Transcriptional regulation (part 3/3)

A new Drosophila Pax gene, sparkling, implicated in eye development, has been isolated and shown to encode the homolog of the vertebrate Pax2, Pax5, and Pax8 proteins. It is expressed in the embryonic nervous system, and in cone, primary pigment, and bristle cells of larval and pupal eye discs. Transcripts are expressed in the posterior portion of the eye disc, with the anterior boundary of expression lagging clearly behind the morphogenetic furrow. In spa(pol) mutants, a deletion of an enhancer abolishes Spa expression in cone and primary pigment cells and results in a severely disturbed development of non-neuronal ommatidial cells. Because Spa is not expressed in R7 cells, its expression in newly recruited cone cells distinguishes their fate from that of R7 cells. Lozenge may be the transcription factor whose synthesis would have to precede that of Spa, which is required for the specification of the R7 equivalence group, including R1/R6, R7 and the cone cells. Lozenge helps define the R7 equivalence group by repressing seven-up (Fu, 1997).

Spa expression is further required for activation of cut in cone cells and of the Bar locus in primary pigment cells. Cut expression is strongly reduced in cone cell of spa(pol) mutants compared to wild type. Interestingly, Cut expression recovers; by 45 hours after pupariation it has risen to levels even above those of wild-type. The lack of Spa protein in cone cells appears to delay the development of the cells, since the shape of their nuclei and the nuclear accumulation of Cut resemble those of earlier stages in wild-type pupal discs. This delay may be caused by a late larval and early pupal requirement of Spa for cut activation, which later becomes independent of Spa. Expression of cut in bristle cells, many of which are mispositioned, appears unaffected during these stages. Expression of both Bar proteins in primary pigment cells is abolished completely in spa(pol) mutants. However, it remains unaffected in the irregularly positioned bristle cells, which continue to express Spa protein. Thus Spa exerts at least part of its control of primary pigment cell development through its regulation of Bar expression. Bar is also expressed in R1 and R6 precuror cells, where Lozenge rather than Spa is one of its activators. It is suggested that close functional analogies exist between Spa and Pax2 in the development of the insect and vertebrate eye. In the absence in Pax2, the optic stalk epithelium develops into pigmented retina and fails to proliferate and differentiate into glial cells, which populate the optic nerve and are essential for guidance of the retinal axons. Thus the cone cell in Drosophila might be considered as a kind of neuronal support, or glial --a cell that may have evolved from a more primitive ancestral glial cell. In favor of such a hypothesis, it is observed that spa is expressed in glial cells in the developing PNS (Fu, 1997).

Cell interactions mediated by Notch-family receptors have been implicated in the specification of tissue boundaries in vertebrate and insect development. Although Notch ligands are often widely expressed, tightly localized activation of Notch is critical for the formation of sharp boundaries. Evidence is presented that the POU domain protein Nubbin contributes to the formation of a sharp dorsoventral (DV) boundary in the Drosophila wing. Nubbin represses Notch-dependent target genes and sets a threshold for Notch activity that defines the spatial domain of boundary-specific gene expression (Neumann, 1998).

Certain features of the abnormal wings in flies mutant for nubbin suggest a possible role for Nubbin protein in spatially limiting Notch activity at the DV boundary of the wing. The row of sensory bristles that makes up the wing margin is disorganized in nubbin wing mutants, suggesting a defect in Wingless or Notch activity. In preparations where the wing margin is viewed edge on, this disorganization reflects a broadening of the region where bristles form. Margin bristles are normally specified in cells very close to the DV boundary, reflecting a requirement for high levels of Wingless signaling activity. The broadening of the margin suggests that Wingless might be ectopically expressed in nubbin mutant wing discs. Wingless is normally expressed in a stripe of two to three cells straddling the DV boundary. In nubbin mutant discs, this stripe is widened considerably. Expression of the Notch targets vestigial and cut are similarly expanded at the DV boundary in nubbin mutants (Neumann, 1998).

During Drosophila wing development growth and patterning are mediated by signaling from the dorsoventral (D/V) organizer. In the metathorax, wing development is essentially suppressed by the homeotic selector gene Ubx to mediate development of a pair of tiny balancing organs, the halteres. Expression of Ubx in the haltere D/V boundary down-regulates the haltere's D/V organizer signaling compared to that of the wing D/V boundary. Somatic loss of Ubx from the haltere D/V boundary thus results in the formation of a wing-type D/V organizer in the haltere field. Long-distance signaling from this organizer was analyzed by assaying the ability of a Ubx minus clone induced in the haltere D/V boundary to effect homeotic transformation of capitellum cells (the capitellum is the main body of the haltere) away from the boundary. The clonally restored wing D/V organizer in mosaic halteres not only enhances the homeotic transformation of Ubx minus cells in the capitellum but also causes homeotic transformation of even Ubx plus cells in a genetic background known to induce excessive cell proliferation in the imaginal discs. In addition to demonstrating a non-cell-autonomous role for Ubx during haltere development, these results reveal distinct spatial roles for Ubx during maintenance of cell fate and patterning in the halteres. Ubx modulates the expression of wingless and cut in the haltere D/V boundary and represses vestigial in the capitellum, thereby suggesting a mechanism for the Ubx mediated down-regulation of the D/V organizer activity in the haltere. While the repression of wingless and cut expression is cell-autonomous, that of the quadrant vestigial-lacZ is non cell-autonomous: pouch cells farther away from the D/V boundary show more severe reduction in lacZ expression. Given the fact that quadrant vestigial-lacZ repression is dependent on the formation of the D/V boundary, the non-cell-autonomy in quadrant vestigial-lacZ repression by ectopic Ubx would not be surprising if Ubx function is to negatively regulate D/V signaling. Thus all the results provide strong evidence for the negative regulation of D/V organizer signaling by Ubx during haltere specification. It is likely that during haltere development, repression of wing patterning signals results in the specification of cell shape and volume that are unique to the haltere (Shashidhara, 1999).

A number of wing scalloping mutations were examined to determine their effects on the mutant phenotype of cut mutations and on the expression of the Cut protein. The mutations fall into two broad classes, those which interact synergistically with weak cut wing mutations to produce a more extreme wing phenotype than either mutation alone and those that have a simple additive effect with weak cut wing mutations. The synergistically interacting mutations are alleles of the Notch, Serrate and scalloped genes. These mutations affect development of the wing margin in a manner similar to the cut wing mutations. The mutations inactivate the cut transcriptional enhancer for the wing margin mechanoreceptors and noninnervated bristles and prevent differentiation of the organs (Jack, 1992).

Armadillo, dishevelled and shaggy/zeste white 3 encode elements of a unique wingless signaling pathway used several times throughout development. cut and achaete are targets of shaggy signalling in the wing margin region, reflecting the activity of wg and probably mediating its function. Thus an important aspect of wingless function is the enhancement of neurogenesis, acting through Shaggy to regulate transcription of cut and achaete. The functional relationship between these genes and wg is the same as that which exists during the patterning of the larval epidermis (Couso, 1994).

cut and wingless as possible Notch targets are suggested by the results of Notch overexpression in the wing disc. Notch gain-of-function alleles in which Notch activity is not restricted to the dorsoventral boundary cause misexpression of cut and wingless and overgrowth of the disc, illustrating the importance of localised Notch activation for wing development (de Celis, 1996).

Notch function is required at the dorsoventral boundary of the developing Drosophila wing for its normal growth and patterning. Clones of cells expressing either Notch or its ligands Delta and Serrate in the wing mimic Notch activation at the dorsoventral boundary, producing non-autonomous effects on proliferation and activating expression of the target genes E(spl), wingless and cut. The analysis of these clones reveals several mechanisms important for maintaining and delimiting Notch function at the dorsoventral boundary:

Thus the combined effects of Notch and its target genes cut and wingless regulate the expression of Notch ligands, which restricts Notch activity to the dorsoventral boundary (de Celis, 1997).

Notch-dependent activation of wg, cut and vestigial at the wing margin depends on the activity of Suppressor of Hairless. Su(H)-mutant cells lose expression of the vestigial early enhancer, of wingless and of cut in a cell autonomous manner. Clones of Su(H)-mutant cells cause loss of wing tissue and scalloping of the wing, but only in Notch mutant clones at the D/V boundary. vestigial expression at the D/V boundary does not depend on wingless, since misexpression of wild-type wg cDNA, which results in wing margin bristles, does not cause an expansion of vestigial expression. Likewise, wingless expression does not depend on an early function of vestigial. Both Notch and wingless cooperate to activate cut at the D/V boundary. Later expression of vestigial in the wing pouch is, however, wingless dependent. vestigial is expressed in a broad domain throughout the wing. Removing of Wg activity in late second instar larvae leads to almost complete loss of the secondary expression of vestigial in the wing pouch without affecting expression at the D/V boundary. Taken together with the observation that clones of cells lacking shaggy activity show a cell-autonomous increase of Vestigial expression, these results suggest the vestigial is a direct target of the Wg pathway (Neumann, 1996).

Strawberry notch is a nuclear protein that functions downstream of Notch. Subjecting temperature sensitive strawberry notch to heat shock results in a down regulation of wg at the wing margin. Expression of wg in other regions of the wing disc as well as in other imaginal discs is unaffected by the loss of sno function. Likewise sno is required for the expression of vestigial, cut and E(spl)-m8 at the wing margin (Majumdar, 1997).

The role of the Notch and Wingless signaling pathways has been investigated in the maintenance of wing margin identity through the study of cut, a homeobox-containing transcription factor and a late-arising margin-specific marker. By late third instar, a tripartite domain of gene expression can be identified in the area of the dorsoventral compartment boundary, which marks the presumptive wing margin. A central domain of cut- and wingless-expressing cells is flanked on the dorsal and ventral side by domains of cells expressing elevated levels of the Notch ligands Delta and Serrate. Cut acts to maintain margin wingless expression, providing a potential explanation for the cut mutant phenotype. Notch, but not Wingless signaling, is autonomously required for cut expression. Rather, Wingless is required indirectly for cut expression; the results suggest this requirement is due to the regulation by wingless of Delta and Serrate expression in cells flanking the cut and wingless expression domains. Delta and Serrate play a dual role in the regulation of cut and wingless expression. Normal, high levels of Delta and Serrate can trigger cut and wingless expression in adjacent cells lacking Delta and Serrate. However, high levels of Delta and Serrate also act in a dominant negative fashion, since cells expressing such levels cannot themselves express cut or wingless. It is proposed that the boundary of Notch ligand along the normal margin plays a similar role as part of a dynamic feedback loop that maintains the tripartite pattern of margin gene expression (Micchelli, 1997).

