on EGFR signalling. Thus, EGFR and Dpp signalling seem to cooperate in specifying notum identity to the cells of the proximal part of the disc by activating their respective 'pronotum' downstream genes, Iro-C and tup (de Navascues, 2007).

Apposition of iroquois expressing and non-expressing cells leads to cell sorting and fold formation in the Drosophila imaginal wing disc

The organization of the different tissues of an animal requires mechanisms that regulate cell-cell adhesion to promote and maintain the physical separation of adjacent cell populations. In the Drosophila imaginal wing disc the iroquois homeobox genes are expressed in the notum anlage and contribute to the specification of notum identity. These genes are not expressed in the adjacent wing hinge territory. These territories are separated by an approximately straight boundary that in the mature disc is associated with an epithelial fold. The mechanism by which these two cell populations are kept separate is unclear. This study shows that the Iro-C genes participate in keeping the notum and wing cell populations separate. Indeed, within the notum anlage, cells not expressing Iro-C tend to join together and sort out from their Iro-C expressing neighbours. Apposition of Iro-C expressing and non-expressing cells induces invagination and apico-basal shortening of the Iro-C cells. This effect probably underlies formation of the fold that separates the notum and wing hinge territories. In addition, cells overexpressing UAS-ara or UAS-caup contact one another and become organized in a network of thin strings that surrounds and isolates large groups of non-overexpressing cells. The strings appear to exert a pulling force along their longitudinal axis. In is concluded that apposition of cells expressing and non-expressing the Iro-C, as it occurs in the notum-wing hinge border of the Drosophila wing disc, influences cell behaviour, leading to cell sorting, and cellular invagination and apical-basal shortening. These effects probably account for keeping the prospective notum and wing hinge cell populations separate and underlie epithelial fold formation. Cells that overexpress a member of the Iro-C and that confront non-expressing cells establish contacts between themselves and become organized in a network of thin strings. This is a complex and unique phenotype that might be important for the generation of a straight notum-wing hinge border (Villa-Cuesta, 2007; full text of article).

Several observations support the possibility that arrangements of the cells in strings is an active process: (1) in young third instar discs bridges composed of a few cells arranged in chains that interconnect different overexpressing clones are quite abundant, while they are quite rare in control clones; (2) very long thin strings spanning many dozens of cell diameters can be observed in discs harbouring few clones; (3) pairs of clones born in the A and P compartments and whose main masses of cells are well separated from the compartment boundary can display interconnecting strings; (4) during the growth of the wing dics the cells of a clone normally remain together and usually separated from those of other clones; (5) control clones in the wing pouch grow mainly in a direction roughly perpendicular to the prospective wing margin while strings between overexpressing clones take any possible direction. If indeed cells from different clones actively search for one another and establish contacts, the mechanisms involved are unknown. Other observations pertaining to clones overexpressing UAS-ara, like the distortion of the D/V boundary by clones contacting it, the stretching of the cells in a direction parallel to the plane of the string, and the relative roundness of the domains of wild-type cells surrounded by strings, suggest that the threads of overexpressing cells exert a pulling force along its longitudinal axis. This force could be actively generated by these cells or result from a restraining action of the strings on the growth of the encircled territories of nonexpressing cells. Regardless of the mechanism, it seems most likely that the overexpressing cells display a strong adhesion between themselves. Since clones of cells with a high differential affinity normally have roundish and smooth contours to minimize cell-cell contacts along the interface of the clone, it is of interest that the contour of the UAS-ara overexpressing clones, excepting for the stretched interconnecting strings, generally appear as wiggly as that of the wild type clones. This suggest the presence of a polarized affinity between the overexpressing cells that permits their arrangement in strings or threads a few cells thick, but does not tend to minimize their interface with non-overexpressing cells. This phenotype of clones interconnected by strings is so far unique. It appears to be difficult to explain by simple differential affinity models (Villa-Cuesta, 2007).

