Targets of Activity

Several genes that regulate achaete-scute gene expression have been characterized. For example, hairy (h) and extramacrochaete (emc) act as negative regulators of ac-sc since mutations in these genes result in the generation of ectopic SOPs. Proteins encoded by these genes, as well as AC-SC, contain basic helix-loop-helix domains that have been found in a number of proteins involved in transcriptional regulation. Hairy has been shown to be a direct transcriptional regulator of ac-sc, while Emc appears to down-regulate ac-sc indirectly by interacting with other factors. Pannier (Pnr), a zinc finger protein with homology to the vertebrate transcription factor GATA-1, also acts as a negative regulator of ac-sc. The u-shaped (ush) gene is involved in transregulation of ac-sc in the dorsal region of the notum. Ush, a zinc finger protein, heterodimerizes with Pnr as a cofactor and negatively regulates the transcriptional activity of Pnr. Two clustered genes isolated from the iroquois region, araucan (ara) and caupolican (caup), show similar spatial expression and function in the wing. iroquois (iro) has been recently identified as a candidate for prepattern genes, that is, these genes are expressed in a pattern which preceeds neurogeneis in the wing imaginal disc. Since the Ara protein has been shown to directly bind to an ac-sc enhancer element, it is suggested that the pattern of expression of iro genes determines the pattern of proneural gene expression and thus the pattern of neural development in the wing disc. Therefore, the iro genes fulfill the characteristics of prepattern genes that direct sensory organ formation in the notum. In addition to the eye, mirr is expressed in the wing disc in a similar, but not identical, pattern as seen with iro genes. The role of the mirr gene has been investigated in the formation of alula and sensory organs in the wing. This study suggests that mirr acts together with other iroquois genes in the prepatterning of sensory organs and alula development (Kehl, 1998).

mirr expression was examined in the imaginal discs by in situ mRNA hybridization and immunohistological detection of the lacZ reporter expression. In the wing disc, Mirror mRNA is expressed in several regions, including the notum and pleura. mirr expression is also detected in the alula region, an accessory basal structure of the wing. Since the alula is lost in mirr mutants, it is suggested that mirr is required for alula formation. The expression pattern of mirr is very similar to that of ara and caup (Gomez-Skarmeta, 1996). However, mirr is not expressed in the precursors for L3/L5 wing veins, tegula and dorsal radius while ara and caup are expressed in these regions. The tegula is the most proximal part of the anterior wing margin. lacZ expression in the wing disc is similar to the mRNA expression pattern, suggesting that the lacZ reporter reflects the pattern of mirr expression (Kehl, 1998).

An individual bristle on the notum can be easily identified by its specific position. mirror mutations caused specific loss of macrobristles only in the lateral domain of the notum. mirr Sai1 /TM3 Sb or mirr Sai1 /TM6 heterozygotes show a dominant bristle phenotype: deletion of presutural (PS) and/or posterior supraalar (pSA) bristles. In many cases (31%), both PS and pSA bristles are absent. Approximately 90% of mirr B1-12 /mirr B1-12 flies examined were missing one bristle, although occasionally two bristles were deleted. The deletion of bristles is specifically restricted to two of the seven macrobristles in the region; the pSA and anterior postalar bristles (aPA). The strongest phenotype observed is found in mirr Sai1 /mirr B1-12 , in which up to four lateral bristles were missing. This is consistent with the observation that Mirror mRNA level is greatly reduced in the notum of this mutant wing disc. These results indicate that the elimination of lateral macrobristles by mirr mutations are allele-specific and are mainly restricted to four bristles: the PS, pSA, aPA and pPA. This is consistent with the expression of mirr in this subset of sensory organ precursors (SOPs). It is concluded that like ara and caup, mirror expression establishes the prepattern of several macrobristles in the notum (Kehl, 1998).

