Lyra

Promoter

During sensory organ precursor (SOP) specification, a single cell is selected from a proneural cluster of cells. Evidence is presented that Senseless (Sens), a zinc-finger transcription factor, plays an important role in this process. Sens is directly activated by proneural proteins in the presumptive SOPs and a few cells surrounding the SOP in most tissues. In the cells that express Sens at low levels, Sens acts in a DNA-binding-dependent manner to repress transcription of proneural genes. In the presumptive SOPs that express Sens at high levels, Sens acts as a transcriptional activator and synergizes with proneural proteins. It is therefore proposed that Sens acts as a binary switch that is fundamental to SOP selection (Jafar-Nejad, 2003).

Proneural genes have been shown to be required for sens expression. To determine whether proneurals directly activate sens expression, the putative enhancers of sens were identified and were scanned for proneural protein-binding sites (E boxes). An 11-kb genomic fragment containing the sens locus is able to rescue the sens mutant phenotype. To identify the embryonic and imaginal disc enhancers, three genomic DNA fragments were used to create lacZ reporter transgenes. Both 5.9-kb and 3.4-kb fragments are sufficient to drive expression in the embryonic PNS in a pattern similar to endogenous sens. To refine sens enhancers, the 3.4-kb enhancer was divided into nine overlapping fragments. Fragments 8 and 9 induced lacZ expression in a pattern similar to the original 3.4-lacZ line, indicating that both contain regulatory elements sufficient for sens expression in the embryonic PNS. Fragments 8 and 9 were further divided into overlapping fragments. Only 9-1-lacZ expresses the reporter in a pattern similar to the 3.4-lacZ. Inspection of the 9-1 sequence showed that it contains a single E box. The recently sequenced genome of Drosophila pseudoobscura, a species 25-30 myr divergent from Drosophila melanogaster was used to align the genomic regions. The alignment showed that the E box, as well as several other elements in the 9-1 enhancer, is fully conserved. Upon mutation of this E box from CAGGTG to CCGGTG, most of the PNS cells failed to express lacZ, and staining in other cells was much weaker than for the wild-type transgene. These data indicate that proneural genes directly regulate the transcription of sens (Jafar-Nejad, 2003).

It is thought that the two core nucleotides of the E box as well as its flanking sequences are involved in the specificity of each E box for its cognate bHLH transcription factor. It was intriguing that expression of the lacZ marker is almost abolished in chordotonal organs that are dependent on atonal (ato) as well as in external organs and multiple dendritic organs that are dependent on ac, sc, and amos. Because the 9-1 fragment contains only a single E box, the data suggest that different proneural proteins can bind the same E box in vivo. Therefore EMSA was performed to determine whether a variety of Da-proneural heterodimers can shift a wild-type or an E box-mutated probe taken from the 9-1 sequence. Da homodimer, Ato/Da heterodimer, Ac/Da heterodimer, and Sc/Da heterodimer were all able to bind to this E box. Mutation from A to C in the second position of the E box abolished binding for all protein combinations tested, suggesting that these interactions are sequence specific. It is concluded that at least three proneural proteins (Ac, Sc, and Ato) directly regulate sens expression in the embryonic PNS, and that they may bind the same site in vivo (Jafar-Nejad, 2003).

To examine whether sens regulation in the precursors of the adult PNS is also under direct proneural regulation, the 9-1-lacZ and 9-1-mut-lacZ expression patterns were compared in the SOPs of the thoracic microchaetae. Similar to what was observed in embryos, a single-nucleotide change in the 9-1 E box abolishes most of the lacZ expression in pupae of the same age, again suggesting direct regulation of sens by proneurals (Jafar-Nejad, 2003).

The effects of loss- and gain-of-function of proneural genes on sens expression were assessed in the imaginal discs of third instar larvae. Because fragments 9 and 9-1 do not drive lacZ at this stage, enhancer 8 was used. The 8-lacZ transgene drives lacZ expression in several wing SOPs in late third instar larvae. To determine whether proneural genes are able to control 8-lacZ expression, Sc was overexpressed in the wing pouch using the C5-GAL4 driver. Many more cells express lacZ in the wing pouch than in wild type, indicating that the Sc protein is able to induce lacZ expression ectopically. However, removal of the activity of both ac and sc genes results in loss of lacZ expression in all of the ac/sc-dependent SOPs. The precursors of the ventral radius and the femoral chordotonal organs still express lacZ, since these cells are dependent on Ato expression. Moreover, upon Ato overexpression driven by dpp-GAL4, 8-lacZ is strongly induced at the A/P boundary. Together, these data indicate that proneural proteins regulate sens expression in the precursors of the adult PNS. Fragment 8 contains two E boxes, one of which is fully conserved between D. pseudoobscura and D. melanogaster. Band-shift experiments show that the Ac/Da heterodimer can bind to a radioactive probe that contains the conserved E box of fragment 8, further suggesting that proneurals directly regulate sens expression (Jafar-Nejad, 2003).

E(spl) proteins are known to prevent SOP formation through transcriptional repression of proneural gene expression. Whether they affect sens expression was examined. scabrous (sca)-GAL4 was used to express E(spl)m8 in the SOPs and a few cells around the SOPs in third instar imaginal discs. lacZ expression is abolished in most or all cells. Moreover, misexpression of an 'activator' version of E(spl)m7 (m7^ACT), in which the Gro-binding motif is replaced with the VP16 transactivator domain, caused numerous extra lacZ-positive cells when driven in the wing pouch. These observations suggest that E(spl)m7 and E(spl)m8 proteins are also involved in the transcriptional regulation of sens and that proneural proteins and E(spl) proteins have an antagonistic relationship in transcriptional control of sens. E(spl) proteins are known to bind to proneural gene enhancers and m7^ACT is able to activate ac and sc transcription. Therefore, it is formally possible that m7^ACT is indirectly activating the sens enhancer through its up-regulation of proneural proteins. However, it has been shown that even in the absence of endogenous ac and sc, overexpression of m7^ACT causes extra bristle formation, suggesting that the E(spl) proteins not only regulate proneural gene expression, but also regulate the expression of one or more of proneural target genes. Is m7^ACT able to induce sens expression in the absence of ac and sc? To address this question, it was confirmed that overexpression of m7^ACT can produce several extra bristles in a sc^10-1 background. Staining of the imaginal wing discs of these flies shows that there are many Sens-positive cells in the anterior part of the presumptive notum, where the Eq-GAL4 driver used in this experiment is expressed. The data suggest that sens is one of the targets of the E(spl) proteins. Altogether, sens enhancers seem to be able to integrate the positive and negative inputs from proneural and E(spl) proteins, respectively (Jafar-Nejad, 2003).

Protein-protein interactions play a significant role in determining how a transcription factor regulates its target genes. To identify proteins that bind Sens, a yeast two-hybrid (YTH) screen was performed. Of 38 positives sequenced from the screen, seven correspond to members of the E(spl) complex. To confirm the interactions identified in yeast, coimmunoprecipitation (co-IP) assays were performed using in vitro-translated E(spl) proteins and myc-tagged Sens. A monoclonal anti-myc antibody can precipitate E(spl)m7, E(spl)m8, and E(spl)m5 only in the presence of myc-Sens. The yeast two-hybrid assay was used to identify the interaction motif in each partner. Testing a series of Sens deletion constructs showed that a 25-amino acid fragment of Sens (amino acids 276-300) is necessary and sufficient for Sens/E(spl) interaction. To further delineate the interaction motif, the 25 amino acids were mutated to alanines five at a time and five mutant sens constructs were generated. The YTH assays suggested that a 15-amino acid deletion (Sens-del) would abrogate the interaction for all three members of the E(spl) complex. This was indeed observed (Jafar-Nejad, 2003).

Proteins of the E(spl) complex have several conserved motifs, for example, a basic domain, a Helix Loop Helix (HLH) domain, an Orange domain, a caseine kinase-binding motif (CK), and a WRPW or Gro interaction domain (W). To find the interaction motif in the E(spl) proteins, deletion constructs of E(spl)m8 were created and their ability to bind Sens was tested in yeast. The Orange domain was necessary and sufficient for the Sens/E(spl)m8 interaction. The 25-amino acid motif of Sens in isolation interacts with the Orange domain of E(spl)m8 in isolation in the yeast two-hybrid assay. The Orange domain is conserved in all members of the Hairy-E(spl) family of proteins and there is evidence that this domain is functionally important. Alignment of the Orange domains of E(spl)m5, E(spl)m7, and E(spl)m8 prompted mutation of three amino acids in each of the two conserved motifs to alanine and the ability of mutant m8 proteins to interact with Sens was tested. Replacement of EVS with AAA or THL with AAA is sufficient to abolish the interaction of E(spl)m8 with Sens in the yeast assay. In summary, the data indicate that Sens and E(spl) proteins interact in yeast and in vitro (Jafar-Nejad, 2003).

To explore the mechanism by which Sens promotes SOP specification, how Sens regulates proneural gene expression was studied. In sens mutant clones, proneural proteins fail to accumulate in the SOPs. A strong synergism exists in the ability of Sens and Sc to promote extra bristle formation. Whether there is an in vivo synergy between Sens and Ac was examined. ac^Hw-1 exhibits an occasional extra macrochaetae on the notum because of an increase in ac transcript level. Overexpression of Sens with sca-GAL4 also causes a number of extra micro- and macrochaetae on the notum, and overexpression of Sens in an ac^Hw-1 background causes many more extra macrochaetae than the sum of the two genotypes alone. Therefore, there is a synergy between the bristle-promoting effects of the two proteins in vivo (Jafar-Nejad, 2003).

An assay to determine whether Sens can affect ac gene transcription was established in Drosophila S2 cells. The reporter construct used in this assay was a 470-nucleotide fragment of the ac gene containing the ac promoter region fused to the firefly luciferase. This fragment contains the Hairy-E(spl)-binding site and the three E boxes that are involved in ac regulation by proneural and E(spl) proteins. Moreover, during the course of microchaetae SOP specification, expression driven by the ac proximal enhancer/promoter refines from the proneural cluster to a single cell, supporting the notion that it can serve as an SOP-specific regulatory region. The constitutively active actin5 promoter was used to drive the expression of Da, Ac, Sens, or E(spl)m8. Sens alone does not activate the ac470-luc construct. Cotransfection of minimal amounts of actin5-da and -ac activates the luciferase expression about 10-fold. However, adding an additional 20 ng of the actin5-sens leads to a dramatic activation of the ac promoter (>500-fold). It is concluded that Sens can activate ac transcription through synergism with Ac/Da heterodimer in Drosophila S2 cells. These findings suggest that there is a parallel between the in vivo and transcriptional synergy observed between Sens and Ac (Jafar-Nejad, 2003).

Because E(spl)m8 strongly antagonizes SOP specification, it was postulated that E(spl)m8 may decrease the synergistic activation of ac by Sens. Cotransfection of 100 ng of actin5-E(spl)m8 with 1 ng actin5-da and -ac does not significantly repress the luciferase activity induced by these proneural proteins. However, cotransfection of actin5-E(spl)m8 together with sens, da, and ac constructs inhibits the synergy in a dose-dependent manner. In summary, Sens is able to strongly synergize with the proneural proteins in vivo and in vitro, and this synergism is antagonized by E(spl) proteins in a dose-dependent manner (Jafar-Nejad, 2003).

To determine whether the E(spl) antagonism of the Sens synergism operates in vivo, it was first documented that overexpression of Sens at high levels in the wing pouch produces a vast excess of bristles in the wing. In addition, extra vein tissue and thickening of the wing veins is observed. E(spl)m8 overexpression with the C5-GAL4 driver causes loss of wing vein tissue, as well as loss of some of the dorsal wing margin bristles. When Sens and E(spl) are coexpressed, the two proteins suppress each other's phenotypes; the number of extra bristles is decreased significantly, and many wing veins are restored. A higher magnification shows that there are still extra bristles as well as some aberrant vein tissue. Taken together, these data support the notion that Sens and E(spl)m8 have antagonistic effects at the level of proneural gene expression, in agreement with their in vivo effects on bristle formation. Finally, whether the physical interaction between Sens and E(spl) plays a role in their antagonism on ac enhancer was examined in S2 cells. The Sens-del, which lacks the 15-amino acid E(spl)-interacting motif, can synergize with Ac and Da similar to wild-type Sens. However, the ability of E(spl)-m8 to antagonize the synergy between Sens-del and proneural proteins is impaired when compared with its effect on the wild-type Sens/Ac/Da synergy, suggesting that the physical interaction between Sens and E(spl) plays a role in their antagonistic effect (Jafar-Nejad, 2003).

Sens can bind the consensus binding site of its vertebrate homolog Gfi-1. Examination of the ac proximal enhancer showed that only one putative Sens-binding site is present between two of the E boxes in this enhancer (S box). Band-shift assays show that Sens can bind to S box in a sequence-specific manner. Mutating the core sequence from AATC to GGTC abolished Sens binding in this assay. As a positive control, another oligo with 92% identity to the consensus (R21) was used. Significantly more R21 probe was shifted by Sens than the endogenous oligo. These data indicate that there is a binding site for Sens in the ac promoter (Jafar-Nejad, 2003).

To determine whether S box mutations affect the synergy between Sens and Da/Ac, the transfections were repeated with an S box-mutant version of the ac-luc reporter. Quite unexpectedly, the mutant reporter construct consistently showed a three- to fourfold increase in synergism when compared with wild-type reporter. This suggests that DNA-binding has a negative regulatory role in the transcriptional activation of ac by Sens in S2 cells. To test the in vivo relevance of this observation, whether the S box in the ac enhancer has a role in bristle formation in vivo was examined. It is well established that sc^10-1 mutant flies are devoid of thoracic bristles. It has also been shown that two wild-type copies of a 2.2-kb ac minigene can restore some of the microchaetae on the notum of sc^10-1 mutant flies. Therefore the S box core from AATC to GGTC was mutated in a 2.2-kb ac genomic fragment and transgenic animals were created. Six wild-type and nine mutant transgenic strains were obtained. For each strain, at least five flies containing the transgene in a sc^10-1 background were scored. Comparison of the number of bristles restored by one copy of mutant versus wild-type transgene showed that, in agreement with the transcription assay results, the mutant ac minigene is more potent in promoting bristle formation than the wild-type transgene. Interestingly, one of the mutant transgenes rescued almost all microchaetae on the notum, along with the three ac-dependent macrochaetae on each side. It is worth mentioning that none of the six wild-type transgenic lines show macrochaetae rescue. These data suggest that DNA binding is a negative modulator of the synergy between Sens and Ac/Da heterodimer in vivo (Jafar-Nejad, 2003).

Because proneural gene expression precedes sens expression in most proneural clusters, one can postulate that at least in a transitional period, Sens levels will be lower than proneural protein levels. Because a 1 proneural:20 sens ratio was used in previous experiments, it was decided to reverse the ratio. Ac and Da can strongly induce luciferase gene expression. Since the amount of sens construct is increased, a gradual repression in luciferase activity was observed that reaches 50% of the Ac/Da activation. When the ratio is 1 proneural:20 sens, synergism is observed (~2000-fold activation of baseline). It is concluded that Sens can act both as a repressor and as an activator of ac transcription, depending on the ratio between Sens and Ac/Da (Jafar-Nejad, 2003).

Since previous transfection and in vivo assays suggested a negative role for Sens DNA-binding in ac transcription and bristle promotion, it was of interest to determine whether the repressive role of low-level Sens is mediated via DNA binding. A similar transfection assay was therefore performed using the AATC to GGTC mutated ac enhancer as the reporter. The results show that upon removal of the Sens-binding site, its ability to repress the luciferase level is lost. Moreover, the synergy between Sens and Ac/Da begins at a much lower sens:proneural ratio and reaches significantly higher levels. Therefore, the repressive effect of Sens seems to depend on its DNA binding (Jafar-Nejad, 2003).

The above findings prompted a reexamination of Sens expression and its colocalization with proneural proteins. Sens protein expression is not confined to the SOPs, in which it is abundantly expressed; it is also expressed at lower levels in cells surrounding the SOPs. This domain of expression is smaller than the proneural cluster and seems to be confined to the proneural field or even fewer cells. This occurs in the wing margin, the eye, and the microchaetae field of the pupal notum. In all of these extended proneural fields, low levels of Sens and proneural proteins are expressed in numerous cells that fail to become SOPs. However, in all of these tissues, cells that exhibit high levels of Sens also accumulate large amounts of proneural proteins. It is worth mentioning that it has not been possible to detect similar low-level Sens expression in the typical single-SOP fields of notum macrochaetae, which could either be a technical issue or suggest that in these proneural fields, Sens expression is confined to SOPs (Jafar-Nejad, 2003).

In summary, the data suggest that low levels of Sens are present in cells that surround the presumptive SOPs of the notum microchaetae, wing margin, embryonic PNS, as well as in cells that surround the presumptive R8 photoreceptors. Although all of the cells with low-level Sens expression also express low levels of proneural proteins, many of them will later lose proneural gene expression and adopt a non-neural fate. These observations are in agreement with the hypothesis that whereas high levels of Sens are required for proneural up-regulation in the SOP, low levels of Sens might repress proneural gene expression, and thus suppress neural potential (Jafar-Nejad, 2003).

Because Sens is expressed in the posterior wing margin, and because ac, sc, ato, and amos are not expressed in the posterior wing margin, it was of interest to determine whether expression of Sens in these cells is dependent on proneural gene expression by removing da. Large clones of da do not cause a loss of early Sens expression at the anterior or posterior wing margin, suggesting that early Sens expression in these cells is under the control of other signaling pathways. This early expression of Sens is not affected in a sc^10-1 animal either. However, Sens expression is lost wherever the da clone encompasses an area other than the wing margin from which SOPs would normally arise. Finally, no Sens protein was detected in the developing notum of a 10-12-hour-old sc^10-1 pupa, suggesting that in the pupal microchaetae field, proneural proteins are the primary transcriptional activators of sens. Together, these data suggest that whereas the initiation and up-regulation of Sens in the majority of presumptive SOPs are under direct transcriptional control of proneural proteins, other proteins seem to be involved in the initiation of Sens expression in the wing margin (Jafar-Nejad, 2003).

So far, evidence has been provided that low levels of Sens can act as a transcriptional repressor of ac in cell culture, and that in most proneural fields, low levels of Sens are present in the cells surrounding the presumptive SOP. To strengthen the hypothesis that Sens acts as a transcriptional repressor in the cells that express low levels of Sens, sens clones were generated in the wing imaginal discs of third instar larvae. If low levels of Sens repress proneural gene expression, and high levels promote SOP development, lack of Sens protein should lead to continued or slightly increased expression of proneural proteins in proneural fields, whereas causing a loss of up-regulation of proneural proteins in SOPs. Therefore broad low levels of proneural proteins are expected to be observed in sens mutant clones. Two different clones were selected, one parallel to the dorso-ventral midline and the other perpendicular to this midline. In both cases, the Sc expression fails to become restricted to single cells, as is observed in adjacent heterozygous or wild-type tissue. These observations provide further evidence that Sens is necessary to down-regulate proneural expression in the cells that will not adopt the SOP fate (Jafar-Nejad, 2003).

Finally, to provide additional evidence that Sens may act as a binary switch, varying levels of Sens were ectopically expressed to determine whether low levels of Sens expression prior to its normal onset of expression might cause bristle loss. Expression of Sens in the wing margin using the C96-GAL4 driver can result in wing-margin tissue loss, including bristles, similar to what is observed in Lyra mutants. C96-GAL4 was crossed to the weakest UAS-sens transgene, which is inserted on the X chromosome, and females and males were compared with one copy of the transgene in an otherwise identical genetic background and environment. Because males display dosage compensation, they should express more Sens protein than their sisters. Most male progeny have a few extra bristles along the margin when reared at 25°C. However, most female progeny display patches of wing margin bristle loss. These data suggest that lower amounts of exogenous Sens can preferentially lead to bristle loss (Jafar-Nejad, 2003).

A model for selection of an SOP from a proneural cluster is proposed in which an intricate set of feedback loops between various transcription factors determines, through the action of Sens and E(spl), the selection of the adult SOP. Most cells of a proneural cluster first express relatively low levels of proneural proteins. This leads to transcriptional activation of E(spl) genes in the cluster. E(spl) proteins, together with the corepressor Gro, then prevent the up-regulation of proneural gene expression in the cluster. It is thought that prepattern factors then lead to a higher level of proneural protein expression in a smaller group of cells of the proneural cluster, the proneural field. It is proposed that this higher level of proneural expression, probably together with the prepattern factors, induces low levels of Sens expression in the proneural field or an area that is even smaller. Consistent low levels of Sens staining are observed in groups of cells in the pupal microchaetae field, embryos, wing, and eye discs. These domains that are part of the proneural cluster colabel with proneural proteins, and a single or a few cells are typically selected from these domains to induce higher levels of Sens. It is proposed that Sens plays a critical role in the SOP through transcriptional synergy with proneural proteins. In addition, the data suggest that Sens plays a role in repressing proneural expression in non-SOP cells. Hence, it is proposed that Sens acts as a binary switch in the refinement of the proneural field that will lead to SOP selection (Jafar-Nejad, 2003).

The data also suggest that sens transcription is mediated directly through proneural binding to E boxes in the sens enhancers. In addition, sens enhancers integrate two opposing forces, the positive regulation by proneural and the negative regulation mediated by E(spl) proteins, similar to SOP-specific enhancers of the proneural genes (Jafar-Nejad, 2003).

Because E(spl) prevents the up-regulation of the proneural gene and sens expression, this repressive effect must be overcome if some cells of the proneural field are to be selected as SOPs. In fact, it has been shown that by repressing E(spl)m8 and other repressors of sens, Su(H) plays a positive role in the SOP fate promotion. It is also known that proneural proteins positively regulate E(spl) gene expression, which will prevent further up-regulation of proneural proteins. This negative feedback has prompted the idea that to accumulate large amounts of proneural proteins in the SOP, the equilibrium between the proneural and E(spl) proteins should be displaced in favor of proneurals. It is proposed that the synergy between Sens and Da/Ac on the ac regulatory region is a key mechanism for the up-regulation of ac transcription. In this model, Sens accelerates proneural gene expression and proneural protein accumulation, overruling the negative feedback conferred by E(spl). This hypothesis is supported by the observation that the synergy between Sens and proneurals is highly sensitive to the levels of E(spl) protein in the transcription assay, as well as in vivo. Ac up-regulation will lead to further Sens production and increased synergistic activation of ac transcription. In the absence of Sens, the presumptive SOPs fail to up-regulate proneural gene expression. Hence, Sens will render the presumptive SOP less sensitive to N signaling. This is also supported by the observation that coexpression of Sens and proneurals is able to produce closely spaced bristles, indicating highly inefficient N signaling. In summary, it is proposed that the balance between the levels of the Sens and E(spl) proteins determines the SOP selection (Jafar-Nejad, 2003).

The synergistic model of proneural gene activation predicts that low levels of Sens and proneural proteins may suffice to override the E(spl) inhibition. However, many cells that express sens and proneural genes fail to up-regulate proneural gene expression. At low levels, Sens acts as a repressor of ac transcription, suggesting that in addition to the relative levels of E(spl), the relative levels of proneural proteins and Sens also play a critical role in SOP selection. In those areas of the proneural field in which Sens and proneural protein levels are low, not only is the transcriptional synergy absent, but there is also a weak repression of proneural gene expression. This should lead to a rapid loss of Sens expression and a failure to adopt the SOP fate. Analysis of the Sc expression pattern in sens clones that include the wing margin confirms that in the absence of Sens function, the broad Sc expression in the wing margin persists, and at the same time, the presumptive SOPs fail to up-regulate Sc protein. This is further supported by the observation that overexpression of low levels of Sens causes bristle loss in the wing margin (Jafar-Nejad, 2003).

The mechanism by which Sens represses transcription of proneural genes is probably through DNA binding. When the S box is mutated, Sens is unable to repress ac transcription. This finding is corroborated with in vivo observations that the ac minigene with the mutated Sens-binding site is a more potent inducer of bristle formation than the wild-type minigene. It is therefore concluded that the transcriptional repression of the ac promoter by Sens is mediated through DNA binding (Jafar-Nejad, 2003).

Altogether, these data support a model in which Sens promotes the SOP fate in one cell by activating ac transcription, whereas it prevents SOP fate in the neighboring cells by repressing ac transcription. The relative levels of Sens, proneural, and E(spl) proteins seem to be the major determinants of these fate decisions. Therefore, it is proposed that Sens acts as a binary switch in SOP determination by affecting a series of interconnected positive and negative regulatory loops to refine the potential for a specific fate from a group of cells to a single cell, the SOP (Jafar-Nejad, 2003).