This study has shown that the apposition of cells expressing and not expressing Iro-C causes the non expressing cells to undergo apical-basal shortening and invagination. These observations suggest that this effect has only a short range and that, as cells proliferate, those that are further removed from the interface recover a normal apical-basal length. This would provide a mechanism for the formation of the fold that surrounds the older clones or of that which separates the notum and wing domains of the imaginal disc. Since both these folds appear to be formed in an approximately symmetric way, at one side by Iro-C expressing cells and at the other by non-expressing cells, the apical-basal shortening effect may gradually and actively extend to the Iro-C expressing cells close to the interface. Alternatively, these may passively accommodate to the shortening of the non expresing cells. It should be stressed that previous evidence has already disclosed non-autonomous patterning effects of Iro-C^-clones located in the notum region on the surrounding Iro-C⁺ cells. Moreover, the suppression of the notum-wing hinge Iro-C border of expression negatively affects the growth of the wing disc. Taken together, these lines of evidence suggest that the notum-wing hinge boundary is a source of signals that affect the growth and patterning of the surrounding tissue, an activity reminiscent of the signals that emerge from the apposition of cells at the A/P and D/V boundaries of the disc (Villa-Cuesta, 2007).

The molecules responsible for the communication between Iro-C expressing and non-expressing cells are unknown. Likely candidates were tested, but the results have been negative. For instance, concerning fold formation, the role of the Rho GTPase pathway has been evaluated by producing dRho^GEF24.1 clones. dRho^GEF24.1 is activated by folded gastrulation and reiteratively required for epithelial folding and mesoderm invagination, but not for other processes regulated by Rho1. Contrary to previous evidence, these clones did not interfere with the notum/hinge fold, whose formation is dependent on the apposition of Iro-C expressing and non-expressing cells, or with the other extant folds of the disc. Overexpression of UAS-Rho^GEF2 in clones did induce apical-basal contraction of cells. However, these clones had low viability and the apical contraction might be due to a basal extrusion of the clones from the epithelium. Overexpression of either folded gastrulation or a dominant negative form of Rho strongly disrupted the epithelium of the disc, so no conclusions could be reached. A change in Myosin II localization is needed for the apical constriction that precedes mesodermal invagination. However, Myosin II accumulation was apparently unaffected in either Iro-C^- or UAS-ara overexpression clones (Villa-Cuesta, 2007).

The arrangement of UAS-ara expressing cells in interconnecting clones was not disturbed by reduction of the MAP kinase pathway (coexpression with UAS-raf^DN), which is active in the presumptive notum and regulates cell adhesion, by the loss-of-function of DaPKC (coexpression with UAS-DaPKC^DN), a protein required for apical/basal cell polarity, or by the reduction of function of Ephrin (coexpression with UAS-DaEph^DN), a molecule involved in cell attraction/repulsion, adhesion/de-adhesion and migration in vertebrates (Villa-Cuesta, 2007).

JAK/STAT signaling is required for hinge growth and patterning in the Drosophila wing disc.

JAK/STAT signaling is localized to the wing hinge, but its function there is not known. The Drosophila STAT Stat92E is downstream of Homothorax and is required for hinge development by cell-autonomously regulating hinge-specific factors. Within the hinge, Stat92E activity becomes restricted to gap domain cells that lack Nubbin and Teashirt. While gap domain cells lacking Stat92E have significantly reduced proliferation, increased JAK/STAT signaling there does not expand this domain. Thus, this pathway is necessary but not sufficient for gap domain growth. Reduced Wingless (Wg) signaling dominantly inhibits Stat92E activity in the hinge. However, ectopic JAK/STAT signaling does not perturb Wg expression in the hinge. Negative interactions occur between Stat92E and the notum factor Araucan, resulting in restriction of JAK/STAT signaling from the notum. In addition, this study found that the distal factor Nub represses the ligand unpaired as well as Stat92E activity. These data suggest that distal expansion of JAK/STAT signaling is deleterious to wing blade development. Indeed, mis-expression of Unpaired within the presumptive wing blade causes small, stunted adult wings. It is concluded that JAK/STAT signaling is critical for hinge fate specification and growth of the gap domain and that its restriction to the hinge is required for proper wing development (Ayala-Camargo, 2013).