Ara and Caup are expressed in SOPs, acting as positive transcriptional regulators of achaete in the wing disc epithelium (Gomez-Skarmeta, 1996). Mirr is also expressed in the SOPs in the notum. Mirr and Ac expression overlap only in a subset of bristle SOPs in the lateral heminotum, including the PS and PA bristles, which are affected by mirr mutations. In contrast, SOPs for the notopleural and anterior supraalar bristles are stained with anti-Achaete, while Mirr is either not expressed or is expressed at low levels in the notopleural (NP) and aSA bristles. This is consistent with the finding that these bristles form normally in mirr mutations. These results suggest that Mirr as well as Ara and Caup might control ac-sc expression, and the loss of a subset of bristles in different mirr alleles might result from the loss of the corresponding SOPs rather than the degeneration of bristles (Kehl, 1998).

mirror controls planar polarity and equator formation through repression of fringe expression and through control of cell affinities

The Drosophila eye is divided into dorsal and ventral mirror image fields that are separated by a sharp boundary known as the equator. Mirror, a homeodomain-containing putative transcription factor with a dorsal-specific expression pattern in the eye, induces the formation of the equator at the boundary between mirror-expressing and non-expressing cells. Evidence is provided that suggests mirror regulates equator formation by two mechanisms: (1) mirror defines the location of the equator by creating a boundary of fringe expression at the mid-point of the eye. mirror creates this boundary by repressing fringe expression in the dorsal half of the eye. Significantly, a boundary of mirror expression cannot induce the formation of an equator unless a boundary of fringe expression is formed simultaneously. (2) mirror acts to sharpen the equator by reducing the mixing of dorsal and ventral cells at the equator. In support of this model, it has been shown that clones of cells lacking mirror function tend not to mix with surrounding mirror-expressing cells. The tendency of mirror-expressing and non-expressing cells to avoid mixing with each other is not determined by their differences in fringe expression. Thus mirror acts to regulate equator formation by both physically separating the dorsal cells from ventral cells, and restricting the formation of a fng expression boundary to the border where the dorsal and ventral cells meet (Yang, 1999).

The formation of an ectopic equator, associated with a fng+/fng- border is restricted to the segments of clonal borders that are located within the anterior one third of the eye, even when the borders extend more posteriorly. A Drosophila eye typically consists of 32-34 vertical columns of ommatidia. No ommatidia of ventral polarity have ever been detected at a mirr mutant clonal border beyond column 12, counted from the anterior edge. Since the ectopic expression of fng-lacZ in mirr mutant discs is also restricted to the dorsal anterior region of the eye, this data is consistent with the hypothesis that ectopic fng expression in mirr mutant clones is required to induce ectopic equator formation at the equatorial borders of clones (Yang, 1999).

It was asked directly if the formation of the ectopic equators at the equatorial borders of mirr dorsal clones is dependent on ectopic fng expression within the clones. Ectopic expression of fng within the mirr mutant clones is required to induce ectopic equators at the equatorial borders of the clones. Thus one way in which mirr regulates equator formation is by repressing fng expression in the dorsal region of the eye. Removal of fng in the dorsal region of the eye causes an ectopic mini-equator to form at the polar border of the clone. One to three ommatidia of ventral polarity are often generated at the polar boundaries of fng dorsal clones. The ectopic juxtaposition of the dorsal and ventral ommatidia at the polar boundary of a fng dorsal clone has been called a ‘mini-equator’ because of its short length. Although the expression of fng transcript is restricted to the ventral region of the eye during the early stages of eye development, it is later transiently present in a narrow band of cells associated with the furrow in the dorsal region of the eye. Thus the formation of the ectopic mini-equators may be in part due to the transient fng+/fng- boundaries created at the polar borders of fng dorsal clones as the furrow passes through the clones (Yang, 1999).

