Transcriptional Regulation

Notch activation at the midline plays an essential role both in promoting the growth of the eye primordia and in regulating eye patterning. Specialized cells are established along the dorsal-ventral midline of the developing eye by Notch-mediated signaling between dorsal and ventral cells. D-V signaling in the eye shares many similarites with D-V signaling in the wing. In both cases an initial asymmetry is set up by wingless expression. Both Eye and wing cells then go through a distinct intermediate step: in the wing, Wingless represses the expression of Apterous, a positive regulator of fringe (fng) expression; in the eye, Wingless promotes the expression of mirror (mrr), which encodes a negative regulator of fringe (unpublished observations of McNeill, Chasen, Papayannopoulos, Irvine, and Simon, cited by Papayannopoulos, 1998). Both wing and eye cells share a Fng-Ser-Dl-Notch signaling cassette to effect signaling between dorsal and ventral cells and establish Notch activation along the D-V midline. Local activation of Notch leads to production of diffusible, long-range signals that direct growth and patterning, which in the wing include Wingless, but in the eye remain unknown. At least one downstream target of D-V midline signaling, four jointed (fj), is also conserved. four jointed is also expressed in the wing and its expression there is indirectly influenced by Notch (Papayannopoulos, 1998 and references).

During early eye development, fringe is expressed by ventral cells. This expression appears to be complementary to that of the dorsally expressed gene mrr. During early to mid-third instar, additional expression of fng appears in the posterior of the eye disc. This line of posterior fng expression is just in front of the morphogenetic furrow and moves across the eye ahead of the furrow. In the wing disc, Dl and Ser induce each other's expression, and become up-regulated along the D-V border where they can productively signal. Dl and Ser are also preferentially expressed along the D-V midline during eye development. Ser expression, like fng expression, is complementary to that of mrr, whereas Dl expression partially overlaps that of mrr. The spatial relations among fng, Ser, and Dl expression in the eye are thus similar to those in the wing, although in the wing, their expressions are inverted with respect to the D-V axis (Papayannopoulos, 1998).

The eye imaginal disc displays dorsal-ventral (D-V) and anterior-posterior polarity prior tothe onset of differentiation, which initiates where the D-V midline intersects the posterior margin. As the wave of differentiation progresses anteriorly, additional asymmetry develops as ommatidial clusters rotate coordinately in opposite directions in the dorsal and ventral halves of the disc; this forms the equator, a line of mirror-image symmetry that coincides with the D-V midline of the disc. The currently unanswered question of how D-V pattern is established and how it relates to ommatidial rotation was addressed by assaying the expression of various asymmetric markers under conditions that lead to ectopic differentiation, such as removal of patched or wingless function. D-V patterning is found to develop gradually. wingless plays an important role in setting up this pattern. To determine if positional information associated with equatorial formation is present along the D-V axis of the disc ahead of the MF, expression of an equatorial marker (WR122, a lacZ insertion in an unknown locus) was studied in various genetic conditions that lead to ectopic neuronal differentiation. This expression is dependent on the activity of the gene frizzled, which is required for proper ommatidial rotation. Induction of patched mutant clones activates the Hedgehog pathway and leads to precocious neuronal differentiation.Ectopic ommatidia that arise in clones show that the potential to express WR122-lacZ is restricted to neurons located near the D-V midline, regardless of their position along the A-P axis of the disc. This suggests that the information necessary to restrict WR122 expression exists ahead of the MF (Heberlein, 1998).

The expression of WR122 was examined under conditions that reduce Wingless activity. A temperature sensitve wg allele was used. A reduction in Wg function during the late larval stages promotes precocious differentiation in the eye disc. This differentiation starts from the dorsal (and to a lesser degree the ventral) margin and proceeds inward, roughly perpendicular to the direction of progression of the normal differentiation front. Expression of WR122 is unrestricted among ectopic ommatidia that differentiate as a consequence of reduced Wg function. The normal expression domain of the marker is broadened toward the lateral margins. It is concluded that the expression of WR122 is inhibited by Wg in ommatidia located near the disc's margin, which restricts expression to the equatorial region. Ectopic expression of Wg is sufficient to repress WR122 expression in the more central portions of the retinal epithelium. Thus Wg functions to restrict the expression of the WR122 marker. wingless is necessary and sufficient to induce dorsal expression of the gene mirror prior to the start of differentiation and also to restrict the expression of the WR122 marker to differentiating photoreceptors near the equator. Manipulations in wingless expression shift the D-V axis of the disc as evidenced by changes in the expression domains of asymmetric markers, the position of the site of initiation and the equator, and the pattern of epithelial growth. Thus, Wg appears to coordinately regulate multiple events related to D-V patterning in the developing retina (Heberlein, 1998).