E(spl) proteins activate cut expression

A common consequence of Notch signaling in Drosophila is the transcriptional activation of seven Enhancer of split [E(spl)] genes, which encode a family of closely related basic-helix-loop-helix transcriptional repressors. Different E(spl) proteins can functionally substitute for each other, hampering loss-of-function genetic analysis and raising the question of whether any specialization exists within the family. Each individual E(spl) gene was expressed using the GAL4-UAS system in order to analyse each gene's effect in a number of cell fate decisions taking place in the wing imaginal disk. A focus was placed on sensory organ precursor determination, wing vein determination and wing pattern formation. All of the E(spl) proteins affect the first two processes in the same way: they antagonize neural precursor and vein fates. Yet the efficacy of this antagonism is quite distinct: E(spl)mbeta, which is normally expressed in intervein regions, has the strongest vein suppression effect, whereas E(spl)m8 and E(spl)m7 are the most active bristle suppressors. While E(spl)m8 is more effective in abolishing the notum microchaeta fate, E(spl)m7 is most active against wing margin bristles (Ligoxygakis, 1999).

During wing patterning, Notch activity orchestrates a complex sequence of events that define the dorsoventral boundary of the wing. Two phases within this process have been discerned, based on the sensitivity of N loss-of-function phenotypes to concomitant expression of E(spl) genes. E(spl) proteins are initially involved in repression of the vg quadrant enhancer, whereas later they appear to relay the Notch signal that triggers activation of cut expression. Of the seven proteins, E(spl)mgamma is most active in both of these processes (Ligoxygakis, 1999).

How do E(spl) proteins, implicated in gene processing, come to activate cut expression? The present work suggests that cut expression requires Gro and partly also depends on E(spl)bHLH factors. One possibility is that E(spl) [at least E(spl)mgamma and E(spl)mdelta], like a number of other transcription factors, might have a dual function as either a transcriptional repressor or an activator, depending on context. Such an activation role has never been suggested before for either E(spl) or Gro. Alternatively, two models can be envisaged that reconcile a repressor activity of E(spl) proteins with their role in cut activation. In one, E(spl) can act by repressing a negative regulator of cut transcription. In the other, E(spl) can repress a negative regulator of Notch signaling. In the latter case, E(spl) expression would promote a positive feedback loop to enhance Notch signaling, thus increasing the signaling output from the severely compromised Nts1 receptor at the restrictive temperature. The fact that no restoration is observed in the expression of two other Notch targets, namely wg and E(spl)m8-lacZ , argues against this hypothesis. A direct role of E(spl)mgamma and E(spl)mdelta in cut expression is favored, either as activators or as repressors of a repressor, but not as general positive regulators of Notch signaling (Ligoxygakis, 1999).

Ectopic expression of E(spl)mgamma/E(spl)mdelta is not sufficient for cut expression in a wild-type background. Rather, it appears that the ability of ectopic E(spl)mgamma/E(spl)mdelta to induce cut is spatially restricted to the normal domain of cut expression. Since activated Notch is sufficient to ectopically turn on cut, it follows that some other Notch-responsive event, other than E(spl) expression, must also contribute to cut expression. This is consistent with findings that early reduction of Notch activity abolishes cut expression despite concomitant ectopic expression of E(spl)mdelta. Molecular analysis has shown that cut expression requires the transcription factor Scalloped (Sd). sd is a candidate target gene of Vg, which in turn is initially activated by Notch independent of E(spl). It is possible that expression of vg and sd at the wing margin during early L3 could make these cells competent for cut expression. This would only be initiated later, when a second pulse of Notch signaling during mid-L3 activates (or relieves the repression of) cut via E(spl)mgamma or another Gro-interacting protein. In conclusion, E(spl) proteins have partially redundant functions, yet they have evolved distinct preferences in implementing different cell fate decisions, which closely match their individual normal expression patterns (Ligoxygakis, 1999).

Notch regulations cut expression along the presumptive wing margin

In the Drosophila wing the cut gene is activated by Notch signaling along the dorso-ventral boundary but not in other cell types. Additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway, are expressed specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of Vestigial the cut trans-regulator. Additional regulatory input comes from the Wingless pathway. These data support a model whereby context specific involvement of distinct co-regulators modulates Notch target gene activation (Nagel, 2001).

These data show that the complex regulation of ct along the D/V boundary is based on a bifurcation of the Notch signaling pathway. Most signals from the Notch pathway are mediated by Su(H), which seems to act as a repressor on its own that is converted to an activator by Nact. Since Su(H) has the capacity to bind directly to the ct wing margin enhancer, the repression of ct by Su(H) and the activation by Nact/Su(H) might be direct. However, although sufficient for the activation of ct along the D/V boundary, a number of additional factors downstream of Nact are required. These include the products of wing fate selector genes vg and sd, that seem to be, together with Sno, part of a multi-factor trans-activation complex that binds to the ct wing margin enhancer. Thereby, Sd binds directly to the ct promoter, presumably recruiting the other factors by protein-protein interactions. In agreement with this hypothesis, respective physical interactions are observed between Vg and Sd or Sno. However, all three genes are targets of the Notch signaling pathway and are activated upon the overexpression of Su(H) specifically within presumptive wing tissue. Activation of Vg is also observed also within the wing pouch, although Su(H) acts as a repressor on the vg quadrant enhancer, indicating that the isolated enhancer elements reveal only a subset of the normal pattern and might contribute differently in a wild type context (Nagel, 2001).

A combination of Sd and Su(H) binding sites is sufficient to drive expression along the D/V boundary within the wing anlagen. This synthetic enhancer is too simplified to faithfully model ct regulation. Since the overexpression of Su(H) affects the accumulation of all the important trans-activator components, Vg, Sd, Sno and Su(H) itself, ct expression would be expected. Instead, repression of ct is observed: this might be due to a lack of Nact as co-activator of Su(H). However, repression can be overcome by concurrent expression of Wg resulting in strong ct activation. It is concluded that factors downstream of the Wg signaling cascade are able to convert Su(H) from a repressor to an activator, maybe by supplying a respective co-activator or by a cooperative combinatorial activity, e.g. together with the Wg signaling mediator dTCF, in accordance with a presumptive dTCF binding site within the ct wing margin enhancer (Nagel, 2001).

These signaling events appear to be unique to the activation of ct along the D/V boundary of the wing disc. Another important role of ct is the specification of external sensory organ cells during embryogenesis and imaginal development alike. Although Notch signaling is essential for setting up the correct number of neuronal cells in the peripheral nervous system by lateral specification, it appears not to be involved in the transcriptional activation of ct within these cells. The complex mechanism of ct trans-activation from the wing margin enhancer is, therefore, not a general paradigm for ct gene regulation. Moreover, neither wg, sd nor sno are under the direct regulatory influence of the Notch pathway in various embryonic tissues suggesting that this remarkably complex control is strictly tissue specific (Nagel, 2001).

These data confirm and extend the model of context dependent activity of Notch signaling towards the regulation of ct expression along the presumptive wing margin. The regulation of ct requires the combined input of components downstream of Su(H) and Wg, including Vg, Sd and Sno. The latter three components have the potential to form a multi-protein complex which seems to be a pre-requisite for the trans-activation of the ct wing margin enhancer. Whether Su(H) is part of this specific complex or other, similar complexes has to be elucidated in the future. Although there are no indications for direct interactions between Su(H) and Sd, Vg or Sno, Su(H) has the capacity to bind to the ct wing margin enhancer and act in a combinatorial manner together with the Sd/Vg/Sno transactivation complex and components of the Wg pathway. Presumably, in many instances of Notch signaling, where Su(H) acts as a DNA-binding molecule and signal transducer, a number of additional positive or negative co-regulators confers tissue and cell specificity. Therefore, the identification of corresponding factors should help to further the understanding of the context dependent outcome of Notch signaling events (Nagel, 2001).

Control of a genetic regulatory network by a selector gene: Reduction of sd function reduces or eliminates the expression of the Cut and Wingless (Wg) proteins

The formation of many complex structures is controlled by a special class of transcription factors encoded by selector genes. It has been shown that Scalloped, the DNA binding component of the selector protein complex for the Drosophila wing field, binds to and directly regulates the cis-regulatory elements of many individual target genes within the genetic regulatory network controlling wing development. Furthermore, combinations of binding sites for Scalloped and transcriptional effectors of signaling pathways are necessary and sufficient to specify wing-specific responses to different signaling pathways. The obligate integration of selector and signaling protein inputs on cis-regulatory DNA may be a general mechanism by which selector proteins control extensive genetic regulatory networks during development (Guss, 2001).

The discovery of genes whose products control the formation and identity of various fields, dubbed 'selector genes', has enabled the recognition and redefinition of fields as discrete territories of selector gene activity. Although the term has been used somewhat liberally, two kinds of selector genes have been of central interest to understanding the development of embryonic fields. These include the Hox genes, whose products differentiate the identity of homologous fields, and field-specific selector genes such as eyeless, Distal-less, and vestigial-Scalloped (vg-sd) whose products have the unique property of directing the formation of entire complex structures. The mechanisms by which field-specific selector proteins direct the development of these structures are not well understood. In principle, selector proteins could directly regulate the expression of only a few genes, thus exerting much of their effect indirectly, or they may regulate the transcription of many genes distributed throughout genetic regulatory networks (Guss, 2001).

In the Drosophila wing imaginal disc, the Vg-Sd selector protein complex regulates wing formation and identity. Sd is a TEA-domain protein that binds to DNA in a sequence-specific manner, whereas Vg, a novel nuclear protein, functions as a trans-activator. To determine whether direct regulation by Sd is widely required for gene expression in the wing field, the regulation of several genes that represent different nodes in the wing genetic regulatory network and that control the development of different wing pattern elements were analyzed. Focus was placed in particular on genes for which cis-regulatory elements that control expression in the wing imaginal disc have been isolated, including cut, spalt (sal), and vg (Guss, 2001).

First it was tested whether sd gene function is required for the expression of various genes in the wing field. Mitotic clones of cells homozygous for a strong hypomorphic allele of sd were generated and the expression of gene products or reporter genes was assessed within these clones. Reduction of sd function reduces or eliminates the expression of the Cut and Wingless (Wg) proteins and of reporter genes under the control of the sal 10.2-kb and the vg quadrant enhancers, demonstrating a cell-autonomous requirement for selector gene function for the expression of these genes in the wing field (Guss, 2001).

These results, however, do not distinguish between the direct and indirect regulation of target gene expression by Vg-Sd. To differentiate between these possibilities, whether the DNA binding domain of Sd could bind to specific sequences in cut, sal, and vg wing-specific cis-regulatory elements were tested. Using DNase I footprinting, Sd-binding sites were identified in all of the elements assayed. Thus, Sd may control the expression of these genes by binding to their cis-regulatory elements (Guss, 2001).