Effects of Mutation or Deletion

A Drosophila mutant has been isolated in which the lateral parts of the notum are completely naked, leaving unaffected a median stripe of hairs. This mutation, iroquois (iro), defines a new gene that maps at 69D. In the presumptive lateral notum of mutant discs, sense organ precursor cells fail to form and the proneural gene scute is not expressed. The expression of a reporter gene inserted near iro suggests that iro itself is massively expressed in this region of the disc. It is proposed that iro is a prepattern gene essential to activate the expression of scute in the regions of the disc that will form the lateral notum (Leyns, 1996).

The effects of the absence of ara and caup were examined by inducing mutant clones. Large clones comprising both surfaces of the wing do not differentiate vein L5 and the allula, two sites of IROC expression. When clones are restricted to either the dorsal or ventral surface of the wing, they largely remove the corresponding component of vein L5. Also removed are the dorsal component of vein L3 (except for its distal part) and part of L1, the L2-associated sensilla campaniformia, the sensillum of the anterior cross-vein, and the TSM sensilla. This removal takes place in one deletion mutation but not another. Such different behavior may be explained by the ectopic expression in one of the deletion mutants of an additional gene related to ara and caup. No other sites of the wing show a requirement for IROC. Notum cells lacking Ara and Caup are nonviable and their loss nonautonomously affects the surrounding tissue (Gómez-Skarmeta, 1996a).

Enhanced expression of ara and caup using the L3/TSM enhancer results either in a lateral expansion of the L3 patch plus a reduction of the TSM patch, or with the closing up and fusion of both patches. achaete-scute L3 and TSM proneural clusters are similarly modified. The wings of these flies are small, have thickened and fused veins, and display few sensory organs at the anterior wing margin. The thickened and fused vein phenotype is also induced by the ectopic expression of veinlet, also known a rhomboid, a gene thought to promote vein formation. The accumulation of veinlet transcripts that normally decorate the wing margin does not occur in flies overexpressing ara (Gómez-Skarmeta, 1996b).

Sensory neurons can establish topologically ordered projections in the central nervous system, thereby building an internal representation of the external world. An analysis has been carried out into how this ordering is genetically controlled in Drosophila, using as a model system the neurons that innervate the mechanosensory bristles on the back of the fly (the notum). The Drosophila notum bears 11 pairs of precisely located large bristles, the macrochaetes, and about 200 smaller bristles arranged in rows, the microchaetes. Each bristle is innervated by a single bipolar neuron, which extends its dendrite towards the base of the bristle shaft, and its axon towards and into the central nervous system (CNS). The axons of all neurons innervating the notum bristles enter the thoracic ganglion through the same root and follow a common pathway that extends to the anterior and posterior along the pro- and meso-thoracic leg neuromeres. The details of the central projection depend, however, on the position of the bristle. In the case of macrochaetes, the relative extension of the anterior and posterior branches reflects the position of the bristle along the anteroposterior axis, while the existence and importance of a contralateral projection depends on the laterality of the bristle. The presence of contralateral branches is characteristic of the neurons innervating the medially located macrochaetes (dorsocentrals: DCs, scutellars: SCs, posterior post-alar: pPA) and the vast majority of microchaetes. The neurons innervating the lateral macrochaetes (notopleurals: NPs, supra-alars: SAs, presutural: PS, anterior post-alar: aPA) and the most lateral microchaetes, in contrast, have a projection confined to the ipsilateral half of the CNS. Mosaic analysis has shown that neurons project according to the position of the bristle they innervate even when many or most of the bristles are absent, suggesting that the distribution of the axonal terminals in the CNS does not depend on interactions between growing axons nor on competition for target sites (Ghysen, 1980). These results led to the conclusion that local or even intrinsic determinants provide some sort of positional information to the differentiating sensory neurons. The role of the proneural genes achaete and scute, which are involved in the formation of the medial and lateral bristles, was analyzed. They have no effect on the medial and lateral identities of the neurons. An analysis was made of the role of the prepattern genes araucan and caupolican, two members of the iroquois gene complex. These genes are required for the expression of achaete and scute in the lateral region of the notum. Their expression is responsible for the lateral identity of the projection (Grillenzoni, 1998).