The dorsoventral midline of the Drosophila eye disc is a source of signals that stimulate growth of the eye disc, define the point at which differentiation initiates, and direct ommatidial rotation in opposite directions in the two halves of the eye disc. This boundary region seems to be established by the genes of the iroquois complex, which are expressed in the dorsal half of the disc and inhibit fringe expression there. Fringe controls the activation of Notch and the expression of its ligands, with the result that Notch is activated only at the fringe expression boundary at the midline. The secreted protein Wingless activates the dorsal expression of the iroquois genes. Pannier, which encodes a GATA family transcription factor expressed at the dorsal margin of the eye disc from embryonic stages on, acts upstream of wingless to control mirror and fringe expression and establish the dorsoventral boundary. Loss of pannier function leads to the formation of an ectopic eye field and the reorganization of ommatidial polarity, and ubiquitous pannier expression can abolish the eye field. Pannier is thus the most upstream element yet described in dorsoventral patterning of the eye disc (Maurel-Zaffran, 2000).

Recently, several studies have established that N activation along the dorsoventral midline of the eye disc is critical for eye growth as well as for positioning the equator. This local activation is the consequence of the ventrally restricted expression of fng, which is negatively controlled by the iro-C homeobox genes expressed in the dorsal half of the eye disc. Either loss of fng function, or ubiquitous expression of fng, caup or mirr, abolishes eye growth. The iro-C genes appear to act redundantly, as both ara and caup must be removed from clones of cells to promote the formation of ectopic dorsal eyes similar to those reported for pnr. The similar effects observed for gain or loss of pnr function suggest strongly that pnr might act in the same pathway as the iro-C and fng. To confirm this and to order pnr with respect to these genes, expression of mirr and fng was examined in eye discs mutant for pnr or misexpressing pnr. In eye discs in which pnr function had been removed, mirr expression is greatly reduced, whereas fng is derepressed dorsally. In eye discs expressing constitutively active pnr, mirr expression is expanded ventrally, shifting the point of morphogenetic furrow initiation to the ventral side. fng expression is dramatically reduced in discs overexpressing pnr D4. It thus appears that pnr acts upstream of the iro-C genes, activating their expression dorsally. Consistent with this, it has been found that ubiquitous expression of ara abolishes photoreceptor differentiation, and that removal of pnr function does not restore photoreceptor formation. If pnr were downstream of ara, blocking its function should have induced ectopic eye development even in the presence of ara (Maurel-Zaffran, 2000).

The results above show that pnr acts upstream of the iro-C genes to regulate dorsal eye development. Another molecule that has been shown to act upstream of the iro-C in this context is Wg. wg is required to inhibit the initiation of the morphogenetic furrow at the lateral margins of the eye disc, preventing ectopic eye differentiation there. The dorsal ectopic eyes induced by removing pnr function thus suggest that the functions of pnr and wg may be related. Consistent with this idea, the block in morphogenetic furrow initiation caused by expressing wg throughout the eye disc, like the block caused by expressing pnr D4 , can be rescued by co-expressing an activated form of N. pnr and wg may thus act in the same cascade to prevent eye differentiation (Maurel-Zaffran, 2000).

The role of wg in directing dorsal development is unexpected because wg is also expressed at the ventral anterior margin of the eye disc, although at a lower level than at the dorsal margin; this expression must have an upstream regulator other than pnr. However, the effects of loss of wg are more robust on the dorsal than the ventral side of the eye disc, and misexpression of wg symmetrically at both lateral margins dorsalizes the eye disc. These observations may be explained by the finding that at early stages wg is limited to the dorsal side of the eye disc and may exert its dorsalizing effect at this time (Maurel-Zaffran, 2000).

