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 (m7ACT), 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 m7ACT is able to activate ac and sc transcription. Therefore, it is formally possible that m7ACT 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 m7ACT 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 m7ACT able to induce sens expression in the absence of ac and sc? To address this question, it was confirmed that overexpression of m7ACT can produce several extra bristles in a sc10-1 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. acHw-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 acHw-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 sc10-1 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 sc10-1 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 sc10-1 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 sc10-1 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 sc10-1 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).
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)scB57, 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)y3PC sc8R] 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 neurA101-lacZ. Loss of neurA101-lacZ expression has been attributed to a 'general sickness' of the mutant discs, since the lack of neurA101-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 neurA101. 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 sdETX4 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 sdETX4 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 sdETX4 enhances the wing phenotype to resemble the result of the enhancement of sdETX4 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).
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).
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).
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 roX63 and scaBP2 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).
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).
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
phyl4.1-GFP as a reporter. At 10-12 h APF,
phyl4.1-GFP is expressed in dorsoventral stripes along
the notum in a pattern analogous to early Ac and Sc expression patterns.
At 15 h APF, phyl4.1-GFP expression is
restricted in SOPs.
In sensE2-null clones,
phyl4.1-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,
phyl4.1-GFP expression in sensE2
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 phyl4.1-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
phyl3.4DeltaS-GFP
reporter with all three S boxes
mutated expresses little difference in the GFP pattern and intensity when
compared to phyl3.4-GFP. However, the
reporter with mutations in all four E boxes and three S boxes
(phyl3.4DeltaES-GFP)
enhances GFP intensity by 20%
when compared to phyl3.4DeltaE-GFP
with mutations only in four E boxes. This
20% increase in GFP intensity reflects an increase in the phyl activity
in vivo because phyl3.4DeltaES-ORF
shows stronger abilities than phyl3.4E-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
sensE2 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).
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 targ