The iro¹ mutation is a null allele of caup and a hypomorphic allele of ara, while iro² is a deletion eliminating both transcripts. The iro¹/iro² genotype thus constitutes the strongest viable iro combination and is probably close to a complete loss-of-function of the ara and caup genes. None of the lateral macrochaetes, nor the most lateral of the microchaetes, ever form in this mutant combination, due to the lack of activation of the ac-sc genes in the lateral regions of the notum. In order to examine whether iro plays any role in the specification of the lateral projections, the iro requirement was bypassed by using a gain-of-function allele of the ac-sc complex, Hairy-wing 49c (Hw^49c). In a normal background, Hw^49c leads to the formation of supernumerary bristles at various positions of the notum. In the iro¹ /iro² background, Hw^49c often leads to the formation of a bristle of variable size in the otherwise naked lateral region of the notum, at a position corresponding to that of the pSA macrochaete. Labelling the projection of these bristles reveal the presence of contralateral branches in about 70% of the cases, whether the bristle be microchaete-like or macrochaete-like. Such a branch was never observed in the projection of the most lateral microchaetes in wild-type flies and only once out of 31 cases in the projection of the pSA. Since the genes ac and sc have by themselves no effect on the type of projection, it was not expected that the Hw^49c mutation would be responsible for this effect. Nevertheless, the projections of neurons innervating the most lateral bristles in a Hw^49c; iro + genetic background were examined, and they were never seen to display contralateral branches. Thus, in the absence of Ara and Caup, the lateral bristles that form in Hw^49c flies display sensory projections that have contralateral branches. sensory projections, characteristic of the medial projections (Grillenzoni, 1998).

The ara and caup genes are weakly expressed in the larval CNS, and only in the ventral part of the brain. Nevertheless, one cannot rule out that the formation of contralateral branches by the lateral bristles in Hw^49c; iro¹/iro² flies could be due to an effect of the iro background on the CNS, rather than on the positional identity of the sensory neurons. To examine this possibility the prothoracic (humeral) bristles were used. Their axons enter the CNS in a more anterior position, extend along the same anteroposterior pathway as the mesothoracic (notal) bristles, and do not form contralateral branches. Even though the morphology of the humerus is not completely normal in iro¹/iro² flies, several humeral bristles are invariably present, and their projections are wild-type. Specifically, a contralateral branch was observed only once out of 17 cases. These results suggest that the neurons innervating the lateralmost bristles of the notum lose their lateral identity when they form in the absence of the iro products, in as much as they display contralateral branches typical of the neurons innervating medial bristles (Grillenzoni, 1998).

Given that iro is necessary for the lateral bristles to display the appropriate ipsilateral projection, an examination was carried out to see whether the expression of either iro gene is sufficient to specify this projection. The GAL4-UAS system was used to induce the expression of caup or ara in all proneural clusters of the developing notum, including the medial clusters that do not normally express the iro complex and give rise to contralaterally projecting neurons, and the capability of the iro products to modify the projections of the medial bristles was tested. In flies carrying both sca-GAL4 and UAS-ara or UAS-caup, the ectopic expression of the two genes does not affect the projections of the medial macrochaetes, which display contralateral branches as in the wild-type. On the contrary, the neurons innervating the most medial microchaetes lack all contralateral branches, i.e., their projection has now become typical of the most lateral microchaetes. The presence of Ara or Caup proteins is thus sufficient to change the projection of the neurons innervating the medial microchaetes to one corresponding to the most lateral microchaetes. The fact that no effect on the medial macrochaetes was observed suggests that the transformation of the microchaete projection (disappearance of contralateral branches) is probably not due to an effect on the CNS (Grillenzoni, 1998).