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

Polarity of the Drosophila compound eye is established at the level of repeating multicellular units (known as ommatidia), which are organized into a precise hexagonal array. The adult eye is composed of ~800 ommatidia, each of which forms one facet. Sections through the eye reveal that each ommatidium contains eight photoreceptor cells in a stereotypic trapezoidal arrangement that has two mirror-symmetric forms: a dorsal form present above the dorsoventral (DV) midline, and a ventral form below. An axis of mirror-image symmetry runs along the DV midline and is known as the equator. By analogy to the terrestrial equator, the extreme dorsal and ventral points of the eye are referred to as the poles. Differentiation of ommatidia begins during the third instar larval stage when a furrow moves from posterior to anterior over the epithelium of the eye imaginal disc. Each ommatidial unit is born as a bilaterally symmetrical cluster of photoreceptor precursors, that is polarized on its anteroposterior axis. The clusters then become polarized on the DV (or equatorial-polar) axis, by the process of proto-ommatidium rotation via two 45° steps away from the DV midline, forming the equator. It has been suggested that the direction of this rotation is a consequence of a gradient of positional information emanating from either the midline or the polar regions of the disc (Zeidler, 1999 and references).

A number of recent studies have shed light on some of the mechanisms involved in the positioning of the equator on the DV midline of the eye imaginal disc. It is now clear that a critical step is the activation of Notch activity in a line of cells along the midline, and that this localized activation of Notch is a consequence of the restricted expression of the fringe (fng) gene product in the ventral half of the disc and the homeodomain transcription factor Mirror (Mirr) in the dorsal half of the disc. Furthermore, an important role for Wingless (Wg) in polarity determination on the DV axis has been demonstrated. Wg is a secreted protein (and the founder member of the Wnt family of morphogens) that is expressed at the poles of the eye disc. Wg has been shown to act as an activator of mirr expression; increasing the levels of Wg expression in the eye disc shifts mirr expression and the position of the equator ventrally, whereas reduction of wg function shifts mirr expression dorsally. Additionally, it has been shown convincingly that a gradient of Wg signaling across the DV axis of the eye disc regulates ommatidial polarity such that the lowest point of Wg signaling coincides with the equator (Zeidler, 1999 and references).

The JAK/STAT pathway is central to the establishment of planar polarity during Drosophila eye development. A localized source of the pathway ligand, Unpaired/Outstretched, present at the midline of the developing eye, is capable of activating the JAK/STAT pathway over long distances. A gradient of JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized Unpaired influences the position of the equator via repression of mirror (Zeidler, 1999).

The data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localized activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, since fng loss of function clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity. Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals (most likely detected by the receptor Frizzled) that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal (Zeidler, 1999 and references).

Given the role of Upd in restricting mirr expression, one possible mechanism by which JAK/STAT LOF clones might induce ectopic axes of mirror-image symmetry would be through the generation of ectopic boundaries of mirr expression. The expression of mirr-lacZ was examined in hop clones. Many clones lying both dorsally and ventrally were examined in eye discs, and in no case was an alteration in mirr-lacZ expression observed. Additionally, hundreds of adults carrying mirr-lacZ were examined, in which hop clones had been induced, and, again, in no case was a change in mirr-regulated white+ expression observed (Zeidler, 1999).

Thus, ommatidial polarity inversions generated by hop clones are mirr independent. It is therefore concluded that the process of midline equator definition by dorsally restricted mirr expression and the regulation of ommatidial polarity by the JAK/STAT pathway are separable processes. It is also noted that these results suggest that Upd might act independently of Hop to regulate mirr expression (Zeidler, 1999).

The ommatidial polarity phenotype produced by removal of JAK activity in mosaic clones has a number of important features: (1) the phenotype observed is an inversion of ommatidial polarity in which either the dorsal rotational form is seen in the ventral hemisphere of the eye or vice versa; (2) the phenotype is only observed on the polar boundary of the mosaic tissue; (3) the strength of the phenotype (in terms of the number of inverted ommatidia seen) is dependent on the size and shape of the clone; (4) the phenotype is cell nonautonomous as either fully mutant, fully wild-type, or as mosaic clusters that can manifest the phenotype (Zeidler, 1999).

From these characteristics, the following can be deduced: the nonautonomy of the phenotype produced by removal of the autonomously acting pathway component JAK, and its dependence on clone size and shape, suggests that JAK/STAT affects ommatidial polarity via a secreted downstream signal (which subsequently will be referred to as a second signal, most likely detected by Frizzled). The direction of the nonautonomy (only in a polar direction) and the strict DV nature of the polarity inversions indicates that this second signal must be graded in its activity along the DV axis, with a change in direction of the gradient at the equator. The direction of this gradient would then be the instructive cue to which ommatidia respond when rotating to establish their mature polarity (Zeidler, 1999).