Two-step selection of a single R8 photoreceptor: a bistable loop between senseless and rough locks in R8 fate

Patterning of sensory organs requires precise regulation of neural induction and repression. The neurocrystalline pattern of the adult Drosophila compound eye is generated by ordered selection of single founder photoreceptors (R8s) for each unit eye or ommatidium. R8 selection requires mechanisms that restrict R8 potential to a single cell from within a group of cells expressing the proneural gene atonal (ato). One model of R8 selection suggests that R8 precursors are selected from a three-cell 'R8 equivalence group' through repression of ato by the homeodomain transcription factor Rough (Ro). A second model proposes that lateral inhibition is sufficient to select a single R8 from an equipotent group of cells called the intermediate group (IG). This study provides new evidence that lateral inhibition, but not ro, is required for the initial selection of a single R8 precursor. In ro mutants ectopic R8s develop from R2,5 photoreceptor precursors independently of ectopic Ato and hours after normal R8s are specified. Ro directly represses the R8 specific zinc-finger transcription factor senseless (sens) in the developing R2,5 precursors to block ectopic R8 differentiation. These results support a new model for R8 selection in which lateral inhibition establishes a transient pattern of selected R8s that is permanently reinforced by a repressive bistable loop between sens and ro. This model provides new insight into the strategies that allow successful integration of a repressive patterning signal, such as lateral inhibition, with continued developmental plasticity during retinal differentiation (Pepple, 2008).

Ro is a homeodomain-containing protein and has been shown to bind DNA at two sites in its own enhancer containing an ATTA core sequence. To explore the possibility that Ro directly represses sens, the R8 specific sens enhancer was identified and the mechanisms regulating sens expression was characterized. A 645 bp fragment within the second intron of the sens genomic locus named F2 was identified that is sufficient to drive reporter expression specifically in photoreceptors of the developing eye-antennal imaginal disc. To test whether the F2 region is necessary for R8-specific sens expression, the 645 bp region was specifically deleted from the sens-L genomic rescue construct generating DeltaF2. In sens-null mutants, one copy of DeltaF2 rescues the null phenotype in all tissues except the eye. Thus, F2 is the sens eye enhancer and is necessary and sufficient for R8-specific sens expression (Pepple, 2008).

F2 contains two potential Ro-binding sites known as H1 and H2, for homeodomain 1 and 2. To test for a direct interaction, electrophoretic mobility shift assays (EMSAs) were performed. A probe containing H1 and H2 is bound specifically by Ro protein in vitro. Complete loss of binding occurs with mutation of H2. Mutation of H1 does not prevent Ro binding, but there may be a mild decrease in binding compared with the wild-type probe. To test the in vivo significance of these interactions, each site was mutated in a reporter generated with the minimal R8-specific enhancer, B-short-GFP, and the effect on GFP was evaluated. Although H1 is not required for Ro binding in vitro, mutation of H1 in B-short (termed H1^*) leads to consistent expression of GFP in two extra cells per ommatidium. These two cells were identified as the R2,5 photoreceptor pair by co-localization of GFP with β-galactosidase from the R2,5-specific enhancer trap RM104. GFP expression is also expanded into the R2,5 pair with the H2 mutation (H2^*). Mutation of both H1 and H2 (H1,2^*) results in a GFP expression pattern indistinguishable from H2^*. To test whether the loss of ro function has the same effect on B-short-GFP expression as does mutation of the Ro-binding sites, ro^X63 clones were generated. In the absence of ro function, both Sens and B-short-GFP expression are detected in two to three cells per ommatidium. Together with the in vitro binding data, these in vivo results suggest that Ro directly represses sens expression in R2,5 photoreceptors (Pepple, 2008).

Therefore, this work shows that Ro directly represses sens in developing R2,5 cells and that de-repression of Sens is sufficient to initiate R8 cell fate in the absence of ectopic Ato. Although there are a small number of ectopic Ato-expressing cells in column 3 in ro^x63 mutants, it is not likely that the additional 'R8' cells are due to misregulation of Ato since the great majority of ectopic 'R8s' never express detectable Ato protein after the intermediate group stage. It is more likely that the extra Ato-positive cells are due to secondary Sens activation of proneural gene expression, a previously reported phenomenon (Pepple, 2008).

sens is required for R8 differentiation to occur through repression of Ro in R8, and that ectopic Sens is sufficient to repress endogenous Ro expression. Thus, in the absence of sens, three R2,5 cells develop and in the absence of ro up to three R8 cells form per ommatidium. This reciprocal phenotype supports the existence of the three cell R8 equivalence group and a mechanism of mutual repression between sens and ro that specifies opposite cell types. Although one mechanism regulating this mutual repression is the direct repression of sens by Ro, other roles for Ro may exist. The Ro-binding site mutations do not produce the same level of GFP reporter protein expression elevation in R2,5 precursors that would be predicted from the level of GFP expressed in ro mutants. This suggests that Ro may also regulate sens by repressing an activator of sens expression in R2,5 precursors (Pepple, 2008).

Regardless of the mechanism, the negative-feedback loop between sens and ro is secondary to the initial force driving R8 selection in which Ato and Sens are transiently repressed by lateral inhibition in all but one cell within an IG. Thus, lateral inhibition transiently represses neural differentiation in the eye, establishing the patterned array of precisely spaced ommatidia while retaining the potential for later recruitment of undifferentiated cells to the photoreceptor cell fate. If the effects of lateral inhibition were to repress permanently the potential for neuronal differentiation, further retinal development would be blocked. Therefore, the effects of lateral inhibition must be limited, and the data indicate that column 3 is the boundary of its influence. Since the effects of lateral inhibition diminish, the negative-feedback loop between sens and ro reinforces the pattern of selected R8s and ensures that only one Sens-expressing cell from the R8 equivalence group develops as an R8. This simple bistable loop translates the transient developmental signal of lateral inhibition into a committed irreversible fate (Pepple, 2008).

In later R8 differentiation, another bistable loop is used to specify the 'pale' or 'yellow' subtypes of R8 photoreceptors. During this late developmental step, the bias for the 'pale' R8 fate is provided by a signal from a 'pale' R7. It is proposed that the bias signal that tips the fate decision in the sens-ro loop is provided by resolution of Ato to a single cell by lateral inhibition. Ato then directly activates Sens expression and biases that cell to the R8 cell fate. It is not yet known what activates Ro expression and thereby establishes the R2,5 cell fates. However, it has been suggested that epidermal growth factor receptor (EGFR) or Hedgehog signaling may be required for Ro expression. As a result, after the R8 bias is established, a signal such as the EGFR ligand Spitz could be sent from R8 to the two neighboring cells that bias their sens-ro loop towards Ro expression and the R2,5 fate. Once Ro expression is initiated in the R2,5 pair, the pattern of a single Sens-expressing R8 per ommatidium becomes irreversible (Pepple, 2008).

Proper patterning of the Drosophila eye requires precise selection of R8 precursors in a highly ordered array. Previously, the potential to assume the R8 fate was generally believed to reside in the single cell that achieved the highest balance of proneural induction by ato and escaped repression by lateral inhibition. This concept has influenced the interpretation of mutants that exhibit multiple R8 phenotypes, such as ro, by linking the extra R8s that form to cells that inappropriately maintain Ato expression. However, the data show that the expression pattern of Ato and Sens in a ro-null mutant is not altered in a manner consistent with this model. This re-evaluation of the ro phenotype suggests the intriguing possibility that undifferentiated cells posterior to the furrow retain the developmental plasticity to develop as R8s even in the absence of ongoing Ato expression (Pepple, 2008).

The ro phenotype demonstrates that, despite initial repression of the R8 cell fate by lateral inhibition, at least two additional cells have the potential to develop as R8s starting in column 3 if Sens expression is de-repressed. One of the subfragments of the sens eye enhancer, fragment C-GFP, is expressed in nearly all cells posterior to the MF, suggesting that sens could be de-repressed in cells other than the R2,5 cell precursors and initiate R8 development. The widespread expression of fragment C-GFP suggests that it lacks an important negative regulatory region distinct from Ro repression. One potential mechanism that may explain the fragment C-GFP expression pattern is that the stripe of Ato expression in the MF confers R8 potential to all cells and that this potential is only transiently repressed by lateral inhibition during patterning. Then, as the effects of lateral inhibition fade, secondary mechanisms repress sens expression and R8 differentiation in cells posterior to the MF. This model, demonstrated by the function of Ro and suggested by fragment C-GFP expression, is distinct from the previous concept that R8 cell fate is limited to cells of the intermediate group (Pepple, 2008).

The minimal eye specific enhancer of sens, fragment B-long, contains at least four potentially discreet regulatory elements that balance the positive and negative inputs required to specify a single R8 precursor per ommatidium. The first positively acting element is under the direct control of Ato/Da heterodimers and contains E-boxes 1 and 4. This element is required for Ato-dependent sens expression in the IGs and in columns 1-3. Although ato is at the top of the genetic cascade required for eye differentiation, sens is only the third direct target identified in the eye after bearded (brd) and dacapo (dap). Ato/Da heterodimers bind to two E-boxes (E1 and E4) to drive early sens expression in R8. This is in contrast to the previously described direct regulation of sens in SOPs of the embryonic and developing adult PNS by Ato and Scute at a single E-box in their common enhancer (Pepple, 2008).

The second positively acting regulatory element resides within the boundaries of fragment E1^*, although the minimal necessary sequence was not specifically identified. This element responds to an Ato-independent mechanism that is sufficient to maintain Sens expression in selected R8 cells after column 3. Sens is known to respond to Ato-independent inductive cues much later in R8 development (48 hours after pupation) when Sens expression requires the spalt genes. However, larval expression of Sens is not disrupted in spalt mutants, suggesting the existence of yet another unidentified positive regulator (Pepple, 2008).

In addition to these two positively acting elements, there are also at least two negative regulatory elements. The Ro-binding element H2, that is responsible for repressing Sens expression in R2,5 cells, was specifically identified. The second element was not specifically identified, but its presence is suggested by the nearly ubiquitous expression of fragment C-GFP. Together these positive and negative regulatory elements outline an elegant strategy for the multi-staged selection of a single R8 per ommatidium and highlights a model where blocking R8 cell fate potential with sequential, independent, repressive mechanisms is an important strategy for patterning and cell fate development in the Drosophila eye (Pepple, 2008).

Transcriptional Regulation

Lyra/Senseless is expressed in some cells of proneural clusters and SOPs, when and where proneural genes are expressed. Whether Sens expression is dependent on proneural activity was determined by staining embryos that lack daughterless (da) or atonal or the genes of the AS-C (Df(1)sc^B57, a deficiency of ac, sc, l'sc, and ase). Embryos that lack da exhibit a loss of all PNS cells, except the SOPIs. The Daughterless protein has been shown to form heterodimers with many proneural proteins, and this dimerization is essential for neuronal determination or differentiation of many SOP lineages. Embryos homozygous for a deficiency that removes da (Df(2L)J27) and da¹ fail to express Sens protein or mRNA. Similarly, embryos that lack genes of the AS-C fail to express Sens in all the PNS cells that are affected by loss of the AS-C. Finally, homozygous atonal (ato¹) mutant embryos fail to express Sens in chordotonal SOPs except in those derived from P cells. Interestingly, the P cell is an SOP that gives rise to the only embryonic chordotonal organ that is not dependent on the activity of the atonal gene (Nolo, 2000).

To determine whether proneural gene expression is required for Sens expression in imaginal discs, eye-antennal discs of atonal mutants were stained for Sens protein. Eye-antennal imaginal discs of ato¹ are devoid of Sens expression, and in the absence of sens, photoreceptor development is aberrant. Similarly, wing discs of achaete mutants [In(1)y^3PC sc^8R] lack most SOPs and Sens expression. In summary, Sens expression is essentially confined to cells of the PNS and is dependent on proneural gene expression. No Sens expression is observed in the CNS of embryos or larvae (Nolo, 2000).

The expression pattern of sens can be almost fully reconstructed in transgenic flies that carry the 3.4 kb and 5.9 kb genomic fragments upstream of lacZ in the pP{CaSpeR-lacZ} vector. Both fragments contain numerous E boxes, including a GCAGGTG E box, shown to be highly preferred by Scute/Daughterless heterodimers. Hence, combined with the temporal and spatial expression data, these data suggest that sens expression may be directly controlled by proneural genes (Nolo, 2000).

The receptor protein Notch plays a conserved role in restricting neural-fate specification during lateral inhibition. Lateral inhibition requires the Notch intracellular domain to coactivate Su(H)-mediated transcription of the Enhancer-of-split Complex. During Drosophila eye development, Notch plays an additional role in promoting neural fate independent of Su(H) and E(spl)-C, and this finding suggests an alternative mechanism of Notch signal transduction. Genetic mosaics were used to analyze the proneural enhancement pathway. Proneural enhancement involves upregulation of proneural gene expression in single cells that will become neurons. In Drosophila eye development, Notch (N) is required for proneural enhancement in addition to lateral inhibition. The molecular mechanism of proneural enhancement has not been determined. As in lateral inhibition, the metalloprotease Kuzbanian, the EGF repeat 12 region of the Notch extracellular domain, Presenilin, and the Notch intracellular domain are required. By contrast, proneural enhancement becomes constitutive in the absence of Su(H), and this leads to premature differentiation and upregulation of the Atonal and Senseless proteins. Ectopic Notch signaling by Delta expression ahead of the morphogenetic furrow also causes premature differentiation. It is concluded that proneural enhancement and lateral inhibition use similar ligand binding and receptor processing but differ in the nuclear role of Su(H). Prior to Notch signaling, Su(H) represses neural development directly, not indirectly through E(spl)-C. During proneural enhancement, the Notch intracellular domain overcomes the repression of neural differentiation. Later, lateral inhibition restores the repression of neural development by a different mechanism, requiring E(spl)-C transcription. Thus, Notch restricts neurogenesis temporally to a narrow time interval between two modes of repression (Li, 2001).

In the developing eye, lateral inhibition restricts the proneural gene atonal (ato) to individual R8 photoreceptor cells, which found each ommatidium. Earlier, ato must first have reached levels of activity sufficient to sustain expression by autoregulation, in conjunction with its bHLH heterodimer partner encoded by daughterless (da) and with a zinc-finger protein encoded by senseless (sens). Such 'proneural enhancement' depends on N and Dl but not on Su(H) or E(spl)-C. Clones of cells mutant for the E(spl)-C or for Su(H) lead to neural hyperplasia because they lack lateral inhibition, but clones of cells mutant for N or Dl show reduced neural differentiation because they lack proneural enhancement. These divergent phenotypes show that proneural enhancement occurs by a mechanism distinct from that of lateral inhibition (Li, 2001).

Clones homozygous for the Su(H)^Delta47 allele are neurogenic. In addition, however, Su(H)^Delta47 mutant cells differentiate prematurely. Ato expression begin earlier in Su(H)^Delta47 clones than in neighboring tissue, and it soon reaches high levels. The senseless gene is expressed in response to ato activity. Senseless is also expressed prematurely in Su(H)^Delta47 clones. Daughterless protein is ubiquitous but upregulated in ato-expressing cells of the furrow. It was hard to see premature elevation of Daughterless in Su(H)^Delta47 clones, and this must be subtle if it occurs (Li, 2001).

These findings suggest a model for proneural enhancement. The release of N intracellular domain in response to Dl derepresses genes that are repressed by Su(H). The relevant targets do not require Su(H)-mediated transcriptional activation, so deletion of Su(H) mimics N signaling. The mechanism contrasts with lateral inhibition. N signaling provides N intracellular domain as a coactivator for Su(H), which is essential for the transcription of E(spl)-C. Lateral inhibition cannot proceed in the absence of Su(H) because blocking repression by Su(H) is not sufficient for E(spl)-C transcription (Li, 2001).

The ato gene could be a direct target of proneural enhancement. ato regulatory sequences have been examined for activity control regions, but possible repression sites have not been assessed. Another candidate is daughterless, which encodes a bHLH heterodimer partner of Ato that is required for Ato function in eye development. A third candidate is senseless, a zinc finger protein that enhances and maintains proneural gene expression. Expression of ato and sens is prematurely elevated in the absence of Su(H), which is consistent with regulation by Su(H)-R. However, each might depend on Su(H)-R only indirectly because elevated expression of ato or sens requires the function of all three genes (Li, 2001).

The secreted glycoprotein Wingless (Wg) acts through a conserved signaling pathway to regulate expression of Wingless pathway nuclear targets. Wg signaling causes nuclear translocation of Armadillo, the fly ß-catenin, which then complexes with the DNA-binding protein TCF (Pangolin), enabling it to activate transcription. Though many nuclear factors have been implicated in modulating TCF/Armadillo activity, their importance remains poorly understood. A ubiquitously expressed protein, Pygopus, is required for Wg signaling throughout Drosophila development. Pygopus contains a PHD finger at its C terminus, a motif often found in chromatin remodeling factors. Overexpression of pygopus also blocks the pathway, consistent with the protein acting in a complex. The pygopus mutant phenotype is highly, though not exclusively, specific for Wg signaling. Epistasis experiments indicate that Pygopus acts downstream of Armadillo nuclear import, consistent with the nuclear location of heterologously expressed protein. These data argue strongly that Pygopus is a new core component of the Wg signaling pathway that acts downstream or at the level of TCF (Parker, 2002).

To examine targets that are positively regulated by Wg in the wing, the zinc-finger protein Senseless (Sens) and the homeodomain protein Distal-less (Dll) were chosen. Sens is expressed in the proneural clusters on either side of the dorsoventral border, immediately adjacent to the Wg expression domain. Inhibition of Wg signaling with a dominant-negative TCF blocks Sens expression, demonstrating that it is a short-range target of Wg action. pygo-expressing cells outside the Wg expression domain completely lack Sens expression. The long-range target Dll is also always lost in clones overexpressing pygo. For reasons that are not clear, occasionally some expression persists just inside the clonal border (Parker, 2002).

Su(H)/CBF1 is a key component of the evolutionary conserved Notch signalling pathway. It is a transcription factor that acts as a repressor in the absence of the Notch signal. If Notch signalling is activated, it associates with the released intracellular domain of the Notch receptor and acts as an activator of transcription. During the development of the mechanosensory bristles of Drosophila, a selection process called lateral inhibition assures that only a few cells are selected out of a group to become sensory organ precursors (SOP). During this process, the SOP cell is thought to suppress the same fate in its surrounding neighbours via the activation of the Notch/Su(H) pathway in these cells. Although Su(H) is required to prevent the SOP fate during lateral inhibition, it is also required to promote the further development of the SOP once it is selected. Importantly, in this situation Su(H) appears to act independently of the Notch signalling pathway. Loss of Su(H) function leads to an arrest of SOP development because of the loss of sens expression in the SOP. These results suggest that Su(H) acts as a repressor that suppresses the activity of one or more negative regulator(s) of sens expression. This repressor activity is encoded by one or several genes of the E(spl)-complex. These results further suggest that the position of the SOP in a proneural cluster is determined by very precise positional cues, which render the SOP insensitive to Dl (Koelzer, 2003).

Thus Su(H) is required to promote SOP development. This is based on the fact that most cells of proneural clusters in the notum that lack Su(H) function do not express SOP markers such as Sens, Hindsight (Hnt) and partially neur^A101-lacZ. Loss of neur^A101-lacZ expression has been attributed to a 'general sickness' of the mutant discs, since the lack of neur^A101-lacZ expression has only been observed in the late developing proneural clusters. The data argue against such an explanation: Presenilin (Psn) mutant wing imaginal discs exhibit a stronger neurogenic phenotype than do Su(H) mutants. Similar to Su(H) mutants, homozygous Psn mutant animals also die during the early pupal phase. Nevertheless, the cells of the proneural clusters of these mutants express all tested markers, indicating that SOP development is not affected. The same is true for kuzbanian (kuz) mutants, whose mutant phenotype is comparable with that of Su(H) mutants. Hence, general sickness of the wing imaginal disc cells is not likely to explain the arrest of SOP development in Su(H) mutants (Koelzer, 2003).

A role of Su(H) in development of the SOP is surprising, because it is a core element of the Notch signalling pathway and the activity of this pathway is required to prevent SOP development in cells of the proneural clusters. Importantly, in this new role, Su(H) seems to function independently of the Notch signalling pathway. This is indicated by the finding that the Su(H) mutant phenotype is epistatic over that of Psn mutants (Koelzer, 2003).

The data presented here indicate that Su(H) appears to be required to suppress the activity of one or more members of the E(spl)-C, that in turn suppress the expression of genes such as hnt and sens. This conclusion is based on: (1) the failure of Su(H)VP16 to activate sens; (2) the fact that Psn H double mutants display a similar loss or reduction of sens expression as Su(H) and Su(H); Psn double mutants, and (3) the fact that expression of a Su(H) construct that is unable to bind H (UAS Su(H)DeltaH) leads to an arrest of SOP development in Psn mutant wing imaginal discs. Several reports show that H is involved in Su(H)-related suppression of gene expression in the absence of Notch signalling. Recently, it has been shown that H acts as a bridge between Su(H) and the general co-repressors CtBP and Gro. It is therefore likely, that this Su(H)/H/Gro/dCtBP complex mediates the repressor function required during SOP development (Koelzer, 2003).

Repression by Su(H) is not strictly required in all proneural clusters to allow expression of sens and other late SOP markers. Examples are the clusters in the wing region, such as the clusters of the dorsal radius. However, even in these clusters, sens and hnt are not expressed in all cells that express early markers, such as neur^A101. Therefore, it appears that the activity of Su(H) promotes SOP development also in these clusters. The clusters of the dorsal radius give rise to other types of sense organs, such as companiforme sensilla, and it is possible that there are different requirements for the activity of Su(H) for the development of the different types of sense organs (Koelzer, 2003).

In Su(H) mutant cell clones induced during the first larval instar stage, hnt is expressed in a fraction of cells of specific proneural clusters, such as the scutellar cluster, but absent or strongly reduced in other clusters. It was further found that in Su(H) mutant wing imaginal discs, expression of sens and hnt is either lost or strongly reduced when compared to mutant cell clones induced during the first larval instar (Koelzer, 2003).

Altogether, these observations suggest that the Notch pathway might have two separable functions during SOP development. During early phases of a proneural cluster, the activity of the pathway keeps the cells of the cluster undecided, perhaps by mutual repression. Owing to positional cues, one cell becomes insensitive to the inhibitory signal and adopts the SOP fate. Subsequently the SOP inhibits its immediate neighbours by sending an inhibitory signal through Dl (Koelzer, 2003).

The Drosophila homolog of the human TEF-1 gene, scalloped (sd), is required for wing development. The Sd protein forms part of a transcriptional activation complex with the protein encoded by vestigial (vg) that, in turn, activates target genes important for wing formation. One sd function involves a regulatory feedback loop with vg and wingless (wg) that is essential in this process. The dorsal-ventral (D/V) margin-specific expression of wg is lost in sd mutant wing discs while the hinge-specific expression appears normal. In the context of wing development, a vg::sdTEA domain fusion produces a protein that mimics the wild-type SD/VG complex and restores the D/V boundary-specific expression of wg in a sd mutant background. Further, targeted expression of wg at the D/V boundary in the wing disc is able to partially rescue the sd mutant phenotype. It is inferred from this that sd could function in either the maintenance or induction of wg at the D/V border. Another functional role for sd is the establishment of sensory organ precursors (SOP) of the peripheral nervous system at the wing margin. Thus, the relationship between sd and senseless (sens) in the development of these cells was also examined, and it appears that sd must be functional for proper sens expression, and ultimately, for sensory organ precursor development (Srivastiva, 2003).

When the sd gene is mutated, the phenotype includes not only the wing margins but also the sensory organs that are found at the wing margins. In addition to the loss of wing margin bristles, there is also a reduction in the number of cells, which results in notching of the wings. This reduction in the number of cells is thought to be a result of apoptosis. In addition, overexpression of sd is also associated with apoptotic cell death. Lyra (Ly) mutations, in contrast, result in the loss of the anterior and posterior margin bristles and this is not associated with apoptotic cell death. However, there is a reduction in the number of cells in the wing margin that manifests itself by erosion of the wing margin. Ly mutations have been shown to be dominant gain of function alleles of sens, in that in a Ly background sens is ectopically expressed. To see if Ly and sd interact genetically, wings were examined from sd^ETX4 males that were also heterozygous for Ly. Flies harboring mutants of both genes show a significant enhancement of the wing phenotype compared to flies with either mutant alone. In the transheterozygous fly, the margin bristles are completely absent, suggesting that these two genes work through a common pathway (Srivastiva, 2003).