The Iroquois complex (Iro-C) homeodomain proteins allow cells at the proximal part of the Drosophila imaginal wing disc to form mesothoracic body wall (notum). Cells lacking these proteins form wing hinge structures instead (tegula and axillary sclerites). Moreover, the mutant cells impose on neighboring wild-type cells more distal developmental fates, like lateral notum or wing hinge. These findings support a tergal phylogenetic origin for the most proximal part of the wing and provide evidence for a novel pattern organizing center at the border between the apposed notum (Iro-C-expressing) and hinge (Iro-C-nonexpressing) cells. This border is not a cell lineage restriction boundary (Diez del Corral, 1999).

At the notum, clones of cells homozygous for the Iro-C deletions Df(3L)iro^DFM1 or Df(3L)iro^DFM3 and induced during the first and second larval instars were always associated with extensive malformations. The notum cuticle was replaced by a mostly naked, corrugated cuticle with sclerotized structures. In 52 of 116 cases [Df(3L)iro^DFM3 clones], these structures were clearly identifiable as components of an ectopic wing hinge, for example, axillary sclerites and tegula-like cuticle, with characteristic bristles and sensilla trichoidea and campaniformia. The multiple wing hairs (mwh) marker indicated that these sensilla arose within homozygous mutant tissue. Forty three of the 52 malformations affected the lateral notum. The ectopic axillary sclerites were always arranged in a mirror-image disposition with respect to the extant ones. Malformations at more medial regions of the notum, which did not affect the lateral notum, most frequently contained disorganized groups of tegula-like sensila trichoidea and campaniformia (7 of the 52 malformations) and, in 2 cases, almost complete ectopic tegulae. Malformations reaching the central-most regions of the notum caused defects in the fusion of heminota, which were separated by an undefined cuticle. Clones not associated with malformations appeared in flies irradiated 96-120 hr after egg laying. They developed normally in the central notum or induced invaginating cuticle vesicles in the lateral regions (not shown). Altogether, these results demonstrate an early requirement of the Iro-C for notum specification, because its absence changes the fate of its cells to wing hinge or impedes their terminal differentiation (Diez del Corral, 1999).

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 Iroquois complex (Iro-C) genes are expressed in the dorsal compartment of the Drosophila eye/antenna imaginal disc. Previous work has shown that the Iro-C homeoproteins are essential for establishing a dorsoventral pattern organizing center necessary for eye development. In addition, the Iro-C products are required for the specification of dorsal head structures. In mosaic animals, the removal of the Iro-C transforms the dorsal head capsule into ventral structures, namely, ptilinum, prefrons and suborbital bristles. Moreover, the Iro-C minus cells can give rise to an ectopic antenna and maxillary palpus, the main derivatives of the antenna part of the imaginal disc. These transformations are cell-autonomous, which indicates that the descendants of a dorsal Iro-C minus cell can give rise to essentially all the ventral derivatives of the eye/antenna disc. These results support a role of the Iro-C as a dorsal selector in the eye and head capsule. Moreover, they reinforce the idea that developmental cues inherited from the distinct embryonic segments from which the eye/antenna disc originates play a minimal role in the patterning of this disc (Cavodeassi, 2000).

The expression of genes required to make antenna was examined in the Iro-C minus clones, such as the homeobox gene Distal-less (Dll). Using Dll-lacZ, Dll expression was found to start at the early third instar and is confined to a small group of cells in the centre of the antennal primordium. Later expression of Dll encompasses all the antennal segments with the exception of the most proximal one. In the non-overproliferating Iro-C minus clones, Dll is activated, although this only occurs in clones located at the dorsal/posterior quadrant of the eye disc. In the antennal primordium, activation of Dll depends on simultaneous signaling by Dpp and Wg. wg is expressed by most of the cells of dorsal Iro-C minus clones. Many of these clones also express Dpp, which could be provided by either the morphogenetic furrow or, in clones that are near the disc margin, by a source of Dpp induced by the ectopic eye organizer associated with the clone. Thus, the clone cells were exposed to both Dpp and Wg, which in combination should activate Dll. Iro-C minus cells at the dorsal/anterior region, which are also exposed to Wg and Dpp, probably fail to activate Dll because they are located within the domain of expression of homothorax (hth). The removal of the Iro-C does not appreciably modify the expression of this gene. The presence of Hth might impair the activation of Dll since, in the leg disc, this protein, by driving the nuclear localization of Extradenticle (a cofactor of many homeoproteins), appears to reduce the sensitivity of cells to Wg and Dpp signaling. Thus, at the dorsal eye disc, Iro-C and hth probably cooperate to repress Dll. Large outgrowths resulting from Iro-C minus clones encompass hth-expressing cells in their anterior part and display ectopic Dll expression in the posterior part. In summary, the results suggest that, upon removal of Iro-C in dorsal cells, the cells become exposed to Wg and Dpp, and this and the absence of Iro-C products promote Dll activation. The interaction of Dll with hth, a gene with an antenna-selector function, should allow the growth of an ectopic antenna (Cavodeassi, 2000).