The simplest model would be that there is a single second signal secreted from the equator, which is downstream of mirr/fng/Notch, and that Wg and Upd/JAK/STAT feed into this pathway upstream of Notch. This is consistent with the roles of Wg and Upd as regulators of mirr expression and, thus, in positioning the endogenous equator. However, it is not consistent with the observed ommatidial polarity inversions produced in the eye field both dorsally and ventrally by Wg-pathway and JAK/STAT-pathway LOF and GOF clones. These phenotypes indicate that second-signal concentration is dependent on Wg pathway and JAK/STAT pathway activity across the whole of the eye field, and thus the second signal cannot be only secreted from the DV midline as a consequence of localized Notch activation. It is conceivable that Notch is activated on the polar boundary of JAK/STAT LOF clones, but in this context the only known mechanism of Notch activation is via mirr/fng interactions, and this possibility has been ruled out (Zeidler, 1999).

Instead, the data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localize activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, becausefng LOF clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity (Zeidler, 1999).

Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal. A number of observations provide a great deal of support for such a model. (1) It is consistent with the known timing of the events involved. The requirement for fng function has been shown to lie between late first instar and mid second instar, which coincides with the first appearance of high levels of Upd expression at the optic stalk. However, the ommatidia are not formed (and thus do not respond to the polarity signal) until the start of the third instar, a stage when localized Upd expression still persists. Furthermore, extracellular Upd protein can be seen in a concentration gradient many cell diameters from the optic stalk at the early third instar stage, consistent with Upd being at least partly responsible for setting up the long-range gradient of JAK/STAT activity across the DV axis of the eye disc that is revealed by the stat92E-lacZ reporter. (2) This model does not require that a single source of second signal secreted by a narrow band of cells at the equator should be capable of determining ommatidial polarity across the whole of the DV axis of the disc during the third instar stage of development. Instead, the band of activated Notch at the equator would serve to draw a straight line between the fields of dorsally and ventrally polarized ommatidia, and need only secrete a localized source of second signal to polarize ommatidia in this region. Further from the equator, the opposing gradients of Upd and Wg signaling would provide a robust mechanism for maintenance of correct ommatidial polarity across the DV axis. Conversely, without the mirr/fng/Notch mechanism to draw a straight line, it would be impossible to imagine how Upd at the posterior margin and Wg at the poles alone could provide the perfectly straight equator that is ultimately formed. (3) The phenotypes that are observed are consistent with multiple competing mechanisms responsible for determining ommatidial polarity. When inversions of ommatidial polarity are induced by generating hop clones or ectopically expressing Upd, straight equators are not produced, such that two cleanly abutting fields of dorsal and ventral ommatidia are produced. Instead, there is usually some confusion of ommatidial identities as if they might be receiving conflicting signals. Additionally, when upd activity is removed from the optic stalk, an equator still forms (albeit at the ventral edge of the disc), but some ommatidia dorsal to the equator still adopt a ventral fate as if the determination of ommatidial polarity is less robust in the absence of Upd (Zeidler, 1999).

The posteriorly expressed signaling molecules Hedgehog and Decapentaplegic drive photoreceptor differentiation in the Drosophila eye disc, while at the anterior lateral margins Wingless expression blocks ectopic differentiation. Mutations in axin prevent photoreceptor differentiation and leads to tissue overgrowth; both these effects are due to ectopic activation of the Wingless pathway. In addition, ectopic Wingless signaling causes posterior cells to take on an anterior identity, reorienting the direction of morphogenetic furrow progression in neighboring wild-type cells. Signaling by Dpp and Hh normally blocks the posterior expression of anterior markers such as Eyeless. Wingless signaling is not required to maintain anterior Eyeless expression and in combination with Dpp signaling can promote Ey downregulation, suggesting that additional molecules contribute to anterior identity. Along the dorsoventral axis of the eye disc, Wingless signaling is sufficient to promote dorsal expression of the Iroquois gene mirror, even in the absence of the upstream factor pannier. However, Wingless signaling does not lead to ventral mirror expression, implying the existence of ventral repressors (Lee, 2001).