Because Ly mutations are gain of function alleles of sens and because Ly interacts with sd, it is possible that this could result in alterations of Sens protein levels in sd mutant wing discs. Wing discs derived from wild-type flies and from flies harboring sd⁵⁸ were stained with an anti-Sens antibody. In wild-type discs, Sens is localized to the region fated to become the wing margin with higher levels at the anterior margin in SOP cells. In addition, sens is also expressed in other SOPs distributed throughout the wing disc. In sd⁵⁸ discs, the wing margin-specific expression of sens is completely lost, but expression in other SOPs is unaffected. Substantial margin-specific expression is restored when the vg::sdTEA fusion construct is expressed in sd⁵⁸ discs using a vg-Gal4 driver. That this restoration of Sens is not complete could be attributed to the amount of the fusion VG::SD TEA protein being produced from the transgene. However, this level of restoration is consistent with the notion that the fusion construct can restore the margin-specific expression of wg, and emphasizes the involvement of wg in specifying the formation of SOPs. The mutual enhancement of mutant wing phenotypes by sd and Ly mutations can also be explained based on the role of wg in SOP formation. Because sd mutations affect the margin-specific expression of wg, and in Ly mutations there is a repression of wg expression, it is predictable that in transheterozygotes the overall Wg signal is further reduced at the margin, resulting in the phenotypic enhancement of wing margin loss (Srivastiva, 2003).

sens has been shown to be both necessary and sufficient for the formation of organs of the peripheral nervous system (PNS). Ectopic expression of sens can result in the formation of extrasensory bristles on the wing and thorax. This ectopic formation of sensory bristles can also happen in the absence of genes of the achaete-scute complex, though to a lesser extent. To see if sd has any role in formation of sensory bristles by ectopic expression of sens, and to confirm that sens is necessary and sufficient for formation of the sensory bristles, sens was expressed in a sd mutant background. The UAS-sens transgene was expressed in both sd^ETX4 and sd⁵⁸ mutant backgrounds using a vg-Gal4 driver and expression from the UAS-sens transgene was determined by staining wing discs with the anti-Sens antibody as a control. If sd has no role in ectopic bristle formation by sens, then expression of sens should result in formation of the sensory bristles missing in the margin of the sd mutants. However, sens expression is unable to restore the margin-specific bristles in sd mutants, suggesting that sens may need sd function for formation of bristles and for proper SOP differentiation. Instead of the formation of ectopic bristles, expression of sens in sd^ETX4 enhances the wing phenotype to resemble the result of the enhancement of sd^ETX4 caused by a Ly mutant. To test this further, UAS-sens was also expressed under the control of a dpp-Gal4 construct that drives expression at the A/P compartment border away from the margin. Wild-type wings expressing sens at the A/P border fail to inflate properly upon eclosion but exhibit numerous ectopic bristles at the position of the A/P border as well as numerous ectopic bristles on the thorax. Expression of sens in a sd⁵⁸ mutant background, however, results in very little to no ectopic bristle formation at the A/P border, again suggesting that sens possibly needs sd function for formation of SOPs (Srivastiva, 2003).

In conclusion, a further characterization of the functions of the SD/VG complex during wing development is reported by analyzing the roles of sd, via the vg::sdTEA fusion during patterning by wg, during growth and during SOP development. In the narrow context of the D/V specific expression of wg, the SD/VG complex appears to act upstream of wg as evidenced by the rescue of the D/V WG stripe by the fusion construct and the rescue of sd wing mutations by the expression of exogenous WG. In addition, the relationship between sd and sens in the development of margin-specific bristles is clarified and the results show that sens needs sd function for proper development of the PNS organs. The current model for actions of the SD/VG complex during wing development, incorporating the new data herein, is that the SD/VG complex either induces or maintains the expression of Wg. This, in turn, causes expression of Sd and Vg to promote cell proliferation in the wing pouch. At the D/V boundary Wg also mediates the expression of sens via its actions on the achaete scute (AS-C) complex that, in the presence of Sd, helps to specify the SOP fate (Srivastiva, 2003).

Regulation of R7 and R8 differentiation by the spalt genes: spalt is required for senseless expresson

Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of the developing ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt has been proposed to promote R7 and R8 terminal differentiation. spalt is shown to be required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specification or differentiation in the larval disc but promotes terminal differentiation during pupation. spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. spalt and senseless are part of a genetic network that regulates rhodopsin 6 and rhodopsin 1. Taken together, these results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8 differentiation during pupal stages, including the regulation of rhodopsin expression (Domingos, 2004).

Photoreceptor cell (PRC) development has been used as a paradigm to understand neuronal specification and differentiation. In the absence of the sal genes, inner PRCs R7 and R8 are transformed into the outer PRC subtype, and this phenotype has been interpreted as a result of the role of sal in R7 and R8 terminal differentiation during pupal stages. As a consequence, a model has been proposed in which PRC differentiation occurs as a two-step process. In the first step, during larval stages, the cells adopt their fate as neurons, become committed and send specific axonal projections. In the second step, during pupal stages, these neurons execute their terminal differentiation program and become mature photoreceptors. In this model, sal is required for the second step of differentiation in R7 and R8. This study shows that sal has distinct roles during R7 and R8 differentiation. In R7, sal is necessary for the expression of the larval markers pros, E(spl)mdelta, and runt. In addition, misexpression of sal during larval stages is sufficient to induce ectopic expression of Pros (R7 marker) and suppress BarH1 (R1/R6 marker). These results demonstrate that sal is required for R7 differentiation during larval stages. However, the majority of sal mutant presumptive R7 cells do not get transformed into the outer PRC subtype during larval stages since the expression of outer PRC markers (Svp, Ro, and BarH1) is not induced. Moreover, R7 specification is not disrupted in sal mutants since R7 still acquires a neuronal fate (expresses Elav), expresses detectable levels of the R7 marker H214-klg, and projects to the medulla. Therefore, it is concluded that the requirement for sal during R7 differentiation occurs soon after R7 specification in a continuum rather than in temporally distinct steps (Domingos, 2004).

In R8, sal is not required for specification or early differentiation in the larval imaginal disc but is necessary for its terminal differentiation during pupation. During pupal stages, sal is necessary for sens expression in R8 and is sufficient to induce ectopic sens in R1-R6. Misexpression of salm, salr, or sens is sufficient to induce ectopic expression of Rh6 and partial suppression of Rh1 in the outer PRCs. Furthermore, the results place sens genetically downstream of sal during R8 pupal development and show that the regulation of Rh1 and Rh6 by sal can occur both via sens-dependent and -independent mechanisms. These findings raise a number of interesting issues with respect to the differentiation of R7 during larval stages, the terminal differentiation of R8 at pupation, and the role of sal and sens in these processes (Domingos, 2004).

Current models account for three developmental stimuli in R7 specification and differentiation during larval stages: EGFR pathway activation, which is required for neuronal differentiation; Sevenless (Sev) receptor signaling, which is required for R7 fate assumption since in Sev mutants the presumptive R7 is transformed into a nonneural cone cell, and Notch signaling, which is also required for R7 fate assumption since loss of Notch function causes the presumptive R7 to be transformed into the R1/R6 subtype (Domingos, 2004).

In salm/salr mutant tissue, the presumptive R7 becomes a neuron since it expresses Elav. This result implies that activation of EGFR and Sev signaling is not significantly affected by the loss of salm/salr function, placing sal downstream of EGFR and Sev activation during R7 differentiation. sal is required for activation of the Notch signaling pathway in R7 since expression of E(spl)mdelta is lost in salm/salr mutants. However, since expression of H214-klg is only partially suppressed in salm/salr mutants and BarH1 is ectopically expressed in only 4.8% of the mutant ommatidia, it is possible that some residual Notch signaling is present in salm/salr mutant R7 cells. Following Notch loss of function, all presumptive R7 cells that are transformed into the R1/R6 subtype show ectopic BarH1 and complete loss of H214-klg expression in larvae. Thus, in salm/salr loss of function, the expression of E(spl)mdelta is lost in R7 but this is not sufficient to respecify the presumptive R7 into R1/R6 subtype as is observed in Notch loss of function mutants. Only later, during pupal development, does the presumptive R7 mutant for salm/salr acquire features of outer PRCs, including large rhabdomere size and expression of rh1 (Domingos, 2004).

Previous studies led to a model for R7 and R8 rhodopsin regulation in the 'yellow' and 'pale' ommatidial subtypes where the 'yellow' subtype (Rh4 in R7 and Rh6 in R8) corresponds to the default state and the 'pale' subtype (Rh3 in R7 and Rh5 in R8) corresponds to the acquired state. This model was based on the observation that, in sev mutants where R7 is absent, all R8 cells express Rh6, suggesting that communication between an R7 expressing Rh3 and the underlying R8 is responsible for the repression of Rh6 and the induction of Rh5 in R8. This study shows that although salm and sens are expressed in all R8 cells, misexpression of these genes in outer PRCs under the control of the rh1 promoter induces ectopic expression of Rh6 but not Rh5. These results suggest that sal and sens regulate the default state of rhodopsin expression in R8 ('yellow' subtype) and that additional factors may be required to repress Rh6 and activate Rh5 expression in the R8 'pale' subtype. The results suggest a model for the regulation of rhodopsin by sal and sens in R8 during pupal stages. In this model, sal regulates sens expression, which in turn suppresses Rh1 and induces Rh6 expression. In addition, sal can also regulate Rh1 and Rh6 independently of sens, in a direct manner or in conjunction with other target genes (Domingos, 2004).

sal is normally expressed in both R7 and R8, which raises the question as to why sal does not also induce Rh6 expression in R7. A possible explanation for the absence of Rh6 in R7 could be the presence of an Rh6 repressor in R7. In accordance with this hypothesis, it has recently been shown that in pros mutant adult retinae, Rh5 and Rh6 expression expands to R7 and that pros is a direct repressor of rh5 and rh6. In pros mutants, salm but not sens is expressed in R7. These results indicate that in the absence of pros, induction of Rh6 expression in R7 occurs independently of sens, and that sal may be involved in this process. Moreover, in R7 cells mutant for pros, since sal is not sufficient to induce sens, factors other than pros should repress sens expression in R7. Alternatively, cofactor(s) required for sens induction by sal in R1-R6 may be absent in R7. Further investigations are necessary to validate these hypotheses and to determine if the regulation of rh1 and rh6 by sal and sens occurs in a direct or indirect manner (Domingos, 2004).

Ligand-dependent de-repression via EcR/USP acts as a gate to coordinate the differentiation of sensory neurons in the Drosophila wing: broad is required for the activation of sens

Loss of function of either the ecdysone receptor (EcR) or Ultraspiracle (USP), the two components of the ecdysone receptor, causes precocious differentiation of the sensory neurons on the wing of Drosophila. It is proposed that the unliganded receptor complex is repressive and that this repression is relieved as the hormone titers increase at the onset of metamorphosis. The point in development where the receptor complex exerts this repression varies for different groups of sensilla. For the chemosensory organ precursors along the wing margin, the block is at the level of senseless expression and is indirect, via the repressive control of broad expression. Misexpressing broad or senseless can circumvent the repression by the unliganded receptor and leads to precocious differentiation of the sensory neurons. This precocious differentiation results in the misguidance of their axons. The sensory precursors of some of the campaniform sensilla on the third longitudinal vein are born prior to the rise in ecdysone. Their differentiation is also repressed by the unliganded EcR/USP complex but the block occurs after senseless expression but before the precursors undertake their first division. It is suggested that in imaginal discs the unliganded EcR/USP complex acts as a ligand-sensitive 'gate' that can be imposed at various points in a developmental pathway, depending on the nature of the cells involved. In this way, the ecdysone signal can function as a developmental timer coordinating development within the imaginal disc (Schubiger, 2005).

The ecdysone signal is transmitted via the ecdysone receptor to activate a number of direct target genes. It has generally been assume that the hormone and its receptor activate a hierarchy by activating early genes that then activate the many late genes. A large body of work based primarily on larval tissues has supported this model. Thus when the ecdysone receptor is non functional, the first step in the cascade fails, the early genes are not activated, and the tissues are unable to undergo a metamorphic response. In imaginal discs it has been shown that loss of function of USP leads to the inability to activate early genes, such as DHR3, EcR and E75B, but also results in precocious differentiation, rather than in a failure to initiate a particular metamorphic response. It has now been demonstrated that loss of EcR function in the wing discs gives similar results (precocious BR-Z1 expression and sensory neuron differentiation) to the ones reported for loss of USP function, and it is concluded that the unliganded EcR/USP heterodimer is the functional repressor. Thus at least some processes at the onset of metamorphosis are not controlled by the ecdysone-induced hierarchy, but rather through the relief of the repressive function of the unliganded EcR/USP complex once the ecdysone titers rise. The importance of this repressive function of the unliganded receptor is further demonstrated by experiments using a dominant negative EcR, which does not bind the hormone, and as a consequence repression cannot be relieved and target genes are not expressed. The repressive role proposed for the unliganded ecdysone receptor complex would also explain why loss-of-function USP clones, in general, result in the differentiation of normal adult bristle organs since loss of receptor function would only control the timing of differentiation. Such an interpretation is supported by early pigmentation of abdominal bristles in usp³ clones that has been observed on occasions. In vivo studies of activation by EcR/USP suggest that activation plays a major role in the metamorphic response of larval tissues but has only a minor role in the development of the imaginal discs. It remains to be seen which processes are activated by the ecdysone hierarchy and which by loss of the repressive actions of the unliganded receptor (Schubiger, 2005).

Adult chemosensory neurons on the wing margin undergo precocious differentiation in loss-of-USP clones. To understand which step is repressed by the EcR/USP complex the early expression patterns of a set of genes involved in neuron differentiation was examined in such mutant clones. In the absence of USP function the early pattern of Achaete (AC) expression in the margin is unaffected. In contrast, both Neur (as visualized by A101) and Sens are expressed in usp mutant cells before they are detected in the surrounding wild-type tissue. In vitro experiments revealed that A101 expression that is already on at the time cultures were set up, remains on through the culture period, but that there is a block by EcR/USP at the level of sens expression that prevents the maturation of the SOPs of the chemosensory neurons in the wing margin. The block is released once the hormone titers rise (Schubiger, 2005).

The repressive function of the unliganded receptor does not act directly on the genes tested. The block of SOP differentiation is controlled through BR-Z1, and br function is required for the activation of sens, a gene necessary and sufficient for sensory organ differentiation. Thus expressing BR-Z1 or Sens early in the margin allows the inhibition from the unliganded ecdysone receptor to be by-passed and the sensory neurons in the margin to differentiate precociously. When Sens is misexpressed it was found that clusters of extra neurons differentiate in the region of high driver expression. This is in agreement with reports that high levels of Sens activate the proneural genes and promote the formation of SOPs. By contrast, when BR-Z1 is misexpressed in the margin, a more normal pattern of sensory neuron arrangement is observed, that is very similar to what is observed in loss-of-function USP or EcR cells. This indicates that BR-Z1 does not induce the formation of SOPs but rather causes the up-regulation of Sens in cells that have already committed to the SOP fate. Occasionally expressing BR-Z1 in the margin leads to the differentiation of a sensory neuron in the posterior margin, normally devoid of neurons. It is possible that in such a situation BR-Z1 misexpression can at times lead to sufficiently high expression of Sens to cause SOP differentiation (Schubiger, 2005).

Loss-of-function of BR demonstrates the requirement for BR to activate the high levels of Sens in the SOPs, as well as the low levels in the posterior margin, but without molecular data it is not known if br is directly activating sens. Since BR-Z1 normally appears later than the initial low expression of Sens, it is proposed that early Sens expression is most probably controlled by BR-Z2. BR-Z2 is expressed shortly after the molt to the third instar, and ectopic BR-Z2 expression induces low levels of Sens. BR-Z3, which also induces Sens when ectopically expressed may induce low levels of Sens as well, but since BR-Z3 is normally expressed at very low levels in the wing disc, it is thought that Br-Z3 plays a minor role. BR-Z1 then is needed for the accumulation of Sens in the mature SOPs (Schubiger, 2005).

In summary the above genetic interactions suggest that unliganded EcR/USP represses BR expression that is required for sens activation and the formation of the mature SOPs in the margin (Schubiger, 2005).

The SOPs are born in a specific temporal sequence in the wing disc. The first SOPs arise in the third instar, 20-30 hours before pupariation; they include GSR, ACV and L3-2 along the third vein. The SOPs of the margin arise later, at 10-12 hours before pupariation, so they are at a very different stage from that of the early born SOPs at the time metamorphosis begins. Since the unliganded receptor is acting as a repressor it is postulated that the block must be occurring at different times during the progression of sensory organ differentiation for these two groups of sensilla. Based on genetic studies, the ecdysone-sensitive arrest for the chemosensory sensilla of the margin occurs in the up-regulation of Sens, since the SOP is undergoing maturation. For the early born sensilla, however, Sens levels are already elevated before the rise in 20E and are not dependent on BR function. For these early born sensilla the ecdysone-sensitive arrest occurs after high Sens expression but prior to the division of the SOP. It is thought that for different sets of sensilla the imposition of an ecdysone-sensitive arrest at different points in development is important to coordinate the differentiation of the sensilla. Such a mechanism would ensure that the outgrowing axons begin to elongate in a choreographed manner leading to the correct axon pathways and to their finding of the correct targets in the CNS according to their physiological function. This idea is supported by the observation that the axons of sensilla forced to differentiate precociously by the absence of a functional ecdysone receptor or by early expression of BR-Z1 or Sens often take abnormal routes (Schubiger, 2005).

Ecdysone is also acting as a timer for the formation of the chordotonal and Johnston's organs as well as for the initiation of the morphogenetic furrow. These structures arise early in the third instar (80 hours after egg laying) and appear to be under the control of the small ecdysone peak at that time. In the case of the leg chordotonal organ, ecdysone appears to be controlling the proneural gene atonal (ato). It is not known yet if this control also occurs via de-repression as is seen for the wing (Schubiger, 2005).

The subsequent progression of the morphogenetic furrow is also dependent on ecdysone. This action of ecdysone has been proposed not to occur via EcR. However, loss of USP leads to an advancement of the furrow and precocious differentiation of the photoreceptors. It has been reported that the progression of the morphogenetic furrow, as well as the timing of differentiation of the chordotonal organs in the leg, are controlled by the insulin receptor (InR)/Tor pathway, with increased InR signaling leading to precocious differentiation. In the wing margin, by contrast, increasing or decreasing InR signaling does not affect the timing of differentiation of the chemosensory neurons. Thus there must be multiple temporal control mechanisms for sensory structures. The current results have demonstrated repression of sensory organs by the unliganded ecdysone receptor at the end of the third instar, but do not rule out additional steps controlled by ecdysone or other factors. It remains to be elucidated which timer(s) is used when, and for which sensory structures (Schubiger, 2005).

In holometabolous insects functional larval tissues are replaced by the differentiating imaginal ones. The endocrine system is acting on larval tissues composed of differentiated cells that are thus in an equivalent state to initiate programs such as cell death and neuronal remodeling. Here EcR/USP's role is activational. For the differentiation of the imaginal tissues the endocrine system faces a varied cellular landscape where some cells may still be dividing while other have begun to differentiate. In these tissues the unliganded receptor acts as a repressor to interrupt the sequence of differentiation at different points in order to coordinate the response to the rising 20E titers. Release of repression by 20E may therefore function as a 'gate' at the onset of metamorphosis and thus would enable development of imaginal tissues to be coordinated and tightly controlled by the rising ecdysone titers. In metamorphosing amphibians a similar situation is seen with functional larval tissues such as the tail and the gills dying and adult limbs and lungs developing in response to thyroid hormone. It would not be surprising to find that the thyroid hormone receptor is activational in the larval tissues but that the forming adult tissues are controlled through de-repression (Schubiger, 2005).

Targets of Activity

An outstanding model to study how neurons differentiate from among a field of equipotent undifferentiated cells is the process of R8 photoreceptor differentiation during Drosophila eye development. Sens is a Zn finger transcription factor that is expressed and required in the sensory organ precursors (SOPs) for proper proneural gene expression. In senseless mutant tissue, R8 differentiation fails and the presumptive R8 cell adopts the R2/R5 fate. senseless repression of rough (ro) in R8 is an essential mechanism of R8 cell fate determination and misexpression of senseless in non-R8 photoreceptors results in repression of rough and induction of the R8 fate. Surprisingly, there is no loss of ommatidial clusters in senseless mutant tissue and all outer photoreceptor subtypes can be recruited, suggesting that other photoreceptors can substitute for R8 to initiate recruitment and that R8-specific signaling is not required for outer photoreceptor subtype assignment (Frankfort, 2001).

sens is expressed in the R8 photoreceptor during third instar eye development beginning within the MF. Since atonal is expressed throughout the MF and is the earliest known marker of R8 differentiation, Ato expression was used to precisely determine when Sens expression begins. The expression of Ato and Sens colocalize beginning with the R8 equivalence group, indicating that Sens is expressed prior to the selection of a single R8 photoreceptor, and Ato and Sens are detected together in the same cell until Ato expression ceases after the third column of photoreceptor development. The overlapping pattern of Sens and a later R8 marker, BBO2, demonstrates that Sens is expressed in R8 throughout larval eye development. Furthermore, Sens does not colocalize with Rough (Ro), which is never expressed in the cells of the R8 equivalence group or in R8 (Frankfort, 2001).

Does Sens continues to mark R8 in mutant backgrounds that contain multiple R8 photoreceptors per ommatidium, as is the case for other R8-specific markers? Sens expression was examined in both ro^X63 and sca^BP2 eye imaginal discs. In both cases, Sens is detected in multiple R8 cells per ommatidium. Thus, Sens remains a faithful marker of R8 in mutant backgrounds (Frankfort, 2001).

Since the expression pattern of Sens overlaps that of Ato and sens lies downstream of proneural genes in the PNS, this relationship was confirmed in the developing eye. ato¹ mutant clones were generated in the eye using the FLP/FRT system; Sens is not detected within these clones. Similarly, Sens is not detected in eye discs dissected from ato¹ mutant larvae. UAS-ato was expressed under the control of sevenless-GAL4, which is expressed in all photoreceptor cells except R8, R2, and R5, and it was found that expression of Sens is strongly activated in response to ectopic ato. Taken together, these data place sens downstream of ato in the developing eye (Frankfort, 2001).

The genetic relationship between ato and sens, the importance of ato in R8 development, and the early detection of Sens in the developing R8 photoreceptor suggest that sens might play a role in normal R8 differentiation. This was tested by generating large sens mutant clones using the FLP/FRT system in a Minute background. Within a single ommatidium of the adult retina, photoreceptors have a characteristic, highly regular arrangement. The rhabdomeres of the six outer photoreceptors (R1-R6) are large in size and form a trapezoid. Centrally placed within the trapezoid are the small rhabdomeres of R7 and R8. This precise organization allows unambiguous identification of all photoreceptor subtypes within a normally constructed ommatidium. Moreover, cells within a single ommatidium are not derived from a fixed cell lineage. Thus, ommatidia located at the border of clones containing both wild-type and mutant photoreceptors may still be normally constructed. Analysis of such 'mosaic' ommatidia reveals which photoreceptors, if any, require the function of a gene in question for normal ommatidial development. Normally constructed ommatidia containing photoreceptors mutant for sens could form only if the R8 photoreceptor had at least one functional copy of sens. Of 91 mosaic ommatidia scored, no normally constructed ommatidia containing a sens mutant R8 were recovered, and there was no significant preference for any other photoreceptor to retain sens function (Frankfort, 2001).

Large patches of retina containing ommatidia entirely mutant for sens are readily recovered in adults. While ommatidia in such clones are disorganized and of variable size and configuration, the number of ommatidia and the spacing between them is not changed from the surrounding wild-type tissue. However, all ommatidia have one striking similarity: they do not contain morphologically discernable R8 or R7 photoreceptors. Small rhabdomeres are extremely rare and could not be identified as part of either an R8 or an R7 photoreceptor. Since R8 is believed to be required for subsequent photoreceptor recruitment during normal eye development, the presence of outer photoreceptors despite the absence of a morphologically distinct R8 within sens mutant ommatidia is a puzzling observation (Frankfort, 2001).

At least four models could explain the presence of photoreceptors in sens mutant ommatidia despite the apparent absence of R8. (1) The R8 photoreceptor could differentiate, begin the process of photoreceptor recruitment, and then die. This has been ruled out by the absence of unexpected programmed cell death. (2) Recruitment of photoreceptors could occur without a differentiated R8. (3) The R8 photoreceptor could differentiate with characteristics of both R8 and another photoreceptor such that it could not be detected as an R8 cell in the adult, yet could still enable photoreceptor recruitment to occur. (4) The R8 cell could initiate differentiation but then undergo a fate change into another photoreceptor subtype, while still enabling photoreceptor recruitment to occur (Frankfort, 2001).

The second model of recruitment without R8 differentiation was addressed by examining other R8-specific markers within sens mutant clones. Normally, Sca protein can be detected in the earliest identifiable R8 photoreceptor, but only for a few columns. Within sens mutant tissue Sca is expressed, but at a lower level of intensity and in fewer cells than in the wild-type. Expression of another early marker of R8 differentiation, Ato, is also altered. Ato is expressed normally from its early broad expression through the R8 equivalence group. However, at the stage where Ato normally resolves into a single R8 cell, reduced expression is routinely observed in sens mutant clones. Specifically, Ato expression is absent at the single R8 stage in 75% of ommatidia but rarely can be detected in single cells as late as the third column of development. The persistence of the enhancer trap in sca suggests that R8 selection occurs, but the observed patterns of Sca and Ato indicate that the process of R8 differentiation is aborted within the MF, most often during resolution of the R8 equivalence group to a single R8 cell, an interval of about one and a half hours. Later markers for R8, Boss and BBO2, are always absent within sens mutant clones. Thus, while the R8 photoreceptor may initiate differentiation, the process is rapidly aborted and the presumptive R8 always ceases to express R8-specific genes. These findings do not unequivocally rule out the second model of recruitment without a differentiated R8, but they do disprove the third model of dual fate because R8-specific gene expression is rapidly and consistently lost (Frankfort, 2001).