To determine whether Sd binding to these sites is necessary for the function of these cis-regulatory elements in vivo, specific Sd-binding sites within each of the elements were mutated such that they reduced or abolished Sd binding in gel mobility-shift assays. The mutation of tandem Sd-binding sites in the cut and sal elements results in complete loss of reporter gene expression in vivo. Similarly, mutation of the four single Sd-binding sites identified in the vg quadrant enhancer eliminated or dramatically reduced reporter gene expression. These results show that Sd binds to and directly regulates the expression of four genes (cut, sal, vg, and DSRF) in the wing genetic regulatory network. This molecular analysis and the genetic requirement for Sd function for the expression of other genes suggest a widespread requirement for direct Vg-Sd regulation of genes expressed in the wing field (Guss, 2001).

Each of the Sd targets analyzed is activated in only a portion of the wing field, in patterns controlled by specific signaling pathways. For instance, cut is a target of Notch signaling along the dorsoventral boundary, and the sal and vg quadrant enhancers are targets of Dpp signaling along the anteroposterior axis. Binding sites for the transcriptional effectors of the Notch- and Dpp-signaling pathways, Suppressor of Hairless [Su(H)], and Mothers Against Dpp (Mad), and Medea (Med), respectively, have been shown to be necessary for the activity of a number of wing-specific cis-regulatory elements, and occur in these elements. This observation, coupled with the data demonstrating a direct requirement for Sd binding, suggests that gene expression in the wing field requires two discrete inputs on the cis-regulatory DNA: one from the selector proteins that define the field, and one from the signaling pathway that patterns the field (Guss, 2001).

These findings also raised the possibility that the combination of selector and signal inputs may be sufficient to drive field-specific, patterned gene expression. To test this, there were built a number of synthetic regulatory elements comprised of combinations of Sd binding sites with binding sites for Su(H) or Mad/Med. The activity of these elements was compared with those composed of tandem arrays of just selector- or signal effector-binding sites, or combinations of different signal effector sites. Each of the binding sites used in these constructs was selected from sequences found in native Drosophila cis-regulatory elements that have been demonstrated to function in vivo (Guss, 2001).

Elements containing only single classes of binding sites for the selector or signal effectors were unable to drive reporter gene expression in the wing. In contrast, the synthetic elements in which binding sites for both selector and signal effector were combined drove field-specific expression restricted to the wing and haltere discs in patterns predicted by the specific signaling inputs to each element. That is, the [Sd]2 [Su(H)]2 element drove wing-specific expression along the dorsoventral margin, consistent with Notch activation along this boundary, and the [Sd]2 [Mad/Med] element drove expression in a broad domain oriented with respect to the anteroposterior axis of the disc, consistent with Dpp-signaling activity along this boundary. These patterns of expression are similar to those of the native cut and vg quadrant cis-regulatory elements that also respond to Notch- and Dpp-signaling inputs, respectively. However, regulatory elements containing a combination of Su(H) and Mad/Med sites were not active in vivo, demonstrating that combinatorial input in the absence of selector input is not sufficient to drive gene expression. These results suggest that the Vg-Sd complex provides a qualitatively distinct function required to generate a wing-specific response to signaling pathways (Guss, 2001).

Notch-dependent downregulation of the homeodomain gene cut is required for the mitotic cycle/endocycle switch and cell differentiation in Drosophila follicle cells

During Drosophila mid-oogenesis, follicular epithelial cells switch from the mitotic cycle to the specialized endocycle in which the M phase is skipped. The switch, along with cell differentiation in follicle cells, is induced by Notch signaling. The homeodomain gene cut functions as a linker between Notch and genes that are involved in cell-cycle progression. Cut is expressed in proliferating follicle cells but not in cells in the endocycle. Downregulation of Cut expression is controlled by the Notch pathway and is essential for follicle cells to differentiate and to enter the endocycle properly. cut-mutant follicle cells enter the endocycle and differentiate prematurely in a cell-autonomous manner. By contrast, prolonged expression of Cut causes defects in the mitotic cycle/endocycle switch. These cells continue to express an essential mitotic cyclin, Cyclin A, which is normally degraded by the Fizzy-related-APC/C ubiquitin proteosome system during the endocycle. Cut promotes Cyclin A expression by negatively regulating Fizzy-related. These data suggest that Cut functions in regulating both cell differentiation and the cell cycle, and that downregulation of Cut by Notch contributes to the mitotic cycle/endocycle switch and cell differentiation in follicle cells (Sun, 2005).

The switch of cell-cycle programs in Drosophila follicle cells provides an excellent opportunity to study how developmental signals control the intrinsic cell-cycle machinery. A switch from the mitotic cycle to the endocycle in follicle cells is induced by the Notch pathway. Cell-cycle regulators such as CycA, CycB, Stg and Fzr are regulated by Notch during this process. The homeodomain gene cut acts between the Notch pathway and some of these cell cycle regulators. Its expression is downregulated by the Notch pathway in main-body follicle cells during the mitotic cycle/endocycle switch. Cut function was required cell-autonomously for maintenance of the mitotic cycle and an immature cell fate in main-body follicle cells. Cut downregulation by Notch signaling is a key step allowing proper entry into the endocycle and cell differentiation. Fzr, an adaptor of the APC/C that promotes endocycle, is negatively regulated by Cut, but Stg is not regulated by Cut (Sun, 2005).

Several roles for cut during Drosophila oogenesis have been described. (1) cut negatively interacts with Notch for partitioning of individual germline cysts into egg chambers. (2) cut defines a signaling pathway from the follicle cells to the oocyte to maintain the germline integrity. In addition, the rarely produced ctC145 mutant follicle-cell clones have fewer, but larger, cells. This last phenotype agrees with findings that (3) cut is required to maintain the follicle cells in mitotic cycle and that ctdb7 mutation results in premature entry into the endocycle. Study of the involvement of cut in this process required generation of clones after the egg chamber exited the germarium, so that defects caused by germarium requirements of cut would not interfere. Interestingly, during both egg-chamber encapsulation and cell-cycle switch, cut function is related to the Notch pathway. Cut expression is downregulated by Notch signaling during the cell-cycle switch at stages 6-7. In the germarium, cut negatively interacts with Notch; heterozygous cut mutation suppresses the Notch phenotype. Whether Cut acts as a target of Notch signaling at this stage is unclear. Gain- or loss-of-function clones of Notch prior to stage 6 were studied and no obvious change of Cut expression was detected; this argues that Cut is not a downstream target of Notch during early oogenesis (Sun, 2005).

The interaction between Notch and cut is not restricted to the follicle cells during Drosophila development. In wing imaginal discs, cut also interacts with Notch, but the Notch pathway positively regulates Cut expression in DV boundary formation. Clones of Notch eliminate Cut expression cell-autonomously at the DV border, a pattern opposite that of the follicle-cell-cycle switch. Ectopic expression of constitutively active Notch causes Cut to be ectopically activated in the disc. Notch downstream genes, such as strawberry notch and Enhancer of split (E(spl)), also positively regulate Cut expression in the disc, suggesting Notch-regulated Cut expression is indirect. In follicle cells, downregulation of Cut expression by Notch signaling seems to be indirect as well, because NICD/Su(H) usually functions as a transcriptional activator. Downregulation of Cut is probably achieved by a transcriptional repressor activated by NICD/Su(H). E(spl) is unlikely to be the mediator in this process, because loss of E(spl) has no effect on follicle-cell cycle transition and follicle-cell differentiation. Two other nuclear proteins, bHLH transcription factor dMyc (Dm, encoded by diminutive - FlyBase) and bHLH protein Emc (encoded by extra macrochaetae), are both required in follicle cells for proper entry into the endocycle. Removal of the function of dMyc results in lack of endocycle in follicle cells, but whether dMyc expression is regulated by Notch is uncertain, because the expression of dMyc is uniform throughout oogenesis. Emc expression is detected in main-body follicle cells from the germarium to stage 8 of oogenesis; its expression after stage 6 depends on Notch signaling. Loss of emc function results in upregulated CycB and FasIII in follicle cells after stage 6, a phenotype similar to that of loss of Notch. Emc could function as a transcriptional repressor, and it is known to be involved in promoting differentiation, so it is a good candidate for mediating Notch-dependent downregulation of Cut during the mitotic cycle/endocycle switch. No change of Cut expression however, was found in emc loss-of-function clones generated by a null allele, emcAP6, thus excluding the possibility that cut is repressed by emc during the mitotic/endocycle switch. Another transcription factor may therefore be involved (Sun, 2005).

The known role of Cut in Drosophila is mostly related to cell differentiation. During neurogenesis, Cut is involved in cell-fate determination in sensory-organ cells. Loss of cut function causes transformation of the external sensory organ into the chordotonal sensory organ, whereas overexpression of Cut has the opposite effect. In main-body follicle cells, overexpression of Cut maintains expression of immature cell-fate markers FasIII and Eya, whereas loss of cut function in early stages represses their expression. Although Cut is involved in cell differentiation in these two developmental processes, its roles in the two are significantly different. In sensory organs, the role of Cut is post-mitotic, whereas in main-body follicle-cell differentiation, it appears to be correlated with Cut function in the mitotic cycle. The requirement for Cut in main-body follicle-cell differentiation may be related to its function in cell-cycle regulation (Sun, 2005).

The role of cut in polar-cell differentiation is intriguing. Cut expression is normally retained in these specialized cells while its expression in main-body cells is decreased. Consistent expression of Cut leads follicle cells to take the immature main-body cell fate, but these cells eventually take the polar-cell fate. Main-body cell fate and polar/stalk-cell fate are separated in the germarium, which requires Notch activity. Continuous Cut activity seems able to reverse this differentiation process (Sun, 2005).

In contrast to the role of Drosophila Cut in cell differentiation, mammalian Cut has mainly been shown to be involved in regulating cell-cycle progression in some cell types. CDP, the mammalian homolog of Cut, has been shown to be physically associated with the complex regulating the G1/S progression. Cut can functionally replace E2F in forming a complex with RB in regulating cell-cycle progression. The requirement for Cut in maintaining the mitotic cell cycle in Drosophila follicle cells echoes its role in mammalian systems. Whether Drosophila E2F has a function in follicle cell proliferation is not known: weak alleles of E2F1 and E2F2 affect gene amplification, whereas no defect appears in the mitotic cycle. Cut may functionally replace E2F for cell-cycle progression in proliferating follicle cells, but it is not an essential regulator of the cell cycle machinery because the mitotic cycle did not seem to be affected in cut germline clones. In addition, cut function has been extensively studied during embryogenesis and in imaginal discs, but no reported function is related to cell-cycle regulation in these developmental stages. The requirement for Cut in cell-cycle regulation is therefore probably specific to follicle cells in Drosophila (Sun, 2005).