Early transplantation experiments and other studies have suggested that the region of the eye disc anterior to the morphogenetic furrow contains intrinsic positional information; the subsequent finding that Hh is essential for furrow movement has been taken to mean that all this information originates posterior to the furrow. The observations presented here challenge this view by showing that Wg signaling can generate a source of anterior positional information that appears to attract the morphogenetic furrow toward itself. Cells mutant for axin autonomously express ey and other markers for the region anterior to the furrow, including hth, tsh and mirr. Cells adjacent to an axin mutant clone show a reorientation of the pattern of Atonal expression and cells at the internal border of the clone express Hairless, which is likely to be activated by Dpp signaling from adjacent wild-type cells. This suggests that axin mutant cells produce a non-autonomous signal that maintains nearby cells in an Ato-expressing state; since wg-lacZ expression is activated in axin mutant clones, the signal could itself be Wg (Lee, 2001).

In addition to providing anterior information to the eye disc, Wg acts early in development to define its dorsal domain. Dorsal wg expression is controlled by pnr, and ectopic eye formation caused by loss of pnr. This can be blocked by restoring wg, suggesting that wg is a downstream effector of pnr. In addition, Wg signaling is necessary to maintain the expression of mirr and can induce ectopic mirr expression along the ventral margin. axin mutation has been used to clarify the relationships between pnr, wg and mirr. Small clones of cells mutant for pnr maintain mirr expression, showing that pnr does not have a direct effect on mirr, but acts through one or more non-autonomous factors. This is consistent with the expression of pnr in a smaller domain than mirr. Restoring Wg signaling to pnr mutant eye discs, by making pnr;axin double mutant clones in a Minute background, allows the expression of mirr; thus, no other factor downstream of pnr can be essential for mirr expression (Lee, 2001).

hh was expressed dorsally in early eye discs and activation of the Hh pathway in ventral ptc clones leads to ectopic mirr expression. The results are consistent with two possible roles for Hh. Dorsal hh expression could be independent of pnr and contribute to mirr activation in the absence of both pnr and axin. The dorsal domain of hh expression in the eye disc is indeed not stably established until the second larval instar, while wg and pnr are expressed dorsally from late embryonic stages. Alternatively, Hh could act downstream of pnr but upstream of wg to activate mirr. In support of this hypothesis, anterior ventral ptc clones activate mirr non-autonomously, and have also been shown to activate wg expression. Because ventral axin clones do not activate mirr expression, this mechanism would imply that Hh activates factors in addition to wg that allow ventral expression of mirr (Lee, 2001).

The restriction of mirr to the dorsal domain in axin clones must reflect either a requirement for Hh (in addition to Wg) for its activation, or the existence of ventral repressors. Ventral repression could explain the sharp expression boundary of mirr, which would otherwise be difficult to accomplish in a region of low and graded Wg activity. The most likely candidate for a ventral repressor is the Unpaired (Upd) ligand for the Janus kinase/signal transducer and activator of transcription (STAT) pathway. At third instar, upd is expressed at the optic stalk, in the center of the posterior margin, and also at the anterior ventral margin, but its removal from the optic stalk region is sufficient to derepress mirr ventrally. Consistent with the findings of this study, upd expression is not affected by ectopic Wg; it is not known whether it can be regulated by Hh, although the phenotypes of loss of upd and overexpression of hh are very similar (Lee, 2001).

Wg has three roles in early eye disc development: establishment of anterior identity, establishment of dorsal identity, and promotion of growth. Prior to furrow initiation, Pnr, expressed at the dorsal margin, activates wg expression in a broader domain; Wg then activates mirr and the other Iro-C genes throughout the dorsal compartment. Hh may contribute to the activation of these genes through Wg or act independently of Pnr. Upd, which is present at the optic stalk also contributes to the ventral repression of mirr. Mirr represses fng, forming a boundary of fng expression at the DV midline that leads to activation of N in this region. During furrow progression, Hh is expressed in the differentiating photoreceptors and Dpp in a stripe in the morphogenetic furrow. These two signals act to downregulate genes expressed in the anterior such as ey, and allow an anterior to posterior transition. Wg establishes the anterior state, probably at an earlier stage, while another factor (X) contributes to its maintenance. Other factors are necessary to modify the response to Wg in order to determine which cell fate should be specified; this is consistent with data suggesting that Wg signaling alters chromatin structure to allow access to transcription factors. A requirement for multiple signaling systems also ensures accuracy in cell fate determination (Lee, 2001).