The sharpness displayed by the path of the wild-type equator is a poorly understood aspect of equator formation. Several observations have suggested that mirr is involved in controlling the sharpness of the equator. (1) A reduction in mirr expression causes a reduction in the sharpness of the equator. One role of mirr in sharpening the equator is to create a difference in cell affinities between dorsal and ventral cells. In a clonal analysis, it was noted that cells that reside inside a mirr mutant dorsal clone tend to minimize interactions with the surrounding mirr-expressing cells, resulting in a rounded clone shape. In addition, the border where mirr-expressing and non-expressing cells juxtapose appears to be significantly smoother compared to the border where no difference in mirr exists across the border. (2) mirr mutants that occasionally survive until adulthood display a dramatic dorsal protrusion from the surface of their eyes. Such a protrusion is suggestive of a group of cells attempting to sort out from the epithelium due to differences in cell affinities. (3) When mirr is overexpressed in dorsal regions of the eye through overexpression of wg, a visible indentation of the epithelium is observed at the novel boundary formed between mirr -expressing and non-expressing cells. This suggests that increasing the differences in mirr expression between dorsal and ventral cells causes them to further minimize contacts with each other, forming a physical groove between them. One important aspect of these findings is that the segregation of dorsal and ventral cells appears to be a process that is independent of the difference in their expression of fng. The shapes of mirr::fng dorsal clones remains significantly rounder than those of wild-type clones, thus the ectopic expression of fng within mirr mutant cells is not likely to be the cause for the reduction of cell-cell mixing between mirr-expressing and non-expressing cells. In addition, the shapes of fng mutant ventral clones are irregular and are not significantly different from those of wild-type ventral clones, thus the difference in fng expression between dorsal and ventral cells is unlikely to be the cause for the sharpness of the wild-type equator. Such a finding is in contrast to fng’s role in D/V border formation in the developing wing disc. In wing discs, removal of fng function in a clone of cells in the dorsal half of the wing disc, where fng is normally expressed, results in a very round clone with a smooth clonal border. In addition, ectopically expressing fng in a clone of cells in the ventral part of the dics where fng is typically absent also results in round clones with a very smooth border. It has been suggested that fng might have a role in controlling cell adhesion in the developing wing disc. Although the possibility that fng is also important in regulating cell adhesion in the eye disc cannot be ruled out, the data strongly suggests that additional components regulated by mirr must be involved. One possible model is that mirr might be regulating some adhesion molecules that are differentially expressed between dorsal and ventral cells. It is concluded that mirr regulates equator formation in the eye by two independent yet complementary pathways. mirr acts to sort the dorsal cells from ventral cells by reducing cell-cell mixing at the boundary where the dorsal and ventral cells juxtapose. In addition, it restricts the activation of Notch signaling to the point where the dorsal and ventral cells meet by repressing fng in the dorsal cells. These two functions of mirr lead to a co-ordination of morphology and signaling in the process of equator formation (Yang, 1999).

The homeobox gene mirror links EGF signaling to embryonic dorso-ventral axis formation through Notch activation

Recent studies in vertebrates and Drosophila have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. This may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis. Ectopic Notch activation has a similar long-range effect on pip. These results suggest that Mirror and Notch induce secretion of diffusible morphogens; a TGF-beta (encoded by dpp) has been identified as one such molecule in the germarium. mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and mirr then represses pipe expression in all but the ventral follicle cells, connecting Egfr activation in the dorsal follicle cells to repression of pipe in the dorsal and lateral follicle cells. These results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signaling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation (Jordan, 2000).

In oogenesis the expression patterns of mirr and fng are complementary. The expression patterns define borders between cells with specific developmental roles: the encapsulation of 16-cell germline cysts that culminates in their separation from the germarium; the boundary between terminal and lateral follicle cells at stage 6, and the boundary between dorsal anterior and all other follicle cells at stages 8-10. In the germarium, mirr is expressed in the inner sheath cells and the anterior-most follicle cells, whereas fng is expressed in the follicle cells in the posterior part of the germarium. The follicle cells at the expression boundary encapsulate the 16-cell germline cysts and subsequently separate the newly formed egg chamber from the germarium. At stage 6, when the follicle cells in the termini of the egg chamber differentiate from the lateral follicle cells and establish the oocyte anterior-posterior (A-P) polarity, mirr expression is detected in the lateral region, complementary to fng expression in the termini. As the follicle cells migrate posteriorly at stages 8 and 9, mirr expression is detected in the most dorsal anterior follicle cells, whereas fng is expressed in all other follicle cells. At this point, signaling between the oocyte and the follicle cells establishes the dorsal-ventral axis of the follicle-cell layer and the future embryo. The complementary expression patterns of mirr and fng throughout oogenesis are likely to be a consequence of Mirr repression of fng expression. Follicle-cell clones made with a loss-of-function allele of mirr result in an expansion of the fng expression pattern into dorsal anterior follicle cells, and, conversely, overexpression of mirr results in the loss of fng expression (Jordan, 2000).