Hindsight mediates the role of Notch in suppressing Hedgehog signaling and cell proliferation

Temporal and spatial regulation of proliferation and differentiation by signaling pathways is essential for animal development. Drosophila follicular epithelial cells provide an excellent model system for the study of temporal regulation of cell proliferation. In follicle cells, the Notch pathway stops proliferation and promotes a switch from the mitotic cycle to the endocycle (M/E switch). This study shows that zinc-finger transcription factor Hindsight mediates the role of Notch in regulating cell differentiation and the switch of cell-cycle programs. Hindsight is required and sufficient to stop proliferation and induce the transition to the endocycle. To do so, it represses string, Cut, and Hedgehog signaling, which promote proliferation during early oogenesis. Hindsight, along with another zinc-finger protein, Tramtrack, downregulates Hedgehog signaling through transcriptional repression of cubitus interruptus. These studies suggest that Hindsight bridges the two antagonistic pathways, Notch and Hedgehog, in the temporal regulation of follicle-cell proliferation and differentiation (Sun, 2007).

How developmental signals coordinate to control cell proliferation and differentiation remains largely unknown. These data reveal a molecular mechanism that links signal-transduction pathways and the cell-cycle machinery. Hnt is induced by Notch signaling and mediates most, if not all, Notch functions in the downregulation of Hh signaling and the M/E switch in follicle cells during midoogenesis. Loss of hnt function in follicle cells results in an extra round of the mitotic cycle after stage 6 and a delayed entry into the endocycle. In contrast, misexpression of Hnt at an earlier stage causes the follicle cells to differentiate prematurely and enter the endocycle. Hnt suppresses both stg and Cut, whose expression must be downregulated to ensure the M/E switch. In addition, Notch signaling appears to act through Hnt to downregulate Hh signaling by suppressing ci transcription, so Hnt links the two antagonistic signaling pathways in follicle-cell development. The transcriptional repression of ci is probably not mediated by Hnt alone, because ttk exhibited a similar defect in transcriptional regulation of ci and stg (Sun, 2007).

Studies have shown that downregulation of Cut mediates part of Notch function during the M/E switch. Specifically, Cut promotes cell proliferation and maintains an immature-cell fate, but Stg, the Cdc25 homolog, is not regulated by Cut. To induce the mitotic division ectopically during midoogenesis in follicle cells, both Cut and Stg must be misexpressed. The current study suggests that both Cut and Stg are suppressed by Hnt. Without Stg activity, a major regulator of G2/M transition, follicle cells are arrested before they enter the M phase, and downregulation of Cut allows accumulation of Fzr, causing degradation of CycA and CycB by the UPS, thus lowering CDK activity. This process allows endocycling follicle cells to by-pass the M phase and enter the next S phase. Repeated gap phases and S phases constitute the endocycle (Sun, 2007).

The finding that hnt follicle cells enter the endocycle after one additional round of the mitotic cycle suggests that hnt mutation causes a delay in the M/E switch. Mutations of the Notch pathway may also result in only a delay in entering the endocycle. In Notch mosaics, the cell number in mutant clones is approximately twice that of the twin spots, suggesting that an additional cell cycle also takes place. Further testing of this hypothesis requires a detailed analysis of the DNA content and clone size in Notch pathway mutants. Alternatively, Hnt may not be the sole mediator of the Notch effect; for example, Su(H)-independent Notch signaling may also be required in the M/E switch. Although hnt mutant cells can enter the endocycle late, they could not enter the chorion-gene-amplification program even much later, suggesting that Hnt function is also required for chorion-gene amplification (Sun, 2007).

The removal of negative components of the Hh pathway such as ptc causes overproliferation in follicle cells. Loss-of-function analyses of fu, a positive regulator of the pathway, revealed fewer cells in the mutant clones than in twin spots. The nuclear sizes of fu mutant cells were similar to those of the wild-type at the same developmental stage, and no fragmentation of the chromosomes was observed. Hh signaling therefore promotes cell proliferation in follicle cells during early oogenesis. Thus, Hnt-mediated downregulation of Hh signaling through suppression of ci transcription plays an important role in the M/E switch. Hh signaling is probably not involved in regulating Cut or Stg expression, because ectopic expression of Ci-155 in follicle cells during midoogenesis did not extend Stg-lacZ or Cut expression beyond stage 6, and fu mutant follicle cells showed normal Cut expression during early oogenesis. Other factors may therefore mediate the role of Hh signaling to modulate proliferation of follicle cells (Sun, 2007).

Hnt is not only required to mediate the role of Notch in regulating the M/E switch in follicle cells, but it is also sufficient to drive premature entry into the endocycle. Only a few cells misexpressing Hnt at the early stages of oogenesis were recovered, consistent with the role of Hnt in terminating the mitotic phase. In an extreme case, a stage-4 egg chamber contained only ~20 follicle cells, most of which misexpressed Hnt. Hnt misexpression suppresses Cut and stg-lacZ expression, suggesting that Hnt acts as a transcriptional repressor. Consistent with this interpretation, the mammalian homolog of Hnt, RREB1, also acts as a transcriptional repressor in several cellular contexts (Sun, 2007).

An interesting observation from these studies is that ttk clones have a phenotype similar to that of hnt clones. As in Notch regulation of Hnt, ttk is possibly downstream of Notch, but the current analysis of Notch mutants in stage-1 to stage-10 egg chambers showed no obvious change in Ttk expression. It was also found that Hnt has no role in regulating ttk expression. The findings that ttk expression is not regulated by Hnt or Notch during midoogenesis is perhaps not surprising given that Ttk69 is evenly expressed throughout early and midoogenesis. The phenotypic similarity between hnt and ttk mutants suggests that ttk and hnt act cooperatively to suppress gene expression at the M/E transition. Ttk may act as a permissive signal for Hnt to regulate Ci expression and the M/E switch. In the absence of either one, the M/E switch cannot take place properly. Consistent with this hypothesis, Ttk is known to act as a transcriptional repressor in the Drosophila eye. Whether Hnt and Ttk bind directly to the regulatory sequence of the cell-cycle genes and/or ci remains unclear (Sun, 2007).

Several lines of evidence suggest that the role of Hnt in promoting the M/E switch is not universal. First, during embryogenesis, a hnt-deficiency line enters the G1 arrest normally after cycle 16 in epidermal cells and undergoes normal M/E switch in the salivary gland, although Fzr is required for this process. Second, nurse-cell endoreplication does not require Hnt; no obvious defect was detected in hnt germline clones. The specific role of Hnt in follicle-cell-cycle regulation may stem from its role in regulating cell differentiation. For example, Hnt expression may cause upregulation of Fzr through the downregulation of Cut. This indirect role of Hnt suggests that the cell-cycle regulation may be a by-product of cell differentiation (Sun, 2007).

Both Notch and Hh signaling pathways are implicated in the regulation of differentiation and proliferation, but precisely how the two interact in regulating cellular processes is poorly understood. Depending on the cellular environment, their effects on proliferation and differentiation differ. In Drosophila eye imaginal discs, Notch triggers the onset of proliferation during the second mitotic wave (SMW), the opposite of its role in follicle-cell development. In the SMW, Notch positively affects dE2F1 and CycA expression and promotes S phase entry. In these cells, Hh signaling, along with Dpp, activates Dl expression, thereby activating the Notch pathway. Hh and Notch therefore act sequentially and positively during the SMW, whereas, in follicle cells, they act antagonistically. Hh signaling is active in the mitotic follicle cells in early oogenesis, but it is downregulated during the M/E switch when Notch signaling is activated. Notch appears to be superimposable on Hh signaling; mutation of the negative regulator of the Hh pathway, ptc, in follicle cells cannot interfere with the activation of Notch signaling as long as these cells are in direct contact with the germline cells. These ptc mutant cells show no accumulation of Ci-155, consistent with the finding that Notch signaling suppresses ci transcription through Hnt. The ptcS2 cells that were out of contact with germline cells remained in the mitotic cycle because they could not receive Dl signaling from them, suggesting that Hh signaling is sufficient to keep these cells in the undifferentiated and mitotically active state (Sun, 2007).

Notch-dependent activation of Hnt and downregulation of Ci may be involved in another follicle-cell process, the migration of a specialized group of anterior follicle cells toward the border between the nurse cells and the oocyte at stage 9. These so-called border cells showed downregulation of ci during migration. When slbo-Gal4 was used to drive Ci overexpression in border cells, ~66% of egg chambers showed defects in border-cell migration. Notch signaling, as well as ttk, has been reported to be required for border-cell migration. Hnt was found to be expressed in the border cells and depended on Notch signaling. The occasional hnt border-cell clones observed also showed defects in border-cell migration, so the crosstalk between Hh and Notch through Hnt may go beyond the regulation of the M/E switch in follicle cells (Sun, 2007).

Defective proventriculus specifies the ocellar region in the Drosophila head: Dve represses wg and cut in the wing

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

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

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

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

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

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

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

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

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

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

Requirements for mediator complex subunits distinguish three classes of notch target genes at the Drosophila wing margin

Spatial and temporal gene regulation relies on a combinatorial code of sequence-specific transcription factors that must be integrated by the general transcriptional machinery. A key link between the two is the mediator complex, which consists of a core complex that reversibly associates with the accessory kinase module. Genes activated by Notch signaling at the dorsal-ventral boundary of the Drosophila wing disc fall into three classes that are affected differently by the loss of kinase module subunits. One class requires all four kinase module subunits for activation, while the others require only Med12 and Med13, either for activation or for repression. These distinctions do not result from different requirements for the Notch coactivator Mastermind or the corepressors Hairless and Groucho. It is proposed that interactions with the kinase module through distinct cofactors allow the DNA-binding protein Suppressor of Hairless to carry out both its activator and repressor functions (Janody, 2011).

Intercellular signaling pathways drive many processes during development. Their activation results in changes in transcription factor activity that lead to the activation or repression of specific target genes. An important goal is to understand the transcriptional regulatory codes that allow the combinations of proteins bound to enhancer elements to direct precise patterns of gene expression. One well-characterized developmental paradigm is the specification of the Drosophila wing margin by Notch signaling. The Notch receptor is specifically activated at the dorsal-ventral boundary of the larval wing imaginal disc, due to the restricted expression of its ligands Delta and Serrate and of the glycosyltransferase Fringe. Notch activation results in expression of the target genes Enhancer of split m8 (E(spl)m8), cut, wingless (wg), and vestigial (vg), the last through a specific enhancer element known as the boundary enhancer (vgBE). Wg signaling then leads to the differentiation of characteristic sensory bristles adjacent to the margin of the adult wing (Janody, 2011).