Dorsoventral (DV) patterning is crucial for eye development in invertebrates and higher animals. DV lineage restriction is the primary event in undifferentiated early eye primordia of Drosophila. In Drosophila eye disc, a dorsal-specific GATA family transcription factor pannier (pnr) controls Iroquois-Complex (Iro-C) genes to establish the dorsal eye fate whereas Lobe (L), which is involved in controlling a Notch ligand Serrate (Ser), is specifically required for ventral growth. However, fate of eye disc cells before the onset of dorsal expression of pnr and Iro-C is not known. L/Ser have been shown to be expressed in entire early eye disc before the expression of pnr and Iro-C is initiated in late first instar dorsal eye margin cells. The evidence suggests that during embryogenesis pnr activity is not essential for eye development. Evidence that loss of L or Ser function prior to initiation of pnr expression results in elimination of the entire eye, whereas after the onset of pnr expression it results only in preferential loss of the ventral half of the eye. Dorsal eye disc cells also become L or Ser dependent when they are ventralized by removal of pnr or Iro-C gene function. Therefore, it is proposed that early state of the eye prior to DV lineage restriction is equivalent to the ventral half and requires L and Ser gene function (Singh, 2003).

Previously, L/Ser were thought to be required for ventral eye growth after the DV lineage restriction boundary was established, which corresponds to the onset of expression of dorsal eye selectors. The results, however, clearly suggest that L/Ser are required much earlier for the growth of the entire early eye disc, even before the DV patterning is established. In contrast to the function of dorsal selector genes in eye patterning, L and Ser have been shown to play a distinct role in controlling ventral-specific growth of eye disc (Singh, 2003).

Loss-of-function phenotypes of L or Ser are restricted to the ventral eye. The spatial as well as temporal requirement of these genes in the ventral eye pattern formation were examined. Extent of loss of ventral eye pattern in loss-of-function clones of L/Ser varies along the temporal scale. During early eye disc development, prior to onset of pnr expression in dorsal eye, removal of L or Ser function results in complete elimination of the eye field, whereas later when dorsal eye selector genes starts expressing the eye suppression phenotype becomes restricted only to the ventral eye. Therefore, DV lineage border in the eye can also be interpreted as the border between the cells sensitive and insensitive to the L/Ser gene function (Singh, 2003).

The eye antennal disc has the most complex origin in the embryo. The eye disc is initiated from a small group of ~70 precursor cells on each side contributed by six different head segments of the embryo. These embryonic precursors do not physically separate from the surrounding larval primordia and are therefore difficult to discern morphologically (Singh, 2003 and references therein).

Once the cells for the eye-antennal disc are committed, these discs proliferate and undergo differentiation into an adult eye, which requires generation of DV lineage restriction in eye. There are possibly three different ways by which genesis of DV lineage in the eye can be explained. Early first instar larval eye disc may initiate either from only dorsal, only ventral or from both DV lineages. Based on results from studies of expression patterns and analysis of mutant phenotypes, it is proposed that larval eye primordium initially comprises only the ventral-equivalent state rather than well-defined DV or dorsal states alone. The initial state of eye is referred to as ventral equivalent state because, at this stage, dorsal and ventral identity is not yet generated. DV lineage restriction is established later after the onset of pnr expression. The cells of the initial ventral-equivalent state are similar to the ventral eye cells that are generated after DV specification. The similarity is in terms of their requirement of L/Ser for growth and maintenance, and the absence of the dorsal selector expression. How dorsal lineage is initiated in the early eye disc is not yet clear. Once the DV lineage restriction is established, N signaling is initiated at the equator, a border between dorsal and ventral compartments. Activation of N signaling promotes proliferation, which is followed by differentiation of eye disc into adult compound eye (Singh, 2003).

The ventral-equivalent state model is supported by two observations: 1) presence of Ser and L expression in the dorsal and ventral eye disc of the early first instar larva and 2) change of dorsal eye fate to ventral upon removal of dorsal selectors. The mutants, which affect ventral eye development, show two major phenotypes in eye: either there is no or very small eye, or there is a preferential loss of ventral eye based on the time they affect their function but none of the mutants for dorsal eye selectors show phenotypes of loss of only dorsal eye. Conversely, loss-of-function clones of pnr or Iro-C causes dorsal eye enlargement or ectopic eye formation rather than loss of only dorsal eye clonal tissue. This phenotype is probably due to generation of ectopic boundary of pnr-expressing and non-expressing cells (rather than absence of pnr), which could be important for promoting eye growth. Overexpression of Ush or Fog proteins in eye discs results in loss of pnr activity, causing complete elimination of eye development. By removing pnr activity at different time points it was found that pnr activity in embryo and early first instar is not essential for eye disc development. Later, pnr becomes essential for DV patterning consistent with its strong expression in dorsal margin of eye disc after the early first instar stage (Singh, 2003).