The eye-antenna disc has a complex origin. Its cells, which appear to be derived from five embryonic segments and the acron, fuse into the disc primordium. Thus, in principle, cells derived from different segments might inherit specific cues that would commit them to their respective developmental fates. Cells that would ordinarily give rise to dorsal head capsule, on losing the Iro-C products, give rise to ventral eye plus ventral head and appendages thought to be derived from different segments. This suggests that the origin of each cell within the disc primordium does not irreversibly commit it to a specific fate. Hence, the eye/antenna disc, as with the wing and leg discs, largely develops as an integrated unity despite its multisegmental origin. In support of this interpretation, transplantation experiments have shown that a head-eye fragment of the disc can regenerate the ablated antennal part. Moreover, cell lineage analyses have shown that a single Minute + clone can include the maxillary palpus, antenna and ventral part of the eye, precisely those structures that can be generated by Iro-C minus clones (Cavodeassi, 2000).

It is of interest to compare the functions of the Iro-C in the development of the head and mesothorax. In the precursors of the mesothorax, the wing and second leg imaginal discs, early Iro-C expression is restricted to the dorsalmost region of the wing disc, which will give rise to the notum. Removal of this early expression in Iro-C minus clones transforms the notum into proximal wing hinge. This has a correlate in the eye disc, because Iro-C is expressed early in the dorsalmost part of the disc and is required for development of the disc's dorsal structures. Moreover, interactions between Iro-C expressing and non-expressing cells at the notum/hinge border and at the DV eye compartment border establish organizing centers that help pattern the neighboring tissue. (Note, however, that the notum/hinge border does not coincide with a restriction of cell lineage). Thus, the early function of the Iro-C genes appears to specify dorsal body wall in both the head and the mesothorax. In both cases, the Iro-C genes may be repressing genetic functions that allow cells to give rise to appendages (wing, antenna and maxillary palpus). Iro-C appears to repress Dll, and thereby antennal development, in the dorsal part of the eye disc, a region that early in development expresses wg and receives Dpp from the disc margin. Consistent with this repressing function, the Iro-C genes are not expressed in the antenna disc, nor are they expressed in the leg discs (except for a late expression in the prospective tibia), and their overexpression in either the antenna or the leg disc truncates the corresponding appendage. It should be of interest to examine whether the Iro-C genes have a similar dorsal specifying function in other parts of the fly’s body wall (Cavodeassi, 2000).

In Drosophila the eye-antennal disc gives rise to most adult structures of the fly's head. Yet the molecular basis for its regionalization during development is poorly understood. homothorax is shown to be required early during development for normal eye development and is necessary for the formation of the ventral head capsule. In the ventral region of the disc are homothorax and wingless involved in a positive feedback loop necessary to restrict eye formation. homothorax is able to prevent the initiation and progression of the morphogenetic furrow without inducing wingless, which points to homothorax as a key negative regulator of eye development. In addition, the iroquois-complex genes are shown to be required for dorsal head development, antagonizing the function of homothorax in this region of the disc (Pichaud, 2000).