As the R8 cell may initiate differentiation but does not subsequently die in sens mutant clones, the fourth model, that R8 might undergo a fate change, was considered. To test this, the expression of photoreceptor subtype-specific markers within sens mutant clones was studied using an R2/R5-specific enhancer trap to mark R2 and R5, Spalt (Sal) to mark R3 and R4, and BarH1 to mark R1 and R6. The most significant finding is that the R2/R5-specific enhancer trap is expressed in three cells per ommatidium instead of the usual two in > 99% of sens mutant ommatidia. In contrast, although photoreceptors of the R3, R4, R1, and R6 subtypes were indeed recruited, they are generally present in reduced numbers. These data suggest that, despite rapid loss of R8-specific gene expression in sens mutant clones, all outer photoreceptor subtypes can be successfully recruited. It was also found that levels of dual phosphorylated extracellular signal regulated kinase (dpERK), the activated MAP kinase of the Ras signaling cascade, are reduced but not eliminated within sens mutant clones. Since EGFR signaling is responsible for nearly all Ras signaling in the developing eye and is required for all non-R8 photoreceptor recruitment, the reduction in dpERK levels is consistent with, and may be the cause of, diminished photoreceptor recruitment in sens mutant tissue (Frankfort, 2001).

The fourth model predicts that the extra R2/R5 photoreceptor observed in sens mutant ommatidia might be the presumptive R8 cell. To test this hypothesis, advantage was taken of the R8-specific enhancer trap in sca, which persists in the presumptive R8 photoreceptor even within sens mutant tissue. The expression of both the R2/R5- and the R8-specific enhancer traps were determined in the same sens mutant clone, and it was found that in the neighboring wild-type tissue the two enhancer traps together mark three cells per ommatidium at comparable levels of intensity. Although three cells per ommatidium continue to express the two enhancer traps within sens mutant clones, the centrally positioned cell is consistently marked with greater intensity Therefore, the R2/R5 marker is misexpressed in the presumptive R8 photoreceptor (Frankfort, 2001).

In addition to the R2/R5-specific enhancer trap described above, another R2/R5 marker, Rough (Ro), was examined to confirm and extend the hypothesis that the presumptive R8 photoreceptor becomes an R2 or R5 photoreceptor. Ro is normally expressed in the R2/R5 photoreceptors and later expands to include the R3/R4 photoreceptors. Within sens mutant clones, Ro is detected abnormally in three cells per ommatidium at the time when it should only be detected in the R2/R5 photoreceptor pair. Moreover, when the presumptive R8 cell is marked with the R8-specific enhancer trap in sca, the enhancer trap is consistently expressed in one of the three Ro-expressing cells. These data suggest that sens normally represses Ro in the differentiating R8 photoreceptor (Frankfort, 2001).

Since ro is not normally expressed in R8, and misexpression of Ro induces changes in cell fate, it was hypothesized that Ro misexpression in the presumptive R8 cell is responsible for the loss of R8 observed in sens mutant clones. To test whether ro is epistatic to sens, sens mutant clones were generated in a ro mutant background. Many ommatidia mutant for both sens and ro contain photoreceptors with small rhabdomeres, suggesting the presence of either R8 or R7 photoreceptors. Furthermore, some double mutant ommatidia contain more than one photoreceptor with a small rhabdomere, similar to the ro mutant phenotype. To determine if the photoreceptors with small rhabdomeres were R8 cells, the expression of two late markers of R8 differentiation, Boss, and an R8-specific enhancer trap, BBO2, were examined in sens mutant clones generated in a ro mutant background. While Boss is never expressed in tissue mutant only for sens, Boss expression is restored in many ommatidia in the double mutant tissue. BBO2 expression is also restored in the double mutant tissue, sometimes in two photoreceptors within the same cluster, suggesting the presence of more than one R8 photoreceptor. Thus, inappropriate Ro expression in the presumptive R8 cell is likely responsible for the early abortion of R8 differentiation and adoption of the R2/R5 fate in sens mutant ommatidia (Frankfort, 2001).

Since sens-mediated repression of ro plays a critical role in R8 differentiation, whether sens misexpression is sufficient to repress ro and induce additional photoreceptors to adopt the R8 fate was tested. sens was misexpressed in clones posterior to the MF using a variation on the MARCM system. Within sens misexpression clones, Ro is repressed in those cells showing the highest levels of Sens. sens misexpression is also sufficient to induce Boss expression in multiple cells per ommatidium and at higher levels than it is normally expressed. Finally, in the adult retina, misexpression of sens causes ommatidial disruption near the center of the clone and induces the formation of ectopic small rhabdomeres within ommatidia near the clonal border. Thus, sens is capable of repressing Ro when misexpressed, and is sufficient to induce both R8-specific gene expression and rhabdomere morphology (Frankfort, 2001).

Thus sens lies downstream of ato and sens expression, which begins in the R8 equivalence group, is maintained in R8 at least through the completion of larval development. Consistent with this expression pattern, there is a cell-autonomous requirement in R8 for sens, but no requirement for sens in any other photoreceptor. Interestingly, in sens mutant ommatidia, a presumptive R8 photoreceptor is selected, but it appears that R8 differentiation does not occur in the majority of ommatidia, and in the minority of ommatidia where R8 differentiation may initiate, the process is quickly aborted within the MF. Since R8-specific gene expression is never observed or is rapidly lost in sens mutant tissue, sens function is thus required to ensure proper R8 differentiation and to maintain the R8 fate immediately following the stage of the R8 equivalence group, but is not required for R8 selection (Frankfort, 2001).

In sens mutant ommatidia, the presumptive R8 cell rapidly expresses R2/R5-specific genes and adopts the fate of an R2/R5 photoreceptor in essentially all cases. One R2/R5-specific marker that is abnormally expressed in the presumptive R8 within sens mutant ommatidia is Ro. ro acts as a repressor of R8 differentiation and Ro is not normally expressed in R8. The consistent misexpression of Ro within the presumptive R8 photoreceptor in sens mutant tissue suggests that sens represses Ro in R8. Such a relationship between sens and ro is further supported by the observation that sens misexpression causes the repression of Ro in outer photoreceptors. Moreover, it is clear that repression of Ro in R8 is of functional significance because loss of ro function is sufficient to rescue the R8 loss observed in sens mutant clones. These data imply that ro is epistatic to (downstream of) sens and that sens-mediated repression of Ro is essential for R8 differentiation. Thus, repression of a cell-fate repressor is identified as a major mechanism of R8 differentiation. These findings are also consistent with the observations that sens acts as a repressor in the Drosophila CNS and that the sens homologs Gfi-1 (murine) and pag3 (C. elegans) function as repressors as well (Frankfort, 2001).

In addition to its role as a repressor of R8 differentiation, ro is sufficient to induce changes in subtype specification, and it is thought that ro acts downstream of photoreceptor recruitment to specify photoreceptor subtype identity as an R2/R5 cell. Moreover, it is clear that in ro mutant ommatidia the presumptive R2/R5 photoreceptors adopt the fate of an R1, R3, R4, or R6 photoreceptor. Similarly, both loss- and gain-of-function experiments reveal that sens function is not required for establishment or maintenance of neural fate in the developing eye, but specifically for directing a cell to follow the R8 differentiation pathway. Thus, sens and ro seem to have analogous roles in directing the specification of specific photoreceptor cell fates. The transcriptional and genetic relationships identified between sens and ro imply that the process of R8 differentiation involves a hierarchical interaction where sens normally represses ro to prevent both ro repression of R8 and ro induction of R2/R5. When sens function is removed, ro is abnormally expressed in the presumptive R8 cell and the R2/R5 fate is adopted (Frankfort, 2001).

Thus a new model is proposed for the genetic regulation of R8 differentiation that includes the relationships among ato, sens, and ro. In this model, ato induces sens within the R8 equivalence group and R8, and sens is in turn required for maintenance of ato expression. Since R8 may transiently differentiate in sens mutant clones, ato is likely sufficient to confer specificity to R8 differentiation, whereas sens is required to 'lock-in' and maintain this program of R8 differentiation, primarily via the repression of ro. Thus, mutual antagonism of ato and ro is likely mediated by sens. sens presumably has a ro-independent role in R8 differentiation as well, because loss of ro function does not completely rescue the sens mutant phenotype (Frankfort, 2001).

Two of these findings suggest that while R8 differentiation and SOP selection are similar in principle, there are fundamental differences between the two: (1) there is no direct evidence that repression of a repressor is a mechanism used during SOP selection to specify neuronal fates, while this mechanism is of great importance during R8 differentiation; (2) while the relationships between sens and proneural genes are maintained both in the developing eye and the emerging SOP, the sens loss-of-function phenotype is quite different in the eye and the embryonic nervous system. In the embryonic nervous system, loss of sens function results in cell death and complete neural loss. However, loss of sens function in the developing eye leads to altered cell fate decisions, but cells remain viable as neurons. These differences between the eye and the embryonic nervous system are not entirely unexpected, because successful R8 selection and differentiation hinges on the unique phenomenon of differential patterns of gene expression with the passage of the MF. For example, whereas the SOP is surrounded by largely equivalent cells on all sides as it is selected, the emerging R8 cell is surrounded by a graded environment -- a field of R8 competent cells immediately anterior to the equivalently staged regions of R8 differentiation dorsally and ventrally, and more mature ommatidia to the posterior. Moreover, cells in each of these environments exert specific effects upon the process of R8 differentiation. The continued analysis of this unusual developmental strategy may thus unveil principles of nervous system development that are not accessible by the study of SOP development or other systems (Frankfort, 2001).

The analysis of sens mutant ommatidia reveals that the process of R8 differentiation fails very early in development. Despite this, recruitment and differentiation of outer photoreceptors occurs. These findings are unique and paradoxical because R8 is thought to initiate the recruitment of all other photoreceptors, although ato-independent photoreceptor differentiation has been observed. Moreover, loss-of-function mutations in all other genes known to be cell-autonomously required in R8 for normal eye development lead to the complete failure of photoreceptor recruitment. How can photoreceptor recruitment, a process known to require R8, occur without an R8 cell? At present, R8 is believed to have two distinct functions in the process of photoreceptor recruitment. First, R8 is thought to recruit photoreceptors by providing the initial source of Spitz (Spi), a positive ligand for the EGFR during eye development. Spi activates the EGFR which induces Ras signaling and differentiation of all photoreceptors (except R8), cone cells, and pigment cells. Second, R8 recruits the R7 photoreceptor via direct Boss/Sevenless interactions. The second function of R8 is clearly abrogated in sens mutant clones. This is expected because R7 is induced by physical contact with the Boss ligand, which is normally expressed solely on R8, and Boss is never expressed within sens mutant clones. However, the first function of R8, while somewhat compromised, is not eliminated (Frankfort, 2001).

Current models predict that after Spi is first secreted from R8, it binds to the EGFR and induces Ras signaling in two neighboring cells, the emerging R2 and R5 photoreceptors. Subsequently, Spi is also secreted from R2 and R5 (and later R3 and R4) and the increased Spi concentration leads to recruitment of all later photoreceptors. In one model, it is specifically the timing of induction of the EGFR pathway that determines photoreceptor subtype. However, an equally plausible model for the recruitment of R2 and R5 (and perhaps later photoreceptors) is one that is similar to R8-mediated induction of R7, where both activation of the EGFR pathway by Spi and ligand/receptor interactions (Boss/Sevenless) are required together for induction of the R7 fate. All outer photoreceptor subtypes can be recruited in sens mutant ommatidia. Because sens mutant ommatidia lack a differentiated R8 cell, these observations rule out all models for subtype specification that involve any R8-specific signaling, either in the form of spatial cues or ligand/receptor interactions. Other models that rely on timing, signaling from other photoreceptors, retinal prepatterning, combinatorial signaling, the actions of surrounding undifferentiated cells, or instructive signaling from the presumptive R8 prior to overt R8 differentiation remain possible, likely in combination with one another (Frankfort, 2001).

In sens mutant ommatidia, the selection of the presumptive R8 cell is not affected, but the presumptive R8 differentiates as an R2/R5 cell at approximately the same time as R8 would normally differentiate. Thus, it is likely that while the identity of the cell initially producing Spi is different in the absence of sens function, the timing of initiation of Spi secretion remains more or the less the same. Because all photoreceptors are recruited, it therefore appears that an R2 or R5 photoreceptor can largely fulfill the previously presumed function of R8 in outer photoreceptor recruitment. Thus, it has been specifically demonstrated that R8 is dispensable for photoreceptor recruitment and it is likely that Spi produced from an alternate source (in this case R2/R5) at roughly the same time is entirely sufficient to initiate the process of recruitment. However, as fewer photoreceptors are recruited in sens mutant tissue, it is clear that activation of recruitment from this alternative source is suboptimal. Indeed, decreased levels of dpERK expression in sens mutant ommatidia reflect a reduction in Ras signaling, which is perhaps due to decreased secretion of Spi. Nevertheless, it is now certain that activation of the recruiting pathway mediated by Spi occurs independently of R8 differentiation (Frankfort, 2001).

Senseless blocks nuclear transduction of Egfr activation through transcriptional repression of pointed

The Epidermal growth factor receptor (Egfr) pathway controls cell fate decisions throughout phylogeny. Typically, binding of secreted ligands to Egfr on the cell surface initiates a well-described cascade of events that ultimately invokes transcriptional changes in the nucleus. In contrast, the mechanisms by which autocrine effects are regulated in the ligand-producing cell are unclear. In the Drosophila eye, Egfr signaling, induced by the Spitz ligand, is required for differentiation of all photoreceptors except for R8, the primary source of Spitz. R8 differentiation is instead under the control of the transcription factor Senseless. High levels of Egfr activation are incompatible with R8 differentiation; the mechanism by which Egfr signaling is actively prevented in R8 is described. Specifically, Senseless does not affect cytoplasmic transduction of Egfr activation, but does block nuclear transduction of Egfr activation through transcriptional repression of pointed, which encodes the nuclear effector of the pathway. Thus, Senseless promotes normal R8 differentiation by preventing the effects of autocrine stimulation by Spitz. An analogous relationship exists between Senseless and Egfr pathway orthologs in T-lymphocytes, suggesting that this mode of repression of Egfr signaling is conserved (Frankfort, 2004).

In this analysis of sens function in R8 differentiation, it was found that the extra R2/R5 cell that develops from the pre-R8 in sens mutants expresses Ro, which is normally expressed in R2/R5 but not R8. Ro is expressed downstream of Egfr pathway activation, and both ro function and high levels of Egfr pathway activation are required for R2/R5 differentiation. Since the pre-R8 cell consistently expresses Ro and differentiates as an R2/R5 cell in sens mutants, it was hypothesized that this transformation occurs as a consequence of high levels of Egfr activation in the pre-R8 cell (Frankfort, 2004).

This hypothesis was tested by simultaneously removing sens function and blocking Egfr activation in the developing Drosophila eye. Egfr activation was blocked by removing function of both rhomboid-1 (rho-1) and rhomboid-3 (rho-3; FlyBase: roughoid, ru). Loss of both rho-1 and rho-3 function prevents processing of secreted Egfr ligands, including Spi, and results in the loss of all ERK (MAP kinase) activation. Furthermore, loss of rho-1 and rho-3 phenocopies Egfr loss-of-function in that only R8 cells differentiate. Loss of sens function results in pre-R8 differentiation as a founder R2/R5 cell which is sufficient to recruit a reduced number of photoreceptors. However, the absence of rho-1, rho-3 and sens together causes total photoreceptor loss, except for a few photoreceptors near the clonal boundary that are rescued non-autonomously by neighboring wild-type cells that produce and process Spi appropriately. A similar phenotype is detected in tissue mutant for both spi and sens. This loss of photoreceptors seen in rho-1 rho-3 sens and spi sens mutants is not due to cell death because apoptosis was prevented in these experiments by expression of GMR-p35. Furthermore, pre-R8 selection still occurs in both rho-1 rho-3 and rho-1 rho-3 sens mutant tissue, suggesting that a potential founding photoreceptor is present. Therefore, these results are interpreted to mean that, in the absence of sens function, pre-R8 differentiation as a founder R2/R5 photoreceptor requires activation of the Egfr signaling pathway via the Spi ligand. In other words, in sens mutants, the pre-R8 switches from a Spi/Egfr-independent R8 differentiation pathway to a Spi/Egfr-dependent R2/R5 differentiation pathway (Frankfort, 2004).

This work suggests that Sens acts to ensure that the organizing center of each ommatidium is refractory to the developmental signals it produces -- the R8 cell can secrete Spi and even activate Egfr on its own cell membrane, yet remains protected from the deleterious effects of activation of Pnt and other Egfr targets, such as Ro, in R8 (Frankfort, 2004).

The mechanism by which Sens regulates the discrepancy between levels of Egfr activation at the receptor/cytoplasmic and nuclear levels in R8 is probably through repression of pnt transcription. This is supported by the observation that pnt transcription is not induced by misexpression of an activated form of Egfr when sens is co-misexpressed. Furthermore, expression of the pnt-P1 isoform in R8 disrupts R8 differentiation. Since misexpression of pnt-P2 has no effect on R8 differentiation, this suggests that Sens negatively regulates transcription of pnt-P1, but not pnt-P2. This mode of regulation is consistent with established models for transduction of the Egfr signal to the nucleus. Specifically, ERK phosphorylates Pnt-P2, which is thought to be a transient positive regulator of pnt-P1 transcription. In this model, transduction of Egfr activation occurs all the way into the nucleus of R8, but Sens represses the pathway at the final step -- positive regulation of pnt-P1 by Pnt-P2. When sens function is removed, the block on pnt-P1 transcription is relieved, and Pnt-P1 can exert its transcriptional effects on the nucleus, including ro induction (Frankfort, 2004).

There is evidence that pnt-P1 transcription can be regulated by Egfr signaling independently of pnt-P2 during Drosophila embryogenesis. If this is the case during eye development, the model would remain essentially the same -- Sens would still act as a negative regulator of pnt-P1 in R8. However, this regulation would occur independently of pnt-P2 rather than downstream of pnt-P2 (Frankfort, 2004).

Sens is also a potent negative regulator of ro and this relationship appears to specifically affect the cell fate decision between R8 and R2/R5 differentiation. Several lines of evidence suggest that Sens-mediated repression of ro is distinct from other effects of Sens in R8: (1) loss of ro function does not rescue R8 differentiation in all ommatidia in sens mutants; (2) even those R8 cells that do differentiate in sens ro double mutants require Spi/Egfr pathway activation; (3) misexpression of ro in R8 causes a different phenotype than misexpression of pnt-P1 in R8. Specifically, even though Egfr pathway activation is necessary and sufficient for Ro expression, misexpression of pnt-P1 in R8 does not cause an obvious cell fate transformation from R8 to R2/R5, while misexpression of ro in R8 does. Indeed, R8 markers are still expressed when pnt-P1 is misexpressed in R8. However, aberrant nuclear movements and the absence of small rhabdomeres at the level of R8 in adults suggest that misexpression of pnt-P1 does perturb R8 differentiation. Together, these results suggest that Sens repression of pnt-P1 occurs independently of Sens function as a repressor of ro, and that Sens-mediated repression of pnt-P1 is probably required for normal R8 differentiation upstream or independently of cell fate determination (Frankfort, 2004).

Since Sens acts as a transcription factor and its mammalian homolog, Gfi-1, binds directly to enhancer regions of Ets1 and Ets3, two mammalian orthologs of pnt, it is possible that Sens repression of pnt-P1 expression occurs directly. Gfi-1 also interacts with nuclear matrix proteins to repress transcription. Thus, it is possible that Sens represses transcription of Egfr nuclear effectors via a similar mechanism. Future experiments are required to determine which of these or other mechanisms are important during R8 differentiation. However, it is likely that Sens does not act as a positive regulator of Edl/Mae, a proposed cell-autonomous repressor of Egfr signaling, because edl/mae function is not required for normal R8 differentiation. Finally, it is also unlikely that sens functions as an activator of yan, which encodes a nuclear repressor of the Egfr pathway, because yan loss-of-function mutations also do not impact R8 differentiation (Frankfort, 2004).

The positioning of Sens repression downstream of ERK activation may help explain interactions observed between sens and Egfr pathway homologs in T-lymphocytes. In Jurkat T-cells, activation induced cell death (AICD), a process that is required to prevent non-specific activation of T-cells, is dependent, in part, on ERK1/2 activation. Intriguingly, high levels of Gfi-1 have been shown to inhibit AICD despite high levels of ERK1/2 activation (Karsunky, 2002). The antagonistic relationship between Sens and the Egfr pathway in R8, in conjunction with the observation that Gfi-1 can bind to the enhancer regions of Ets1 and Ets3, suggest that this inhibition of AICD may occur via Gfi-1-mediated repression of ERK1/2 targets (such as Ets/pnt) in T-cells (Duan, 2003). Thus, these results may establish R8 development as a powerful and novel system with which to study mechanisms of lymphomagenesis, apoptosis and cancer (Frankfort, 2004).

Evolution of neural precursor selection: functional divergence of proneural proteins: Differential interactions with different types of zinc (Zn)-finger proteins mediate the divergence of ATO and NGN activities

How conserved pathways are differentially regulated to produce diverse outcomes is a fundamental question of developmental and evolutionary biology. The conserved process of neural precursor cell (NPC) selection by basic helix-loop-helix (bHLH) proneural transcription factors in the peripheral nervous system (PNS) by atonal related proteins (ARPs) presents an excellent model in which to address this issue. Proneural ARPs belong to two highly related groups: the ATONAL (ATO) group and the NEUROGENIN (NGN) group. A cross-species approach was used to demonstrate that the genetic and molecular mechanisms by which ATO proteins and NGN proteins select NPCs are different. Specifically, ATO group genes efficiently induce neurogenesis in Drosophila but very weakly in Xenopus, while the reverse is true for NGN group proteins. This divergence in proneural activity is encoded by three residues in the basic domain of ATO proteins. In NGN proteins, proneural capacity is encoded by the equivalent three residues in the basic domain and a novel motif in the second Helix (H2) domain. Differential interactions with different types of zinc (Zn)-finger proteins mediate the divergence of ATO and NGN activities: Senseless is required for ATO group activity, whereas MyT1 is required for NGN group function. These data suggest an evolutionary divergence in the mechanisms of NPC selection between protostomes and deuterostomes (Quan, 2004).

NPC formation in Drosophila requires the Zn-finger protein Senseless (SENS). Fly proneural proteins first induce sens expression and then synergize with it in a positive feedback loop. This appears to enhance the ability of proneural genes to downregulate Notch signaling in the presumptive NPC. In vertebrates, Senseless-like proteins appear not to act in NPC formation, although they are expressed in the PNS. To test the possibility that SENS shows group specific interactions with bHLH proteins during NPC selection, the abilities of ATO and NGN1 to induce SENS were examined. SENS expression in wild-type L3 wing discs marks NPC formation. Ectopic SENS induction is detected along the AP axis of wing discs when ATO is misexpressed. However, SENS expression is not induced by NGN1. These data suggest that unlike ATO, NGN1 does not efficiently induce SENS expression. Whether lowering endogenous levels of Notch would allow NGN1 to induce SENS was examined. Expression of NGN1 in Notch heterozygous animals, although significantly increasing the number of induced bristles, fails to induce SENS expression when compared with N^+/– controls, arguing that NPCs induced by NGN proteins are specified via a different mechanism not normally used in Drosophila. Although NGN1 does not induce SENS, it is possible that synergy might occur if the requirement for SENS induction is bypassed. Therefore the ability of NGN1 and MATH1 to synergize with SENS in vivo was compared by co-expressing either NGN1 or MATH1 with SENS using a moderate scutellar Gal4 driver (C5-Gal4). Neural induction was examined by counting the ectopic bristles induced on the scutellum. Wild-type flies have four large bristles, or macrochaete, on their scutella. Expression of SENS or MATH1 alone with C5-Gal4 induces a number of ectopic microchaete, or small bristles, on the scutellum. No ectopic sensory bristles were found when NGN1 was expressed alone. Co-expression of NGN1 and SENS has the same effect on the scutellum as the misexpressing SENS alone. Co-expression of MATH1 and SENS, however, causes the appearance of a large number of both micro- and macrochaete. Finally, NGN1 or MATH1 were co-expressed in the absence of one copy of sens. No effect on NGN1 activity in a sens^+/– background was observed. By contrast, the average number of sensory bristles produced by MATH1 along the AP axis was reduced by 42% if a single copy of sens was removed suggesting dose-sensitive interactions. Thus, neither by loss nor gain of function criteria does NGN1 appear to interact with SENS, thus explaining its weak proneural activity and inability to efficiently antagonize Notch signaling in Drosophila. Therefore, SENS is a key extrinsic difference in how ATO proteins and NGN proteins regulate NPC selection (Quan, 2004).

In Xenopus, the C2HC-type Zn-finger protein X-MyT1 is expressed in primary neurons and can be induced by NGN proteins. In addition X-MyT1 has been suggested to play a role in NPC formation and to synergize with NGN proteins. In order to test if X-MyT1, like SENS, shows specificity in its interaction with ARP proteins, its ability to interact with NGN1 and ATO in Xenopus was compared. X-MyT1 mRNA was injected alone or co-injected with either Ngn1 or Ato mRNA. As expected, the injection of X-MyT1 increases the number of N-tubulin-expressing cells in the neural plate domains where neurons normally form, while the injection of Ngn1 mRNA alone leads to induction of N-tubulin expression. Co-injection of Ngn1 and X-MyT1 mRNAs results in very strong N-tubulin induction, pointing to a synergistic interaction between the two proteins. By contrast, co-injection of Ato and X-MyT1 mRNAs does not cause a detectable increase in N-tubulin expression compared with the injection of X-MyT1 mRNA alone. Similarly, the few ectopic N-tubulin-expressing cells observed when Math1 mRNA is injected are not increased by co-injection of Math1 and X-MyT1. Thus, X-MyT1 interacts specifically with NGN1 and not with ATO or MATH1. The data above demonstrate that the correct combination of ARP protein and Zn-finger protein is necessary for NPC induction (Quan, 2004).