In contrast to enlargements or ectopic eyes induced by loss-of-function clones of dorsal selectors, the loss-of-function clones of L or Ser always resulted in the elimination of the eye fate. L/Ser are primarily required for the maintenance and development of ventral or ventral-equivalent state of the eye, whereas dorsal genes establish the DV border. This suggests that dorsal genes and L/Ser, although involved in a common goal of generation of DV lineage in eye, probably affect eye development at two different tiers (Singh, 2003).

Fng, another essential component of DV patterning in eye, is expressed preferentially in the ventral domain of early eye disc and is required for restriction of N signaling to the DV border. Although fng is known to act upstream of Ser in the wing and eye discs, there is also an apparent difference between the two genes. Unlike L/Ser, the main function of fng seems to affect DV ommatidial polarity but not the growth. This suggests that fng may be selectively required for DV patterning after dorsal selectors initiate domain specification. This may be the reason why phenotypes of loss-of-function clones of fng are different from those of L and Ser in the eye. It has been observed that the pattern of fng expression is not altered in L mutants, and vice versa, supporting the independent functions of these two genes in controlling DV border formation and growth of ventral domain (Singh, 2003).

The function of Pnr in organizing the DV pattern from an initial ventral-equivalent state raises an interesting question of whether similar patterning processes occur in other developing tissues and organs. Interestingly, Pnr is expressed in a broad dorsal domain in early embryos, but later refined in a longitudinal dorsal domain extending along the thoracic and abdominal segments. During this stage, Pnr has an instructive and selector-like function, determining the identity of the medial dorsal structures. It has been shown that loss of pnr eliminates the dorsomedial pattern in the larval cuticle whereas the dorsolateral pattern extends dorsally without cell loss. This suggests that DV pattern in the larval cuticle is established with the onset of Pnr expression in the dorsomedial domain, and ventral may be the initial fate of epidermal cells (Singh, 2003).

The Drosophila ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from one another. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. This study shows that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment (Buescher, 2006).

Row 1/2 NBs are distinguished from NBs in adjacent rows by the expression of Mirror (Mirr). Mirr expression is initiated during stage 7 in transverse stripes in the NE. Across the ventral midline, the neuroectodermal expression of Mirr extends further anteriorly by 2-3 rows of cells. All row 1/2 NBs, the MNB and NB6-1 are at least transiently Mirr-positive. Staining of stage 7 midGA174 mutant embryos revealed a strong reduction of neuroectodermal Mirr expression. Loss of Mirr expression is more pronounced in odd-numbered segments. In stage 10 midGA174 mutant embryos, a strong loss of Mirr is observed in the row 1/2 NB layer predominantly in odd-numbered abdominal segments; this loss arises through the additive effects of the loss of Mirr expression in the NE (which occurs prior to NB formation) and the general loss of row 1/2 NBs. The severity of the phenotype was not enhanced in mid/H15-deficient embryos. It is noteworthy that the loss of Mirr function itself (mirrcre2) does not result in defects in NB formation suggesting that mid does not promote NB formation via Mirr. In summary, Mirr expression represents a further aspect of row 1/2 NB identity and mid, but not H15, contributes to the specification of this identity by promoting the expression of Mirr in odd- and in (to a lesser extent) even-numbered abdominal segments. The consequences of the loss of Mirr function in NB specification and lineage elaboration are as yet unknown (Buescher, 2006).

Capicua regulates follicle cell fate in the Drosophila ovary through repression of mirror

The dorsoventral axis of the Drosophila egg is established by dorsally localized activation of the epidermal growth factor receptor (Egfr) in the ovarian follicular epithelium. Subsequent positive- and negative-feedback regulation generates two dorsolateral follicle cell primordia that will produce the eggshell appendages. A dorsal midline domain of low Egfr activity between the appendage primordia defines their dorsal boundary, but little is known about the mechanisms that establish their ventral limit. This study demonstrated that the transcriptional repressor Capicua is required cell autonomously in ventral and lateral follicle cells to repress dorsal fates, and functions in this process through the repression of mirror. Interestingly, ectopic expression of mirror in the absence of capicua is observed only in the anterior half of the epithelium. It is proposed that Capicua regulates the pattern of follicle cell fates along the dorsoventral axis by blocking the induction of appendage determinants, such as mirror, by anterior positional cues (Atkey, 2006; full text of article).