The role of Mirr has been investigated by examining egg chambers from females with altered Mirr function. In mirr-mutant females, defects in encapsulation of the 16-cell cyst and separation from the germarium are observed. At stage 6 mirr expression is excluded from the follicle cells at the termini of the egg chamber; ectopic expression of mirr throughout the follicle cells, beginning at this stage, reduces the size of the expression domain of the anterior (dpp and L53B) and posterior (pntP1) terminal markers. Thus, mirr expression must be excluded from the termini for proper differentiation of those regions. Ectopic expression of Mirr also perturbs the oocyte anterior-posterior axis, consistent with the function of posterior terminal follicle cells in the establishment of this axis. Later in oogenesis, when dorsal and ventral follicle cells are differentiating, mirr is expressed in dorsal anterior follicle cells and reduction or loss of dorsal structures is evident in mirr loss-of-function egg chambers. Loss of mirr function results in expansion of the ventral expression of pip, a gene required for embryonic dorsal-ventral axis formation, to the lateral and dorsal follicle cells in stages 9 and 10. In contrast, ectopic expression of mirr in the ventral follicle cells at this stage causes dorsalization of the eggshell and embryo. Thus Mirr is required for proper dorsal-ventral axis formation and mirr expression must be restricted to the dorsal region for correct ventral patterning to occur. In summary, either loss of Mirr function or ectopic expression of Mirr disrupts follicle-cell differentiation at the three stages of oogenesis in which a Mirr-Fng expression border is observed (Jordan, 2000).

In a number of developmental systems, regulation of fng by a homeobox gene has a role in establishing a domain in which Notch is activated. Thus the phenotypes observed in mirr and Notch (N) mutants during oogenesis have been compared. In oogenesis, Notch activity is required in the germarium and for the formation of the termini at stage 6. A test was performed to see whether Notch function is also required for dorsal-ventral patterning of follicle cells by analysing the eggs laid by Nts females at the restrictive temperature. The strongest phenotype observed in eggs laid by Nts females is similar to that observed in eggs laid by mirr loss-of-function females: a complete loss of the dorsal appendages. In addition, the ventral pip expression domain is defective in Nts females and restricted due to expression of constitutively active Notch. Thus Notch, like Mirr, functions to restrict pipe expression to the ventral region and to organize dorsal structures; loss of either Mirr or Notch function affects follicle cells on both sides of the Mirr-Fng expression border (Jordan, 2000).

Activation of Notch at a fng expression border has been observed in wing and eye development. In the wing this border acts as an organizing center by producing a morphogen, Wingless, that acts on cells on both sides of the border. At stage 9 in oogenesis the mirr-fng expression border and a region of localized Notch activation are approximately 10 cell diameters from the ventral pip expression border. Nevertheless, reduction of mirr expression expands the pip domain laterally. If a Mirr-Fng border activates Notch locally to produce a morphogen that represses pip, a reduction of pip expression should be seen upon expansion of the mirr expression domain or ectopic activation of Notch. To examine this, mirr was expressed ectopically in anterior follicle cells. pip repression occurs 5-7 cell diameters beyond the mirr expression domain, showing that the effect of Mirr on pip is non-cell autonomous and supporting the idea that a Mirr-Fng border generates a pip-repressing agent. To further test the effect of ectopic Mirr expression, Mirr was expressed in the posterior follicle and the effect on pip and rho, which is normally expressed as two stripes on the dorsal region at stage 10, was tested. Such ectopic mirr expression induces a ring of rho expression and represses pip at a distance. Expression of constitutively active Notch in the posterior follicle cells also represses pip expression at a distance. These results suggest that Mirr and Notch induce secretion of a diffusible molecule that represses pip. Although it is not known what the Notch-dependent diffusible molecule is at stage 9, it was found that dpp is expressed in follicle cells in the mid-germarium near a stripe of cells showing localized Notch activity in a Notch-dependent manner. Furthermore, in follicle cell clones of MAD or MEDEA (downstream effectors of the Dpp pathway), encapsulation defects of 16-cell cysts are seen. This phenotype is similar to Notch- and mirr-mutant phenotypes in the germarium, suggesting that Dpp may be a morphogen induced by Notch activity in the germarium (Jordan, 2000).