Genes common to protostomes and deuterostomes (including atonal, ngn genes, Notch signaling genes, sens and X-MyT1) most probably derive from the last common bilaterian ancestor. This implies that such an ancestor already possessed all the tools to specify a large diversity of neural cell types and lineages, suggesting a structurally, and consequently behaviorally, complex animal (Quan, 2004).

Proneural basic helix-loop-helix (bHLH) proteins initiate neurogenesis in both vertebrates and invertebrates. The Drosophila Achaete (Ac) and Scute (Sc) proteins are among the first identified members of the large bHLH proneural protein family. phyllopod (phyl), encoding an ubiquitin ligase adaptor, is required for ac- and sc-dependent external sensory (ES) organ development. Expression of phyl is directly activated by Ac and Sc. Forced expression of phyl rescues ES organ formation in ac and sc double mutants. phyl and senseless, encoding a Zn-finger transcriptional factor, depend on each other in ES organ development. These results provide the first example that bHLH proneural proteins promote neurogenesis through regulation of protein degradation (Pi, 2004).

The promoter analysis suggests that phyl expression in SOPs might be activated by factors other than Ac and Sc. Within the 4.1-kb promoter region, eight putative Sens-binding sites (AAATCA, S box) were identified, with three sites distributed within the 3.4-kb proximal region and five sites in a cluster located in a very distal region. Whether Sens plays a role in phyl activation in SOPs was tested, using phyl^4.1-GFP as a reporter. At 10-12 h APF, phyl^4.1-GFP is expressed in dorsoventral stripes along the notum in a pattern analogous to early Ac and Sc expression patterns. At 15 h APF, phyl^4.1-GFP expression is restricted in SOPs. In sens^E2-null clones, phyl^4.1-GFP is expressed in dorsoventral stripes, and this expression is quickly restricted to single SOPs at 16 h APF, identical to that in wild-type tissue. At 20 h APF, when wild-type SOPs have divided to two daughter cells, phyl^4.1-GFP expression in sens^E2 clones is still maintained in single SOPs, and mostly in two cells at 23 h APF when wild-type cells are in GFP-positive clusters containing three or four cells. Therefore, these results suggest that, in the absence of sens activity, SOP development is delayed, but phyl^4.1-GFP expression is minimally affected (Pi, 2004).

To determine the contribution of Sens binding sites to phyl expression, the 3.4-kb phyl promoter region (whose expression pattern is analogous to the 4.1-kb promoter in both wild-type and sens mutant background) was tested. The phyl^3.4DeltaS-GFP reporter with all three S boxes mutated expresses little difference in the GFP pattern and intensity when compared to phyl^3.4-GFP. However, the reporter with mutations in all four E boxes and three S boxes (phyl^3.4DeltaES-GFP) enhances GFP intensity by 20% when compared to phyl^3.4DeltaE-GFP with mutations only in four E boxes. This 20% increase in GFP intensity reflects an increase in the phyl activity in vivo because phyl^3.4DeltaES-ORF shows stronger abilities than phyl^3.4E-ORF in rescuing both the viability and the ES organ number of phyl⁴/phyl²²⁴⁵ flies. Therefore, these data suggest that these S boxes play a negative role in regulation of phyl activity (Pi, 2004).

To test whether phyl regulates sens expression, Sens protein expression was examined in phyl mutants. In phyl²-null clones, Sens expression was almost diminished in all stages examined, including the single-SOP stage, the two-cell stage and the four-cell stage, suggesting that phyl is required for Sens expression in ES organ development (Pi, 2004).

To analyze the functional relationship between phyl and sens further, rescue experiments were performed. Misexpression of sens by Eq-GAL4 fails to induce ES organ formation in phyl² mutant clones. Similarly, ES organ formation induced by phyl misexpression is blocked in sens^E2 mutant clones. This result suggests that although Sens expression depends on phyl activity, Sens and Phyl function in parallel to promote ES organ development (Pi, 2004).

It is concluded that phyl is a non-bHLH gene that can functionally substitute for proneural bHLH genes to execute neural developmental program. This ability of phyl is also manifested from the analysis of phyl loss-of-function phenotypes: sens and ase, required for SOP differentiation, are inactivated, and in addition, neuralized (A101 insertion locus), implicated in the activation and E(spl)-m8 in the transduction of the Notch pathway, is not expressed. Furthermore, SOP cell division, a prerequisite step to generate distinct daughter cells for constructing a complete ES organ, is blocked in phyl mutants. This defect likely reflects a role for phyl in controlling cell cycle progression, because CycE expression in SOPs maintains at a basal level. Therefore, although SOPs have been selected from proneural clusters in phyl hypomorphs, they are associated with several defects as described (Pi, 2004).

Studies of proneural genes have shown that ac and sc promote ES organ identity, whereas ato promotes CH organ identity. cut is the selector gene to specify the ES organ identity; in its absence ES organs are transformed into CH organs and misexpression of cut transforms CH organs into ES organs. The absence of Cut expression in phyl mutants suggests that specification of ES organ identity may be through a regulation of cut expression by Phyl. Although phyl is expressed in SOPs for both ES and CH organs, it was found that, in phyl²/phyl⁴ and phyl¹/phyl⁴ mutants, A101 expression in leg CH organ precursors remained normal. Also, misexpression of phyl fails to rescue ato mutants in CH organ formation. These results suggest that phyl mediates functions of ac and sc only in ES organ development (Pi, 2004).

One well characterized function of Phyl is to bring the Ttk protein to the ubiquitin-protein ligase Sina for degradation. During SOP development, phyl is expressed in SOPs, and Ttk is expressed ubiquitously except in the SOPs and the proneural clusters. Genetic studies among phyl, sina, and ttk suggest that phyl and sina promote ES organ development by antagonizing ttk activity. Ttk contains a BTB/POZ domain and functions as a transcriptional repressor. Therefore, degradation of Ttk can lead to the derepression of SOP-specific genes. These studies suggest that degradation of a general transcriptional repressor plays a crucial role in regulating gene expression in different aspects of neural precursor differentiation (Pi, 2004).

Coordinate control of synaptic-layer specificity and rhodopsins in photoreceptor neurons: Senseless regulatesR9 targeting and Capricious transcription

How neurons make specific synaptic connections is a central question in neurobiology. The targeting of the Drosophila R7 and R8 photoreceptor axons to different synaptic layers in the brain provides a model with which to explore the genetic programs regulating target specificity. In principle this can be accomplished by cell-type-specific molecules mediating the recognition between synaptic partners. Alternatively, specificity could also be achieved through cell-type-specific repression of particular targeting molecules. This study shows that a key step in the targeting of the R7 neuron is the active repression of the R8 targeting program. Repression is dependent on NF-YC (CG3075), a subunit of the NF-Y (nuclear factor Y) transcription factor (Mantovani, 1999). In the absence of NF-YC, R7 axons terminate in the same layer as R8 axons. Genetic experiments indicate that this is due solely to the derepression of the R8-specific transcription factor Senseless (Sens) late in R7 differentiation. Sens is sufficient to control R8 targeting specificity and Sens directly binds to an evolutionarily conserved DNA sequence upstream of the start of transcription of an R8-specific cell-surface protein, Capricious (Caps) that regulates R8 target specificity. R7 targeting requires the R7-specific transcription factor Prospero (Pros) in parallel to repression of the R8 targeting pathway by NF-YC. Previous studies demonstrated that Sens and Pros directly regulate the expression of specific rhodopsins in R8 and R7. It is proposed that the use of the same transcription factors to promote the cell-type-specific expression of sensory receptors and cell-surface proteins regulating synaptic target specificity provides a simple and general mechanism for ensuring that transmission of sensory information is processed by the appropriate specialized neural circuits (Morey, 2008).

The compound eye comprises about 750 simple eyes (ommatidia), each containing a cluster of eight photoreceptor neurons (R1-R8). These neurons form synaptic connections in two regions of the optic lobe, the lamina and the medulla. The R1-R6 neurons innervate the lamina; the R7 and R8 neurons form connections in the M6 and M3 medulla layers, respectively. Genetic studies have led to the identification of cell-surface proteins regulating R7 and R8 target specificity. Notably, mis-targeting mutant R7 neurons terminate selectively in M3, the layer in which wild-type R8 axons terminate, suggesting a close relationship between the genetic programs controlling R7 and R8 target specificity. This study describes transcriptional regulatory pathways that control the differential targeting specificity of these neurons (Morey, 2008).

In a screen for R7 targeting mutants, a strong loss of function mutation was identified in the NF-YC gene, which encodes a subunit of NF-Y, an evolutionarily conserved heterotrimeric transcription factor. Although NF-Y function has not been studied extensively in the fly, it has been shown to act as both an activator and a repressor in other organisms. The targeting of visual-system neurons was assessed in mosaic animals to generate large patches of mutant retinal tissue early in development. About 75% of NF-YC mutant R7 axons terminated in M3, the same layer as wild-type R8 axons. This phenotype was fully rescued by an NF-YC complementary DNA. In contrast with the marked effect of NF-YC mutations on R7, targeting of R8 to the M3 layer and targeting of R1-R6 to the lamina were unaffected (Morey, 2008).

To assess whether NF-YC is required in a cell-autonomous fashion in R7 neurons, mosaic flies were generated in which a fraction of R7 neurons was rendered mutant and labelled with green fluorescent protein (GFP), whereas the remaining R7 neurons and all the R8 neurons were wild-type and unlabelled. About 17% of the mutant R7 neurons (n = 144 of 807) mis-targeted to M3. The decrease in penetrance of the phenotype, in comparison with mutant R7 neurons generated by mitotic recombination induced earlier in the eye primordium, probably reflects perdurance of NF-YC protein present in precursor cells. NF-YC is therefore required autonomously for R7 targeting but not for the targeting of other classes of photoreceptor neurons. As NF-YC is expressed in all R cells, NF-YC must function in combination with other factors or signals selectively activating NF-YC function in R7 (Morey, 2008).

Given that NF-YC is part of a transcription factor complex and is expressed in the nucleus of R7 neurons, it is likely that the change in targeting specificity reflects a change in gene expression. Wild-type R7 neurons initially target to the temporary R7 layer in the medulla and then, during mid-pupal development, extend to their final target. Targeting of NF-YC mutant R7 neurons to the temporary layer is indistinguishable from the wild type. Extension to the final target layer at 70% after puparium formation (APF) is frequently disrupted, with many R7 neurons terminating in the layer within which R8 terminates. Consistent with this finding was the observation that NF-YC mutant R7 neurons expressed all five early R7 markers tested in wild-type patterns. It was reasoned, then, that NF-YC might repress a subset of R8-specific genes in the R7 neuron that later in development control final target layer selection. Indeed, the R8-specific transcription factor Sens was expressed ectopically in NF-YC mutant R7 neurons (Morey, 2008).

sens is a key regulator of R8 development. In wild-type larval eye discs, Sens is expressed in two or three cells that have the potential to become R8 before becoming restricted to a single differentiating R8 neuron. Sens remains expressed in R8 into the adult. It is required at a very early stage of eye development to regulate R8 specification and, much later during pupal development and in the adult, to regulate the transcription of R8-specific rhodopsins directly. In NF-YC mutant larval eye discs, Sens expression in R8 begins before overt R8 differentiation as in the wild type. By contrast, Sens mis-expression in mutant R7 neurons was first observed 15-20 h after the onset of differentiation as assessed by the expression of the R7-specific marker pros. Expression of Sens in mutant R7 neurons persists throughout pupal development and into the adult and is cell-autonomous. As Sens mis-expression occurs after the onset of R7 differentiation and NF-YC mutant R7 neurons mis-target to the M3 layer during the late phase of R7 targeting, sens may promote an R8 targeting program that is distinct from the role of sens in cell fate earlier in development (Morey, 2008).

If upregulation of Sens in NF-YC mutant R7 neurons is responsible for targeting to the M3 layer, removal of Sens from NF-YC mutant cells should suppress the targeting defect. To test this, mitotic recombination was induced on two different chromosomes (namely chromosomes X and 3) to generate R7 neurons that were simultaneously mutant for both NF-YC and sens, and their targeting was assessed in an otherwise wild-type background. Removing sens from NF-YC mutant R7 neurons completely suppresses the mis-targeting phenotype. Thus, during wild-type development the NF-YC mediated repression of sens in R7 is necessary to prevent inappropriate targeting to M3 (Morey, 2008).

To test whether Sens is sufficient to implement an R8 targeting program, sens was mis-expressed in R7 neurons. Under these conditions about 25% of the R7 neurons were redirected to the M3 layer, thus phenocopying NF-YC loss-of-function mutations. Additional experiments using the method in which Sens was provided conditionally early in development to promote R8 cell fate, but removed later, support the view that Sens functions at later stages of R8 development to promote targeting. Taken together, these data raise the possibility that Sens could directly control the expression of cell-surface proteins regulating R8 target specificity (Morey, 2008).

Caps is the only cell-surface molecule that has been shown to be both specifically expressed in the R8 neuron and required for R8 targeting and it is therefore an excellent candidate for direct regulation by Sens. Indeed, like Sens, Caps is expressed ectopically in R7 in NF-YC mutants. Expression of Caps, as detected with an enhancer trap, is specifically activated in NF-YC mutant R7 neurons about 9 h after the onset of Sens expression. Furthermore, a previous study showed that ectopic expression of Caps in R7 respecified their connections to the R8 layer. Both NF-YC mutant R7 neurons and R7 neurons mis-expressing Caps initially target correctly but then select the inappropriate M3 layer during mid-pupal development. Taken together, these observations indicate that caps could be a downstream target of Sens (Morey, 2008).

Examination of the DNA sequences 1 kilobase upstream of caps and within the first large intron led to the identification of four and three putative Sens-binding sites, respectively. An evolutionarily conserved Sens-binding site was identified 500 base pairs upstream of the caps transcriptional start site. Sens protein binds specifically to this site in gel-shift assays, making it likely that caps is a direct target of Sens. However, Sens must regulate R8 target specificity by controlling the expression of other genes in addition to caps, because loss of caps does not suppress the NF-YC mutant phenotype. This is consistent with the finding that loss of caps, in an otherwise wild-type background, results in targeting defects in about 50% of the R8 neurons. Together, these data suggest that Sens directly regulates the expression of Caps, a cell-surface protein controlling R8 target specificity, and must also regulate the expression of other genes involved in this process (Morey, 2008).

Specific repression of sens in R7 neurons could arise through interactions between NF-YC and the R7-specific transcription factor Pros. Like NF-YC, Pros is also required for R7 target specificity. It is expressed in R7 from an early stage of its development through to the adult in a similar fashion to Sens expression in R8. About 20% of the pros-null mutant R7 neurons terminate in M3. Two lines of evidence support the view that Pros works in parallel with NF-YC: first, the loss of pros in R7 neurons does not lead to ectopic expression of Sens, and second, the frequency of mis-targeting R7 axons in single pros-null mutant cells is markedly increased by removing NF-YC. Thus, Pros could either promote R7 targeting directly or, like NF-YC, act to repress an R8 targeting program, or both (Morey, 2008).

Thus, R7 targeting requires NF-YC and, in parallel, Pros, whereas R8 targeting relies on Sens-dependent regulation of caps and other genes. Mutations in many other genes required for R7 targeting cause R7 neurons to mis-target to the M3 layer specifically rather than terminating promiscuously in the medulla. This underscores a tight inter-relationship between the mechanisms regulating targeting to these two layers. On the basis of the strong M3 mis-targeting phenotype of NF-YC mutant R7 neurons and complete suppression of the phenotype by the removal of sens, a key mechanism regulating R7 layer specificity is repression of an R8 targeting program. More generally, repression of inappropriate pathways may promote differential targeting in closely related neurons (Morey, 2008).

The roles of Pros and Sens in target layer selection are analogous to their function in controlling the expression of R7-specific and R8-specific rhodopsins. R7 and R8 neurons express different rhodopsins and hence detect different wavelengths of light. In R8, Sens directly represses the transcription of R7 rhodopsins and directly activates the transcription of an R8 rhodopsin. In the R7 neuron, Pros binds to an upstream regulatory sequence in the R8 rhodopsin genes and represses their expression. NF-YC mutant R7 neurons no longer express R7 rhodopsins, and all express R8 rhodopsins. This is consistent with the finding that NF-YC mutant R7 neurons in adults express Sens but no longer express Pros. Thus, transcription of both R8-specific rhodopsins and an R8-specific targeting protein Caps is directly regulated by Sens (Morey, 2008).

These observations suggest a simple solution to the mechanisms by which sensory neurons connect to the neural circuits specialized for the reception of different sensory stimuli (for example, different wavelengths of light or different odours). Although the molecular basis of this coupling is understood in considerable detail for vertebrate olfactory neurons, in which odorant receptors have a direct function in controlling target specificity, little is known about the coupling in other sensory systems. Coupling is likely to be regulated in a different fashion in other neurons, because even in the fly olfactory system, for example, targeting is independent of sensory receptor expression. On the basis of these studies on Sens it is proposed that the same transcription factors directly control both rhodopsin expression and the cell-surface proteins that control target layer specificity. More generally, it is speculated that in many sensory neurons a common set of transcription factors may directly control, and thereby coordinate, the expression of cell-surface proteins regulating target specificity and the receptors detecting specific sensory stimuli (Morey, 2008).

Atonal, senseless, and abdominal-A regulate rhomboid enhancer activity in abdominal sensory organ precursors

The atonal (ato) proneural gene specifies different numbers of sensory organ precursor (SOP) cells within distinct regions of the Drosophila embryo in an epidermal growth factor-dependent manner through the activation of the rhomboid (rho) protease. How ato activates rho, and why it does so in only a limited number of sensory cells remains unclear. A rho enhancer (RhoBAD) has been identified that is active within a subset of abdominal SOP cells to induce larval oenocytes and it has been shown that RhoBAD is regulated by an Abdominal-A (Abd-A) Hox complex and the Senseless (Sens) transcription factor (Li-Kroeger, 2008). This study shows that ato is also required for proper RhoBAD activity and oenocyte formation. Transgenic reporter assays reveal RhoBAD contains two conserved regions that drive SOP gene expression: RhoD mediates low levels of expression in both thoracic and abdominal SOP cells, whereas RhoA drives strong expression within abdominal SOP cells. Ato indirectly stimulates both elements and enhances RhoA reporter activity by interfering with the ability of the Sens repressor to bind DNA. As RhoA is also directly regulated by Abd-A, a model is proposed for how the Ato and Sens proneural factors are integrated with an abdominal Hox factor to regulate region-specific SOP gene expression (Witt, 2010).

This study found that the Atonal proneural factor is required for both normal rho enhancer function and the proper specification of abdominal oenocytes. In addition, it was determined that two distinct regions of the RhoBAD enhancer contribute to gene activity within the C1 SOP cells. The RhoA element preferentially drives gene expression within abdominal SOP cells, whereas RhoD drives weaker gene expression within the C1 SOP cells of both the thoracic and abdominal segments. Using a combination of genetic and biochemical analyses, it was found that the Ato, Sens, and Abd-A inputs contribute to proper rho enhancer activity. In particular, it was shown that RhoA, but not RhoD, is directly responsive to the Abd-A Hox factor. In addition, Ato was found to indirectly stimulate RhoBAD activity through both the RhoA and RhoD elements. Although it is currently not understood how Ato stimulates RhoD, it was found that Ato limits the DNA binding activity of the Sens repressor protein to RhoA. Coupled with other recent findings on proneural gene function, these results have two major implications: 1) A model is described for how Ato and Sens inputs are integrated to differentially regulate gene expression during SOP cell lineage development, and 2) How proneural input (Ato) and a Hox factor (Abd-A) cooperate to regulate Rho enhancer activity, at least in part, by limiting Sens-mediated repression is discussed (Witt, 2010).

Sens and the proneural factors are intricately linked during PNS development in Drosophila. Loss-of-function mutations in proneural genes disrupt sens expression resulting in a decrease in sensory organ formation and sens mutations result in decreased proneural gene expression and widespread sensory organ deficits. While both encode transcription factors required for PNS development, they have opposite effects on gene expression when bound to DNA. Proneural factors bind E-box DNA sequences with Daughterless to activate gene expression, whereas Sens binds a distinct DNA sequence to repress gene expression. However, recent data revealed that proneural proteins can convert Sens from a transcriptional repressor to a co-activator. Three different proneural factors (Ac, Sc, and Ato) interact with Sens in GST-pulldown and/or co-immunoprecipitation assays. In addition, cell culture assays showed that Sens stimulates the activation potential of proneural factors bound to E-Box sequences. Thus, Sens is a transcriptional repressor when directly bound to DNA through its zinc finger motifs whereas it is a potent co-activator when recruited to DNA by proneural proteins (Witt, 2010).

This study provides two pieces of information that add to understanding of how Sens and proneural factors regulate gene expression. First, purified Sens and Ato/Da proteins were used to show that Ato decreases the ability of Sens to bind the RhoA enhancer element. As RhoA contains a relatively low affinity Sens site, a parallel experiment was performed using a high affinity Sens site (SensS), and it was found that Ato does not significantly alter Sens binding to an optimized site. This data reveals that Ato's ability to interfere with Sens binding to DNA is site-specific and dependent upon binding affinity. How might Ato interfere with Sens binding to DNA? It has been shown that Ato, Ac, and Sc all directly interact with Sens through the second and third Sens zinc finger motifs. Since Sens requires these motifs to bind DNA, it is likely that the proneural factors compete with DNA for the same zinc fingers. Thus, the following model is proposed: if the binding affinity of Sens to DNA is high, Ato cannot interfere with Sens-mediated repression. However, if the binding affinity of Sens to DNA is low, Ato binds Sens and interferes with its ability to repress gene expression (Witt, 2010).

Secondly, expression analysis revealed that cells of the C1 SOP lineage differentially express Ato and Sens during their maturation. The initial SOP cell (SOPI) expresses both Ato and Sens during sensory organ specification. However, Ato protein is rapidly extinguished and no longer detectable once the SOP cell divides, whereas Sens persists into the SOPII cells. The rapid loss of Ato, even when it is expressed using a Gal4 driver, is consistent with recent findings that proneural proteins activate an E3 ubiquitin ligase pathway to trigger their own degradation. Thus, these findings suggest that the early SOP cell expresses both Ato and Sens and that Ato can alter Sens function in two ways: 1) by recruiting Sens to E-Box sequences as a co-activator, and 2) by interfering with Sens's ability to bind low affinity DNA sites (Witt, 2010).

It has been reported that rho is initially weakly expressed in C1 SOP cells in both the thorax and abdomen, and is only up-regulated in the abdominal SOP cells by the Abd-A Hox factor. This study found that the RhoBAD-lacZ reporter is also expressed in this pattern; it is proposed that Ato is part of an initiator pathway that allows rho expression in early C1 SOP cells. Ato does so in two ways: 1) by inhibiting Sens binding to RhoA through direct protein-protein interactions, and 2) by indirectly stimulating RhoD through an unknown mechanism. In total, these interactions result in the initiation of rho expression in early C1 SOP cells of both thoracic and abdominal segments. Ato's subsequent degradation releases Sens to bind RhoA and repress gene expression in thoracic SOP cells. Consistent with this idea, mutations that abolish Sens binding (SensM) result in de-repression of Rho reporters in the thorax. In the abdomen, however, an Abd-A complex out-competes Sens for RhoA to allow continued rho expression, subsequent EGF signaling, and the specification of additional cell types. Thus, Ato cooperates with the Abd-A Hox factor to stimulate EGF signaling by up-regulating rho expression via interfering with Sens-mediated repression (Witt, 2010).

While these findings provide insight into how rho is up-regulated in abdominal SOP cells, they uncover an interesting question: why is rho activated at all within thoracic SOP cells? Currently, there is no known function for rho activity within the thorax as rho mutant embryos show no phenotypic defect in cells surrounding the thoracic SOPs. As the lack of oenocyte production within the thorax is solely due to insufficient Spi secretion (oenocytes form in the thorax if rho is ectopically expressed), these data suggest that Rho levels are too low to trigger enough Spi secretion to affect neighboring cell fate. Consistent with this prediction is that the levels of an activated kinase downstream of EGF signaling (phospho-ERK) are very low in cells neighboring the thoracic C1 SOP cells compared to the abdominal SOP cells. So, why is rho activated within the thorax if it has no functional consequences? One interpretation is that Ato may provide competency for rho expression so that an additional positional factor such as Abd-A can fully stimulate rho and trigger Spi secretion and EGF signaling. In support of this idea, the widespread expression of Abd-A within the thorax activates RhoBAD-lacZ expression only within the C1 SOP cells and oenocytes form only in close proximity to these thoracic SOP cells. Thus, weak rho expression downstream of ato may provide a flexible and responsive system for activating Spi secretion in different body regions (Witt, 2010).