In both cic homozygous egg chambers and cic mutant follicle cell clones, ectopic mirr expression is restricted to the anterior half of the epithelium, indicating that mirr is also regulated by positional information along the AP axis. Although in principle a posterior repressor could account for this effect, on the basis of prevailing models of follicular epithelium AP patterning, the hypothesis that expression of mirr requires positive input from an anterior positional cue is favored. It is propose that, in wild-type ovaries, Cic blocks the induction of mirr by this anterior signal. However on the dorsal side, where Cic becomes downregulated, this signal is not blocked, leading to the induction of mirr expression and appendage-producing fate. In cic mutant ovaries, the anterior signal induces mirr expression throughout the DV axis (Atkey, 2006).

A likely candidate for an anterior signaling molecule required for mirr expression is Dpp, which is produced by the anterior-most follicle cells and regulates gene expression along the AP axis. Coordinate regulation of mirr along the DV and AP axes provides a molecular explanation for the observation that appendage-producing fates are determined at the intersection of Egfr and Dpp signaling. Regulation of mirr by an anterior cue such as Dpp could also explain the observation that mirr expression in cic mutant ovaries is normal until stage 10B; although the cic mutant cells are competent to express mirr, detectable levels may not be induced until the posterior migration of anterior follicle cells in mid-oogenesis brings the source of Dpp to the anterior margin of the oocyte (Atkey, 2006).

In addition to invoking an anterior signal in the regulation of mirr, the data indicate that mirr is also positively regulated by dorsally restricted Egfr signaling, independent of Cic. cic mutant egg chambers exhibit ectopic mirr throughout their anterior circumference, but mirr levels remain highest dorsally. In grk;cic double mutant egg chambers this dorsal high point of mirr expression is abolished, suggesting that the wild-type dorsal anterior mirr expression pattern is the result of both dorsal and anterior inputs (Atkey, 2006).

Collectively, the data support a model in which Cic blocks the induction of mirr expression and appendage-producing fates in response to an anterior signal, for example Dpp. Egfr-mediated downregulation of Cic in dorsal anterior follicle cells therefore allows these cells to respond to Dpp, contributing to the dorsal anterior mirr expression pattern, whereas the presence of Cic in ventral and lateral follicle cells blocks their response to this cue. Within the dorsal Cic-free domain, the Rhomboid/Spi/Aos autocrine-feedback loop would regulate Egfr activity to resolve two distinct appendage primordia. In cic mutant egg chambers, all follicle cells would be competent to respond to the anterior signal, resulting in ectopic mirr expression and appendage-producing fate in the anterior follicle cells that receive the signal (Atkey, 2006).

Previous work has shown that the appendage primordia are determined at the intersection of dorsal and anterior signals, and the simplest interpretation has been that these signals function additively to specify appendage-producing fate. Instead, however, the demonstration that the distribution of Cic along the DV axis determines the competence of follicle cells to respond to AP patterning signals reveals unexpected crosstalk between DV and AP patterning signals, and indicates that Cic integrates these pathways. Along the DV axis, it is proposed that the pattern of the follicular epithelium is determined by the function of two Egfr targets, Cic and Aos, in distinct domains. High levels of Egfr activity induce production of Aos at the dorsal midline, where it antagonizes Spi, thus splitting the initial dorsal domain of Egfr activity and defining the dorsal limits of the appendage primordia. Lower levels of Egfr signaling are sufficient to downregulate Cic, defining a dorsal domain that lacks Cic and is therefore competent to adopt dorsal fates. Cic remains present in ventral and lateral follicle cells, where it blocks the induction of crucial transcriptional targets, such as mirr, by Dpp. The dorsal limit of the Cic domain thus defines the ventral limit of the appendage primordia. Cic-mediated repression of target genes may represent a general mechanism for the integration of multiple spatial inputs in a developing tissue (Atkey, 2006).

Antagonistic and cooperative actions of the EGFR and Dpp pathways on the iroquois genes regulate Drosophila mesothorax specification and patterning

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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