Upon ligand binding, Notch is cleaved by the γ-secretase complex, and its intracellular domain (Nintra) enters the nucleus, where it interacts with the DNA-binding protein Suppressor of Hairless (Su(H)). In the absence of Notch activation, Su(H) represses target gene expression through interactions with the corepressor Hairless (H), which binds to Groucho (Gro) and C-terminal binding protein (CtBP). Nintra displaces these corepressors from Su(H) and recruits coactivators such as Mastermind (Mam). It has been proposed that only a subset of Notch target genes require Su(H) to recruit coactivators, while others require Notch signaling only to relieve Su(H)-mediated repression, allowing transcription to be activated by other factors. However, the mechanisms by which Su(H) directs both activation and repression are not fully understood (Janody, 2011).

The mediator complex is thought to promote transcriptional activation by recruiting RNA polymerase II (Pol II), the general transcriptional machinery, and the histone acetyltransferase p300 to promoters, and by stimulating transcriptional elongation by Pol II molecules paused downstream of the promoter. The 'head' and 'middle' modules of the core complex bind to Pol II and general transcription factors, while the 'tail' module consists largely of adaptor subunits that bind to sequence-specific transcription factors. This core complex reversibly associates with a fourth 'kinase' module that consists of the four subunits Med12, Med13, Cdk8, and Cyclin C (CycC). Several studies have implicated the kinase module in transcriptional repression, which can be mediated by phosphorylation of Pol II and other factors by Cdk8, by histone methyltransferase recruitment, and by occlusion of the Pol II binding site. However, this module also appears to function in activation in some contexts; for example, it promotes Wnt target gene expression during Drosophila and mouse development, in mammalian cells, and in colon cancer. Although all four subunits have very similar mutant phenotypes in yeast, loss of Med12 or Med13 has more severe effects on Drosophila development than loss of Cdk8 or CycC, suggesting that Med12 and Med13 have evolved additional functions in higher eukaryotes (Janody, 2011).

This study shows that Notch target genes at the wing margin can be divided into three classes based on their requirements for kinase module subunits. An E(spl)m8 reporter requires all four subunits for its activation, cut requires only Med12 and Med13 (known as Kohtalo [Kto] and Skuld [Skd], respectively, in Drosophila) for its activation, and wg and the vgBE enhancer require Med12 and Med13 for their repression in cells close to the wing margin. Because Med12 and Med13 coimmunoprecipitate with Su(H), regulate an artificial reporter driven by Su(H) binding sites, and can be replaced by a VP16 activation domain or a WRPW repression signal fused to Su(H), it is proposed that the kinase module directly regulates Notch target genes. All four Notch target genes fail to be expressed in the absence of Mam and are similarly affected by the loss of Hairless or Gro, suggesting that other more specific cofactors might recruit kinase module subunits to these genes (Janody, 2011).

The kinase module of the mediator complex is conserved throughout eukaryotes, yet its functions in transcription remain poorly understood. In yeast, loss of any of the four subunits has a very similar effect. In Drosophila, however, loss of Med12 or Med13 has more dramatic effects than loss of Cdk8 or CycC. The kinase module was originally thought to be primarily important for transcriptional repression, mediated by the kinase activity of Cdk8. However, Med12 and Med13 appear to directly activate genes regulated by Wnt signaling in Drosophila and mammalian systems, and also play a positive role in gene activation by the Gli3 and Nanog transcription factors. The data presented in this study confirm that Med12 and Med13 have functions distinct from Cdk8 and CycC. In addition, evidence is provided that all four kinase module subunits contribute to the activation of E(spl)m8 (Janody, 2011).

The human Mastermind homologue MAM has been shown to recruit Cdk8 and CycC to promoters of Notch target genes, where Cdk8 phosphorylates the intracellular domain of Notch, leading to its ubiquitination by the Fbw7 ligase and degradation (Fryer, 2004). This mechanism would be expected to reduce Notch target gene expression, consistent with the increase in E(spl)mβ expression seen in clones lacking the Drosophila Fbw7 homologue Archipelago (Nicholson, 2011); thus it cannot explain the positive effects of Cdk8 and CycC on E(spl)m8. A function for Cdk8 and CycC in Notch-mediated activation would be analogous to recent findings showing that Cdk8 phosphorylation of Smad transcription factors and of histone H3 promotes activation. Cdk8 phosphorylation of RNA polymerase II (Pol II) is also important for transcriptional elongation (Janody, 2011).

Of interest, the current data also suggest that Med12 and Med13 are involved in the repression of wg and the vgBE enhancer in the absence of Notch signaling. The kinase module has been proposed to inhibit transcription through steric hindrance of Pol II binding, independently of Cdk8 kinase activity. Removal of this module on the C/EBP promoter is thought to convert the mediator complex to its active form. In contrast, this study find that wg and vgBE require Med12 and Med13 for their repression but not their activation, while cut and E(spl)m8 require Med12 and Med13 only for their activation, arguing that the two functions occur on different promoters. It cannot be ruled out that Med12 and Med13 have only indirect effects on some of the genes examined; however, their physical association with Su(H) and the requirement for Su(H) binding sites for misexpression of an artificial reporter in skd and kto mutant clones are consistent with a direct effect of Med12 and Med13 on the Su(H) complex (Janody, 2011).

Med12 and Med13 are found associated with both active and inactive promoters in genome-wide chromatin immunoprecipitation studies, suggesting that they can have different effects on transcription when bound to distinct interaction partners. Although both are very large proteins, they contain no domains predicted to have enzymatic activity, and may instead act as scaffolds for the assembly of transcriptional complexes (Janody, 2011).

It has been proposed that Notch target genes could be categorized into two classes: permissive genes, for which the primary function of Notch is to relieve repression by the Su(H) complex, and instructive genes, for which Notch plays an essential role in activation by recruiting specific coactivators. These differences presumably depend on the combinatorial code of transcription factors that regulate each promoter. This study shows that vgBE, an enhancer previously placed in the permissive category, as well as wg, require Med12 and Med13 for their repression but not their activation. During eye development, the proneural gene atonal is likewise regulated permissively by Notch, and ectopically expressed in skd or kto mutant clones. Unexpectedly, this study found that Gro, previously thought to be a cofactor through which Hairless mediates repression, is not required for the repression of vgBE or wg. Hairless may repress target genes at the wing margin through CtBP, its other binding partner. Alternatively, Gro may affect the expression of other upstream regulators of wing margin fate, masking its repressive effect on the genes that were examined (Janody, 2011).

It was also show in this study that instructive Notch target genes can be further subdivided into two classes based on their requirement for kinase module subunits; E(spl)m8 requires all four subunits, while cut requires Med12 and Med13, but not Cdk8 and CycC. Cdk8 and CycC may simply increase the ability of the mediator complex to recruit Pol II or promote transcriptional initiation; this model would suggest that E(spl)m8 has a higher activation threshold than cut. Alternatively, Cdk8 and CycC might enhance the function of a transcription factor that is specifically required for the expression of E(spl)m8 but not cut. Good candidates for such factors would be the proneural proteins Achaete or Scute or their partner Daughterless (Janody, 2011).

The mechanism by which the kinase module is recruited to promote the activation of instructive target genes is not yet clear. Although Mam proteins are well-characterized coactivators for Nintra, this study found that Mam is necessary for the activation of both instructive and permissive genes. It may thus have a general function in transcriptional activation, such as recruiting histone acetyltransferases or stabilizing the Notch-Su(H) complex. A coactivator that recruits Med12 and Med13 specifically to instructive target genes to promote activation may remain to be identified. The current results, like recent reports demonstrating that the arrangement of Su(H) binding sites can affect the interactions between Notch and its coactivators, highlight the complexity in the mechanisms through which promoter elements respond to Notch signaling (Janody, 2011).

Segregation of eye and antenna fates maintained by mutual antagonism in Drosophila

A general question in development is how do adjacent primordia adopt different developmental fates and stably maintain their distinct fates? In Drosophila, the adult eye and antenna originate from the embryonic eye-antenna primordium. These cells proliferate in the larval stage to form the eye-antenna disc. The eye or antenna differs at mid second instar with the restricted expression of Cut (Ct), a homeodomain transcriptional repressor, in the antenna disc and Eyeless (Ey), a Pax6 transcriptional activator, in the eye disc. This study shows that ey transcription in the antenna disc is repressed by two homeodomain proteins, Ct and Homothorax (Hth). Loss of Ct and Hth in the antenna disc resulted in ectopic eye development in the antenna. Conversely, the Ct and Hth expression in the eye disc was suppressed by the homeodomain transcription factor Sine oculis (So), a direct target of Ey. Loss of So in the eye disc caused ectopic antenna development in the eye. Therefore, the segregation of eye and antenna fates is stably maintained by mutual repression of the other pathway (Wang, 2012).

In l-L3 eye-antenna disc, although the expression domain of Ct/Hth and Ey/So are juxtaposed, so3 clones showed derepression of Ct and Hth only in the most posterior region (zone 4) but not in the more anterior regions behind MF (zones 2 and 3). Thus, there may be an additional mechanism to repress Ct and Hth expression. For Hth, the repression is by Dpp and Hh signaling in L3 eye disc. It has not been tested whether Ct is also repressed by Dpp and Hh (Wang, 2012).

For individual cells in the eye-antenna disc, the mutual repression provided a mechanism for a choice of bistable states, either eye or antenna fate. A bistable state can often be maintained by positive-feedback loop, in addition to mutual repression. Such a positive-feedback loop is known for the eye pathway, but has not been reported for the antenna pathway (Wang, 2012).

The mutual transcriptional repression mechanism is expected to work at the level of individual cells. Therefore, a salt-and-pepper mosaic pattern would be predicted unless there is additional patterning influence. The patterning gene dpp is expressed in the posterior margin of e-L2 eye disc, and is required for Eya expression at this stage. However, dpp is not required for the restricted expression of Ct and Ey. It is proposed that there is another patterning gene that biases the antenna disc to express Ct. Thus, the difference between eye and antenna primordia may be predetermined before the onset of Ct and Eya at e-L2 (Wang, 2012).

The subdivision of a developmental primordium into subprimordia with specific fates is a common requirement in development. For example, the mammalian ventral foregut endoderm differentiates into the adjacent liver and pancreas, and a bipotential population of foregut endoderm cells give rise to both liver and pancreas. The maintenance of such division by mutual antagonism has been reported before. For example, the division between the presumptive thalamus and prethalamus in Xenopus is due to the mutual repression by the Irx homeodomain proteins and the Fezf zinc-finger proteins. The boundary between optic cup and optic vesicle is maintained by mutual transcriptional repression between Pax6 and Pax2. The current findings provide a new example, with clear correlation, both temporal and causal, of gene expression changes and developmental fate specification (Wang, 2012).