Local activation of Notch in a number of developmental systems is achieved by spatially restricted expression of a homeodomain protein that either represses or induces fng expression, generating a border of fng expressing and non-expressing cells. It is less clear how the initial asymmetric expression of the homeobox protein is generated. Because the dorsal anterior expression of mirr is characteristic of a number of genes regulated by the Egfr pathway, mirr expression was analyzed in mutants that lack Gurken, one of the ligands for Egfr. In these egg chambers, the dorsal anterior pattern of mirr expression is reduced or lost, showing that activation of the Egfr pathway is necessary for mirr expression. However, the patterns of oogenesis in the germaria at stage 6 and in the centripetally migrating cells are unaltered, indicating that either another Egfr ligand or another pathway regulates mirr expression at these stages (Jordan, 2000).

Results from several developmental systems have led to the idea that the trio of a homeobox gene, FNG and Notch are fundamental to organogenesis. It is suggested that Mirr, Fng and Notch are part of a conserved mechanism for dividing epithelial cell layers into domains; it is thought that such a mechanism is not restricted to organogenesis. Furthermore, the data suggest that Mirr integrates the Egfr and Notch pathways in oogenesis: mirr transcription is induced by the Egfr pathway, and Mirr in turn spatially regulates fng expression leading to a Notch activation border. Finally, it is proposed that the link between Egfr pathway signaling in the dorsal follicle cells and the differentiation of the ventral follicle cells suggested by genetic studies is mediated by Mirr. The Egfr pathway induces mirr expression, which leads to creation of a Notch-Fng border in lateral follicle cells from which molecules are secreted that repress pipe expression. Pipe regulates the activity of a protease cascade that activates Toll and ultimately determines the dorsal-ventral pattern of the Drosophila embryo. These data show that expression of pip in the ventral follicle cells is not a direct consequence of a graded germline signal by Gurken, but depends on Mirr-dependent long-range signals from dorsal follicle cells. Mirr therefore connects the well-studied events in early and late Drosophila dorsal-ventral axis formation (Jordan, 2000).

Iroquois transcription factors recognize a unique motif to mediate transcriptional repression in vivo

Iroquois transcription factors regulate diverse aspects of developmental patterning in all metazoans. Despite their widespread importance, the direct targets of the Iroquois are poorly understood. This study used in vitro site selection to define the DNA-binding preference of the Drosophila Iroquois Mirror. Electrophoretic mobility shift assays were used to determine the critical nucleotides for Mirror binding and to show that this site is recognized by other Drosophila Iroquois transcription factors. This site also is recognized by vertebrate Iroquois transcription factors. Transgenic analysis demonstrates that Drosophila Iroquois proteins recognize this site in vivo to mediate transcriptional repression. It was further shown that Iroquois transcription factors form homodimers and heterodimers, suggesting that combinatorial binding may contribute to gene regulation by this family (Bilioni, 2005).

Visual inspection of selected sequences has revealed an enrichment of ACA and TGT sequences, with a preponderance of palindromic sequences of ACAnnTGT. There were very few examples of the putative Iroquois binding site TAAT in the sequences. To further analyze the pool of sequences, MEME analysis was used. The MEME identified motif was [A/G],A,[A/T],[A/T]-ACA-[C/T],[G/A]-TGT-[T/A],A,[A/T] (e = 3.6e-062; bold type signifies strong bias). Another way of analyzing the results of the site selection is the use of a positional weight matrix (PWM). From both the MEME and the PWM analyses, it is evident that the in vitro site selection has identified a Mirr consensus motif [A/G]AAAACACGTGTTAA (Bilioni, 2005).

To determine which nucleotides are essential for Mirr binding, 32P-labeled oligonucleotides with point mutations were tested in EMSA. Analysis of the sequences derived from both experiments indicated a common core sequence of ACACGTGT, with an A/T-rich region on either side of the palindrome. Whether the A/T-rich flanking region was essential was tested by EMSA and it was found that Mirr can bind ACACGTGT in the context of several different flanking sequences. The complex can be supershifted by the addition of Abs to Mirr or to the FLAG tag, demonstrating that ACACGTGT is sufficient for specific Mirr binding (Bilioni, 2005).