Post-transcriptional regulation; miR-9a targets senseless

MicroRNAs (miRNAs) have been implicated in regulating various aspects of animal development, but their functions in neurogenesis are largely unknown. Loss of miR-9a function in the Drosophila peripheral nervous system leads to ectopic production of sensory organ precursors (SOPs), whereas overexpression of miR-9a results in a severe loss of SOPs. A strong genetic interaction occurs between miR-9a and senseless (sens) in controlling the formation of SOPs in the adult wing imaginal disc. Moreover, miR-9a suppresses Sens expression through its 3' untranslated region. miR-9a is expressed in epithelial cells, including those adjacent to SOPs within proneural clusters, suggesting that miR-9a normally inhibits neuronal fate in non-SOP cells by down-regulating Sens expression. These results indicate that miR-9a ensures the generation of the precise number of neuronal precursor cells during development (Li, 2006).

miR-9a is one of the miRNAs that are highly expressed in the mammalian brain and 100% conserved at the nucleotide sequence from flies to humans, suggesting an important role in brain development and/or function. miR-9a loss-of-function alleles were generated; homozygous mutant flies developed into adulthood at the expected Mendelian ratio. Adult mutant flies are grossly normal and fertile, indicating that miR-9a is not required for viability or fertility. This finding is different from the reported severe dorsal closure defects and embryonic lethal phenotype generated by antisense 2 O-methyl oligoribonucleotide-mediated depletion of miR-9a. Interestingly, Drosophila miR-9a is not expressed in mature neurons, but is expressed in epithelial cells, including the proneural clusters that give rise to SOPs. Detailed analysis of embryonic PNS development revealed an unexpected finding that miR-9a mutants have an increased number of sensory neurons that elaborate extensive dendritic arbors underneath the epithelial cell layer, such as ddaE and ddaF neurons. The duplicated neurons occupy the same dendritic field and appear to have similar dendritic branching patterns. Indeed, the average numbers of dendritic ends of ddaE and ddaF neurons in abdominal segments 3-5 were similar in wild-type and miR-9a mutant larvae and MARCM clones, indicating that loss of miR-9a activity affected the number of these sensory neurons only but had no cell-autonomous effect on their dendritic branching patterns (Li, 2006).

The effect of miR-9a on the number of embryonic sensory neurons has two major features: (1) the ectopic ddaE or ddaF neurons were generated as a result of ectopic SOPs and not cell fate transformation within a cell lineage, suggesting miR-9a affects an early step in neurogenesis, consistent with its embryonic expression pattern; (2) both the expressivity and penetrance of this defect were relatively low. This finding supports the idea that miRNAs, at least in this particular case, are not developmental switches, but instead function as a fine-tuning mechanism to ensure the accuracy of a particular developmental process. In this study, focus was placed on the formation of SOPs in adult flies. Like embryos, only 14% of miR-9a mutant flies exhibited ectopic SOPs on the notum, again indicating a fine-tuning role for miR-9a in controlling SOP formation. However, analysis of the miR-9a mutant phenotype in adult flies also indicates that miRNAs can have dramatic effects on some other developmental processes. For instance, miR-9a is widely expressed in the wing disc, and 100% of miR-9a mutant flies exhibited a severe posterior wing margin defect, suggesting that cell proliferation and/or survival are much more sensitive to changes in the expression levels of the proteins regulated by miR-9a (Li, 2006).

How does miR-9a exert its effect on SOP formation? Sens is a zinc finger transcription factor required to maintain high-level expression of proneural gene in SOPs and to suppress their expression in non-SOP cells. Several findings in this study demonstrate that Sens is a key target of miR-9a regulation and is essential for mediating miR-9a function in SOP formation. (1) The wing margin defects in miR-9a mutant flies were remarkably similar to that caused by overexpression of Sens by the UAS-Gal4 system or in Lyra1 mutants. (2) miR-9a was expressed at a much lower level in SOPs than in adjacent epithelial cells, correlating with the high level of Sens expression in SOPs and the low level of Sens in non-SOP cells in proneural clusters. The inability to use immunostaining to detect subtle changes of Sens expression level in non-SOP cells due to miR-9a loss of function could be attributed to the following reasons: Sens expression is primarily down-regulated at the transcriptional level in the non-SOP proneural cells, and miR-9a's function is limited to preventing translation of the leaky/residual sens mRNA. The alteration in Sens level, due to loss of miR-9a function in the non- SOP cells, is sufficient to initiate the production of ectopic SOPs, but it may not be dramatic enough to be detected by immunostaining. (3) The sens 3' UTR contains three miR-9a-binding sites and is the best predicted target of miR-9a. (4) Wild-type but not mutant miR-9a precursors down-regulated reporter gene expression through the sens 3' UTR in transfected cells. (5) Overexpression of miR-9a in vivo inhibited Sens expression. It was observed that Sens expression along the wing margin in the dorsal compartment is lower than in the ventral compartment in some wing discs when miR-9a is expressed by ap-Gal4. The failure to completely suppress Sens expression in the dorsal compartment is probably due to the fact that, at this developmental stage, Sens expression in the wing margins is controlled by proneural genes, unlike SOPs in the notum region where Sens expression is maintained by itself. (6) miR-9a and sens showed strong genetic interactions in controlling SOP formation (Li, 2006).

These findings provide an experimental example to support the notion that miRNAs and their mRNA targets are often expressed in cells adjacent to each other. The differential expression of Sens in SOPs and adjacent neuroepithelial cells is essential for the production of a precise number of SOPs during development. A model is proposed in which miR-9a functions in non-SOPs cells to further suppress Sens expression at the translational level, as a complementary mechanism to the transcriptional inhibition of Sens expression by E(spl). Loss of miR-9a function increases Sens protein level, not so dramatically but just enough to convert Sens in some neuroepithelial cells from a transcription repressor into an activator of proneural genes, therefore resulting in the formation of a small number of ectopic SOPs. However, unlike many other genes essential for neurogenesis, such as Notch and Delta, miR-9a does not function as an absolute switch. Instead, it only ensures the accurate differential Sens expression and fine-tunes this developmental. Overexpression of miR-9a in the wing imaginal disc could dramatically inhibit the formation of sensory organs on the notum, suggesting that misregulation of miR-9a expression itself could potentially have severe developmental consequences. Since both miR-9a and E(spl) have similar functions in non-SOP cells, it is possible that both genes may be regulated by a similar transcriptional mechanism. Indeed, binding sites for the Achaete-Scute complex and Su(H) are present in the regulatory region of miR-9a. Taken together, these studies presented here have uncovered another layer of gene regulation during early neurogenesis in the Drosophila PNS. miR-9a is 100% conserved at the nucleotide level from flies to humans. Moreover, the human miR-9a is highly expressed in fetal but not in adult brains. Therefore, a similar mechanism of miR-9a function may operate during mammalian neurogenesis as well (Li, 2006).

Drosophila Ataxin-2 mediates expanded Ataxin-1-induced neurodegeneration in a Drosophila model of SCA1

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of neurodegenerative disorders sharing atrophy of the cerebellum as a common feature. SCA1 and SCA2 are two ataxias caused by expansion of polyglutamine tracts in Ataxin-1 (ATXN1) and Ataxin-2 (ATXN2), respectively, two proteins that are otherwise unrelated. This study used a Drosophila model of SCA1 to unveil molecular mechanisms linking Ataxin-1 with Ataxin-2 during SCA1 pathogenesis. Wild-type Drosophila Ataxin-2 (dAtx2) is a major genetic modifier of human expanded Ataxin-1 (Ataxin-1[82Q]) toxicity. Increased dAtx2 levels enhance, and more importantly, decreased dAtx2 levels suppress Ataxin-1[82Q]-induced neurodegeneration, thereby ruling out a pathogenic mechanism by depletion of dAtx2. Although Ataxin-2 is normally cytoplasmic and Ataxin-1 nuclear, it was shown that both dAtx2 and hAtaxin-2 physically interact with Ataxin-1. Furthermore, expanded Ataxin-1 induces intranuclear accumulation of dAtx2/hAtaxin-2 in both Drosophila and SCA1 postmortem neurons. These observations suggest that nuclear accumulation of Ataxin-2 contributes to expanded Ataxin-1-induced toxicity. This hypothesis was tested by engineering dAtx2 transgenes with nuclear localization signal (NLS) and nuclear export signal (NES). NLS-dAtx2, but not NES-dAtx2, was found to mimic the neurodegenerative phenotypes caused by Ataxin-1[82Q], including repression of the proneural factor Senseless. Altogether, these findings reveal a previously unknown functional link between neurodegenerative disorders with common clinical features but different etiology (Al-Ramahi, 2007).

This study reports functional interactions between the proteins causing two distinct Spinocerebellar ataxias. A Drosophila model of SCA1 was used to show that wild-type dAtx2 (the fly homolog of the protein that when expanded causes SCA2) mediates, at least in part, neuronal degeneration caused by expanded Ataxin-1 (the protein triggering SCA1). Ataxin-1[82Q]-induced toxicity is worsened by increasing the levels of dAtx2. More significantly, decreasing the levels of dAtx2 suppresses expanded Ataxin-1-induced neuronal degeneration as shown in several independent assays. The suppression of Ataxin-1[82Q] phenotypes by partial loss of function of dAtx2 argues against a possible mechanism by which sequestration and depletion of Ataxin-2 contributes to expanded Ataxin-1-induced neurodegeneration. This is further supported by lack of cerebellar or other neuronal abnormalities in mice that are deficient for Ataxin-2 (Al-Ramahi, 2007).

The human expanded Ataxin-1 interacts with the dAtx2 and human Ataxin-2 proteins in co-AP assays. Furthermore, overexpressed Ataxin-1 pulls down endogenous hAtaxin-2 in cultured cells. These results suggest that Ataxin-1 and Ataxin-2 may be functional interactors in vivo. Consistent with this, it was found that expanded Ataxin-1 induces accumulation of Ataxin-2 in the nucleus, where the two proteins localize in nuclear inclusions (NIs) both in Drosophila neurons and SCA1 human brain tissue. These are surprising observations since Ataxin-2 is normally a cytoplasmic protein both in humans and Drosophila. Interestingly, wild-type Ataxin-1 can cause neurotoxicity when overexpressed, although to a much lesser extent than expanded Ataxin-1. However, nuclear accumulation of dAtx2 is triggered by pathogenic but not wild-type forms of Ataxin-1, at least in detectable amounts. Taken together these data suggested that accumulation of Ataxin-2 in the nucleus contributes to the exacerbated toxicity of expanded Ataxin-1, and is an important mechanism of pathogenesis in SCA1. To investigate this hypothesis dAtx2 was targeted to the nucleus by means of an exogenous NLS signal. It was found that dAtx2^NLS is sufficient to cause a dramatic increase of its toxicity, when compared to either wild-type dAtx2 or dAtx2 with an exogenous nuclear export signal (dAtx2^NES) expressed at similar levels (Al-Ramahi, 2007).

To further test the hypothesis that nuclear accumulation of Ataxin-2 contributes to neurodegeneration caused by expanded Ataxin-1 Sens levels were investigated. Sens and its murine orthologue Gfi1 are proneural factors whose levels are decreased in the presence of expanded Ataxin-1; thus providing a molecular readout for the neurotoxicity of Ataxin-1. In Drosophila, reduction of Sens levels leads to the loss of mechanoreceptors, so Sens was monitored in the context of flies expressing either dAtx2^NLS or dAtx2^NES but not carrying the Ataxin-1[82Q] transgene. It was found that nuclear targeted, but not cytoplasmic, dAtx2 mimics both the Sens reduction and mechanoreceptor loss phenotypes caused by Ataxin-1[82Q] (Al-Ramahi, 2007).

Expanded Ataxin-2 accumulates both in the cytoplasm and the nuclei of SCA2 postmortem brains. In mouse and cell culture models of SCA2, expanded Ataxin-2 accumulates in the cytoplasm and its nuclear accumulation is not necessary to induce toxicity. However, nuclear accumulation of expanded Ataxin-2 also occurs in cultured cells, and is consistently observed in human SCA2 postmortem brainstem neurons. These observations suggest that both nuclear and cytoplasmic mechanisms of pathogenesis contribute to neurodegeneration in SCA2, as it is known to occur in other polyglutamine diseases like HD and SCA3. One possibility is that Ataxin-2 shuttles between the nucleus and the cytoplasm although the protein is normally detected only in the cytoplasm. The data show that accumulation of dAtx2 in the nucleus is more harmful than in the cytoplasm. Thus, neurons with nuclear Ataxin-2 in SCA2 patients may be relatively more compromised than neurons where Ataxin-2 accumulates in the cytoplasm. In agreement with this possibility, expanded Ataxin-2 is found in the nuclei of pontine neurons of SCA2 brains, one of the neuronal groups and brain regions with prominent degeneration in SCA2 (Al-Ramahi, 2007 and references therein).

Reducing Ataxin-2 levels suppresses expanded Ataxin-1 toxicity, strongly arguing against a mechanism of pathogenesis by loss of function of Ataxin-2 in the cytoplasm. Studies of the normal function of Ataxin-2 and its yeast, C. elegans, and Drosophila homologs suggest a role in translational regulation. Thus, an attractive possibility is that Ataxin-1 [82Q] requires dAtx2 to impair Sens translation and induce the loss of mechanoreceptors. Consistent with this hypothesis is the finding that partial loss of function of dAtx2 suppresses the loss of mechanoreceptors phenotype caused by expanded Ataxin-1 (Al-Ramahi, 2007).

The data described in this study uncover unexpected functional interactions between proteins involved in two different SCAs. Nuclear accumulation of Ataxin-2, normally a cytoplasmic protein, is a common denominator of SCA1 and SCA2, and leads to reduced levels of at least one important proneural factor; i.e. Sens, whose mammalian orthologue Gfi1 is required for Purkinje cell survival. Thus neuronal degeneration may take place through common mechanisms in different ataxias, and one of these mechanisms may involve the abnormal accumulation of Ataxin-2 in neuronal nuclei (Al-Ramahi, 2007).

Drosophila dLMO-PA isoform acts as an early activator of achaete/scute proneural expression

The Drosophila bHLH proneural factors Achaete (Ac) and Scute (Sc) are expressed in clusters of cells (proneural clusters), providing the cells with the potential to develop a neural fate. Mediodorsal proneural patterning is mediated through the GATA transcription factor Pannier (Pnr) that activates ac/sc directly through binding to the dorsocentral (DC) enhancer of ac/sc. Besides, the Gfi transcription factor Senseless (Sens), a target of Ac/Sc, synergizes with ac/sc in the presumptive sensory organ precursors (SOPs). This study investigated, through new genetic tools, the function of dLMO (Beadex), the Drosophila LIM only transcription factor that was already known to control wing development. dLMO gene encodes two isoforms, dLMO-RA and dLMO-RB. dLMO null and dLMO-RA⁻ deletions have similar phenotypes, lacking thoracic and wing margin sensory organs (SO), while dLMO-RB⁻ deletion has normal SOs. At early stages, dLMO-RA is expressed in proneural clusters, however later it is excluded from the SOPs. dLMO functions as a Pnr coactivator to promote ac/sc expression. In the late SOPs, where dLMO-PA is not expressed, Pnr participates to the Sens-dependent regulation of ac/sc. Taken together these results suggest that dLMO-PA is the major isoform that is required for early activation of ac/sc expression (Asmar, 2008).

The lack of dLMO protein leads to very distinctive phenotypes. The mutant animals are not able to fly, they have a short life span and show an abnormal gait behaviour. In addition, they show a discreet bristle phenotype. In Drosophila, there are two paralogous LMO factors, dLMO and CG5708. These genes are expressed in the CNS where redundancy is not excluded. However CG5708 is not expressed in the wing discs and presumptive SOPs. Therefore it is concluded that the mild phenotype observed for the adult PNS in dLMO mutants, is not attributed to gene redundancy. dLMO encodes two distinct isoforms, dLMO-PA and -PB, which only differ from their N-terminus. Only dLMO-RA is broadly expressed in the notum, and contributes to the PNS phenotype. dLMO function is also critical in the developing central nervous system for the activity of the ventral lateral neurons, LN_vs. It is highly probable that dLMO-RB has some subtle biological activities in the brain, where it has a specific pattern (Asmar, 2008).

In vertebrate, multiproteic complexes composed by GATA-1, LMO2, Ldb-1 and the bHLHs E47 and SCL, are required for normal differentiation of haematopoietic cells. The current results highlight several evidences in favour of dLMO as a GATA coactivator in Drosophila . (1) A genetic synergism exists between pnr⁻ and dLMO⁻ null alleles. (2) dLMO modulates the activity of a DC:ac-lacZ reporter, the model target of Pnr, in vivo. Loss of function dLMO mutants show reduced level of the DC:ac-lacZ expression, whereas in gain-of-function dLMO mutants the DC:ac-lacZ expression is increased. (3) dLMO-PA isoform directly interact with Pnr in GST pull down assay. Therefore it is concluded that dLMO might enhance the proneural activity of Pnr through direct interaction with the GATA factor. Consistently, dLMO expression overlaps with the dorsal-most domain of Pnr during third instar larval stages. Though Pnr controls the development of both DC and SC bristles, dLMO null alleles affect only DC bristles. dLMO expression, that overlaps both SC and DC proneural clusters in the notum, is significantly weaker in the SC region, suggesting that regulation of proneural ac/sc expression is differentially sensitive and responds to local combinations of transcription factors. These data support previously published studies demonstrating that the proneural activity of Pnr is prominently repressed in the SC region by the LIM-HD transcription factor Isl (Asmar, 2008).

At later stages, dLMO expression is excluded from the corresponding SOP and its derivative cells. In contrast, the proneural factor Sens, that plays an important role for sensory organ specification, is first broadly expressed in proneural clusters at low levels where it functions as a repressor of ac/sc, and then later, is expressed at high levels in the presumptive SOPs, where it acts as a transcriptional activator that directly interacts and synergizes with the proneural proteins, Ac and Sc. It has been shown that both Gfi-1 and GATA-1, the mammalian ortholog of Sens and Pnr respectively, are essential for development of the closed related erythroid and megakaryocytic lineages. The Sens/Pnr interaction is evolutionary conserved in Drosophila neurogenesis. It is suggestd that Pnr could participate to the Sens-dependent positive autoregulation of Ac/Sc in late SOPs where dLMO is not expressed. The synergism between Pnr and Sens would need more detailed investigations. Taken together, these studies have shown dLMO-PA as a co-activator for Pnr during the establishment of proneural fields and revealed another level of proneural ac/sc regulation during late neurogenesis in the Drosophila PNS (Asmar, 2008).

Hox and senseless antagonism functions as a molecular switch to regulate EGF secretion in the Drosophila PNS

Hox factors are key regulators of distinct cells, tissues, and organs along the body plan. However, little is known about how Hox factors regulate cell-specific gene expression to pattern diverse tissues. This study shows an unexpected Hox transcriptional mechanism: the permissive regulation of EGF secretion, and thereby cell specification, by antagonizing the Senseless transcription factor in the peripheral nervous system. rhomboid expression in a subset of sensory cells stimulates EGF secretion to induce hepatocyte-like cell development. A rhomboid enhancer was identified that is active in these cells; an abdominal Hox complex directly competes with Senseless for enhancer binding, with the transcriptional outcome dependent upon their relative binding activities. Thus, Hox-Senseless antagonism forms a molecular switch that integrates neural and anterior-posterior positional information. As the vertebrate Senseless homolog is essential for neural development as well as hematopoiesis, it is proposed Hox-Senseless antagonism will broadly control cell fate decisions (Li-Kroeger, 2008).

Hox genes have long been known to specify distinct cell types along the body axes of both vertebrates and invertebrates. However, it has remained elusive how Hox factors regulate transcription in a tissue- or cell-specific manner. In this study, a Hox-regulated enhancer (Rho654) active within a subset of PNS cells was identified. Rho654 drives gene expression in abdominal C1-SOP cells to induce oenocytes, and an Exd/Hth/Abd-A complex stimulates gene expression by directly competing with Sens for this enhancer. These findings have three main implications: (1) They demonstrate how a Hox selector gene integrates A-P positional information with a PNS factor to differentially regulate gene expression along the body plan. (2) They uncover a permissive rather than instructive role for Hox factors in regulating transcription. (3) As Hox and Sens binding sites share a common core sequence, they suggest that additional target genes will be regulated through this mechanism. Moreover, genetic studies in mice have linked Gfi1 and Hox factors to both neural and blood cell development, and this study found that vertebrate Hox and Gfi1 factors compete for binding sites in blood cells (Li-Kroeger, 2008).

Sensory organs within the fly head, thorax, and abdomen require sens for their development. However, the type, location, and number of sensory organs that form in different body regions are regulated, at least in part, by Hox factors. The results provide new insight into how Hox factors provide positional information to modify gene expression in sensory cells. A series of point mutations was used to demonstrate that Hox-Sens competition forms a molecular switch whose outcome correlates with the binding activity of each factor. Intrinsic to this model is the following prediction: If Hox factors differ in their ability to interact with composite sites, then A-P differences in Hox-Sens target expression will be observed. Previous biochemical studies revealed that posterior Hox factors have higher affinity for DNA when bound with Pbx (Exd) than anterior Hox proteins (LaRonde-LeBlanc, 2003). Consistent with these results, this study found that a posterior Hox complex (Abd-A/Hth/Exd) that stimulates Rho654 binds 5-fold more RhoA than an anterior Hox complex (Antp/Hth/Exd) that fails to stimulate Rho654. Thus, differences in binding activities between Hox factors for Hox-Sens composite sites result in the differential regulation of gene expression along the A-P axis of the sensory system (Li-Kroeger, 2008).

Hox proteins instructively regulate gene expression by either activating and/or repressing transcription. In fact, the same Hox factor can perform both functions. Abd-A directly binds regulatory elements to activate wingless (wg) and repress decapentaplegic (dpp) in the same cells of the visceral mesoderm. So what determines if a Hox factor activates or represses transcription? Two recent studies revealed that the transcriptional outcome depends upon the binding of additional transcription factors (Gebelein, 2004; Walsh, 2007). The repression of Distal-less (Dll) by the Abd-A and Ultrabithorax (Ubx) Hox factors requires the binding of two transcription factors in addition to Exd and Hth. In posterior compartment cells, the Engrailed (En) protein collaborates with Abd-A/Exd/Hth to bind DNA and repress Dll. In anterior compartment cells, the Sloppy-paired (Slp) protein binds DNA near the Hox complex to repress Dll (Gebelein, 2004). As both En and Slp interact with the Groucho (Gro) corepressor, their recruitment by Hox factors suggests a mechanism to repress transcription. Similarly, Walsh and Carroll found that Ubx and Smad binding are required to repress spalt-major (salm) in the wing. In this case, the Smad proteins recruit the Schnurri corepressor to inhibit transcription. Thus, Hox factors collaborate with additional factors to determine the transcriptional outcome (Li-Kroeger, 2008).

Studies on Abd-A stimulation of a rho enhancer reveal an unexpected mechanism by which Hox factors control gene expression: through competition with the Sens repressor for DNA binding sites. Sens binds RhoA to repress thoracic gene expression, whereas in the abdomen Exd/Hth/Abd-A is permissive for activation by out-competing Sens. Importantly, mutations that disrupt both Sens and Hox binding to RhoA (SensM/HoxM) are expressed in the thorax and abdomen, revealing that Exd/Hth/Abd-A binding is not required to activate gene expression. In addition, coexpression of Exd, Hth, and Abd-A in cultured cells failed to stimulate Rho654- or RhoAAA-luciferase unless Abd-A is fused to a potent activation domain. Thus, unlike other Hox target genes, Hox complexes on RhoA are permissive rather than instructive and stimulate Rho654 by interfering with the binding of a transcriptional repressor (Li-Kroeger, 2008).

A comparison of consensus Sens, Hox/Exd, and Exd/Hth sites reveal a shared core sequence, suggesting that additional target genes will be regulated through Hox-Sens antagonism. In fact, bioinformatics reveals many Hox-Sens composite sites throughout the Drosophila and mammalian genomes. However, both the Sens and Hox sites extend beyond this core sequence, indicating that only a subset of target genes will comprise composite sites. Thus, three types of target genes for those factors are proposed: (1) those regulated by only Hox factors, (2) those regulated by only Sens/Gfi1, and (3) those regulated by both Hox and Sens/Gfi1. For example, many of the previously characterized Hox target genes in the Drosophila embryo are controlled in tissues that do not express Sens, suggesting they are only regulated by Hox genes. However, the Hox and Sens/Gfi1 factors are coexpressed in many neural cells of the developing PNS in both flies and vertebrates, indicating that similarly to rho regulation in abdominal SOP cells, additional targets will be coregulated by Hox and Sens (Li-Kroeger, 2008).

Like Hox genes, the Sens gene family is conserved in C. elegans (Pag-3), Drosophila, and vertebrates (Gfi1 and Gfi1b). These zinc finger transcription factors are essential for nervous system development in all three organisms. In addition, Gfi1 plays a critical role in hematopoiesis, where it participates in regulating stem cell renewal as well as specific blood cell lineages. Interestingly, Hox factors also regulate blood cell differentiation, proliferation, and stem cell renewal. HoxA9, for example, is required for normal hematopoiesis in mice, and alterations in HoxA9 expression have been implicated in acute myeloid leukemia (AML). In fact, a study analyzing the expression profile of 6817 genes in AML patients who either responded or did not respond to treatment found the highest correlated gene associated with poor prognosis is HoxA9. To determine if the Hox-Sens mechanism uncovered in Drosophila is conserved in mammals, in vitro DNA binding assays were used to show that HoxA9 forms a complex with Pbx and Meis that competes with Gfi1 for common binding sites. Moreover, mouse genetic studies support the hypothesis that Hox-Gfi1 factors antagonize each other to regulate gene expression and blood cell development. Thus, Hox-Sens/Gfi1 competition for composite binding sites is likely a conserved mechanism for the regulation of gene expression in organisms from flies to humans (Li-Kroeger, 2008).