The maxillary palp and ocelli are derived from specific regions in the eye-antenna disc. The maxillary palp fate does not become segregated from the rest of eye-antenna disc as late as late L3. The timing of ocelli fate decision is not clear. otd is required for ocelli development, and is the first marker for the ocellar region: it is ubiquitously expressed in the early L2 eye-antenna disc, and becomes restricted to the ocellar region in the eye disc in early L3. Thus, the palp and ocelli may be determined as subfields of the antenna disc and eye disc, respectively. This is consistent with the finding that hth>mi-ct+mi-hth (knocking down both ct and hth in their endogenous expression domain) resulted in the loss of palp, whereas so affected ocelli but not palp (Wang, 2012).

The results showed that Ct and Hth are repressed by So. Previous studies also found induction of Ct and Hth expression in so3 clones in a region far posterior to the MF in l-L3 eye disc. The fact that So represses Ct and Hth in two spatially and temporally distinct situations suggest that this is a conserved function of So (Wang, 2012).

Whether the repression of Ct and Hth by So is direct transcriptional repression is not clear. Ectopic So expression caused cell-autonomous repression of Ct and Hth, suggesting that the repression could be direct. Recently it was shown that So acts as a transcriptional repressor to repress ct transcription (Anderson, 2012). So may interact with a repressor and Groucho (Gro) is a likely candidate. So can bind to Gro and the So-Gro complex was postulated to repress Dac transcription in eye disc. The zebrafish So homologue Six3 interacts with Groucho and functions as a transcriptional repressor. The transcriptional co-repressor CtBP has been shown to functionally and physically interact with Ey, Dac and Dan. Whether the protein complex also involves So has not been determined. Overexpression of CtBP caused eye and antenna defect, but the phenotype was not affected by reducing so dose. Therefore, CtBP is probably not the co-repressor for So (Wang, 2012).

so3 clones caused non-autonomous induction of Ct in its surrounding wild-type cells. Similar non-autonomous induction of Dac has been reported in L3 disc. Elevated Delta was observed within the mutant clone and elevated activated N at the border of mutant clone, thus suggesting that the non-autonomous induction is due to N signaling to surrounding cells. Whether a similar mechanism operates in the L2 disc remains to be tested (Wang, 2012).

The finding that ey and toy do not repress Ct and Hth, in both gain-of-function and loss-of-function experiments, was initially perplexing. Clonal ey expression in the antenna disc did not repress Ct and Hth. In these clones, so-lacZ was induced, but not in all ey+ cells and at a level lower than the endogenous level in most cells in the eye disc. When ey was clonally induced at 29°C, Ct level was reduced. These results suggested that the ectopic ey and toy at 25°C induced so at a level not sufficient to repress Ct. The strength of Ey has been shown to be crucial for its ability to induce ectopic eye development. In the double knockdown of ey and toy in the eye disc, Ct and Hth were not induced. Judging from the eye disc phenotype and residual neuronal differentiation, the knockdown was not complete and may account for the failure to detect Ct and Hth derepression. Alternatively, additional factors, independent of ey and toy, may also repress Ct and Hth expression. This would be consistent with the weak effect of so3 clones in inducing Ct and Hth expression (Wang, 2012).

Hth expression is initially uniform in the eye-antenna disc but becomes restricted to the antenna disc in e-L2. In L3 eye disc, Hth expression is downregulated by Dpp and Hh, produced from the progressing MF and developing photoreceptors, respectively. However, Hth expression retracted from the posterior part of the eye disc in e-L2, even before the initiation of MF and photoreceptor differentiation. At e-L2, dpp and hh are expressed in the posterior region of the eye disc. It is possible that the early Hh and Dpp contributed to the repression of Hth from the eye disc, in addition to the repression by So (Wang, 2012).

The results showed that Ct and Hth represses ey transcription. The binding sites for both Hth and Ct in ey3.6 are required for its repression in the antenna disc, suggesting that both Hth and Ct bind to the ey3.6 enhancer directly. The ChIP assay results showed that both Hth and Ct can bind to the ChIP-1 fragment, which contains the binding site for Ct but not for Hth. This suggests that the Hth may bind through a Hth-Ct complex. However, as ectopic expression of either Hth or Ct is sufficient to repress ey transcription, the repression does not require the formation of the Hth-Ct complex (Wang, 2012).

In the RNAi experiments, knocking down Ct or Hth individually did not cause de-repression of the eye pathway genes. However, when the Ct- or Hth-binding site in ey3.6 was separately mutated, the repression of ey3.6 in the antenna disc was partially lost. One possible explanation for the discrepancy is that the RNAi knockdown was not complete. When the binding sites for both Ct and Hth were mutated, the de-repression of ey3.6 in the antenna disc was strongly enhanced. It is possible that both Ct and Hth contributed to the repression of ey transcription, and a threshold net amount of these repressors is required (Wang, 2012).

Hth physically interacts with Exd through the MH domain of Hth and the PBC-A domain of Exd to promote Exd nuclear localization. Hth generally acts as a transcriptional activator , but Hth and Exd can interact with En or UbxIa to repress transcription. Thus, Hth would need to interact with a repressor to repress ey. Ct can serve such a role. Ct can act as a transcriptional repressor by direct binding to a target gene. The human and mouse Ct homologues generally function as transcriptional repressor. However, as ectopic expression of Hth alone in the eye disc, in the absence of Ct, is sufficient to repress ey, Hth must be able to interact with an additional repressor (Wang, 2012).

This study found that Ct can also block the function of Ey when co-expressed with Ey. It is possible that the block resulted from the repression of toy transcription, which may reduce the strength of the feedback regulation of the retinal determination gene network (Wang, 2012).

Although Ct is expressed in L2 in the entire antenna disc, the phenotype caused by ct clones affected only restricted domains, perhaps owing to its later restricted expression. This study reports a novel function of Ct in antenna development. Ct and Hth function redundantly to repress the retinal determination pathway. Because of this functional redundancy, this Ct function was not revealed in ct clones (Wang, 2012).

hth or exd mutations caused antenna-to-leg transformation. Hth has a role in blocking eye development at the anterior margin of the eye disc, where Ct is not expressed. In the antenna disc, this function is masked because of the functional redundancy with Ct revealed in this study (Wang, 2012).

Even when both ct and hth were knocked down in their endogenous expression domain (hth>mi-hth+mi-ct), no significant transformation of the antenna to eye was seen in adult. One possible reason is that the hth>mi-hth+mi-ct caused lethality and the flies have to be raised at a lower temperature, thereby excluding a stronger phenotype. Another possibility is that the Dll expression in the antenna disc served to block eye development. Dll and hth are required in parallel for normal antenna development. Co-expression of Dll and hth can induce the formation of antenna structures in many ectopic sites. It may be the presence of Dll that blocked eye development and provided a leg identity to cause the distal antenna-to-leg transformation found in hth>mi-hth+mi-ct flies (Wang, 2012).

Deadpan contributes to the robustness of the Notch response

Notch signaling regulates many fundamental events including lateral inhibition and boundary formation to generate very reproducible patterns in developing tissues. Its targets include genes of the bHLH hairy and Enhancer of split [E(spl)] family, which contribute to many of these developmental decisions. One member of this family in Drosophila, deadpan (dpn), was originally found to have functions independent of Notch in promoting neural development. Employing genome-wide chromatin-immunoprecipitation, this study has identified several Notch responsive enhancers in the bHLH hairy and Enhancer of split (Espl) family gene dpn, demonstrating its direct regulation by Notch in a range of contexts including the Drosophila wing and eye. dpn expression largely overlaps that of several Espl genes and the combined knock-down leads to more severe phenotypes than either alone. In addition, Dpn contributes to the establishment of Cut expression at the wing dorsal-ventral (D/V) boundary; in its absence Cut expression is delayed. Furthermore, over-expression of Dpn inhibits expression from Espl gene enhancers, but not vice versa, suggesting that dpn contributes to a feed-back mechanism that limits Espl gene expression following Notch activation. Thus the combined actions of dpn and Espl appear to provide a mechanism that confers an initial rapid output from Notch activity which becomes self-limited via feedback between the targets (Babaoglan, 2013).

HES genes are well-known targets of Notch activity. However, in Drosophila only the bHLH genes within the E(spl) Complex were originally thought to be directly downstream of Notch. The expression of another HES genes, dpn, appeared independent of Notch and indeed was associated with cells where Notch activity is considered to be down-regulated (embryonic neuroblasts). More recently it has emerged that dpn expression is under Notch regulation in some contexts (San Juan, 2012; Zacharioudaki, 2012). The current results extend these findings by demonstrating that dpn is directly bound by Su(H) in vivo. As the Su(H) occupied regions differ according to the tissue-type, it appears that dpn contains several Notch responsive enhancers, and the results demonstrate that these direct Notch-dependent expression in different subsets of tissues. Nevertheless, it is striking that a single dpn enhancer, dpn[b] exhibits Notch related expression in both the eye and the wing discs yet these patterns are characteristic of distinct genes/enhancers from the E(spl) Complex (Babaoglan, 2013).

Despite the clear regulation by Notch, there is however relatively few phenotypes resulting from loss of dpn in many tissues. For example, both the wing and eye disc exhibit robust expression of dpn but neither exhibit phenotypes when dpn function was ablated. However, genetic interactions demonstrate that dpn function is related to Notch and both the current evidence, and that from recent studies (San Juan, 2012; Zacharioudaki, 2012), indicate that it has partially redundant functions with the E(spl) genes. This is exemplified by the fact that absence of dpn or of E(spl) Complex alone has little effect on the D/V boundary, but the combined knock down leads to loss of key gene expression (Babaoglan, 2013).

What then is the relevance of dpn in these contexts, especially given that there are 7 E(spl)bHLH genes that also appear to have largely redundant functions? Two components to dpn function are proposed to explain its importance in the Notch response. Clues for the first come from the fact that subtle phenotypes were detected from reductions in dpn when early developmental stages were analyzed. Thus the absence of dpn led to a delay in the ability of Notch to up-regulate cut. Earlier studies also demonstrated a subtle decrease in cut expression in cells lacking E(spl) genes. These results could be explained if both E(spl) and dpn make a contribution to cut regulation. It is suggested that this must be indirect, via the inhibition of a repressor, since both dpn and E(spl)bHLH are thought to be dedicated repressors. So far, no other repressor has been identified that could act as an intermediary (Babaoglan, 2013).

The second component of dpn function is suggested by the observation that Dpn can repress the enhancers derived from E(spl)bHLH genes but not vice versa. Futhermore, it was observed that cells with high levels of Dpn often had lower levels of E(spl)bHLH on a cell by cell level. It is therefore proposed that there is a direct regulatory relationship between dpn and E(spl)bHLH, whereby dpn represses E(spl)bHLH expression. This could set a maximum threshold for E(spl) gene expression since, in previous studies, it was found that dpn shows a less rapid up-regulation following Notch activation than the E(spl) genes. This is reminiscent of the differences seen between HES gene responses in the oscillatory clock involved in somitogenesis and suggests that similar HES gene cross-regulatory network may underpin other Notch dependent processes (Babaoglan, 2013).