The experiments that implicated a TAAT sequence had been performed using a construct of HD containing fragment of Ara, rather than the full-length protein. It was reasoned that a construct of HD alone might have reduced specificity. This hypothesis was tested by generating a FLAG-tagged Mirr HD-only construct, containing only the 63-aa HD of Mirr, and binding on the L3 enhancer (502-bp region flanking the Fng-Iro binding site) was examined. Strikingly, it was found that the Mirr HD construct binds equally well to both the L3 enhancer and to the ACAnnTGT site. Binding is specific, because the complex can be supershifted by the addition of FLAG Abs. Mirr HD fails to bind an L3 enhancer that carries a point mutation within the half-site (S1), indicating it is recognizing the ACA motif. The Mirr HD construct fails to bind a classic HOX site, suggesting that some specificity is retained (Bilioni, 2005).

The palindromic nature of the IBS suggests that Iro proteins might form dimers on DNA. Dimer formation also is supported by the observation that a 2-nt spacer is essential for high-affinity binding. To directly test whether Iro proteins form dimers, a hemagglutinin (HA)-tagged version of Mirr was created and cotranslated with FLAG-tagged Mirr. Addition of Abs to either the HA-tag or the FLAG-tag supershifted the complex. Addition of both Abs produced a super-supershift, indicating that both FLAG- and HA-tagged Mirr were present in the DNA-protein complex (Bilioni, 2005).

A FLAG-tagged Mirr construct that lacks the N-terminal domain but includes the HD binds weakly and fails to show a super-supershift when mixed with full-length Mirr, suggesting that the N-terminal domain is important for dimer formation. Incubation of FLAG-tagged Ara and HA-tagged Mirr also resulted in a super-supershift, indicating that Mirr can form heterodimers with Ara on the Iro binding site (IBS). Pull-down experiments indicate that Mirr can form dimers independent of the presence of DNA. The formation of Iro homodimers and heterodimers, together with the differences in strength of binding of the different Iro for the IBS, suggests that the overlapping expression patterns of the Iro seen in both vertebrates and invertebrates may result in a combinatorial control of gene expression by the Iro (Bilioni, 2005).

The EMSA data show that Mirr binds specifically and with high affinity to the IBS. However, these data do not demonstrate that this site has in vivo relevance. To directly test whether the IBS is functional in vivo, transgenic flies were generated carrying four repeats of the IBS in a ß-gal reporter construct. Because mirr functions in vivo to repress expression of fng, these IBSs were placed in a vector that carries binding sites for the transcriptional activator Grainyhead (GBEs). The GBEs provide a general transcriptional activation, allowing a predicted transcriptional repression in response to Mirr to be detected (Bilioni, 2005).

Mirr and the other Iro are expressed in the dorsal half of the eye where they act to repress fng, resulting in the ventral fng expression. Inclusion of four IBSs in the LacZ reporter results in repression of ß-gal expression in the dorsal region, where Mirr is expressed. In contrast, the parental reporter vector has ubiquitous ß-gal expression. To directly test whether Mirr binding leads to repression of ß-gal, the effects of Mirr overexpression on the IBS reporter were examined. The Gal4 system was used to ectopically express Mirr in the ventral half of the eye. Significantly, overexpression of Mirr results in a dramatic reduction in ß-gal expression, indicating that Mirr can recognize the IBS to mediate transcriptional repression in vivo (Bilioni, 2005).

To determine whether Mirr also acts as a repressor on endogenous enhancers, the fng genomic region was examined. There is an IBS located 193 bp downstream of fng. EMSA analysis confirms that this IBS binds Mirr. The 500-bp downstream region containing the fng-IBS was cloned into a luciferase reporter vector and the effects of altering Mirr levels on reporter expression were examined. S2 cells contain endogenous mirr mRNA and protein. Depletion of mirr by RNAi leads to significant increases in reporter activity, indicating that physiological levels of Mirr repress transcription. Mutation of the fng-IBS increases reporter activity to the same level as removal of Mirr, consistent with Mirr repressing transcription through the IBS. Depletion of an irrelevant protein (GFP) had no effect on Mirr levels or reporter activity. Together, these data strongly support the IBS as a biologically relevant Mirr-binding site and suggest that the Iro directly represses fng expression in vivo (Bilioni, 2005).