To analyze the role of iro-C in dorsal head development, iro-C function was removed in clones of cells carrying a deficiency for ara, caup and mirr (iroDFM3), or only ara and caup (iroDFM1), and the phenotypic consequences were examined in adult heads. Only results for iroDFM3 clones will be described, since iroDFM1 clones give similar results. iro-C^- clones cause a series of phenotypes, adding progressively more 'ventral-type' tissue in the following order: dorsal eye overgrowth or ectopic dorsal eyes; overgrowth of ventral type of cuticle (ptilinum and rostral membrane); ectopic antennal pouches; antennae and maxillary palps. The extra head structures are produced autonomously, but the eyes can be composed of both mutant and wild-type ommatidia. The ectopic structures, which can duplicate the full complement of ventral structures, all grow from the orbital region of the head. The rest of the dorsal head is displaced by the overgrown tissue. The orbital region fate maps to the domain where hth and iro-C expressions overlap. These results show that the iro-C genes are required to repress the proliferation of a group of dorsal cells, that otherwise would grow with ventral head identity, and may contribute to assign them a dorsal head ('orbital-region') identity (Pichaud, 2000).

iro-C^- clones in discs frequently produce overgrowths, in agreement with the structures produced dorsally. However, hth and wg expressions are not substantially altered in iro-C^- clones. Since removal of ara, caup and mirr produces the same phenotypes as removal of only ara and caup, it is concluded that mirr is dispensable for suppressing ventral identity in the dorsal head. Alternatively, mirr expression could be under the control of ara and caup (Pichaud, 2000).

In Drosophila, imaginal wing discs, Wg and Dpp, play important roles in the development of sensory organs. These secreted growth factors govern the positions of sensory bristles by regulating the expression of achaete-scute (ac-sc), genes affecting neuronal precursor cell identity. Earlier studies have shown that Dally, an integral membrane, heparan sulfate-modified proteoglycan, affects both Wg and Dpp signaling in a tissue-specific manner. dally is required for the development of specific chemosensory and mechanosensory organs in the wing and notum. dally enhancer trap is expressed at the anteroposterior and dorsoventral boundaries of the wing pouch, under the control of hh and wg, respectively. dally affects the specification of proneural clusters for dally-sensitive bristles and shows genetic interactions with either wg or dpp signaling components for distinct sensory bristles. These findings suggest that dally can differentially regulate Wg- or Dpp-directed patterning during sensory organ assembly. For pSA, a bristle on the lateral notum, dally shows genetic interactions with iroquois complex (IRO-C), a gene complex affecting ac-sc expression. Consistent with this interaction, dally mutants show markedly reduced expression of an iro::lacZ reporter. These findings establish dally as an important regulator of sensory organ formation via Wg- and Dpp-mediated specification of proneural clusters (Fujise, 2001).

Dally, Dpp, and IRO-C genetically interact with each other during the formation of the pSA macrochaete. Although interactions between Dpp signaling and IRO-C have been suggested, evidence is provided that Dpp signaling components interact with the genes of IRO-C. Ectopic Dpp signaling using a constitutively active type I receptor, tkv, leads to an ectopic induction of the pSA macrochaete, supporting the idea that Dpp signaling is required for prepatterning for this bristle. Significant reductions in the expression of iro enhancer trap is observed in dally mutant wing discs. Expression of the iro at the lateral notum region is critical for the proneural cluster formation and bristle development in this region. Taken together, these findings suggest that dally mediates Dpp signaling to control expression of the genes of IRO-C during the formation of the pSA bristle (Fujise, 2001).

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) iro^DFM3/+ 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, DL^DN. bi>Dl^DN causes reduction of eye on both dorsal and ventral margins whereas coexpression of tsh and Dl^DN (bi>tsh+Dl^DN) further enhances this phenotype. A dorsal Act>tsh+Dl^DN clone suppresses eye development. Act>tsh+Dl^DN clones also non-autonomously suppress eye development, a phenotype also seen in Act>Dl^DN 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, Ser^DN was used to block Ser function. In bi>Ser^DN, the eyes are suppressed on both dorsal and ventral margins. This phenotype is partially blocked in bi>tsh+Ser^DN eye. Similar results were observed in Act>tsh+Ser^DN 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).

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 (mirr^G8. 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 caup^G3) and null (or strong hypomorph) mutations of ara that do not affect significantly caup or mirr (e.g. TSI or ara^G4) (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 (caup^G3) 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 ara^G4/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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