Double-bromo and extraterminal (BET) domain proteins regulate dendrite morphology and mechanosensory function

A complex array of genetic factors regulates neuronal dendrite morphology. Epigenetic regulation of gene expression represents a plausible mechanism to control pathways responsible for specific dendritic arbor shapes. By studying the Drosophila dendritic arborization (da) neurons, this study discovered a role of the double-bromodomain and extraterminal (BET) family proteins in regulating dendrite arbor complexity. A loss-of-function mutation in the single Drosophila BET protein encoded by female sterile 1 homeotic [fs(1)h] causes loss of fine, terminal dendritic branches. Moreover, fs(1)h is necessary for the induction of branching caused by a previously identified transcription factor, Cut (Ct), which regulates subtype-specific dendrite morphology. Finally, disrupting fs(1)h function impairs the mechanosensory response of class III da sensory neurons without compromising the expression of the ion channel NompC, which mediates the mechanosensitive response. Thus, these results identify a novel role for BET family proteins in regulating dendrite morphology and a possible separation of developmental pathways specifying neural cell morphology and ion channel expression. Since the BET proteins are known to bind acetylated histone tails, these results also suggest a role of epigenetic histone modifications and the 'histone code,' in regulating dendrite morphology (Bagley, 2014).

Dendrites are the primary site of information input to neural circuits, and the shape of dendritic arbors influences the electrophysiological responses of neurons. Due to the existence of highly diverse morphologies among different neuronal subtypes, a question of the relationship between form and function arises: By understanding how the shape of a neuron is specified, it is possible to understand how morphology relates to neural function and how altered morphology relates to dysfunction (Bagley, 2014).

Neurons can be defined by their physiology, morphology, and gene expression. Neuronal diversity is thought to arise from the combinatorial expression of genetic determinants. The dendritic arborization (da) sensory neurons of the Drosophila peripheral nervous system (PNS) constitute a powerful system to study genetic determinants of dendritic arbor morphology. In particular, the use of Drosophila genetic techniques to study the specification of stereotyped, subtype-specific dendritic arbor shapes resulted in the identification of multiple transcription factors, encoded by abrupt (ab), knot/collier (kn/col), spineless (ss), and cut (ct), which regulate dendritic arbor morphology. However, large-scale genomic analyses comparing the transcriptomes of various neural subtypes indicate a daunting amount of varied gene expression and implicate regulation by multiple transcription factors. Thus, a particular neuronal morphology is likely the result of coordination between multiple genomic programs (Bagley, 2014). Epigenetic modifications are one mechanism that could allow coordinated, genome-wide expression profiles. Chromatin is packaged into nucleosomes, where DNA nucleotides wrap an octamer of histone proteins. The chromatin structure can be altered through three main types of modifications, consisting of direct methylation of DNA nucleotides, post-translational histone-tail modifications such as acetylation and methylation, and ATP-dependent chromatin remodeling. ATP-dependent chromatin remodelers were first shown to regulate dendrite morphology when RNAi knockdown of brahma (brm)-associated protein 60kD (Bap60), Bap55, and the ATPase brm altered the dendritic arbors of class I da sensory neurons. In mammalian neurons, the neural-specific Brg/Brm-associated factor (BAF) complex (nBAF), which contains BAF53b and the ATPase Brg, regulates activity-dependent dendrite growth. In addition, the Drosophila BAF53a/b homolog Bap55 regulates dendritic targeting of olfactory projection neurons (PNs) (Bagley, 2014).

The post-translational modification of histone tails involves three types of molecules: The 'writers' add methyl, acetyl, or phospho groups and consist of histone methyl transferase (HMT), histone acetyltransferase (HAT), and kinase enzymes. The 'erasers' remove these modifications and include demethylases (DMTs), histone deacetyltransferases (HDACs), and phosphatases. Finally, the 'readers' are scaffolding proteins that recognize and bind acetyl, methyl, or phosphate modifications to position the 'writer' and 'eraser' enzymes along with transcriptional machinery to the correct genomic position and thereby modify gene expression. The discovery that the Polycomb repressor complex, which binds methylated histone tails, regulates da sensory neuron dendrite morphology indicates a role of histone methylation in dendrite development and a notion supported by the recent finding that the chromodomain Y-like (CDYL) protein negatively regulates dendritic complexity (Qi, 2014). Regarding a role of histone acetylation in dendrite morphogenesis, both HDAC and HAT activities have been implicated in regulating dendrite morphology. Specifically, the Drosophila HDAC1/2 homolog Rpd3 regulates class I da sensory neuron morphology and olfactory PN dendritic targeting. In addition, HDAC2 suppresses dendritic spine density of hippocampal CA1 and dentate granule neurons. The HAT enzyme Pcaf also regulates class I da sensory neuron dendrite morphology. A different HAT enzyme, CREB-binding protein (CBP), regulates the developmental pruning of class IV da sensory neuron dendrites, and mutations in the human homolog CREBBP cause the mental retardation syndrome Rubenstein-Taybi. While these studies indicate a definite role of 'writers' and 'erasers' of histone modifications in regulating dendrite morphogenesis, the role of 'reader' scaffolding proteins associated with histone acetylation has not been thoroughly investigated (Bagley, 2014).

Double-bromo and extraterminal (BET) domain-containing proteins bind acetylated histone tails (Umehara 2010a; Umehara 2010b) and modulate gene expression. In mice, mutations in one BET family member, BRD2, cause neural tube closure defects, behavioral abnormalities, and altered interneuron numbers. In addition, in certain human genomic population studies, mutations in BRD2 have been associated with juvenile myoclonic epilepsy and photosensitivity, which is frequently observed in idiopathic generalized epilepsies. In the current study, evidence is provided for a role of the Drosophila homolog of BRD2, encoded by female sterile 1 homeotic [fs(1)h], in regulating dendrite morphology and sensory function (Bagley, 2014).

This study examined the role of fs(1)h in dendritic development. The effect of a loss-of-function allele [fs(1)h1112] was examined on the morphology of class III da sensory neurons in the Drosophila PNS. Overall, fs(1)h1112 causes a reduction in dendritic arbor complexity, most notably in the finer, higher-order branches. It was possible to partially rescue this reduced morphological complexity by reintroducing Drosophila Fsh-S or the human homolog (huBRD2) proteins. Furthermore, one aspect of the genetic mechanism of action for fs(1)h was found to be regulating the expression of ct (and possibly other genes in the pathway) in multiple da neuron subtypes as well as subtype-specific transcription factors, such as Abrupt for class I and Knot/Collier for class IV da neurons, which in turn affect subtype-specific dendrite development. The data show that fs(1)h regulates genetic pathways controlling dendritic arbor development but does not specify which ion channels are expressed. Finally, the results suggest that the subtype-specific spike morphology is important for an optimal response to relevant sensory stimuli in the mechanosensitive class III da neurons (Bagley, 2014).

The development of a dendritic arbor involves multiple steps beginning with differentiation, where a neuronal precursor acquires a neural fate. Next, neurites begin to extend, and a neuron becomes polarized as neurites are designated as axon or dendrite. The immature axons and dendrites continue to grow as the neuron and the nervous system develop. Initially, the dendritic arbors are simple, with only a few primary dendrites, but as development progresses, the number of branches and overall arbor size increase. The terminal dendrite branches are dynamic throughout development, exhibiting growth, retraction, or stability. In addition, as the animal body size increases, the dendritic field area increases, and therefore a dendritic arbor must scale accordingly. Thus, dendritic development involves a complex plethora of processes, and dendritic morphology could be altered by affecting any of these processes. For instance, if the balance of dendrite dynamics is shifted such that retraction is greater than growth, then dendritic branching will become reduced over time. This appears to be the case in fs(1)h1112 mutants, since an increase was observed in retracting branches with no change in growth as well as a decrease in the proportion of stable branches in dendritic arbors of fs(1)h1112 mutant da neuron clones compared with wild-type clones. Alternatively, if scaling of the dendritic arbor is affected, the size of the dendritic arbor will become disproportionately small as the body size of the animal increases throughout development. This does not seem to occur in fs(1)h1112 mutants because the primary dendrites of fs(1)h1112 arbors exhibited growth throughout development, although at a delayed rate. Instead, the number of spikes in class III da neuron arbors was reduced early in development and remained reduced throughout development, probably due to the increased amount of dendritic branch retraction and reduced stability. Since the primary dendritic branches were not affected to a large degree by loss of fs(1)h function, it is concluded that the major role of fs(1)h in dendritic development is to regulate dendritic complexity at the level of higher-order dendritic spikes. Moreover, the data suggest that fs(1)h affects dendritic arbor complexity by modulating the dynamics of terminal dendritic branches (Bagley, 2014).

In the da neurons, many molecules are known to regulate dendrite morphology. In particular, Ct, Ss, Ab, and Kn have been shown to regulate subtype-specific morphology of the four classes of da sensory neurons, and these proteins act in parallel genetic pathways. Moreover, the expression of Ct and Ss regulates class III da neuron spike morphology. This study observed a loss of Ct expression in fs(1)h1112 mutant class III da neuron clones, which suggests that fs(1)h regulates the induction or maintenance of Ct expression throughout class III da neuron development. However, reintroducing Ct expression to class III da neuron fs(1)h1112 clones did not rescue the nearly absent spike morphology. Therefore, the class III da neuron dendritic phenotype caused by loss of fs(1)h cannot be solely attributed to the loss of Ct protein. Since it is thought that Ct is a component of a genetic pathway responsible for subtype-specific dendritic arbor development, it is possible that fs(1)h regulates Ct expression as well as expression of genes necessary for the Ct pathway to affect dendritic morphology. Therefore, the relationship between ct and fs(1)h does not appear to be a linear pathway, and fs(1)h might regulate both upstream and downstream components of ct. These data indicating that fs(1)h is necessary for the Ct-induced overbranching and spike formation in class I da neuron dendrites support the idea that fs(1)h regulates the expression of downstream components of the Ct pathway, which are necessary for Ct-induced overbranching and spike formation. This hypothesis also explains why reintroducing Ct expression to fs(1)h1112 clones fails to rescue the dendrite phenotype. It is also known that Ct and Rac1 act synergistically to produce spike morphology. This study examined Rac1 overexpression in a fs(1)h1112 mutant background and found that Rac1 expression significantly rescued the loss of spikes in class III da neurons. However, Rac1-induced overbranching in class I da neurons was not affected by fs(1)h1112. Therefore, fs(1)h does not appear to regulate genes downstream from Rac1 but does regulate genes downstream from ct. Since these pathways are known to converge in order to regulate dendritic spike formation, the current data suggest that fs(1)h may be a crucial link between these two pathways. One possible scenario is that ct and Rac1 regulate parallel pathways, but ct may regulate the level of Rac1 expression such that increased Rac1 expression facilitates the formation of spikes. In this model, the results support the hypothesis that fs(1)h is necessary for the ct potentiation of Rac1 expression, which explains why increased expression of Rac1 with UAS-Rac1 causes a rescue of the class III da neuron dendritic phenotype in fs(1)h1112 mutants. Recent evidence indicates a role for reduced Rac1 expression in social defeat and depressive behavior in mice, possibly through regulating dendritic spine morphology (Golden, 2013). In these behavioral paradigms, reduced Rac1 expression occurred with altered epigenetic marks such that transcriptionally permissive histone H3 acetylation was reduced, while repressive histone H3 methylation was increased. Moreover, administering a class 1 HDAC inhibitor mitigated the reduced Rac1 expression. Thus, these data suggest that Rac1 expression can be regulated by histone acetylation. It is possible that epigenetic reader proteins, such as BET family proteins like fs(1)h, bind acetylated histone marks in the Rac1 promoter to recruit transcriptional machinery and in turn enhance Rac1 expression (Bagley, 2014).