Specificity of Atonal and Scute bHLH factors: analysis of cognate E box binding sites and the influence of Senseless

The question of how proneural bHLH transcription factors recognize and regulate their target genes is still relatively poorly understood. It has been shown that Scute (Sc) and Atonal (Ato) target genes have different cognate E box motifs, suggesting that specific DNA interactions contribute to differences in their target gene specificity. This study shows that Sc and Ato proteins (in combination with Daughterless) can activate reporter gene expression via their cognate E boxes in a non-neuronal cell culture system, suggesting that the proteins have strong intrinsic abilities to recognize different E box motifs in the absence of specialized cofactors. Functional comparison of E boxes from several target genes and site-directed mutagenesis of E box motifs suggests that specificity and activity require further sequence elements flanking both sides of the previously identified E box motifs. Moreover, the proneural cofactor, Senseless, can augment the function of Sc and Ato on their cognate E boxes and therefore may contribute to proneural specificity (Powell, 2008).

The proneural proteins exhibit very precise specificity in activation of different neurogenesis programmes. It has been suggested that utilization of different E-box motifs as binding sites may partly underlie this specificity. This was based on the finding that E boxes from Sc- and Ato-specific target genes conform to different consensus motifs. This study found further support for observation. In a cell culture assay, artificial enhancers of concatemers of E_Ato or E_Sc sequences generally show specific activation by Ato or Sc proteins, respectively. Nevertheless, the results also show that E box activity and specificity depends on complex features of the DNA surrounding the proneural-specific motifs both in cell culture and in vivo. The task of predicting by sequence analysis how proneural proteins regulate targets remains formidable (Powell, 2008).

Transcription factor activity depends on a complex interplay of interactions with DNA and with other protein factors, including those bound to other sites within the enhancer. To concentrate on the role that proneural protein interaction with E-box binding sites plays in specificity, synthetic enhancers of concatemers of E-box-containing sequences were studied in a cell culture reporter gene assay. A previous study of Ato or Sc-specific enhancers relied on the analysis of expression patterns produced in transgenic flies carrying GFP reporter gene constructs. In that study, specific regulation by Sc or Ato was inferred indirectly from patterns of GFP expression. This study showed that much of this inferred specificity is also seen in a cell culture reporter gene assay, strongly supporting the conclusion that Ato and Sc directly use different E box motifs. Thus, in general, the specificity of E box response (ratio of response to Sc and Ato) could be predicted from matches to E_Sc or E_Ato motifs identified previously. In most cases, this specificity also corresponded to the specificity of the native enhancer from which the E box was taken. An interesting exception is sens-E1: while this E box is proposed to respond to both Ato and Sc in vivo, it responds slightly better to Sc than to Ato in culture, which is more consistent with its E_Sc motif. It will be important to determine what other enhancer features allow such an E box to function as a common target of Ato and Sc in vivo (Powell, 2008).

Importantly, E box specificity is achieved without the appropriate cellular and developmental context of neurogenesis: S2 cells are embryonic, non-neural cells of likely hematopoietic origin and are not expected to contain neural-specific factors. The results therefore indicate that proneural factors have intrinsic ability to use different E box motifs without the need for interactions with neural specific cofactors. The E_Sc and E_Ato motifs differ most notably in the bases immediately flanking the 5' end of the 6-bp core sequence (NG vs. AW). There is evidence from the crystal structure of the MyoD bHLH domain–DNA complex that protein contacts are made with bases in this position, suggesting that similar direct contacts may influence E box utilization by proneural proteins. The basic region amino acids making these contacts (R₁₁₀, R₁₁₇ and E₁₁₈) are conserved in the proneural proteins, but in Ato the arginines are separated by three amino acids (LAA, equivalent to MyoD KAA) that are absent in Sc. Thus despite the apparent conservation of DNA-contacting residues, one might predict strong differences in how the proneural proteins interact with the flanking nucleotides. SPR analysis shows Ato/Da to bind to ato-E1 and sc-E1 with similar affinity. Rather than affecting E box affinity, it is possible that subtle differences in binding contacts may cause conformational effects that affect the transactivation ability of the proneural protein (Powell, 2008).

The above results point to the importance of distinct Ato and Sc E box motifs for proneural specificity. Several findings, however, demonstrate that these motifs are heavily dependent on the wider DNA context. For instance, the E(spl)mγ-E2(C₄ > G), sens-E1 and sc-E1 binding sites show very large differences in activity in cell culture, even though they have identical perfect E_Sc motifs at their core (gCAGGTGt). The effect of DNA context is also seen in the general inability, in the cell culture assay, to swap the proneural specificities of sc-E1 and ato-E1 by mutating the immediate 5' flanking bases of the core E box. Such changes generally result in loss of E box activity rather than a clear change in specificity. These results indicate that the E_Sc and E_Ato motifs are generally not sufficient for activity or specificity in the cell culture assay and that the surrounding DNA context is important (even within the short 20-bp sequences used) (Powell, 2008).

Interestingly, in some circumstances specificity could be manipulated more successfully in vivo: (sc-E1 GG > AA)₆-GFP transgenic flies showed GFP expression consistent with strongly reduced activation by Sc and a gain of activation in some specific locations by Ato. However, this mutated motif did not respond to ectopically expressed Ato, perhaps suggesting that improved specificity in vivo results from cofactors expressed in locations of endogenous Ato expression and function (Powell, 2008).

Overall, the results above show that further sequences on both flanks of the E_Sc and E_Ato box motifs are also important for specificity and activity. One possibility is that the 20-bp DNA sequences used to construct the concatemers may include flanking sequences that interact with other protein factors to influence proneural specificity. Such adjacent sites have been identified for some mouse proneural E box binding sites. Moreover, in its native enhancer, ato-E1 is adjacent to an Ets-domain transcription factor binding site (although this site is mutated in the constructs used in this study). However, such cofactors would need to be expressed in S2 cells. Moreover, although the flanking sequences of the ato-E1 and sc-E1 sites are strongly conserved among Drosophila species, no obvious shared sequence motifs were found in the 5' and 3' flanks of known Drosophila E boxes that might be cofactor binding sites. Whilst there is a potential POU factor binding sequence 5' of the ato-E1 site, no members of the Drosophila POU family appear to be expressed during early neurogenesis. Alternatively, the further flanking bases may influence bHLH heterodimer interaction either through direct contacts or through indirect conformational effects. It is interesting that 3' bases appear important as these may be predicted to affect Da interaction. It is notable that the Da homologue, E2A, has different half-site preferences when bound to Twi or MyoD (Powell, 2008).

The specificity of E-box concatemer constructs is generally more complete in vivo than in the S2 luciferase assay -- notably proneural proteins can generally activate non-cognate E boxes to some extent in cell culture but not in vivo. One possibility is that the intrinsic specificity of proneural proteins must normally be enhanced by interaction with cofactors that are not present in S2 cells. In the cell culture assay, at high proneural levels it was found that Sens enhanced proneural activity in a general manner. None of the constructs tested contain Sens binding motifs, so it is likely that enhancement occurs in a DNA-binding independent manner via protein–protein interactions. At low proneural concentrations, however, the effect of Sens enhancement becomes selective. For many of the constructs tested, Sens only enhanced the activity of proneural proteins for concatemers consisting of their cognate E box. It is suggested that proneural–Sens interaction may enhance the specificity of proneural–E box interaction. Thus, this is an interesting case in which proneural specificity can be influenced by a common cofactor, rather than requiring interaction with different subtype-specific cofactors. It remains to be determined whether Sens would enhance specificity on native enhancers as well as concatemer constructs. Moreover, it seems unlikely that Sens is a specificity factor for all proneural target genes. However, the results are consistent with Sens acting as a specificity cofactor in certain contexts -- such as the proneural autoregulatory enhancers active in SOPs where there are high levels of Sens and proneural proteins present. Other non-DNA binding proneural protein interactors, such as Chip may have a similar effect in other contexts (Powell, 2008).

The effect of Sens could be explained by two models. First, interaction of a proneural protein with a specific E-box motif may give rise to a specific conformation which results in an increased affinity for Sens protein. Alternatively, the Sens–proneural protein interaction may alter the proneural bHLH domain conformation thereby increasing its affinity for its cognate binding site (i.e., an induced fit model). Indeed, variation in MyoD bHLH protein DNA sequence preferences have been previously observed to be the result of effects on basic region conformation arising because of binding partner differences or amino acid composition of the basic region. In this view, proneural specificity relies on a combination of cognate DNA sequence recognition and protein–protein interactions. Important future work will be the identification of the amino acid residues of Ato and Sc necessary for their interaction with Sens and the determination of whether these influence DNA recognition (Powell, 2008).

Protein Interactions

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by an expanded glutamine tract in human Ataxin-1 (hAtx-1). The expansion stabilizes hAtx-1, leading to its accumulation. To understand how stabilized hAtx-1 induces selective neuronal degeneration, Drosophila Atx-1 (dAtx-1), which has a conserved AXH domain but lacks a polyglutamine tract, was studied. Overexpression of hAtx-1 in fruit flies produces phenotypes similar to those of dAtx-1 but different from the polyglutamine peptide alone. The Drosophila and mammalian transcription factors Senseless/Gfi-1 interact with Atx-1's AXH domain. In flies, overexpression of Atx-1 inhibits sensory-organ development by decreasing Senseless protein. Similarly, overexpression of wild-type and glutamine-expanded hAtx-1 reduces Gfi-1 levels in Purkinje cells. Deletion of the AXH domain abolishes the effects of glutamine-expanded hAtx-1 on Senseless/Gfi-1. Interestingly, loss of Gfi-1 mimics SCA1 phenotypes in Purkinje cells. These results indicate that the Atx-1/Gfi-1 interaction contributes to the selective Purkinje cell degeneration in SCA1 (Tsuda, 2005).

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by the expansion of a translated CAG repeat encoding glutamine in the ataxin-1 (Atx-1) protein. This autosomal-dominant disorder typically has its clinical onset in adulthood, when patients exhibit a progressive loss of balance and coordination and gradually develop swallowing and breathing difficulties. The primary sites of neurodegeneration in SCA1 include cerebellar Purkinje cells (PCs), brain-stem nuclei, the inferior olive, and the spinocerebellar tracts. At least eight other inherited neurodegenerative diseases, including Huntington's disease (HD), are caused by a similar mutational mechanism. A common feature of polyglutamine (polyQ) diseases is the loss of specific subsets of neurons in spite of broad expression of the mutant gene product. For example, both Atx-1 and huntingtin are expressed in PCs and striatal medium spiny neurons, yet in SCA1, PCs are the prominent sites of pathology, whereas in HD, the spiny neurons are predominantly affected. Furthermore, although mutant Atx-1 is expressed throughout brain development, the clinical symptoms of SCA1 appear during adulthood, suggesting a temporal restriction of the pathology despite prolonged protein expression (Tsuda, 2005).

The mechanisms mediating SCA1 pathogenesis are still not fully understood, but some general principles have emerged. Genetic studies in mice and Drosophila support a toxic gain-of-function mechanism. Mice lacking Atx-1 do not develop ataxia or PC pathology, arguing against a loss-of-function mechanism. In contrast, overexpression of mutant Atx-1 with an expanded polyQ tract causes neurodegeneration in mice and flies (Burright, 1995 and Fernandez-Funez, 2000). Interestingly, even human Atx-1 with a nonpathogenic repeat of (CAG)14CAT-CAG-CAT(CAG)₁₅, hereafter hAtx-1[30Q], causes neuronal degeneration if expressed at sufficiently high levels in either mice or Drosophila (Fernandez-Funez, 2000), suggesting that protein domains other than the expanded polyQ tract might contribute to SCA1 pathogenesis. Also, a single serine (S776) is critical for Atx-1-induced degeneration, since neuronal dysfunction is dramatically dampened in mice expressing a glutamine-expanded hAtx-1[82Q] in which S776 is replaced by alanine (Emamian, 2003). Phosphorylation of Atx-1 at S776 allows the 14-3-3 protein to bind to both wild-type (wt) and mutant Atx-1 (Chen, 2003). Glutamine-expanded Atx-1 binds more strongly to 14-3-3 than wt Atx-1, and this in turn leads to the accumulation of mutant Atx-1 and enhances neurotoxicity in the SCA1 Drosophila model. These data indicate that regions outside the polyQ expansion are critical for SCA1 pathogenesis (Tsuda, 2005 and references therein).

Several reports have suggested that Atx-1 may play a role in the regulation of gene expression. (1) Genetic studies in mice have shown that nuclear localization of mutant Atx-1 is critical to the course of the disease (Klement, 1998). (2) Alterations in gene expression occur very early in SCA1-transgenic mice, prior to the onset of any detectable neurological or pathological changes. (3) Heterozygosity for loss-of-function alleles of several genes encoding transcriptional coregulators, including Sin3A, Rpd3, dCtBP, and dSir2, enhances the neurodegeneration in a SCA1 Drosophila model (Fernandez-Funez, 2000). (4) Several reports have suggested that Atx-1 may interact with or modulate the function of transcriptional coregulators, including polyQ binding protein 1 (PQBP1) and silencing mediator of retinoid and thyroid hormone receptors (SMRT) (Okazawa, 2002 and Tsa, 2004). How these interactions might contribute to the disease process and how they might cause toxicity in only a subset of neurons in SCA1 is not fully understood (Tsuda, 2005 and references therein).

To gain insight into how Atx-1's function contributes to SCA1 pathogenesis, the Drosophila Atx-1 homolog (dAtx-1), which lacks a polyQ tract, was studied; its in vivo effects and interactions were compared to those of the human protein. This study shows that overexpression of hAtx-1 induces phenotypes similar to those of dAtx-1 overexpression but distinct from those observed upon overexpression of polyQ chains in flies. Furthermore, the Drosophila zinc-finger transcription factor Senseless (Sens) and its mammalian homolog growth factor independence-1 (Gfi-1) have been identified as physical and genetic interactors of Drosophila and mammalian Atx-1, respectively. It is proposed that the Sens/Gfi-1 interaction provides a mechanism for SCA1 pathogenesis and the selective vulnerability of PCs in this disease (Tsuda, 2005).

The expanded glutamine tract is clearly the initiating event in the pathogenesis of polyQ neurodegenerative disorders. In SCA1 and related disorders, the length of the glutamine tract correlates inversely with the age of onset and directly with the severity of the phenotype. Overexpression of a polyQ peptide in cell-culture, invertebrate, and mouse models is quite toxic and causes progressive dysfunction and cell death. However, there are several pieces of data that argue that the expanded glutamine tract is not the sole factor that induces pathology and determines disease severity. For example, several genetic studies support the idea that sequences outside of the glutamine tract are critical for causing neurodegeneration phenotypes in SCA1. (1) Overexpression of wt hAtx-1[30Q] in flies and mice causes pathology (Fernandez-Funez, 2000). (2) Replacing serine 776 with an alanine in Atx-1[82Q] dampens SCA1 pathology (Emamian, 2003). Also, a CAG repeat length of 45 causes severe disease in SCA2, mild disease in SCA1, and no symptoms in SCA3 (Zoghbi, 2000). These genotype-phenotype correlation studies argue that protein sequences outside of the glutamine tract as well as protein-protein interactions are critical for pathology (Tsuda, 2005 and references therein).

These data indicate that hAtx-1 induces phenotypes similar to (but more severe than) those of dAtx-1 and distinct from those induced by overexpression of a polyQ peptide. The effects of hAtx-1[82Q] on Sens/Gfi-1 protein levels are abolished in the absence of the AXH domain in both flies and mammalian cells. Furthermore, overexpression of wt hAtx-1[30Q] in mice decreases Gfi-1 levels and induces degeneration. This is noteworthy given that the 30Q allele does not contain 30 consecutive Qs but is comprised of two stretches of 14 and 15 Qs interrupted by two histidine residues. These data collectively argue that the AXH domain but not the expanded polyQ tract is necessary to generate the Atx-1 gain-of-function phenotype in flies or mice. The findings that glutamine-expanded hAtx-1 produces more severe phenotypes than wt hAtx-1 and dAtx-1 suggest that the polyQ tract enhances the activity of the AXH domain in a quantitative and/or qualitative manner (Tsuda, 2005).

Because the AXH domain is the likely mediator of the effects of Atx-1 overexpression, the prediction would be that this domain might mediate the biochemical and genetic interactions that modulate or contribute to the SCA1 phenotypes. Indeed, the data on the genetic and biochemical interactions of fly and hAtx-1 proteins with Drosophila Sens and its mammalian homolog Gfi-1 strongly support this prediction. The finding that the AXH domain of dAtx-1/hAtx-1 is necessary and sufficient for physical interactions with Sens/Gfi-1 underscores the cross-species functional conservation of this domain and the potential role of Sens/Gfi-1 in mediating Atx-1-induced phenotypes. In support of Atx-1's specific effect on Sens, it was found that (1) Atx-1 has direct effects on Sens protein (not RNA) levels through the AXH domain; (2) Atx-1 antagonizes the activity of Sens, not that of Ac or Da, in a transcription assay, and (3) Sens overexpression suppresses the Atx-1-induced bristle phenotypes in flies. Similarly, hAtx-1 and Gfi-1 also genetically interact in mice, as evidenced by the worsening of PC pathology in a background heterozygous for a Gfi-1 loss-of function allele. Moreover, hAtx-1 reduces Gfi-1 protein levels in the absence of cell loss. Exactly how Atx-1 promotes the degradation of Sens/Gfi-1 is not known at this time, but the data suggest that Atx-1 enhances the proteasome-mediated degradation of Gfi-1. It is proposed that Atx-1 and Sens/Gfi-1 interactions alter the conformation, modification, or other protein interactions of Sens/Gfi-1, rendering them less stable (Tsuda, 2005).

Given the decline of Gfi-1 levels in PCs of SCA1-transgenic mice, a key question is whether this decrease contributes to SCA1 pathogenesis. Indeed, it is found that loss of Gfi-1 phenocopies SCA1 and causes PC degeneration after several months of normal PC development. Furthermore, loss of one allele of Gfi-1 enhances the motor-incoordination phenotype of SCA1 mice at 4 weeks of age, well before the PCs of these mice develop obvious morphological changes. These data, together with the finding that Gfi-1 and Atx-1 physically interact, highlight the important role of Gfi-1 in PC degeneration and SCA1 pathogenesis. The finding that a transcriptional regulator like Gfi-1 is a major modifier of SCA1 phenotypes and an interactor of Atx-1 is interesting given the proposal that Atx-1 might function in the regulation of gene expression (Tsuda, 2005).

Several neurodegenerative diseases are caused by a gain-of-function mutational mechanism, which renders the host protein toxic to neurons. Examples include mutations in amyloid precursor protein (APP) causing Alzheimer's disease (AD), mutations in tau causing frontotemporal dementia (FTD), and mutations in α-synuclein causing Parkinson's disease (PD). It is interesting that an extra dose of wt APP in Down's syndrome causes early onset AD-like changes and that duplications as well as triplications of wt α-synuclein cause PD. Finding that overexpression of wt Atx-1 causes neurodegeneration is reminiscent of these observations. In fact, accumulation of wt Atx-1 has been noted in patients with neuronal intranuclear inclusion disease in the absence of a CAG expansion of the SCA1 gen. These data collectively suggest that higher than wt levels of proteins such as Atx-1 or α-synuclein are toxic to specific groups of neurons. It is proposed that expansion of the glutamine tract in Atx-1 (and possibly other polyQ proteins) will stabilize them and/or enhance their interaction with other proteins. Thereby, enhancement of wt functions results in the neurodegeneration (Tsuda, 2005).

To date, several Atx-1-interacting proteins have been identified, including leucine-rich acidic nuclear protein, PQBP1, and SMRT. Gfi-1 is the first protein to fulfill the criteria to be a mediator of the SCA1 pathogenesis: it interacts physically and genetically with Atx-1 (as do the fly orthologs), loss of Gfi-1 copies the SCA1 phenotype in PCs, and Gfi-1 and SCA1 show a dose-dependent interaction. The molecular mechanisms described in the current study underscore the importance of studying the wt function and interactors of proteins involved in neurodegenerative diseases (Tsuda, 2005).

Senseless and Daughterless confer neuronal identity to epithelial cells in the Drosophila wing margin

The basic helix-loop-helix (bHLH) proneural proteins Achaete and Scute cooperate with the class I bHLH protein Daughterless to specify the precursors of most sensory bristles in Drosophila. However, the mechanosensory bristles at the Drosophila wing margin have been reported to be unaffected by mutations that remove Achaete and Scute function. Indeed, the proneural gene(s) for these organs is not known. This study shows that the zinc-finger transcription factor Senseless, together with Daughterless, plays the proneural role for the wing margin mechanosensory precursors, whereas Achaete and Scute are required for the survival of the mechanosensory neuron and support cells in these lineages. Evidence is provided that Senseless and Daughterless physically interact and synergize in vivo and in transcription assays. Gain-of-function studies indicate that Senseless and Daughterless are sufficient to generate thoracic sensory organs (SOs) in the absence of achaete-scute gene complex function. However, analysis of senseless loss-of-function clones in the thorax implicates Senseless not in the primary SO precursor (pI) selection, but in the specification of pI progeny. Therefore, although Senseless and bHLH proneural proteins are employed during the development of all Drosophila bristles, they play fundamentally different roles in different subtypes of these organs. The data indicate that transcription factors other than bHLH proteins can also perform the proneural function in the Drosophila peripheral nervous system (Jafar-Nejad, 2006).

In 1978, García-Bellido and Santamaria reported that ac and sc are required for the generation of the majority of the Drosophila bristles. The large body of work that followed this discovery led to the realization that Ac and Sc are members of the bHLH proneural protein family, which are involved in early steps of neurogenesis in flies and vertebrates. Later, two other bHLH genes, atonal and amos, were shown to play the proneural role for almost all SOs that did not depend on Ac and Sc function, with the notable exception of the wing margin (WM) mechanosensory bristles (Garcia-Bellido, 1978). This study shows, based on multiple lines of evidence, that Sens plays the proneural role for these bristles: sens expression in the WM begins before the selection of mechanosensory pIs in a proneural cluster, similar to other proneural proteins; sens expression is upregulated in presumptive pIs and is downregulated in ectodermal cells, just like ac and sc expression is refined to pIs in thoracic proneural clusters; loss and gain of sens function result in loss and gain of SOs in the wing; and Sens synergizes with the Da protein in vivo and in transcription assays, and binds Da in a GST pull-down assay. Unexpectedly, overexpression of the anti-apoptotic protein P35 in the WM results in the generation of a large number of neurons along the PWM, uncovering the neural identity of the PWM bristle precursors. Similar to the AWM (anterior wing margin), the expression pattern and loss of-function phenotype of sens in the PWM (posterior wing margin) indicate a proneural role for sens for the PWM bristles as well. However, the neural potential of the PWM bristles is not realized in the wild-type situation because of apoptosis of the pI progeny, providing an example of the role of apoptotic machinery in diversifying the various sensory lineages. In summary, Sens satisfies all the genetic and developmental criteria for being a proneural protein for the WM bristles, and is the only zinc finger protein shown to play a proneural role in SO development in flies (Jafar-Nejad, 2006).

As for other proneural proteins, the proneural function of Sens requires the function of Da. Da serves as the binding partner for the bHLH proneural proteins to bind E-box sequences and is also able to bind DNA as homodimers. No function has been assigned to Da homodimers in Drosophila, largely because of the identification of tissue-specific bHLH proteins in most contexts in which Da functions. In the WM mechanosensory precursors, however, none of the known tissue-specific bHLH proneural proteins is expressed, suggesting a proneural role for Da homodimers. One might argue that there is probably an unknown dimerization partner for Da in these sensory precursors, and this possibility cannot be excluded. However, two groups have independently identified all Drosophila genes encoding bHLH proteins using database searches of the complete Drosophila genome and none of the newly identified bHLH proteins are predicted to be a transcriptional activator of the Ac-Sc or Atonal families. Also, none of these genes shows an embryonic expression pattern compatible with a proneural function for the CNS. Because da is required for mechanosensory organ formation, and as it can efficiently generate bristles in the absence of ASC, it is proposed that Da homodimers cooperate with Sens to endow neural identity to AWM mechanosensory organs and PWM bristle precursors. The physical interaction of these two proteins and the strong transcriptional synergy between them strongly favors a role in activating key target genes in SO development (Jafar-Nejad, 2006).