Although these data indicate that Mirr acts as a transcriptional repressor in the eye, the possibility that Mirr may act as a transcriptional activator in other contexts cannot be excluded. Recent work has shown that phosphorylation can convert the vertebrate Iro, IRX2, from a transcriptional repressor to an activator, suggesting that regulation of transcription by Mirr also may be context dependent. A major goal for the future is to understand how Iro transcription factors regulate gene expression to direct the development of complex structures (Bilioni, 2005).

Dpp of posterior origin patterns the proximal region of the wing

The decapentaplegic (dpp) gene encodes a long-range morphogen that plays a key role in the patterning of the wing imaginal disc of Drosophila. The current view is that dpp is transcriptionally active in a narrow band of anterior compartment cells close to the anterio-posterior (A/P) compartment border. Once the Dpp protein is synthesised, it travels across the A/P border and diffuses forming concentration gradients in the two compartments. A new site of dpp expression has been found in the posterior wing compartment that appears during the third larval period. This source of Dpp signal generates a local gradient of Dpp pathway activity that is independent of that originating in the anterior compartment. This posterior tier of Dpp activity is functionally required for normal wing development: the elimination of dpp expression in the posterior compartment results in defective adult wings in which pattern elements such as the alula and much of the axillary cord are not formed. Moreover, these structures develop normally in the absence of anterior dpp expression. Thus the normal wing pattern requires distinct Dpp organizer activities in the anterior and posterior compartments. It was further shown that, unlike the anterior dpp expression domain, the posterior one is not dependent on Hedgehog activity but is dependant on the activity of the IRO complex gene mirror. Since there is a similar expression in the haltere disc, it is suggested that this late appearing posterior Dpp activity may be an attribute of dorsal thoracic discs (Foronda, 2009).

The approach taken to identify the factor/s behind this posterior Dpp expression was to look for candidate genes or signalling pathways which are expressed in the corresponding place in the posterior compartment. The first one was vein, a ligand of EGFR signalling pathway, which coexpresses with Dpp in late 3rd instar wing disc. vein is the only EGFR ligand required for a proper wing development, so it was a good candidate. The elimination of all posterior vein function has effect neither on posterior Dpp function nor on hinge morphology. Other genes were tested based on expression and/or mutant phenotype in the alula, i.e., homothorax, Zfh2 and empty spiracles, among others. None of them affected Dpp expression (Foronda, 2009).

Another likely candidate was mirror, a member of the Iroquois complex (iro-C), for which a role in alula and axillary cord formation has been described. mirr expression was examined using the mirr-lacZ line and it was found that mirr is expressed in the presumptive alula and axillary cord region (Foronda, 2009).

M+ mirr clones were made to generate posterior compartments that were wholly mirr. They show an adult phenotype more extreme than that of dpp compartments: the alula and the axillary cord are entirely missing. In the discs dpp expression (shown by pMad staining) in the posterior compartment is lost, and consequently brk expression is up-regulated in the presumptive alula region. This result indicates that mirr is necessary for posterior dpp expression. In contrast, Dpp is not required for mirr expression, since the lack of posterior Dpp does not have an effect on mirr-LacZ transcription (Foronda, 2009).

Since the preceding results might suggest a mirr-mediated dpp activation gain of function clones of mirr were generated and whether they gave rise to ectopic Dpp activity was checked. NI significant up-regulation of dpp associated with those clones was detected. These experiments demonstrate that mirr activity is necessary for posterior dpp expression, but it is not sufficient to induce it. Therefore there must be other factors involved in posterior dpp activation (Foronda, 2009).

Transcriptional interpretation of the EGF receptor signaling gradient

Epidermal growth factor receptor (EGFR) controls a wide range of developmental events, from body axes specification in insects to cardiac development in humans. During Drosophila oogenesis, a gradient of EGFR activation patterns the follicular epithelium. Multiple transcriptional targets of EGFR in this tissue have been identified, but their regulatory elements are essentially unknown. This study reports the regulatory elements of broad (br) and pipe (pip), two important targets of EGFR signaling in Drosophila oogenesis. br is expressed in a complex pattern that prefigures the formation of respiratory eggshell appendages. This pattern is generated by dynamic activities of two regulatory elements, which display different responses to Pointed, Capicua, and Mirror, transcription factors involved in the EGFR-mediated gene expression. One of these elements is active in a pattern similar to pip, a gene repressed by EGFR and essential for establishing the dorsoventral polarity of the embryo. This similarity of expression depends on a common sequence motif that binds Mirror in vitro and is essential for transcriptional repression in vivo (Fuchs, 2012).