In addition, overexpression of UAS-Fsh-S in class I da neurons did not cause an overbranching phenotype similar to UAS-Ct. In fact, there was no alteration of class I morphology, suggesting that Fsh-S is not sufficient to induce necessary components of the ct pathway to alter dendrite morphology. However, overexpression of Fsh-S in class III and class IV da neurons did cause a decrease in dendritic spike numbers. These data indicate that dendrite morphology may be sensitive to the amount of Fsh-S expression, which was confirmed by modulating the amount of overexpression by reducing GAL4/UAS activity with lower temperature. This may explain why it is possible to achieve only a partial rescue of the fs(1)h1112 dendritic phenotype with UAS-Fsh-S expression and why overexpression causes a dendritic phenotype similar to the phenotype caused by loss of Fsh-S. In support of this expression level hypothesis, it was observed that Fsh-S overexpression can reduce Ct-induced branching in class I da neurons. Since BRD2 is known to be part of a protein complex (Denis 2006), it is possible overexpression causes a gain-of-function or dominant-negative effect by altering the availability of complex components (Bagley, 2014).

Another possible explanation for the partial rescue of Fsh-S expression concerns the developmental timing of expression. Since these experiments were completed using MARCM, GAL80 is expressed until mitotic recombination occurs to generate the mutant clones. It is likely that GAL80 protein may persist for some time after the clones are formed, and the presence of GAL80 would block GAL4/UAS activity. Therefore, UAS-induced Fsh-S expression may occur at a delayed stage in embryonic development, which could produce a partial rescue. In actuality, a combination of both expression level and developmental timing probably explains the partial rescue of the fs(1)h1112 phenotype (Bagley, 2014).

While this study focused on the role of fs(1)h in regulating class III da neuron dendrite morphology, phenotypes were observed in other classes of the da neurons as well as expression of Fsh-S in all da neuron classes. In fs(1)h1112 mutants, a loss of Ct expression was observed in all da neurons that normally express Ct (classes II, III, and IV), suggesting that fs(1)h regulates Ct expression broadly among different neural subtypes. A loss of the class I-specific transcription factor Ab and the class IV-specific transcription factor Kn/Col wer also observed. Thus, it appears that fs(1)h can regulate the expression of subtype-specific gene expression among various neuron subtypes. The loss of Ct or the loss of Kn/Col could explain the reduction in class IV da neuron dendritic arbor complexity, and this further illustrates the pleiotropic nature of the fs(1)h1112 phenotype. The loss of Ab from class I da neurons should produce an increase in dendritic complexity, but interestingly, this did not occur in fs(1)h1112 mutants. Thus, these results consistently suggest that fs(1)h is necessary for dendritic arbor complexity, probably by regulating the expression of many different genes. In this manner, fs(1)h could act as a necessary gate for the gene expression responsible for establishing dendritic complexity (Bagley, 2014).

How can fs(1)h regulate gene expression? Histone modifications are a diverse set of post-translational modifications that produce a code whereby epigenetic reader proteins bind these modified histone tails with specificity for particular modifications, such as methylation, acetylation, or phosphorylation. Previous structural studies have shown that the bromodomains of BET family proteins form a hydrophobic pocket enveloping acetylated histone tails (Umehara 2010a; Umehara, 2010b). Moreover, histone acetylation is largely, but not exclusively, regarded as a mark for transcriptional activation. Therefore, fs(1)h may be required for transcriptional activation of gene expression, which has been shown in vitro with respect to Ubx (Chang, 2007). The current data suggest that fs(1)h is required for ct expression and is in agreement with the hypothesis that fs(1)h is a transcriptional activator. It is possible that expression of other genes in the ct pathway also depends on histone acetylation modifications for transcriptional activation, and this activation may require Fsh-S. This would explain the observed nonlinear genetic relationship between ct and fs(1)h. In addition, the results indicate a necessary, but not sufficient, role of fs(1)h in regulating gene expression. This may indicate that BET family proteins require histone acetylation marks to be established but that these scaffold reader proteins do not actively alter histone tail modifications (Bagley, 2014).

Histone modifications, termed the histone code, vary among different cell types and constitute a genome-wide mechanism for coordinating gene expression programs. This is intriguing because fs(1)h contains bromodomains that require histone acetylation to be first established at specific genomic regions in order to influence transcription at these regions. The current results suggest BET family proteins as candidates for reading this histone code to allow the development of dendritic complexity. It is important to note that although many proteins are observed with altered expression in fs(1)h1112 mutant da neurons, some proteins were unaltered, such as the mechanosensitive ion channel NompC. Furthermore, even though the Ct-induced overbranching in class I da neurons was blocked by fs(1)h1112, the Ct-induced NompC expression was normal. These data indicate some specificity to the action of fs(1)h in regulating dendritic morphology but not ion channel specification. It is possible that epigenetic 'reader' proteins, such as the BET proteins, coordinate the activity of many genetic pathways but with relevance to a specific outcome, such as regulating dendritic arbor morphology. In this model, the epigenetic 'readers' provide coordination and specificity of genome-wide histone marks to regulate particular aspects of neural cell biology. Moreover, it is conceivable that the specific genes regulated by fs(1)h could vary among different cell types depending on the cell type-specific histone code. This is supported by the different effects of UAS-Fsh-S overexpression in class I versus class III and IV da neurons as well as the loss of expression of cell type-specific transcription factors (Ab and Kn/Col) in fs(1)h1112 mutants. Currently, there is no atlas of the histone code for individual neural subpopulations. However, as the technology for conducting these types of analyses improves for distinct cell populations, it is conceivable that future studies can provide an answer to how cell type-specific histone modifications affect neural subtype-specific dendritic arbor morphologies (Bagley, 2014).

Finally, the results suggest that the specific morphological shape of the class III da neuron dendrites is important for their ability to appropriately respond to sensory stimuli. The results indicate that pathways regulating dendrite morphology, such as the ct pathway, are reduced in fs(1)h mutants, but other pathways involved in axon morphogenesis or cell type-specific physiology, such as NompC channel expression, remain active. Moreover, the number of spike protrusions correlates with the number of APs produced in response to a mechanosensitive stimulus. This was also observed in another study (Tsubouchi 2012) involving manipulation of the number of spiked protrusions through modulating Rac1 activity. In that study, the gentle touch response increases as spike numbers increase, causing elevated calcium activity detectable with GCaMP fluorescence imaging. Conversely, decreasing the spike numbers results in a decrease of the gentle touch response and calcium activity. One potential caveat to this study is that Rac1 can modulate many aspects of dendritic cell biology through modulating actin cytoskeletal dynamics, and therefore it is unclear whether manipulating Rac1 activity alters the electrophysiological properties or localization of ion channels such as NompC. The finding of a correlation between dendritic spike number and gentle touch/electrophysiological responses in fs(1)h mutant neurons with normal appearance of NompC expression implicates dendritic morphology in regulating touch sensitivity (Bagley, 2014).

Interestingly, NompC is expressed in fs(1)h1112 mutants, and its distribution throughout the dendritic arbor resembles that of wild-type neurons. While nompC mutants lack a mechanosensory response, neurons lacking fs(1)h still respond to mechanical stimuli, but the magnitude of the response (number of APs) is reduced for a given stimulus intensity. At the behavioral level, this manifests as a reduced response to gentle touch. Therefore, the data suggest that the unique dendritic spike morphology of class III dendrites contributes to their mechanical sensitivity (Bagley, 2014).

While various proteins involved in epigenetic regulation of gene expression have been implicated in dendrite morphogenesis, this study provides evidence that 'readers' of acetylated histone marks regulate dendrite morphology by demonstrating the involvement of BET family proteins in this process. Given the complexity of achieving a comprehensive view of molecularly defined neural subtypes, it is necessary to identify genome-wide mechanisms for molecular diversity that regulate dendritic morphology in order to further understand how morphological diversity is specified. Epigenetic regulators are an intriguing possibility in this endeavor, and future studies comparing gene expression profiles in mutants for regulators of histone modifications among neurons with varied morphologies may be one step forward in answering this fundamental question (Bagley, 2014).

Drosophila eyes absent is required for normal cone and pigment cell development

In Drosophila, development of the compound eye is orchestrated by a network of highly conserved transcriptional regulators known as the retinal determination (RD) network. The retinal determination gene eyes absent (eya) is expressed in most cells within the developing eye field, from undifferentiated retinal progenitors to photoreceptor cells whose differentiation begins at the morphogenetic furrow (MF). Loss of eya expression leads to an early block in retinal development, making it impossible to study the role of eya expression during later steps of retinal differentiation. Two new regulatory regions have been developed that control eya expression during retinal development. These two enhancers are necessary to maintain eya expression anterior to the MF (eya-IAM) and in photoreceptors (eya-PSE), respectively. Deleting these enhancers affects developmental events anterior to the MF as well as retinal differentiation posterior to the MF. In line with previous results, reducing eya expression anterior to the MF was found to affect several early steps during early retinal differentiation, including cell cycle arrest and expression of the proneural gene ato. Consistent with previous observations that suggest a role for eya in cell proliferation during early development, deletion of eya-IAM was found to lead to a marked reduction in the size of the adult retinal field. On the other hand, deletion of eya-PSE leads to defects in cone and pigment cell development. In addition it was found that eya expression is necessary to activate expression of the cone cell marker Cut and to regulate levels of the Hedgehog pathway effector Ci. In summary, this study uncovers novel aspects of eya-mediated regulation of eye development. The genetic tools generated in this study will allow for a detailed study of how the RD network regulates key steps in eye formation (Karandikar, 2014, PubMed ID: 25057928).

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cut: Biological Overview | Evolutionary Homologs | Targets of Activity | Developmental Biology | Effects of Mutation | References

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