These data also reveal that Ac and Sc promote the survival of the WM mechanosensory neurons and support cells independently of pI selection. The more severe loss of neurons compared with support cells associated with the loss of Ac and Sc in sc^10-1 suggests either that the neurons (or their precursors) are more sensitive to the lack of ac and sc function, or that the loss of support cells is secondary to the neuronal death. The observation that adding or removing one copy of wild-type sens strongly modifies the sensory lineage apoptosis observed in sc10-1 animals indicates that, in addition to a proneural function, Sens also plays an anti-apoptotic role in these cells; this is in agreement with many reports on the role of sens and its homologues in mammals and C. elegans in preventing apoptosis. It is interesting to note that although Ac and Sc are not detected in the PWM by antibody staining, P35 overexpression rescues many more neurons in the PWM of wildtype flies than in sc^10-1 animals. This indicates a requirement for Ac and Sc in these cells (Jafar-Nejad, 2006).

During the third instar larval period, low levels of Sens are expressed in the proneural clusters along the AWM that will give rise to the pI cells of the AWM chemosensory bristles. Using in vivo and in vitro assays, it has been shown that low levels of Sens repress, and high levels of Sens activate, ac and sc expression in these proneural clusters, and thereby that Sens is involved in pI selection. Given the similar low-level expression of Sens in thoracic microchaetae proneural clusters and the severe loss of microchaetae in adult sens clones, it had been hypothesized that Sens also functions during proneural upregulation and in the selection of the microchaetae pIs. It was therefore surprising to find that microchaetae pI selection does not require Sens function. Data has been presented on the function of the adaptor protein Phyllopod and its relationship with Sens in microchaetae development. Sens was shown to be required for the function of Phyllopod in the pIs, as well as for timely downregulation of phyllopod expression in epidermal cells. This suggests a dual role for Sens in pIs and surrounding epidermal cells, in agreement with the binary switch model. In contrast, phyllopod expression can still be upregulated in single cells in sens mutant clones, suggesting that pI selection is not disrupted. This study now presents evidence that microchaetae pIs are indeed selected in sens clones and that they divide to generate progeny. However, the mutant pIs exhibit an abnormal division pattern, and a pIIa-to-pIIb transformation is observed, as evidenced by a gain of neurons at the expense of support cells. These data indicate that Sens regulates several aspects of microchaetae precursor development after the pIs are selected (Jafar-Nejad, 2006).

In summary, the normal development of all adult bristles in flies relies on the function of Ac and Sc, Da and Sens. The data indicate that despite the structural and functional similarities between various adult bristles, sens functions at four distinct steps in different lineages. First, in the WM mechanoreceptor and noninnervated lineages, very high levels of Wingless induce the expression of Sens, which assumes a true proneural role and specifies SO fate independently of the typical proneural proteins Ac and Sc. Second, in the WM chemosensory lineages, for which ac and sc are the proneural genes, Sens is required for pI selection, as evidenced by the observation that it represses proneural gene expression in ectodermal cells and activates proneural gene expression in presumptive pIs. Third, even though gain-of-function studies show that Sens is able to induce pI formation in the thorax in the absence of Ac and Sc function, it normally plays a later role in specification of the pIIa versus the pIIb of microchaetae lineages. Fourth, Sens is required for the survival of the pI progeny in the WM mechanosensory lineages. It was also found that ac and sc prevent apoptosis in this lineage independently of pI specification. Finally, the data suggest that a typical Da heterodimeric complex is not required during the formation of the WM mechanosensory and noninnervated bristle pIs. Hence, the cooperation between the same group of genes is adapted in different ways to ensure the proper development of various SOs (Jafar-Nejad, 2006).

The Sens homolog Gfi1 plays important roles in several developmental processes, including inner ear hair cell development, hematopoietic stem cell self-renewal rate, intestinal cell fate specification and neutrophil differentiation. Moreover, Gfi1 has an oncogenic potential and has been implicated in several human diseases, such as hereditary neutropenia, spinocerebellar ataxia type 1 and small cell lung carcinoma. Therefore, given the structural and functional similarities between Gfi1 and Sens, further analysis of the various aspects of Sens function in Drosophila SO development will continue to help unravel the mechanisms of Gfi1 function in health and disease (Jafar-Nejad, 2006).

Senseless physically interacts with proneural proteins and functions as a transcriptional co-activator

The zinc-finger transcription factor Senseless is co-expressed with basic helix-loop-helix (bHLH) proneural proteins in Drosophila sensory organ precursors and is required for their normal development. High levels of Senseless synergize with bHLH proteins and upregulate target gene expression, whereas low levels of Senseless act as a repressor in vivo. However, the molecular mechanism for this dual role is unknown. This study shows that Senseless binds bHLH proneural proteins, including Achaete, Scute, and Daughterless, via its core zinc fingers and is recruited by proneural proteins to their target enhancers to function as a co-activator. Some point mutations in the Senseless zinc-finger region abolish its DNA-binding ability but partially spare the ability of Senseless to synergize with proneural proteins and to induce sensory organ formation in vivo. Therefore, it is proposed that the structural basis for the switch between the repressor and co-activator functions of Senseless is the ability of its core zinc fingers to interact physically with both DNA and bHLH proneural proteins. Since Senseless zinc fingers are ~90% identical to the corresponding zinc fingers of its vertebrate homologue Gfi1, which is thought to cooperate with bHLH proteins in several contexts, the Senseless/bHLH interaction might be evolutionarily conserved (Acar, 2006).

The Sens protein has been shown to act as a transcriptional repressor and activator, depending on its relative abundance in relation to proneural proteins. The reporter construct used in that study consists of the ac proximal enhancer/promoter region upstream of the firefly luciferase coding sequence. This ac enhancer contains a Sens-binding site (S-box: AATC) and three E-boxes, known binding sites for proneural proteins. Proneural proteins heterodimerize with Daughterless (Da) via their bHLH domains and bind to the E-boxes on ac-luc to upregulate transcription. Depending on the amount of ac and da expression constructs transfected, the luciferase expression from this reporter can be increased 10 to 1000 times the basal level. To obtain the optimal sensitivity in the transcription assays, low levels of proneural expression constructs (1-2 ng) were used to assess the transcriptional activation potential of Sens (activation assay), and higher levels of proneurals (10 ng) to assess the transcriptional repression potential of Sens (repression assay). In the absence of Ac and Da, Sens does not activate or repress ac transcription (Acar, 2006).

Based on evolutionary conservation with its vertebrate homologues, Sens can be divided into two domains: an N-terminal domain of 414 amino acids, which shows little homology with other GPS proteins, and a C terminal domain of 127 amino acids, which exhibits strong homology with other GPS proteins and contains four highly conserved C2H2-type Zn fingers. Sens was aligned to its closest homologue from the mosquito Anopheles gambiae, which is thought to have diverged from Drosophila about 180 million years ago, and nine conserved stretches of 6-10 amino acids were found in the Sens N-terminal domain. Mutational analysis of the conserved stretches followed by transcription assays indicate that the individual conserved motifs in the N-terminal domain are not important for the activation and repression mediated on ac by Sens (Acar, 2006).

Four C2H2-type Zn-finger domains of the GPS proteins mediate DNA binding. Deletion analysis of Gfi1 Zn fingers has shown that Zn fingers 3-5 of Gfi1, which correspond to Zn fingers 1-3 of Sens, are required for DNA binding. To begin to assess the precise role of individual Zn fingers in the repressor and activator functions of Sens, each Zn finger in Sens was mutated and the ability of the mutant Sens proteins to bind DNA in electromobility shift assays (EMSA) was assayed. Two types of mutants were generated for each Zn finger. In the first group (Sens-1CC, Sens-2CC, Sens-3CC and Sens-4CC), the two cysteines in the C2H2 structure were mutated to alanines. These mutations probably disrupt the structure of the individual Zn fingers. In the second group (Sens-1RTT, Sens-2QDK, Sens-3QNT and Sens-4RDR), the amino acids were altered that have been predicted to directly contact DNA to alanines. Since these amino acids are not crucial for the Zn-finger structure these mutations should abolish direct contact with specific DNA targets but at least partially preserve the overall Zn-finger structure (Acar, 2006).

To determine protein-DNA interactions and relative binding affinities of the mutant Sens proteins for DNA, two different probes were used in EMSA assays. To detect weak protein-DNA interactions, a previously characterized Gfi1-binding site called R21, to which the wild-type Sens is able to bind strongly, was used as a probe. Sens-1CC, Sens-2CC and Sens-3CC proteins lose their ability to bind the R21 probe, suggesting that Zn fingers 1, 2 and 3 are required for DNA binding. However, in agreement with Gfi1 data, Sens-4CC can bind DNA, suggesting that Zn finger 4 is not essential for DNA binding. The second group of Sens Zn-finger mutants behave somewhat differently in the EMSA. Sens-2QDK, Sens-3QNT and Sens-4RDR behave similarly to their CC counterparts, indicating that the amino acids predicted to directly contact the R21 probe in Zn fingers 2 and 3 are crucially important for DNA binding. However, unlike Sens-1CC, Sens-1RTT is still able to bind the R21 probe, albeit weaker than wild-type Sens and Zn-finger 4 mutants. This difference suggests that although Zn finger 1 is required for DNA binding, its role in DNA binding is more complex than a direct contact between the RTT amino acids and DNA (Acar, 2006).

The S-box in the ac promoter was used as a probe in the EMSA assay to determine the binding affinities of mutant Sens proteins for the endogenous Sens-binding site. Wild-type Sens and Sens-4CC but not Sens-1CC, Sens-2CC nor Sens-3CC are able to bind to the S-box probe. Moreover, in line with the R21 data, the Sens-1RTT binds much weaker than wild-type Sens and Sens-4CC. Note that the Sens-4CC binding affinity for the S-box is weaker than wild-type Sens, suggesting that although Zn finger 4 is not essential for DNA binding, it may increase the strength of Sens-DNA interaction (Acar, 2006).

To determine the importance of each Zn-finger domain for the activation and repression mediated by Sens, the mutants were tested in the S2 cell transcription assay. In the activation assay (ac-da, 2ng), wild-type Sens can synergize with Ac-Da and increase the transcription induced by Ac-Da about 18 times. Sens-2CC and Sens-3CC failed to synergize with Ac-Da. Sens-4CC and especially Sens-1CC exhibited significantly less synergism than wild-type Sens. Similar results were obtained for Sens-1RTT, Sens2-QDK, Sens-3QNT and Sens-4RDR. These data indicate that all Zn fingers cooperate in the Sens/bHLH synergism. However, Zn fingers 2 and 3 are indispensable for this process (Acar, 2006).

To test the ability of the Zn-finger mutants to repress ac transcription, the 'repression assay' was used. Low levels of wild-type Sens repress transcription in this assay and as the Sens to proneural ratio increases, the Sens activity switches from a repressor to an activator. Sens-4CC and Sens-4RDR behave essentially as wild-type proteins in this assay. By contrast, mutations in Zn fingers 1, 2 or 3 abolish the repression function of Sens, corroborating the correlation between Sens DNA binding and repression. Interestingly, Sens-1CC and Sens-1RTT display transcriptional activation at a lower Sens to proneural ratio compared with the wild-type Sens, providing further evidence for the negative contribution of Sens DNA-binding to its ability to synergize with proneural proteins. Similar to the data obtained from the 'activation assay.' Sens proteins with mutations in Zn finger 2 or 3 do not show any premature synergism with Ac-Da, highlighting the role of these core Zn fingers in both synergism and repression. Together, these data indicate specific roles for the Zn fingers in repression and activation (Acar, 2006).

Based on the current data, the following model is proposed for the role of Sens in transcriptional regulation of proneural target genes in sensory precursors. Early in the proneural cluster, proneural gene expression is under the control of proneural and E(spl) proteins. At this stage, proneural genes start to engage in a positive autoregulatory loop by binding to the E-boxes in their own enhancers. Initially, low levels of Sens bind DNA rather than the proneural proteins via its Zn fingers because it has a higher affinity for DNA. When bound to DNA, Sens acts as a repressor. Since Sens interacts with several E(spl) proteins, recruitment of E(spl) through Sens might contribute to the negative regulation of proneural target enhancer. As the level of proneural proteins increases, proneural proteins induce more Sens expression. This will lead to saturation of the S-boxes. Additional Sens will bind proneural proteins via its core Zn-finger domains and act as a co-activator to increase the transcription induced by proneural proteins. It is proposed that the switch between the repressor and co-activator functions of Sens depends on the conformational state of its Zn fingers. In this model, binding to proneural proteins will allow the Sens Zn fingers to adopt an alternative conformation compared to the DNA-bound state. This will enable Sens to cooperate with co-activators already recruited by proneural proteins, or to recruit new co-activators to further increase the ability of proneural proteins to increase the expression of their target genes in some contexts. This conformation-based hypothesis is supported by the observation that even point mutations in Sens Zn fingers that are dispensable for proneural interaction still cause severe reduction in the synergism between Sens and proneural proteins (Acar, 2006).

Multiple lines of evidence suggest that Sens acts as a transcriptional co-activator for bHLH proneural proteins. First, Sens is required for the upregulation and maintenance of proneural gene expression in the wing margin chemosensory SOPs. Second, Sens synergizes with proneural proteins to upregulate the expression of the ac proximal enhancer in S2 cell assays. Third, ectopic expression of Sens induces ectopic proneural gene expression. Fourth, Sens physically binds bHLH proteins via the core region of its Zn-finger domain. Fifth, Sens can not induce transcription in the absence of proneural proteins. It should be mentioned that in vitro and in vivo observations indicate that DNA binding is not essential for the ability of Sens to act as a co-activator and to induce SOP formation. Therefore, since SOPs accumulate high levels of both proneural proteins and Sens, it is likely that proneural target enhancers that do not contain a Sens-binding site might also be a target for proneural-Sens transcriptional synergism (Acar, 2006).

Similar to its vertebrate homologues, Sens can function as a transcriptional repressor when bound to DNA. Mutational analysis of Sens Zn fingers also indicates a link between DNA binding and the repressor function of Sens: those Sens mutants that do not bind DNA (1CC, 2CC and 3CC) fail to repress ac transcription, whereas mutating Zn finger 4, which does not play a major role in DNA binding, does not affect the repressor function of Sens. Although the repressor function seems to be less crucial than the co-activator function in vivo, these data suggest that the repressor function of Sens also contributes to its role in PNS development (Acar, 2006).

Sens physically interacts with proneural proteins via its Zn-finger domains, which are highly conserved between Sens and its vertebrate homologues. In addition, Sens can synergize with the mouse Ato homologue Math1 (Atoh1- Mouse Genome Informatics), when the two proteins are co-expressed in flies. Together, these observations suggest that the Sens-bHLH interaction is evolutionarily conserved. In other words, vertebrate bHLH proteins such as Math1, Mash1 (AScl1- Mouse Genome Informatics) and Math5 (Atoh7- Mouse Genome Informatics), which are co-expressed with Gfi1 in mouse tissues, might be able to recruit Gfi1 to their target enhancers (Acar, 2006).

In conclusion, the data suggest that Sens, a C2H2-type Zn-finger protein, binds to bHLH proneural proteins via its core Zn-finger domains and acts as a co-activator of the expression induced by proneural proteins. Sens can bind to various bHLH proteins and synergize with fly proteins, as well as some of their vertebrate homologues in vivo. These data, together with other examples of Zn-finger/bHLH synergism, suggest that physical and genetic interactions of this type might be a common mechanism for Zn-finger/bHLH cooperation during development (Acar, 2006).

Proneural and abdominal Hox inputs synergize to promote sensory organ formation in the Drosophila abdomen

The atonal (ato) proneural gene specifies a stereotypic number of sensory organ precursors (SOP) within each body segment of the Drosophila ectoderm. Surprisingly, the broad expression of Ato within the ectoderm results in only a modest increase in SOP formation, suggesting many cells are incompetent to become SOPs. This study shows that the SOP promoting activity of Ato can be greatly enhanced by three factors: the Senseless (Sens) zinc finger protein, the Abdominal-A (Abd-A) Hox factor, and the epidermal growth factor (EGF) pathway. First, it was shown that expression of either Ato alone or with Sens induces twice as many SOPs in the abdomen as in the thorax, and does so at the expense of an abdomen-specific cell fate: the larval oenocytes. Second, Ato was shown to stimulate abdominal SOP formation by synergizing with Abd-A to promote EGF ligand (Spitz) secretion and secondary SOP recruitment. However, it was also found that Ato and Sens selectively enhance abdominal SOP development in a Spitz-independent manner, suggesting additional genetic interactions between this proneural pathway and Abd-A. Altogether, these experiments reveal that genetic interactions between EGF-signaling, Abd-A, and Sens enhance the SOP-promoting activity of Ato to stimulate region-specific neurogenesis in the Drosophila abdomen (Gutzwiller, 2010).

How proneural pathways that specify sensory precursor cells throughout the body are integrated with region-specific patterning genes to yield the correct type and number of sensory organs is not well understood. This study shows that three factors enhance the ability of Ato to promote ch organ SOP cell fate in the Drosophila abdomen; the EGF pathway mediated by the Spi ligand, the Abd-A Hox factor, and the Sens zinc finger transcription factor (Gutzwiller, 2010).

EGF signaling is used reiteratively throughout development to specify the formation of distinct cell types along the body plan. In the embryonic Drosophila abdomen, EGF signaling initiated by the activation of rhomboid (rho) in a set of ch organ SOP cells induces the formation of both a cluster of abdomen-specific oenocytes as well as a set of 2° ch organ SOP cells. But how does the EGF-receiving cell know whether to become a larval oenocyte that is specialized to process lipids or a ch organ SOP cell that forms part of the peripheral nervous system? Previous studies have shown that oenocyte specification requires at least two inputs: (1) the reception of relatively high levels of EGF signaling and (2) the expression of the Spalt transcription factors. Hence, oenocytes develop in close proximity to the abdominal C1 SOP cells that lie within a Spalt expression domain and express high levels of rho. In contrast, 2° SOP cells require less EGF signaling and form if the receiving cells lack Spalt. Consistent with this model, genetic studies have shown that oenocytes fail to develop and one to two additional ch organ SOP cells are specified in Spalt mutant embryos, whereas ectopic Spalt expression in the ventral ectoderm inhibits the recruitment of 2° SOP cells. Thus, Spalt promotes oenocyte development and antagonizes 2° ch organ specification in the Drosophila embryo (Gutzwiller, 2010).

Evidence that ato has the opposite effect as Spalt: it promotes ch organ SOP cells at the expense of oenocyte specification. Witt (2010) showed that ato loss-of-function results in decreased expression of activity of the rho enhancer, RhoBAD (Witt, 2010), in C1 SOP cells and induces fewer oenocytes. These data are consistent with EGF signaling being compromised in ato mutant embryos and oenocyte specification being dependent upon the reception of high levels of Spi. This study shows that Ato gain-of-function stimulates RhoBAD expression yet results in the inhibition of oenocyte formation. Importantly, the loss of oenocytes is not due to decreased EGF signaling as similar whorls of phospho-ERK-positive cells and even extra phospho-ERK staining are observed in Ato-expressing segments compared with non-expressing segments. In addition, no difference was detected in cell death between Ato-expressing and non-Ato-expressing segments (using an anti-cleaved Caspase3 marker), indicating the oenocyte loss is not due to apoptosis. Instead, Ato promotes the formation of additional ch organ SOP cells in abdominal segments that normally form oenocytes. Moreover, while the broad activation of EGF signaling (PrdG4;UAS-Rho) induces many extra oenocytes and a few scolopodia, the co-expression of Ato and Rho induces many scolopodia and few oenocytes. These data suggest that if the Spi-receiving cell expresses high Ato relative to Salm then ch organ development occurs whereas if the Spi-receiving cell expresses high Salm relative to Ato then oenocytes are formed. Thus, Ato plays a role in both the Spi-secreting (induction of rho expression) and Spi-receiving cell to dictate the choice of cell fate (Gutzwiller, 2010).

The broad expression of Ato within the ectoderm revealed differences in sensory organ competency between the thorax and abdomen. In particular, it was found that Ato induced approximately twice as many ch organ SOP cells in the abdomen as in the thorax. Moreover, the co-expression of Ato with the Abd-A Hox factor induced significantly more ch organ cell formation than expression of either factor alone (none by Abd-A, four by Ato, and eight by Ato/Abd-A). These data suggest that Ato and Abd-A synergize to enhance ch organ SOP formation in the abdomen, an prompted an examination whethere these SOP cells are predominantly 1° or 2° cells. This problem was first addressed by first showing that the co-expression of Ato and Abd-A stimulates Rho enhancer activity (RhoAAA) within additional cells and results in enhanced phospho-ERK staining. Second, it was shown that Ato and Abd-A require the EGF pathway to enhance ch organ development as co-expression of both factors in a spi mutant embryo failed to promote more ch organs than expression of Ato alone. These data indicate that the co-expression of Ato and Abd-A enhances the ability of 1° ch organ SOP cells to activate rho, stimulates Spi secretion and, since the receiving cell expresses Ato, 2° SOPs form instead of oenocytes. The net result is that Ato and Abd-A synergize to activate the EGF pathway to promote region-specific neurogenesis within the Drosophila abdomen (Gutzwiller, 2010).

The Sens transcription factor is essential for the formation of much of the peripheral nervous system in Drosophila and previous studies revealed that Sens can stimulate the sensory bristle-forming activity of the Scute and Achaete proneural factors in the wing disc. Similarly, it was found that Sens stimulates the ability of Ato to generate internal stretch receptors in the embryo and that Ato and Sens promote more sensory organ development in the abdomen than in the thorax. In addition, while the overall number of ch organs formed by Ato and Sens co-expression is decreased in spi mutant embryos, significantly more ch organ SOP cells in the abdomen than in the thorax are observed in this EGF-compromised genetic background. Thus, Ato and Sens can stimulate abdominal ch organ SOP cell development in the presence or absence of Spi-mediated cell signaling (Gutzwiller, 2010).

So, what is the relationship between Ato, Sens, and Abd-A in regulating both EGF signaling and region-specific sensory organ formation? It was previously found that Ato, Sens, and Abd-A control EGF signaling through the regulation of a cis-regulatory element within the rhomboid (rho) locus (RhoBAD) (Li-Kroeger, 2008; Witt, 2010). RhoBAD acts in abdominal C1 SOP cells to induce oenocyte formation, and Ato and Abd-A both stimulate RhoBAD expression, at least in part, by limiting the ability of Sens to repress RhoBAD activity. Moreover, they do so using different mechanisms. An Abd-A Hox complex containing Extradenticle and Homothorax directly competes with the Sens repressor for overlapping binding sites in RhoBAD (Li-Kroeger, 2008). In contrast, Ato does not directly bind RhoBAD but does directly interact with Sens to limit its ability to bind and repress Rho enhancer activity (Witt, 2010). Consequently, SOPs that co-express Ato and Abd-A are likely to limit the ability of Sens to repress Rho and thereby increase the number of ch organ SOP cells that secrete Spi. Consistent with this prediction, the co-expression of Ato and Sens preferentially stimulates Rho enhancer activity within abdominal segments compared to thoracic segments. Each SOP cell that expresses rho would further enhance sensory organ development through the recruitment of 2° SOP cells via Spi-mediated signaling. Hence, the genetic removal of spi results in a significant decrease in the number of ch organ SOP cells that develop in response to Ato and Sens. Thus, the ato-sens genetic pathway, which is used throughout the body to promote SOP formation, interacts with an abdominal Hox factor to stimulate EGF signaling and promote additional cell fate specification in the abdomen (Gutzwiller, 2010).

While the above model fits well with most of the data, two unexpected findings were observed when comparing the ability of Ato-Sens co-expression to induce ch organ development in the presence and absence of spi function: First, it was predicted that Ato-Sens co-expression in the thoracic regions, which lack Abd-A, should predominantly induce the formation of 1° ch organ SOP cells that do not require EGF signaling for their development. However, it was found that significantly fewer ch organs form in the thorax of spi mutants, indicating that EGF signaling can enhance 2° sensory organ formation within thoracic segments that co-express Ato and Sens. Interestingly, previous studies have shown that both rho and the Rho enhancers are weakly active within thoracic C1 SOP dcells, but their levels do not reach a high enough threshold to induce oenocyte formation. However, it is possible that the co-expression of Ato and Sens sufficiently sensitizes the receiving cells to respond to low levels of EGF signaling and become ch organ SOP cells. The second unanticipated finding is that Ato and Sens co-expression still induced significantly more ch organ development within the abdomen (5-6 extra SOP cells) relative to the thorax (1-2 extra SOP cells) in the absence of Spi-mediated signaling. This finding suggests that Ato and Sens can genetically interact with the Abd-A Hox factor to promote sensory organ development in an Spi-independent manner. Currently, it is not understood how Abd-A enhances the proneural activity of the Ato-Sens factors in the absence of Spi signaling. One possibility is that Abd-A and Ato use similar mechanisms to limit Sens-mediated repression of additional target genes besides rho to stimulate ch organ development. Alternatively, Abd-A could independently regulate other factors such as those involved in the Notch-Delta pathway to enhance the competency of the ectoderm to respond to the Ato-Sens pathway. Intriguingly, a Hox factor (lin-39) in C. elegans has been shown to directly regulate Notch signaling during vulval development, and the vertebrate Hoxb1 factor regulates neural stem cell progenitor proliferation and maintenance by modulating Notch signaling. Since differential Notch-Delta signaling is a key pathway in deciding neural versus non-neural cell fates, the ability of Hox factors to modify this pathway could result in segmental differences in neurogenesis (Gutzwiller, 2010).

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