Current models of pattern formation in Drosophila oogenesis involve multiple components, signaling pathways, and network motifs. Critical tests of these models require direct analysis of the cis-regulatory sequences of genes comprising the network. As a first step in this direction, this study identified the regulatory elements of br, a gene that plays a key role in eggshell patterning and morphogenesis. The dynamic pattern of br was found to be generated by superposition of the activities of two distinct regulatory regions, which drive br expression in nonoverlapping regions of space and display differential sensitivity to three transcription factors that act downstream of EGFR (Fuchs, 2012).

It was shown that loss of Mirr induces ectopic br expression in the dorsal midline follicle cells, but leads to a complete loss of br in the lateral cells, which form dorsal appendages. This region-specific effect can be now explained, and is fully consistent with our finding that Mirr represses the brE and activates brL regions, respectively. Previous studies suggest that Mirr functions as a dedicated repressor. Based on this theory, it is speculated that the activating effect of Mirr on the expression of the brL region is indirect and involves intermediate factors. In contrast, these results strongly suggest that Mirr represses the brE region directly (Fuchs, 2012).

In contrast to the brL region, which generates br expression in a two-domain pattern that is necessary for the formation of two eggshell appendages, the function of the brE region is unclear. At the same time, this regulatory region was instrumental in identification of a critical cis-element that controls the expression of pip, a gene which must be repressed in the dorsal follicle cells for proper induction of the DV polarity of the embryo. The regulatory regions of both br and pip contain a sequence essential for their transcriptional restriction to the ventral follicle cells. Moreover, the data suggest that the identified sequence is a direct sensor of Mirr, which is derepressed by EGFR. Thus, thus this study has upheld an earlier proposal that Mirr connects the EGFR-mediated patterning of the follicle cells to the DV patterning of the embryo. In the emerging transcriptional cascade, EGFR signaling down-regulates CIC, which derepresses Mirr, which in turn represses pip (Fuchs, 2012).

Previous studies have demonstrated that Mirr can repress pip, but suggested that this effect requires a relay mechanism. The current results, based on marked mirr overexpression clones, demonstrate that the effect is cell-autonomous. Other studies argue against Mirr-dependent pip repression, based on the fact that mirr mutant clones did not induce ectopic expression of pip. These results may be because of the fact that the mirr allele that was used is not a complete null and has residual activity sufficient for pip repression. It is argued that the current data, demonstrating pip derepression by deletion of a sequence that binds Mirr, provide a strong support for Mirr-dependent repression of pip. Thus, these findings close a long-standing gap in the chain of events that convert EGFR signaling to pipe repression, a key step in transmitting the DV polarity from the egg to the embryo (Fuchs, 2012).

EGFR-dependent patterning of the follicle cells and the resulting effects for patterning of the embryo represent canonical examples of inductive effects in development. Indeed, genetic connection between EGFR signaling and pipe repression are found in essentially all textbooks of development. However, as discussed above, the identity of transcription factors involved in pipe regulation remained controversial and the cis-regulatory sequences responsible for pipe repression were unknown. The current results, which established Mirr as a direct repressor or pipe and identified the regulatory element responding to Mirr, clearly change this status. Thus, the results provide a significant addition to a very important model of inductive signaling. The regulatory element of pipe was discovered using an approach that harnesses both conventional and modern techniques of gene regulation research and can be extended to other transcriptional targets of EGFR pathway in the follicle cells. Finally, it is noted that most of the available information on the transcriptional effects of EGFR signaling is related to gene activation (mediated by Pnt) or derepression (mediated by Cic). The current work reveals a mechanism for EGFR-dependent gene repression, mediated by Mirr. Given the central role played by the EGFR signaling in development, the identified regulatory sequences can shed light on other EGFR-dependent pattern formation events (Fuchs, 2012).

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

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